Anti-Aβ globulomer 7C6 antibodies
09540432 · 2017-01-10
Assignee
Inventors
- Stefan Barghorn (Mannheim, DE)
- Heinz Hillen (Hassloch, DE)
- Boris Labkovsky (Wales, MA)
- Andreas R. Striebinger (Speyer, DE)
- Patrick Keller (Darmstadt, DE)
- Ulrich Ebert (Mannheim, DE)
Cpc classification
C07K2317/41
CHEMISTRY; METALLURGY
C07K14/4711
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
A61P25/28
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
International classification
Abstract
Anti-A globulomer antibodies, antigen-binding moieties thereof, corresponding hybridomas, nucleic acids, vectors, host cells, methods of producing said antibodies, compositions comprising said antibodies, uses of said antibodies and methods of using said antibodies. The present invention relates to anti-A globulomer antibodies having a binding affinity to A(20-42) globulomer that is greater than the binding affinity of the antibody to A(1-42) globulomer, antigen-binding moieties thereof, hybridomas producing said antibodies, nucleic acids encoding said antibodies, vectors comprising said nucleic acids, host cells comprising said vectors, methods of producing said antibodies, compositions comprising said antibodies, therapeutic and diagnostic uses of said antibodies and corresponding methods relating to Alzheimer's disease and other amyloidoses.
Claims
1. An antibody selected from the group consisting of an isolated antibody, a monoclonal antibody and a recombinant antibody, wherein (a) said antibody has a binding affinity to an A(20-42) globulomer that is greater than the binding affinity of the antibody to an A(1-42) globulomer; (b) the binding affinity of the antibody to the A(20-42) globulomer is at least 10 times greater than the binding affinity of the antibody to the A(1-42) globulomer; (c) the antibody is humanized and binds to the same epitope as a monoclonal antibody 7C6 obtainable from a hybridoma designated by American Type Culture Collection deposit number PTA-7240; (d) the antibody comprises (i) a VH 7C6 CDR1 consisting of amino acid residues 31-35 of SEQ ID NO: 11, (ii) a VH 7C6 CDR2 consisting of amino acid residues 50-65 of SEQ ID NO: 11, (iii) a VH 7C6 CDR3 consisting of amino acid residues 98-107 of SEQ ID NO:11, (iv) a VL 7C6 CDR1 consisting of amino acid residues 24-39 of SEQ ID NO: 12, (v) a VL 7C6 CDR2 consisting of amino acid residues 55-61 of SEQ ID NO: 12, and (vi) a VL 7C6 CDR3 consisting of amino acid residues 94-102 of SEQ ID NO:12.
2. Antigen-binding moiety of an antibody of claim 1.
3. The antigen-binding moiety of claim 2, wherein the moiety is selected from the group consisting of a Fab fragment, a F(ab).sub.2 fragment and a single chain Fv fragment of the antibody.
4. A composition comprising said antibody of claim 1 or said antigen-binding moiety of claim 2.
5. The composition of claim 4, wherein said composition is a pharmaceutical composition and further comprises a pharmaceutical acceptable carrier.
6. An antibody selected from the group consisting of an isolated antibody, a monoclonal antibody and a recombinant antibody, wherein said antibody comprises two variable domains, wherein said two variable domains are SEQ ID NO:11 and SEQ ID NO:12.
7. The antibody of claim 6, wherein the antibody comprises a constant region.
8. The antibody of claim 7, wherein the antibody comprises a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD and IgE constant regions.
9. The antibody of claim 8, wherein the antibody comprises an IgG1 heavy chain constant region.
10. The antibody of claim 8, wherein the constant region is a human constant region.
11. The antibody of claim 7, wherein the constant region comprises the amino acid sequence of SEQ ID NO:39.
12. The antibody of claim 11, wherein the antibody possesses a human glycosylation pattern.
13. Monoclonal antibody (7C6) obtainable from a hybridoma designated by American Type Culture Collection deposit number PTA-7240.
14. Hybridoma designated by an American Type Culture Collection deposit number PTA-7240.
Description
(1) In the drawings:
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(13) TABLE-US-00003 FIG. 11 SEQ ID NO: Sequence Type Chain mAb A1 1 nucleotide variable heavy (VH) 5F7 A2 2 nucleotide variable light (VL) 5F7 A1 3 amino acid variable heavy (VH) 5F7 A2 4 amino acid variable light (VL) 5F7 B1 5 nucleotide variable heavy (VH) 10F11 B2 6 nucleotide variable light (VL) 10F11 B1 7 amino acid variable heavy (VH) 10F11 B2 8 amino acid variable light (VL) 10F11 C1 9 nucleotide variable heavy (VH) 7C6 C2 10 nucleotide variable light (VL) 7C6 C1 11 amino acid variable heavy (VH) 7C6 C2 12 amino acid variable light (VL) 7C6 D1 13 nucleotide variable heavy (VH) 4B7 D2 14 nucleotide variable light (VL) 4B7 D1 15 amino acid variable heavy (VH) 4B7 D2 16 amino acid variable light (VL) 4B7 E1 17 nucleotide variable heavy (VH) 2F2 E2 18 nucleotide variable light (VL) 2F2 E1 19 amino acid variable heavy (VH) 2F2 E2 20 amino acid variable light (VL) 2F2 F1 21 nucleotide variable heavy (VH) 6A2 F2 22 nucleotide variable light (VL) 6A2 F1 23 amino acid variable heavy (VH) 6A2 F2 24 amino acid variable light (VL) 6A2 G1 25 nucleotide variable heavy (VH) 4D10 G2 26 nucleotide variable light (VL) 4D10 G1 27 amino acid variable heavy (VH) 4D10 G2 28 amino acid variable light (VL) 4D10 H1 29 nucleotide variable heavy (VH) 7E5 H2 30 nucleotide variable light (VL) 7E5 H1 31 amino acid variable heavy (VH) 7E5 H2 32 amino acid variable light (VL) 7E5 I1 33 nucleotide variable heavy (VH) 10C1 I2 34 nucleotide variable light (VL) 10C1 I1 35 amino acid variable heavy (VH) 10C1 I2 36 amino acid variable light (VL) 10C1 J1 37 nucleotide variable heavy (VH) 3B10 J1 38 amino acid variable heavy (VH) 3B10
(14) The following examples are intended to illustrate the invention, without limiting its scope
Example 1
Preparation of Globulomers
(15) a) A(1-42) Globulomer
(16) The A(1-42) synthetic peptide (H-1368, Bachem, Bubendorf, Switzerland) was suspended in 100% 1,1,1,3,3,3-hexafluoro-2-propano (HFIP) at 6 mg/mL and incubated for complete solubilization under shaking at 37 C. for 1.5 h. The HFIP acts as a hydrogen-bond breaker and is used to eliminate pre-existing structural inhomogeneities in the A peptide. HFIP was removed by evaporation in a SpeedVac and A(1-42) resuspended at a concentration of 5 mM in dimethylsulfoxide and sonicated for 20 s. The HFIP-pre-treated A(1-42) was diluted in phosphate-buffered saline (PBS) (20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4) to 400 M and 1/10 volume 2% sodium dodecyl sulfate (SDS) (in H.sub.2O) added (final concentration of 0.2% SDS). An incubation for 6 h at 37 C. resulted in the 16/20-kDa A(1-42) globulomer (short form for globular oligomer) intermediate. The 38/48-kDa A(1-42) globulomer was generated by a further dilution with three volumes of H.sub.2O and incubation for 18 h at 37 C. After centrifugation at 3000 g for 20 min the sample was concentrated by ultrafiltration (30-kDa cut-off), dialysed against 5 mM NaH.sub.2PO.sub.4, 35 mM NaCl, pH 7.4, centrifuged at 10000 g for 10 min and the supernatant comprising the 38/48-kDa A(1-42) globulomer withdrawn. As an alternative to dialysis the 38/48-kDa A(1-42) globulomer could also be precipitated by a ninefold excess (v/v) of ice-cold methanol/acetic acid solution (33% methanol, 4% acetic acid) for 1 h at 4 C. The 38/48-kDa A(1-42) globulomer is then pelleted (10 min at 16200 g), resuspended in 5 mM NaH.sub.2PO.sub.4, 35 mM NaCl, pH 7.4, and the pH adjusted to 7.4.
(17) b) Cross-Linked A(1-42) Globulomer
(18) The A(1-42) synthetic peptide (H-1368, Bachem, Bubendorf, Switzerland) was suspended in 100% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 6 mg/mL and incubated for complete solubilization under shaking at 37 C. for 1.5 h. The HFIP acts as a hydrogen-bond breaker and was used to eliminate pre-existing structural inhomogeneities in the A peptide. HFIP was removed by evaporation in a SpeedVac and A(1-42) resuspended at a concentration of 5 mM in dimethylsulfoxide and sonicated for 20 s. The HFIP-pre-treated A(1-42) was diluted in phosphate-buffered saline (PBS) (20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4) to 400 M and 1/10 volume 2% sodium dodecyl sulfate (SDS) (in H.sub.2O) added (final concentration of 0.2% SDS). An incubation for 6 h at 37 C. resulted in the 16/20-kDa A(1-42) globulomer (short form for globular oligomer) intermediate. The 38/48-kDa A(1-42) globulomer was generated by a further dilution with three volumes of H.sub.2O and incubation for 18 h at 37 C. Cross-linking of the 38/48-kDa A(1-42) globulomer was now performed by incubation with 1 mM glutaraldehyde for 2 h at 21 C. room temperature (RT) followed by ethanolamine (5 mM) treatment for 30 min at RT.
(19) c) A(20-42) Globulomer
(20) 1.59 ml of A(1-42) globulomer preparation prepared according to example 1a were admixed with 38 ml of buffer (50 mM MES/NaOH, pH 7.4) and 200 l of a 1 mg/ml thermolysin solution (Roche) in water. The reaction mixture was stirred at RT for 20 h. Then 80 l of a 100 mM EDTA solution, pH 7.4, in water were added and the mixture was furthermore adjusted to an SDS content of 0.01% with 400 l of a 1% strength SDS solution. The reaction mixture was concentrated to approx. 1 ml via a 15 ml 30 kDa Centriprep tube. The concentrate was admixed with 9 ml of buffer (50 mM MES/NaOH, 0.02% SDS, pH 7.4) and again concentrated to 1 ml. The concentrate was dialyzed at 6 C. against 1 I of buffer (5 mM sodium phosphate, 35 mM NaCl) in a dialysis tube for 16 h. The dialysate was adjusted to an SDS content of 0.1% with a 2% strength SDS solution in water. The sample was centrifuged at 10000 g for 10 min and the A(20-42) globulomer supernatant was withdrawn
(21) d) A(12-42) Globulomer
(22) 2 ml of an A(1-42) globulomer preparation prepared according to example 1a were admixed with 38 ml buffer (5 mM sodium phosphate, 35 mM sodium chloride, pH 7.4) and 150 l of a 1 mg/ml GluC endoproteinase (Roche) In water. The reaction mixture was stirred for 6 h at RT, and a further 150 l of a 1 mg/ml GluC endoproteinase (Roche) in water were subsequently added. The reaction mixture was stirred at RT for another 16 h, followed by addition of 8 l of a 5 M DIFP solution. The reaction mixture was concentrated to approx. 1 ml via a 15 ml 30 kDa Centriprep tube. The concentrate was admixed with 9 ml of buffer (5 mM sodium phosphate, 35 mM sodium chloride, pH 7.4) and again concentrated to 1 ml. The concentrate was dialyzed at 6 C. against 1 l of buffer (5 mM sodium phosphate, 35 mM NaCl) In a dialysis tube for 16 h. The dialysate was adjusted to an SDS content of 0.1% with a 1% strength SDS solution in water. The sample was centrifuged at 10000 g for 10 min and the A(12-42) globulomer supernatant was withdrawn.
Example 2
Size-Exclusion Chromatography of Different A(1-42) Monomer and A(1-40) Monomer Preparations
(23) A(1-42), 0.1% NH.sub.4OH:
(24) 1 mg of A(1-42) (Bachem, catalogue no. H-1368) were dissolved in 500 l of 0.1% NH.sub.4OH in H.sub.2O and agitated for 1 min at ambient temperature. The sample was centrifuged for 5 min at 10,000 g. The supernatant was collected. A(1-42) concentration in the supernatant was determined according to Bradford's method (BIO-RAD).
(25) 5 min sample:
(26) 20 l of A(1-42) in the 0.1% NH.sub.4OH containing supernatant were diluted with 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4 to an A(1-42) concentration of 0.2 mg/ml. The sample was incubated for 5 min at ambient temperature. Then 100 l were analyzed by size exclusion chromatography (SEC).
(27) 1 hour sample:
(28) 20 l of A(1-42) in the 0.1% NH.sub.4OH containing supernatant were diluted with 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4 to an A(1-42) concentration of 0.2 mg/ml. The sample was incubated for 1 hour at ambient temperature. Then 100 l were analyzed by size exclusion chromatography (SEC).
(29) A(1-42), 70% HCOOH:
(30) 1 mg of A(1-42) were dissolved in 50 l 70% HCOOH in H.sub.2O and agitated 1 min at ambient temperature. The sample was centrifuged for 5 min at 10,000 g. The supernatant was collected. A(1-42) concentration in the supernatant is determined according to Bradford's method (BIO-RAD).
(31) 5 min sample:
(32) 2 l of A(1-42) in 70% HCOOH were diluted to a concentration of 0.2 mg/ml A(1-42) with 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4 and adjusted to pH 7.4 with 1 M NaOH. The sample was incubated for 5 min at ambient temperature. Then 100 l were analyzed by size exclusion chromatography.
(33) 1 hour sample:
(34) 2 l of A(1-42) in 70% HCOOH were diluted to a concentration of 0.2 mg/ml A(1-42) with 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4 and adjusted to pH 7.4 with 1 M NaOH. The sample was incubated for 1 hour at ambient temperature. Then 100 l were analyzed by size exclusion chromatography.
(35) A(1-42), 0.1% NaOH:
(36) 1 mg of A(1-42) (Bachem, catalogue no. H-1368) were dissolved in 500 l of 0.1% NaOH in H.sub.2O and agitated 1 min at ambient temperature. The sample was centrifuged for 5 min at 10,000 g. The supernatant was collected. A(1-42) concentration in the supernatant is determined according to Bradford's method (BIO-RAD).
(37) 5 min sample:
(38) 20 l of A(1-42) in 0.1% NaOH were diluted to a concentration of 0.2 mg/ml A(1-42) with 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4. The sample was incubated for 5 min at ambient temperature. Then 100 l were analyzed by size exclusion chromatography.
(39) 1 hour sample:
(40) 20 l of A(1-42) in 0.1% NaOH were diluted to a concentration of 0.2 mg/ml A(1-42) with 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4. The sample was incubated for 1 hour at ambient temperature. Then 100 l were analyzed by size exclusion chromatography.
(41) A(1-40), 0.1% NaOH:
(42) 1 mg of A(1-40) (Bachem, catalogue no. H-1194) were dissolved in 500 l of 0.1% NaOH in H.sub.2O and agitated 1 min at ambient temperature. The sample is was centrifuged for 5 min at 10,000 g. The supernatant was collected. A(1-42) concentration in the supernatant was determined according to Bradford's method (BIO-RAD).
(43) 5 min sample:
(44) 20 l of A(1-40) in 0.1% NaOH were diluted to a concentration of 0.2 mg/ml A(1-40) with 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4. The sample was incubated for 5 min at ambient temperature. Then 100 l were analyzed by size exclusion chromatography.
(45) 1 hour sample:
(46) 20 l of A(1-40) in 0.1% NaOH were diluted to a concentration of 0.2 mg/ml A(1-40) with 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4. The sample was incubated for 1 hour at ambient temperature. Then 100 l were analyzed by size exclusion chromatography.
(47) Conditions for size exclusion chromatography (SEC):
(48) SEC column: Superose 12 HR 10/300 GL (Amersham, catalogue no. 17-5173-01)
(49) Flow: 0.5 ml/min
(50) Paper feed: 0.2 cm/min
(51) Extinction at 214 nm: 0-0.2 absorption units
(52) Mobile phase: 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4 Results are shown in
(53) The preparation of a purely monomeric A-solution is a great challenge due to the strong tendency of the A peptide, especially the A(1-42) monomer, to aggregate into fibrils. Nevertheless, for the screening and characterization of anti-A(20-42) globulomers that discriminate A(1-42)-monomers and A(1-40)-monomers the best technically achievable A-monomeric preparation should be used. Here the effect of the initial solvent of the A peptide on the aggregation effect after further dilution into 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4 was tested. The A peptide supplier (Bachem) states in their technical information that the A(1-42) should be solubilized in 0.1% NH.sub.4OH. Five minutes at room temperature (RT) after solubilizing the A(1-42) in NH.sub.4OH and immediate further 1:10 dilution in 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4 a size-exclusion chromatography shows first signs of A(1-42) aggregation to fibrillary precursors with a minor peak at 74 kD. Monomeric A(1-42) runs at a major peak with 11 kD and a shoulder at 6 kD. After incubation for one hour at room temperature (RT) the A(1-42) peptide in NH.sub.4OH has already aggregated to a high extent to A(1-42) fibrils leading to a loss of detectable material that did not enter the size-exclusion chromatographic column. If 70% formic acid is used as the initial solvent for A(1-42) peptide a high extent of aggregation after 1 h at RT occurs with only a minor fraction A(1-42) monomer left (note that the formic acid itself leads to a high background absorption at the protein detection wavelength). The best initial solvent for A(1-42) to prevent aggregation is 0.1% NaOH which even after 1 h incubation of solubilization and further dilution shows only a minor fraction of aggregated A(1-42) with the majority of A(1-42) being still monomeric. A(1-40) solubilized initially in 0.1% NaOH shows no signs at all of aggregation even after 1 h at RT incubation.
Example 3
Semi-Quantitative Analysis Visualized by SDS-PAGE of the Discrimination of A(20-42) Globulomer Selective Antibodies for A(1-42) Fibrils
(54) A(1-42) fibril preparation:
(55) 1 mg of A(1-42) (Bachem, Cat. no.: H-1368) were dissolved in 500 l 0.1% NH.sub.4OH in H.sub.2O and agitated for 1 min at ambient temperature. The sample was centrifuged for 5 min at 10,000 g. The supernatant was collected. A(1-42) concentration in the supernatant was determined according to Bradford's method (BIO-RAD).
(56) 100 l of A(1-42) in 0.1% NH.sub.4OH were mixed with 300 l of 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4 and adjusted to pH 7.4 with 2% HCl. The sample was then incubated at 37 C. for 20 hours. Following which the sample was centrifuged for 10 min at 10,000 g. The supernatant was discarded, and the residue was mixed with 400 l of 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4, resuspended by vigorous agitation (vortexing) for 1 min and centrifuged for 10 min at 10,000 g. The supernatant was discarded and the residue was mixed with 400 l of 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4, resuspended by vigorous agitation (vortexing) for 1 min and centrifuged for 10 min at 10,000 g once more. The supernatant was discarded. The residue was resuspended in 380 l of 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4 and prompted by vigorous agitation (vortexing).
(57) Binding of anti-A antibodies to A(1-42) fibrils:
(58) 40 l of A(1-42) fibril preparation were diluted with 160 l of 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, 0.05% Tween 20, pH 7.4 and agitated 5 min at ambient temperature, then the sample was centrifuged for 10 min at 10,000 g. The supernatant was discarded, and the residue was resuspended in 95 l of 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, 0.05% Tween 20, pH 7.4. Resuspension was prompted by vigorous agitation (vortexing).
(59) Aliquots of 10 l of the fibril preparation were each mixed with: a) 10 l 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4 b) 10 l 0.5 g/l of 5F7 in 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4 c) 10 l 0.5 g/l of 6E10 (Signet Nr.: 9320) in 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4
(60) The samples were incubated at 37 C. for 20 hours, then centrifuged for 10 min at 10,000 g. The supernatants were collected and mixed with 20 l of SDS-PAGE sample buffer. The residues were mixed with 50 l of 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, 0.025% Tween 20, pH 7.4 and resuspended by vortexing, then the samples were centrifuged for 10 min at 10,000 g. The supernatants were discarded, and the residues were mixed with 20 l 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, 0.025% Tween 20, pH 7.4, then with 20 l of SDS-PAGE sample buffer. The samples were applied to a 4-20% Tris/glycine gel for electrophoresis.
(61) Parameters for SDS-PAGE: SDS sample buffer. 0.3 g SDS 4 ml 1 M Tris/HCl pH 6.8 8 ml glycerine 1 ml 1% bromphenol blue in ethanol Fill with H.sub.2O ad 50 ml 4-20% Tris/Glycine Gel: (Invitrogen, Cat. no.: EC6025BOX) Electrophoresis buffer: 7.5 g Tris 36 g Glycine 2.5 g SDS Fill with H.sub.2O ad 2.5 l The gel is run at a constant current of 20 mA. Staining of the gels: Coomassie Blue R250
(62) Results are shown in
(63) Semiquantitative analysis of different anti-A antibodies and their discrimination of A(1-42) fibrils. Positions of antibodies, A(1-42) fibrils and A(1-42) monomers are marked at the edge of the gel. Due to their size, A(1-42) fibrils cannot enter the SDS-PAGE gel and can be seen in the gel slot. 1. Marker 2. A(1-42) fibril preparation; control 3. A(1-42) fibril preparation; +mAb 5F7; 20 h 37 C.; supernatant 4. A(1-42) fibril preparation; +mAb 5F7; 20 h 37 C.; pellet 5. A(1-42) fibril preparation; +mAb 6E10; 20 h 37 C.; supernatant 6. A(1-42) fibril preparation; +mAb 6E10; 20 h 37 C.; pellet
(64) The relative binding to fibril type A was evaluated from SDS-PAGE analysis by measuring the Optical Density (OD) values from the Heavy Chain of the antibodies in the fibril bound (pellet-fraction) and the supernatant fractions after centrifugation. Antibodies that have bound to the As fibrils should be co-pelleted with the A-fibrils and therefore are found in the pellet fraction whereas non-A-fibril bound (free) antibodies are found in the supernatant. The percentage of antibody bound to A-fibrils was calculated according to the following formula:
Percent antibody bound to A-fibrils=OD.sub.fibril fraction100%/(OD.sub.fibril fraction+OD.sub.supernatant fraction).
(65) This procedure was performed for the mAbs 6E10 (Signet, Cat. no.: 9320), 5F7, 2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1.
(66) In the Alzheimer disease brain the A fibrils are a major component of the total A peptide pool. By attacking these fibrils by anti A-antibodies the risk of negative side effects is elevated due to a liberation of high amounts of A which subsequently may increase the risk of microhaemorrhages. An increased risk for microhaemorrhages was observed in an active Immunization approach with fibrillar aggregates of the A peptide (Bennett and Holtzman, 2005, Neurology, 64, 10-12; Orgogozo J, Neurology, 2003, 61, 46-54; Schenk et al., 2004, Curr Opin Immunol, 16, 599-606).
(67) In contrast to the commercially available antibody 6E10 (Signet 9320) which recognizes a linear A-epitope between AA1-17, the A(20-42) globulomer selective antibody 5F7 (which actually has the lowest selectivity for A(20-42) globulomers over other A-forms) does not bind to A(1-42) fibrils in an co-pelleting experiment. This is shown by the fact that the 5F7 antibody after an incubation with A(1-42) fibrils remains after a pelleting step in the supernatant and is not co-pelleted because of being bound to the A(1-42) fibrils.
Example 4
Analysis of Cognitive Performance in Mice by Means of an Object Recognition Test after Active Immunization with A(1-42) Monomer (0.1% NH4OH), A(1-42) Globulomer or A(20-42) Globulomer in Comparison to Wild Type
(68) In these experiments mice overexpressing human APP with a point mutation were used. The point mutation refers to amino acid 717 (substitution of isoleucine for valine) and has been found in a London family where it leads to onset of AD before the beginning of the sixth decade of life (Mullan et al., Nature Genetics 2 (1992) 340-342). The transgenic mice, herein referred to as APP/L, were created by and first described in Leuven (Moechars et al., J. Biol Chem. 274 (1999) 6483-6492). Female APP/L mice were subjected to active immunization at 6 weeks of age.
(69) The mice received either 100 g of A(1-42) monomer (0.1% NH.sub.4OH), A(1-42) globulomer or A(20-42) globulomer in phosphate-buffered saline (PBS) mixed with an equal amount of complete Freund's adjuvant intraperitoneally, followed by booster injections with the same a mount of antigene in incomplete Freund's adjuvant every third week for three months. Throughout the time course of the experiment the animals were kept under standard conditions in a reverted day/night cycle (14 hours of light beginning at 7 pm/10 hours of darkness). Body weight gain over the time of the experiment was as expected and did not differ from a control group which received PBS/adjuvant alone, suggesting that the antigen treatments were well tolerated.
(70) At 4.5 month of age cognitive ability of the mice was tested by an object recognition test as described in the art (Dewachter et al. Journal of Neuroscience 22 (2002) 3445-3453). To this end, mice were accustomed to an arena and then exposed for 10 minutes to an acquisition phase during which they were individually placed in the arena which now contained two identical elements (blue pyramid, green cube, yellow cylinder of similar size, ca. 4 cm). The duration and frequency with which the mouse explored the objects were recorded. During retention phase, 2.5 h later, mice were returned to the arena which now contained, in addition to the known object, an unknown object randomly selected from the other objects. Recognition of the new object was recorded as the time during which the mouse was exploring the old object relative to total time (exploration of old and new object). The recognition index expresses this relation (time for new object/total time). A mouse which does not remember the known object will consider it as equally interesting as the new object and spend an equal amount of time on exploring it, in other words, will show a recognition index of 50%. A mouse which remembers the known object will consider it as not interesting and therefore show a significantly higher recognition index. APP/L mice are known to be cognitively deficient at 4.5 months of age and exhibit a recognition index in the dimension of the random level, i.e. 50%.
(71) Results are shown in
(72) Object recognition test in mice. The test reports recognition of a known object in comparison to an unknown one, measured in terms of explorative behaviour during a 10 minute test phase. The recognition Index is defined as the percentage of time which the mouse spends on exploring the unknown object relative to the time spent on exploring both objects. The known object was explored by the mouse during a 10 minute acquisition phase three hours before the test phase. Five groups of mice (number n given below the columns) were compared. Normal C57Bl/6 mice (wild type) show a high RI significantly different from random level (50%, i.e. equal times of exploration spent on both the known and the unknown object) (**=p<0.001; Student's t-test). The other four groups of APP transgenic mice were subjected to active immunisation three months before. The immunogens used were A(1-42) monomer, A(1-42) globulomer and A(20-42) globulomer. Phosphate-buffered saline (PBS) was used as control. Significant differences between PBS and the other groups are indicated with circles: =p<0.05; =p<0.01 (post-hoc t-test after p<0.05 in ANOVA).
(73) APP/L mice are known to show, in contrast to non-transgenic mice, a cognitive deficiency at 4.5 months of age, scoring results close to random level (i.e. 50% recognition index). In fact, the PBS-treated mice showed random behaviour, in contrast to non-transgenic mice (wild type). Immunization with native A(1-42) globulomer as well as with A(20-42) globulomer resulted in significantly Improved object recognition in APP/L mice.
(74) As both globulomer preparations (native and truncated) resulted in memory improvement in APP transgenic animals and even superior recognition in animals treated with A(20-42) globulomer it is reasonable to assume that induction of antibodies against truncated A(20-42) globulomer will produce the best result, and that passive immunisation with antibodies reacting specifically with this species represents the optimal strategy of treatment.
Example 5
Dot-Blot Analysis of the Antibody Profile for Different A-Forms after an Active Immunization of APP/L Tg mice with A(20-42) Globulomer
(75) After immunization of mice (compare example 4) of APP/L mice (Moechars et al., 1999, J. Biol. Chem. 274, 6483-6492) with different forms of AP, plasma samples were assessed for anti-A antibodies To this end, dilution series of the individual A(1-42) forms ranging from 100 pmol/l to 0.01 pmol/l in PBS supplemented with 0.2 mg/ml BSA were made. 1 l of each sample was blotted onto a nitrocellulose membrane. For detection the corresponding mouse plasma samples were used (diluted 1:400). Immunostaining was done using alkaline phosphatase conjugated anti-mouse-IgG and the staining reagent NBT/BCIP.
(76) A-standards for dot-blot:
(77) 1. A(1-42) Globulomer
(78) The preparation of the A(1-42) globulomer is described in example 1a.
(79) 2. HFIP Pretreated A(1-42) Monomer in Pluronic F68
(80) 3 mg of A(1-42), (Bachem Inc; cat no. H-1368) were dissolved in 0.5 ml HFIP (6 mg/ml suspension) in an 1.7 ml Eppendorff tube and was shaken (Eppendorff Thermo mixer, 1400 rpm) for 1.5 h at 37 C. till a clear solution was obtained. The sample was dried in a SpeedVac concentrator (1.5 h) and resuspended in 13.2 l DMSO, shaken for 10 sec., followed by sonification (20 sec), and shaking (e g. in Eppendorff Thermo mixer, 1400 rpm) for 10 min. 6 ml of 20 mM NaH.sub.2PO.sub.4; 140 mM NaCl; 0.1% Pluronic F68; pH 7.4 were added and stirred for 1 h at room temperature. The sample was centrifuged for 20 min at 3000 g. The supernatant was discarded and the precipitate solved in 0.6 ml 20 mM NaH.sub.2PO.sub.4; 140 mM NaCl; 1% Pluronic F68, pH 7.4. 3.4 ml of H.sub.2O were added and stirred for 1 h at room temperature followed by 20 min centrifugation at 3000 g. Eight aliquots of each 0.5 ml of the supernatant were stored at 20 for further use.
(81) 3. A(20-42) Globulomer
(82) The preparation of the A(20-42) globulomer is described in example 1c.
(83) 4. A(12-42) Globulomer
(84) The preparation of the A(12-42) globulomer is described in example 1d.
(85) 5. HFIP Pretreated A(1-40) Monomer, 5 mM in DMSO
(86) 1 mg A(1-40), (Bachem Inc, cat. no. H-1194) were suspended in 0.25 ml HFIP (4 mg/ml suspension) in an Eppendorff tube. The tube was shaken (e.g. In Eppendorff Thermo mixer, 1400 rpm) for 1.5 h at 37 C. to get a clear solution and afterwards dried in a speed vac concentrator (1.5 h). The sample was redissolved in 46 l DMSO (21.7 mg/ml solution=5 mM), shaken for 10 sec and subsequently sonicated for 20 sec. After 10 min shaking (e.g. in Eppendorff Thermo mixer, 1400 rpm) the sample is stored at 20 C. for further use.
(87) 6. A(1-42) Monomer, 0.1% NH.sub.4OH
(88) 1 mg A(1-42) (Bachem Inc., cat no. H-1368) were dissolved in 0.5 ml 0.1% NH.sub.4OH in H.sub.2O (freshly prepared) (=2 mg/ml) and immediately shaken for 30 sec. at room temperature to get a clear solution. The sample was stored at 20 C. for further use.
(89) 7. A(1-42) Fibrils
(90) 1 mg A(1-42) (Bachem Inc. Catalog Nr.: H-1368) were dissolved in 500 l aqueous 0.1% NH.sub.4OH (Eppendorff tube) and the sample was stirred for 1 min at room temperature. 100 l of this freshly prepared A(1-42) solution were neutralized with 300 l 20 mM NaH.sub.2PO.sub.4; 140 mM NaCl, pH 7.4. The pH was adjusted to pH 7.4 with 1% HCl. The sample was incubated for 24 h at 37 C. and centrifuged (10 min at 10000 g). The supernatant was discarded and the fibril pellet resuspended with 400 l of 20 mM NaH.sub.2PO.sub.4; 140 mM NaCl, pH 7.4 by vortexing for 1 min.
(91) 8. sAPP
(92) Supplied from Sigma (cat.no. 89564; 25 g in 20 mM NaH.sub.2PO.sub.4; 140 mM NaCl; pH 7.4). The sAPP was diluted with 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4, 0.2 mg/ml BSA to 0.1 mg/ml (=1 pmol/l).
(93) Materials Dot Blot:
(94) A-Standards:
(95) Serial dilution of A-antigens in 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4+0.2 mg/ml BSA 1) 100 pmol/l 2) 10 pmol/l 3) 1 pmol/l 4) 0.1 pmol/l 5) 0.01 pmol/l
Nitrocellulose: Trans-Blot Transfer medium, Pure Nitrocellulose Membrane (0.45 m); BIO-RAD
Anti-Mouse-AP: AQ330A (Chemicon)
Detection Reagent: NBT/BCIP Tablets (Roche)
Bovine Serum Albumin, (BSA): A-7888 (SIGMA)
Blocking Reagent: 5% low fat milk in TBS
Buffer Solutions: TBS 25 mM Tris/HCl-buffer pH 7.5 +150 mM NaCl TTBS 25 mM Tris/HCl-buffer pH 7.5 +150 mM NaCl +0.05% Tween 20 PBS+0.2 mg/ml BSA 20 mM NaH.sub.2PO.sub.4 buffer pH 7.4 +140 mM NaCl +0.2 mg/ml BSA
Antibody Solution I: Mouse plasma samples from an active immunization study with A(20-42) globulomer (1:400 diluted in 20 ml 1% low fat milk in TBS)
Antibody Solution II: 1:5000 dilution Anti-Mouse-AP in 1% low fat milk in TBS
Dot-Blot-Procedure: 1) 1 l each of the different A-standards (in their 5 serial dilutions) were dotted onto the nitrocellulose membrane in a distance of approximately 1 cm from each other. 2) The A-standards dots were allowed to dry on the nitrocellulose membrane on air for at least 10 min at room temperature (RT) (=dot blot) 3) Blocking: The dot blot was incubated with 30 ml 5% low fat milk in TBS for 1.5 h at RT. 4) Washing: The blocking solution was discarded and the dot blot was incubated under shaking with 20 ml TTBS for 10 min at RT. 5) Antibody solution I: The washing buffer was discarded and the dot blot was incubated with antibody solution I overnight at RT. 6) Washing: The antibody solution I was discarded and the dot blot was incubated under shaking with 20 ml TTBS for 10 min at RT. The washing solution was discarded and the dot blot was incubated under shaking with 20 ml TTBS for 10 min at RT. The washing solution was discarded and the dot blot was incubated under shaking with 20 ml TBS for 10 min at RT. 7) Antibody solution II: The washing buffer was discarded and the dot blot was incubated with antibody solution II for 1 h at RT 8) Washing: The antibody solution II was discarded and the dot blot was incubated under shaking with 20 ml TTBS for 10 min at RT. The washing solution was discarded and the dot blot was incubated under shaking with 20 ml TTBS for 10 min at RT. The washing solution was discarded and the dot blot was incubated under shaking with 20 ml TBS for 10 min at RT. 9) Development: The washing solution was discarded. 1 tablet NBT/BCIP was dissolved in 20 ml H.sub.2O and the dot blot was incubated for 5 min with this solution. The development was stopped by intensive washing with H.sub.2O.
(96) Results are shown in
(97) Dot blot analysis of anti-A antibodies produced after active immunization of mice with A(20-42) globulomer to assess their specificity towards different forms of AP. The individual forms of A were blotted in serial dilutions and incubated with the corresponding mouse plasma containing anti AP-antibodies produced during immune reaction. The individual dot blots correspond to different individuals of Immunized mice. 1. A(1-42) globulomer 2. A(1-42) monomer, HFIP pretreated, in 0.1% Pluronic F68 3. A(20-42) globulomer 4. A(12-42) globulomer 5. A(1-40) monomer, HFIP pretreated, 5 mM in DMSO 6. A(1-42) monomer, 0.1% NH.sub.4OH 7. A(1-42) fibril preparation 8. sAPP (Sigma); (first dot 1 pmol)
(98) In the active immunization study of APP/L Tg-mice it was shown that immunization with A(20-42) globulomer leads to the best result in alleviating a cognitive impairment in these mice compared to PBS treatment Plasma samples from APP/L Tg mice after being actively immunized with A(20-42) globulomer exhibit an antibody profile (predominant recognition of A(20-42) globulomer and A(12-42) globulomer) which resembles that of the A(20-42) globulomer mAbs claimed herein.
Example 6
Concentration of Soluble and Insoluble A(1-42) and A(1-40) Peptide in Brain ExTracts of Actively Immunized APP/PS1 Tg Mice with Either A(1-42) Monomer, A(1-42) Globulomer, A(20-42) Globulomer or Vehicle as Control
(99) 40 female mice of a double transgenic mouse model of Alzheimer's Disease (APP/PS1 Tg mice) in FVBxC57Bl/6J background with an age of 4 months were used for this study. The APP/PS1 Tg mice contain the 695 amino acid form of human APP with the V7171 mutation (position referring to the longest APP-isoform) and in addition the human Presenilin 1 gene with the A264E mutation. Both genes are under control of the Thy1 promotor. The mice were generated and characterized in one of the founding laboratories of reMYND, the Experimental Genetics Group, Campus Gasthuisberg, Catholic University Leuven, by Prof. Fred Van Leuven et al.
(100) All mice were genotyped by polymerase chain reaction (PCR) at the age of 3 weeks and received a unique identity number, once the PCR results were known.
(101) Mice had free access to pre-filtered and sterile water (UV-lamp) and standard mouse chow. The food was stored under dry and cool conditions in a well-ventilated storage room. The amount of water and food was checked daily, supplied when necessary and by default refreshed twice a week.
(102) Mice were housed under a reversed day-night rhythm: 14 hours light/10 hours darkness starting at 7 p.m., in standard metal cages type RVS T2 (area of 540 cm.sup.2). The cages are equipped with solid floors and layer of bedding litter. The number of mice per cage was limited in accordance with legislation on animal welfare. Five days before the onset of the behaviour test, mice were re-caged in macrolon Type 2 cages and transported to the laboratory in order to adapt to the laboratory environment in preparation to the behaviour test.
(103) The mice received either 100 g of A(1-42) monomer (0.1% NH.sub.4OH), A(1-42) globulomer or A(20-42) globulomer in phosphate-buffered saline (PBS) mixed with an equal amount of complete Freund's adjuvant intraperitoneally, followed by booster injections with the same amount of antigene in incomplete Freund's adjuvant every third week for four months.
(104) Biochemistry
(105) The A(1-40) and A(1-42) in the soluble fraction of 1 hemisphere was determined by ELISA. In addition the A(1-40) and A(1-42) of the insoluble membrane fraction of 1 hemisphere was determined by ELISA
(106) The mice were anaesthetized with a 2:1:1 mixture of Ketalar (ketamin), Rompun (xylazin 2%) and atropin and flushed trans-cardially with physiological serum at 4 C. This was performed to remove blood from the brain vessels, a procedure which has no influence on organ integrity.
(107) Cerebrospinal fluid (CSF) was collected via an incision in the neck muscles, between the skull and the first cervical vertebrae. A puncture into the cistema magna was given with a 26 gauge needle and 10-20 l of CSF was collected with a fine glass pipette.
(108) Blood was collected via a heart puncture and a 1 ml syringe into heparin-coated Eppendorf tubes. The blood was centrifuged at 14, 000 rpm at 4 C. for 5 minutes. The serum was stored at 70 C.
(109) The mice were flushed transcardially with physiological serum at 4 C.
(110) The brain was removed from the cranium and hindbrain and forebrain were separated by a cut in the coronal/frontal plane. The cerebellum was discharged. The forebrain was divided evenly into left and right hemisphere by using a midline sagittal cut
(111) One hemisphere was immediately immersed in liquid nitrogen and stored at 70 C. until biochemical analysis.
(112) Homogenization and Fractionation of One Brain Hemisphere
(113) Brains were homogenized using a Potter, a glass tube (detergent free, 2 cm.sup.3) and a mechanical homogenizer (650 rpm). A volume of 6.5 brain weight of freshly prepared 20 mM Tris/HCl buffer (pH 8.5) with Proteinase Inhibitors (1 tablet per 50 ml Tris/HCl buffer, Complete, Roche, Mannheim, Germany) was used as homogenization buffer.
(114) Samples were transferred from 70 C. into a sample holder with liquid nitrogen and each individual sample was pre-warmed by incubation on the bench for a few seconds prior to homogenization. The homogenates were collected in Beckman centrifuge tubes TLX and collected on ice prior to centrifugation. Between two samples, the Potter and the glass tube were rinsed carefully with distilled water (AD) without detergents and dried with absorption paper.
(115) Samples were centrifuged in a pre-cooled ultracentrifuge (Beckman, Mannheim, Germany) for 1 hour and 20 minutes at 48000 rpm (135.000g) at 4 C. Due to a limited number of centrifuge holders (N=8), samples were sorted by brain weight (to equilibrate the centrifuge) and randomized in order to divide the different treatment groups over the different centrifugation sessions.
(116) The supernatant (soluble fraction containing secreted APP and amyloid peptides) was separated from the pellet (membrane fraction containing membrane-bound APP-fragments and plaque-associated amyloid peptides in case of aged mice). The supernatant was divided over two tubes of which one was stored at 20 C. as back-up and the other was processed further for column chromatography to concentrate the amyloid peptides.
(117) Brain weights, volume Tris/HCl buffer used, centrifugation sessions (marked by colour) and volume soluble fraction used for column chromatography are given exemplary in the following table.
(118) TABLE-US-00004 Weight V Tris 50% Sample brain (W) (=W 6.5) V Tris N.sup.o Treatment Mouse ID N.sup.o (mg) (l) (l) 19 X TAB.TPF 1305 157.8 1026 513 21 X TAB.TPF 1335 160.2 1041 521
(119) Small reversed phase columns (C18-Sep-Pack Vac 3cc cartridges, Waters, Mass., MA) were mounted on a vacuum system and washed with 80% acetonitrile in 0.1% trifluoroacetic acid (A-TFA) followed with 0.1% TFA twice. Then the samples were applied and the columns were washed successively with 5% and 25% A-TFA. Amyloid peptides were eluted with 75% A-TFA and the eluates were collected in 2 ml tubes on ice. Eluates were freeze-dried in a SpeedVac concentrator (Savant, Farmingdale, N.Y.) overnight and resolved in 330 l of the sample diluent furnished with the ELISA kits.
(120) The pellets were further fractionated into different membrane fractions: membrane fraction A (MFA), membrane fraction B (MFB) containing full length APP and membrane fraction C (MFC) containing plaque associated amyloid. Therefore the pellets were dissolved in TBS buffer with proteinase inhibitors (1 tablet per 50 ml TBS buffer, Complete, Roche, Mannheim, Germany) and the MFA was divided over two tubes of which one was stored at 20 C. as backup. 60% of MFA was further processed with addition of NP40 (2% of final volume) and Triton X-100 (2% of final volume) in TBS with proteinase inhibitors and centrifuged for one hour at 27.000 rpm (98,000g) in a Beckman ultracentrifuge at 4 C. using a swing-out rotor (SW60). The supernatant (MFB) was separated from the pellet (MFC) and both were stored at 20 C.
(121) Brain weights, 60% of the brain weight, the volumes TBS+PI+NP40+Triton X-100 buffer used and the centrifugation sessions (marked by colour) are given exemplary in the following table.
(122) TABLE-US-00005 Volume Weight Weight buffer = Sample brain (W) brain W 15 N.sup.o Treatment Mouse ID N.sup.o (mg) (mg) (l) 19 X TAB.TPF 1305 157.8 95 1420 21 X TAB.TPF 1335 160.2 96 1442
ELISA of Human A in the Soluble Fraction of One Hemisphere
(123) To quantify the amount of human A(1-40) and human A(1-42) in the soluble fraction of the brain homogenates and/or in cerebrospinal fluid (CSF), commercially available Enzyme-Linked-Immunosorbent-Assay (ELISA) kits were used (h Amyloid 40 or 42 ELISA high sensitive, The Genetics Company, Zurich, Switzerland). The ELISA was performed according to the manufacturer's protocol. Briefly, standards (a dilution of synthetic A(1-40) or A(1-42)) and samples were prepared in a 96-well polypropylene plate without protein binding capacity (Greiner bio-one, Frickenhausen, Germany). The standard dilutions with final concentrations of 1000, 500, 250, 125, 62.5, 31.3 and 15.6 pg/ml and the samples were prepared in the sample diluent, furnished with the ELISA kit, to a final volume of 60 l. Since amyloid levels increase with the age of the mouse and since the actual evaluation requires that the readings of the samples are within the linear part of the standard curve, the samples for A(1-40) analysis were diluted 1:3, the samples for A(1-42) analysis were diluted 1:6.
(124) Samples, standards and blanks (50 l) were added to the anti-A-coated polystyrol plate (capture antibody selectively recognizes the C-terminal end of the antigen) in addition with a selective anti-An-antibody conjugate (biotinylated detection antibody) and incubated overnight at 4 C. in order to allow formation of the antibody-Amyloid-antibody-complex. The following day, a Streptavidine-Peroxidase-Conjugate was added, followed 30 minutes later by the addition of a TMB/peroxide mixture, resulting in the conversion of the substrate into a coloured product. This reaction was stopped by the addition of sulfuric acid (1 M) and the colour intensity was measured by means of photometry with an ELISA-reader with a 450 nm filter. Quantification of the A content of the samples was obtained by comparing absorbance to the standard curve made with synthetic A(1-40) or A(1-42).
(125) ELISA human A in the insoluble fraction of one hemisphere
(126) To quantify the amount of human A(1-40) and human A(1-42) in the insoluble membrane fraction of the brain homogenates, the MFC samples were further processed and dissolved in 8M Guanidine in 80 mM Tris/HCl. Subsequently samples were incubated for 3 hours in a thermomixer at 25 C. and pipetted up and down with a 100 l pipette every hour to dissolve the MFC pellet into the guanidine buffer Finally samples were centrifuged for only 1 minute at 4000 rpm to remove debris.
(127) Brain weight, the weight of the MFC pellet and the volume of 8M guanidine buffer are given exemplary in the following table.
(128) TABLE-US-00006 Weight Weight pellet of Volume brain MFC 8M guanidine Sample Mouse (W) (WMFC) (WMFC 1.6) N.sup.o Treatment ID N.sup.o (mg) (40% brain) (l) 19 X TAB.TPF 157.8 63 101 1305 2 X TAB.TPF 160.2 64 103 1335
(129) To quantify the amount of human A(1-40) and human A(1-42) in the final samples, commercially available Enzyme-Linked-Immunosorbent-Assay (ELISA) kits were used (h Amyloid 40 or 42 ELISA high sensitive, The Genetics Company, Zurich, Switzerland). The ELISA was performed according to the manufacturer's protocol, except for the preparation of the standards (a dilution of synthetic A(1-40) or A(1-42)). The samples were prepared in the sample diluent, furnished with the ELISA kit, to a final volume of 60 l. Since guanidine influences the OD-values of the standard curve, the standard dilutions with final concentrations of 1000, 500, 250, 125, 62.5, 31.3 and 15.6 pg/ml were prepared in sample diluent with the same concentration guanidine as for the samples. This was performed in a 96-well polypropylene plate without protein binding capacity (Greiner bio-one, Frickenhausen, Germany).
(130) Since amyloid levels increase with the age of the mouse and since the actual evaluation requires that the readings of the samples are within the linear part of the standard curve, the samples for insoluble A(1-40) and insoluble A(1-42) analysis were diluted 1:500.
(131) Samples, standards and blanks (50 l) were added to the anti-A-coated polystyrol plate (capture antibody selectively recognizes the C-terminal end of the antigen) in addition with a selective anti-A-antibody conjugate (biotinylated detection antibody) and incubated overnight at 4 C. in order to allow formation of the antibody-Amyloid-antibody-complex. The following day, a streptavidin-peroxidase conjugate was added, followed 30 minutes later by the addition of a TMB/peroxide mixture, resulting in the conversion of the substrate into a coloured product. This reaction was stopped by the addition of sulfuric acid (1M) and the colour intensity was measured by means of photometry with an ELISA-reader with a 450 nm filter. Quantification of the A content of the samples was obtained by comparing absorbance to the standard curve made with synthetic A(1-40) or A(1-42)
(132) Results are shown in
(133) Concentration of soluble and insoluble A(1-42) and A(1-40) peptide in brain extracts of actively immunized APP/PS1 Tg-mice with either A(1-42) monomer (0.1% NH.sub.4OH), A(1-42) globulomer, A(20-42) globulomer or vehicle as control.
(134) In the soluble and insoluble fraction of a brain extract of APP/PS1 Tg-mice actively Immunized with A(20-42) globulomer the level of A(1-40)- and A(1-42)-peptide is not significantly different to the vehicle control. In contrast, immunization with A(1-42) globulomer and A(1-42) monomer leads to a reduction in brain A(1-40)- and A(1-42)-levels. This shows that an A(20-42) globulomer directed immunization approach does not alter the total A-brain levels significantly but nonetheless is effective in alleviating the A-peptide related cognitive impairments (see example 4).
Example 7
Analysis of Cognitive Performance by Object Recognition Test in APP/L Transgenic Mice after Passive Immunization with Anti-A(20-42) Globulomer Antibodies
(135) In these experiments mice overexpressing human APP with a point mutation were used. The point mutation refers to amino acid 717 (substitution of isoleucine for valine) and has been found in a London family where it leads to onset of AD before the beginning of the sixth decade of life (Mullan et al., Nature Genetics 2 (1992) 340-342). The transgenic mice, herein referred to as APP/L, were created by and first described in Leuven (Moechars et al., J. Biol. Chem. 274 (1999) 6483-6492). Female APP/L mice were subjected to passive immunization at 3 months of age. Mice received 250 g of any of the monoclonal mouse antibodies 5F7, 10F11 or 7C6 in 100 l of phosphate-buffered saline (PBS). Throughout the time course of the experiment the animals were kept under standard conditions in a reverted day/night cycle (14 hours of light beginning at 7 pm/10 hours of darkness). They tolerated passive immunization well, without any signs of adverse effects.
(136) After the third injection (day 15 of experiment) cognitive ability of the mice was tested by an object recognition test as described in the art (Dewachter et al. Journal of Neuroscience 22 (2002) 3445-3453). To this end, mice were accustomed to an arena and then exposed for 10 minutes to an acquisition phase during which they were individually placed in the arena which now contained two identical elements (green cube or orange cylinder of similar size, ca. 4 cm). The duration and frequency with which the mouse explored the objects were recorded. During retention phase, 2.5 h later, mice were returned to the arena which now contained, in addition to the known object, the other object. Recognition of the new object was recorded as the time during which the mouse was exploring the old object relative to total time (exploration of old and new object). The recognition index expresses this relation (time for new object/total time). A mouse which does not remember the known object will consider it as equally interesting as the new object and spend an equal amount of time on exploring it, in other words, will show a recognition index of 50%. A mouse which remembers the known object will consider it as not interesting and therefore show a significantly higher recognition index. APP/L mice are known to be cognitively deficient at 4.5 months of age and exhibit a recognition index in the dimension of the random level, i.e. 50%.
(137) Results are shown in
(138) Object recognition test in mice. The test reports recognition of a known object in comparison to an unknown one, measured in terms of explorative behaviour during a 10 minute test phase. The recognition index is defined as the percentage of time which the mouse spends on exploring the unknown object relative to the time spent on exploring both objects. The known object was explored by the mouse during a 10 minute acquisition phase 2.5 hours before the test phase.
(139) a) APP transgenic mice were immunized once a week for three weeks by intraperitoneal injection of 250 g of the antibody 5F7 (n=9), the antibody 10F11 (n=11) or the antibody 7C6 (n=11); control animals received PBS (n=6). Significant differences from random level (50%, i.e. equal time of exploration spent on the known and the unknown object) are indicated with asterisks. *=p<0.05 (t-test)
(140) b) Comparison of all mice treated with antibodies (5F7, 10F11 and 7C6; (n=31)) and mice treated with phosphate-buffered saline (PBS; n=6). The RI of the antibody-treated group different significantly from random level (**=P<0.01; t-Test).
(141) APP/L mice are known to be cognitively deficient at 4.5 months of age and exhibit a recognition index in the dimension of the random level, i.e. 50%.
(142) Indeed the PBS-treated mice showed random behaviour. Passive immunization with all three antibodies (5F7, 10F11 and 7C6) resulted in a markedly increased recognition index. When compared as a pooled group against the controls, the recognition index is significantly increased. This beneficial effect on memory performance of APP/L mice after administration of all three antibodies suggests that an antibody against truncated A(20-42) globulomer is sufficient to achieve cognitive improvement.
Example 8
Dot-Blot Profile of the Selectivity of the Anti-A(20-42) Globulomer Antibodies
(143) In order to characterize the selectivity of the monoclonal anti A(20-42) globulomer antibodies they were probed for recognition with different A-forms. To this end, serial dilutions of the individual A((1-42) forms ranging from 100 pmol/l to 0.01 pmol/l in PBS supplemented with 0.2 mg/ml BSA were made. 1 l of each sample was blotted onto a nitrocellulose membrane. For detection the corresponding antibody was used (0.2 g/ml). Immunostaining was done using peroxidase conjugated anti-mouse-IgG and the staining reagent BM Blue POD Substrate (Roche).
(144) A-Standards for Dot-Blot:
(145) 1. A(1-42) Monomer, 0.1% NH.sub.4OH
(146) 1 mg A(1-42) (Bachem Inc., cat. no. H-1368) were dissolved in 0.5 ml 0.1% NH.sub.4OH In H.sub.2O (freshly prepared) (=2 mg/ml) and immediately shaken for 30 sec at room temperature to get a clear solution. The sample was stored at 20 C. for further use.
(147) 2. A(1-40) Monomer, 0.1% NH.sub.4OH
(148) 1 mg A(1-40) (Bachem Inc., cat. no. H-1368) were dissolved in 0.5 ml 0.1% NH.sub.4OH In H.sub.2O (freshly prepared) (=2 mg/ml) and immediately shaken for 30 sec. at room temperature to get a clear solution. The sample was stored at 20 C for further use.
(149) 3. A(1-42) Monomer, 0.1% NaOH
(150) 2.5 mg A(1-42) (Bachem Inc., cat. no. H-1368) were dissolved in 0.5 ml 0.1% NaOH in H.sub.2O (freshly prepared) (=5 mg/ml) and immediately shaken for 30 sec. at room temperature to obtain a clear solution. The sample was stored at 20 C. for further use.
(151) 4. A(1-40) Monomer, 0.1% NaOH
(152) 2.5 mg A(1-40) (Bachem Inc, cat. no. H-1368) were dissolved in 0.5 ml 0.1% NaOH in H.sub.2O (freshly prepared) (=5 mg/ml) and Immediately shaken for 30 sec. at room temperature to obtain a clear solution. The sample was stored at 20 C. for further use.
(153) 5. A(1-42) Globulomer
(154) The preparation of the A(1-42) globulomer is described in example 1a.
(155) 6. A(12-42) Globulomer
(156) The preparation of the A(12-42) globulomer is described in example 1d.
(157) 7. A(20-42) Globulomer
(158) The preparation of the A(20-42) globulomer is described in example 1c.
(159) 8. A(1-42) Fibrils
(160) 1 mg A(1-42) (Bachem Inc. cat. no.: H-1368) were solved in 500 l aqueous 0.1% NH.sub.4OH (Eppendorff tube) and the sample was stirred for 1 min at room temperature. 100 l of this freshly prepared A(1-42) solution were neutralized with 300 l 20 mM NaH.sub.2PO.sub.4; 140 mM NaCl, pH 7.4. The pH was adjusted to pH 7.4 with 1% HCl. The sample was incubated for 24 h at 37 C. and centrifuged (10 min at 10000 g) The supernatant was discarded and the fibril pellet resuspended with 400 l 20 mM NaH.sub.2PO.sub.4; 140 mM NaCl, pH 7.4 by vortexing for 1 min.
(161) 9. sAPP
(162) Supplied by Sigma (cat.no. S9564; 25 g in 20 mM NaH.sub.2PO.sub.4; 140 mM NaCl; pH 7.4). The sAPP was diluted to 0.1 mg/ml (=1 pmol/l) with 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4, 0.2 mg/ml BSA.
(163) Materials for Dot Blot:
(164) A-Standards:
(165) Serial dilution of A antigens in 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4+0.2 mg/ml BSA 1) 100 pmol/l 2) 10 pmol/l 3) 1 pmol/l 4) 0.1 pmol/l 5) 0.01 pmol/l
Nitrocellulose: Trans-Blot Transfer medium, Pure Nitrocellulose Membrane (0.45 m); BIO-RAD
Anti-Mouse-POD: Cat no: 715-035-150 (Jackson Immuno Research)
Detection Reagent: BM Blue POD Substrate, precipitating (Roche)
Bovine Serum Albumin, (BSA): Cat no: A-7888 (SIGMA)
Blocking Reagent: 5% low fat milk in TBS
Buffer Solutions: TBS 25 mM Tris/HCl buffer pH 7.5 +150 mM NaCl TTBS 25 mM Tris/HCl-buffer pH 7.5 +150 mM NaCl +0.05% Tween 20 PBS+0.2 mg/ml BSA 20 mM NaH.sub.2PO.sub.4 buffer pH 7.4 +140 mM NaCl +0.2 mg/ml BSA
Antibody Solution I: 0.2 g/ml antibody diluted in 20 ml 1% low fat milk in TBS
Antibody Solution II: 1:5000 dilution Anti-Mouse-POD in 1% low fat milk in TBS
Dot Blot Procedure: 1) 1 l each of the different A-standards (in their 5 serial dilutions) were dotted onto the nitrocellulose membrane in a distance of approximately 1 cm from each other. 2) The A-standards dots were allowed to dry on the nitrocellulose membrane on air for at least 10 min at room temperature (RT) (=dot blot) 3) Blocking: The dot blot was incubated with 30 ml 5% low fat milk in TBS for 1.5 h at RT. 4) Washing: The blocking solution was discarded and the dot blot was incubated under shaking with 20 ml TTBS for 10 min at RT. 5) Antibody solution I: The washing buffer was discarded and the dot blot was incubated with antibody solution I for 2 h at RT 6) Washing: The antibody solution I was discarded and the dot blot was incubated under shaking with 20 ml TTBS for 10 min at RT. The washing solution was discarded and the dot blot was incubated under shaking with 20 ml TTBS for 10 min at RT. The washing solution was discarded and the dot blot was incubated under shaking with 20 ml TBS for 10 min at RT. 7) Antibody solution II: The washing buffer was discarded and the dot blot was incubated with antibody solution II overnight at RT 8) Washing: The antibody solution II was discarded and the dot blot was incubated under shaking with 20 mil TTBS for 10 min at RT. The washing solution was discarded and the dot blot was incubated under shaking with 20 ml TTBS for 10 min at RT. The washing solution was discarded and the dot blot was incubated under shaking with 20 mil TBS for 10 min at RT. 9) Development: The washing solution was discarded. The dot blot was developed with 10 ml BM Blue POD Substrate for 10 min. The development was stopped by intense washing of the dot blot with H.sub.2O. Quantitative evaluation was done using a densitometric analysis (GS800 densitometer (BioRad) and software package Quantity one, Version 4.5.0 (BioRad)) of the dot-intensity. Only dots were evaluated that had a relative density of greater than 20% of the relative density of the last optically unambiguously Identified dot of the A(20-42) globulomer. This threshold value was determined for every dot-blot independently. The calculated value indicates the relation between recognition of A(20-42) globulomer and the respective A form for the antibody given
(166) Results are shown in
(167) Dot blot analysis of the specificity of different anti-A antibodies (6E10, 5F7, 487, 10F11, 6A2, 4D10, 2F2; 3B10, 7C6, 7E5, 10C1) towards different forms of A. The monoclonal antibodies tested were obtained (except for 6E10) by active immunization of mice with A(20-42) globulomer, followed by selection of the fused hybridoma cells. The individual A forms were applied in serial delusions and incubated with the respective antibodies for immune reaction. 1. A(1-42) monomer, 0.1% NH.sub.4OH 2. A(1-40) monomer, 0.1% NH.sub.4OH 3. A(1-42) monomer, 0.1% NaOH 4. A(1-40) monomer, 0.1% NaOH 5. A(1-42) globulomer 6. A(12-42) globulomer 7. A(20-42) globulomer 8. A(1-42) fibril preparation 9. sAPP (Sigma); (first dot: 1 pmol)
(168) The anti A(20-42) globulomer selective mAbs can be divided in 3 classes with respect to the discrimination of A(1-42) globulomer and A(12-42) globulomer. The first class comprising the antibodies 6A2, 5F7 and 2F2 recognizes preferentially A(20-42) globulomer and to some extent A(1-42) globulomer (and also A(12-42) globulomer). The second class comprising the antibodies 10F11, 4D10 and 3810 recognizes preferentially A(20-42) globulomer and also recognizes A(12-42) globulomer but to a lesser extent and do not significantly recognize A(1-42) globulomer. The third class comprising the antibodies 7C6, 4B7, 7E5 And 10C1 recognizes A(20-42) globulomer but shows no significant recognition of the others. All three classes do not significantly recognize monomeric A(1-42), monomeric A(1-40), A$(1-42) fibrils or sAPP.
(169) The selectivity profile of the anti-A(20-42) globulomer antibodies shows that the significantly elevated recognition Index in the passive immunization (in
Example 9
In Situ Analysis of the Specific Reaction of A(20-42) Selective Antibodies to Fibrillary A Peptide in the Form of A Plaques in Old TG2576 Mice and A Amyloid in Meningeal Vessels
(170) For these experiments brain material of 19 month old TG2576 mice (Hslao et al., 1996, Science; 274(5284), 99-102) or 9 month old APP/LxPS1 mice (description as above; ReMYND, Leuven, Belgium) or autopsy material of two Alzheimer's disease patients (RZ16 and RZ55; obtained from BrainNet, Munich) was used. The mice overexpress human APP with the so-called Swedish mutation (K670N/M671L; Tg2576) or with the so-called London mutation (V7171) in addition with the human Presenillin 1 gene with the A264E mutation (APP/LxPS1) and formed amyloid deposits in the brain parenchyma at about 7-11 months of age and P amyloid deposits in larger cerebral vessels at about 18 months of age (Tg2576). The animals were deeply anaesthetized and transcardially perfused with 0.1 M phosphate-buffered saline (PBS) to flush the blood. Then the brain was removed from the cranium and divided longitudinally. One hemisphere of the brain was shock-frozen, the other fixated by immersion into 4% paraformaldehyde. The immersion-fixated hemisphere was cryoprotected by soaking in 30% sucrose in PBS and mounted on a freezing microtome. The entire forebrain was cut into 40 m section which were collected in PBS and used for the subsequent staining procedure. The human brain material was an about 1 cm.sup.3 deep-frozen block of the neocortex. A small part of the block was immersion-fixated in 4% paraformaldehyde and further treated like the mouse brain material.
(171) Individual sections were stained with Congo Red using the following protocol:
(172) Material:
(173) Amyloid dye Congo Red kit (Sigma-Aldrich; HT-60), consisting of alcoholic NaCl solution, NaOH solution and Congo Red solution staining cuvettes microscope slides SuperfrostPlus and coverslips Ethanol, Xylol, embedding medium
Reagents: NaOH diluted 1:100 with NaCl solution yields alkaline saline alkaline saline diluted 1:100 with Congo Red solution yields alkaline Congo Red solution (prepare no more than 15 min before use, filtrate) mount sections on slide and allow them to dry incubate slide in staining cuvette, first for 30-40 minutes in alkaline saline, then for 30-40 minutes in alkaline Congo Red solution rinse three times with fresh ethanol and embed over xylol
(174) Staining was first photographed using a Zeiss Axioplan microscope and evaluated qualitatively. Red colour indicated amyloid deposits both in the form of plaques and in larger meningeal vessels. These Results are shown in
(175) Antibody staining was performed by incubating the sections with a solution containing 0.07-7.0 g/ml of the respective antibody in accordance with the following protocol:
(176) Materials:
(177) TBST washing solution (Tris Buffered Saline with Tween 20; 10 concentrate; DakoCytomation; S3306 1:10 in Aqua bidest) 0.3% H.sub.2O.sub.2 in methanol donkey serum (Serotec), 5% in TBST monoclonal mouse-anti-globulomer antibody diluted in TBST secondary antibody: biotinylated donkey-anti-mouse antibody (Jackson Immuno; 715-065-150; diluted 1:500 in TBST) StreptABComplex (DakoCytomation; K 0377) Peroxidase Substrate Kit diaminobenzdine (=DAB; Vector Laboratories; SK-4100) SuperFrost Plus microscope slides and coverslips xylol free embedding medium (Medite; X-tra Kitt)
Procedure: transfer floating sections into ice-cold 0.3% H.sub.2O.sub.2 and incubate for 30 min wash for 5 min in TBST buffer incubate with donkey serum I TBST for 20 minutes incubate with primary antibody for 24 hours at room temperature wash in TBST buffer for 5 minutes Incubate with blocking serum from the Vectastain Elite ABC peroxidase kit for 20 minutes wash in TBST buffer for 5 minutes incubate with secondary antibody for 60 minutes at ambient temperature wash in TBST buffer for 5 minutes incubate with StreptABComplex for 60 minutes at ambient temperature wash in TBST buffer for 5 minutes incubate with DAB from the Vectastain Elite ABC peroxidase kit for 20 minutes mount the section on slides, air-dry them, dehydrate them with alcohol and embed them
(178) Besides visual inspection of the staining, plaque staining was additionally quantified by graphically excising 10 randomly selected plaques from the histological images using the ImagePro 5.0 Image analysis system and determining their average greyscale value. Optical density values were calculated from the greyscale values by subtracting the mean background density of the stained material from the density of amyloid plaques (0%no plaque staining above surrounding background, 100%no transmission/maximal staining), and the differences between control and antibodies and between 6G1 and the antibodies selective for A(20-42), respectively, were tested for statistical significance with ANOVA.
(179) Results of the staining in Tg2576 and APP/LxPS1 mice are shown in
(180) Binding of different antibodies at a concentration of 0.7 g/ml in transversal section of the neocortices of transgenic TG 2576 mice at 19 months of age: C) Parenchymal A deposits (amyloid plaques) were stained only with 6G1 and 6E10 but not with the globulomer selective antibodies (i.e. 5F7, 2F2, 6A2, 4010, 10F11, 3B10, 7C6, 7E5 and 10C1) D) All globulomer selective antibodies (i.e. 5F7, 2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1) showed significantly less parenchymal plaque staining compared to the commercially available antibodies 6E10 and 4G8.
(181) Binding of different antibodies at a concentration of 0.07-7.0 g/ml in transversal section of the neocortices of transgenic APP/LxPS1 mice at 11 months of age: E) Parenchymal Adeposits (amyloid plaques) were significantly more and at lower concentrations stained with 6G1, 6E10 and 4G8 than with the globulomer selective antibodies (i.e. 5F7, 2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1).
(182) All amyloid deposits had been verified by congophilic staining before (Congo Red; see
(183) Evaluation of brown DAB deposits showed that the Ap-unselective 6G1 and 6E10 antibodies stained plaques and meningeal vessels, whereas the A(20-42) globulomer selective antibodies 5F7, 2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1 did not. This finding demonstrates that there is no or markedly less binding of these antibodies to A fibrils or other A species present in the amyloid structures in vivo. This reduced binding is supposed to reduce the danger of side effects induced by too fast dissolution of plaques and subsequent increase in soluble A or neuroinflammation due to the interaction of plaque-bound antibodies with microglia.
(184) Results of the staining in human Alzheimer's disease brain are shown in
(185) Binding of different antibodies at a concentration of 0.7 g/ml in transversal section of the neocortex of patient RZ55: B) Parenchymal A deposits (amyloid plaques) were stained only with 6G1 and 6E10 but not with the globulomer selective antibodies (i.e. 5F7, 2F2, 6A2, 4D10, 10F11, 3810, 7C6, 7E5 and 10C1). F) All globulomer selective antibodies (i.e. 5F7, 2F2, 6A2, 4D10, 10F11, 3810, 7C6, 7E5 and 10C1) showed significantly less staining compared to the commercially available antibodies 6E10 and 4G8. H) Vascular A deposits (arrows) were stained only with 6G1 and 6E10 but not with globulomer selective antibodies (i.e. 5F7, 2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1).
(186) Binding of different antibodies at a concentration of 0.07-7.0 g/ml in transversal section of the neocortices of transgenic APP/LxPS1 mice at 11 months of age: G) Parenchymal A deposits (amyloid plaques) were significantly more and at lower concentrations stained with 6G1, 6E10 and 4G8 than with the globulomer selective antibodies (i.e. 5F7, 2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1).
(187) All amyloid deposits had been verified by congophilic staining before (Congo Red; see
(188) Evaluation of brown DAB deposits showed that the A-unselective 6G1 and 6E10 antibodies stained plaques and meningeal vessels, whereas the A(20-42) globulomer selective antibodies 5F7, 2F2, 6A2, 4010, 10F11, 3B10, 7C6, 7E5 and 10C1 did not. Commercially available antibodies 6E10 and 4G8 showed stronger staining compared to globulomer selective antibodies, but less staining than 6G1. This finding confirms the staining pattern in APP transgenic mice where there is no or markedly less binding of the globulomer selective antibodies to AP fibrils or other A species present in the amyloid structures in vivo. This reduced binding to human amyloid is supposed to reduce the danger of side effects induced by too fast dissolution of plaques and subsequent increase in soluble A or neuroinflammation due to the interaction of plaque-bound antibodies with microglia.
Example 10
Anti-AP-Antibody Titer and Dot-Blot Selectivity Profile in Plasma of TG2576 Mice Approximately One Year after Active Immunization
(189) Approximately one year after the last immunization (with A(20-42) globulomer, A(12-42) globulomer, A(1-42) monomer and vehicle) of Tg 2576 mice (from example 9) plasma samples were assessed for anti-A, antibodies produced and still present. To this end, dilution series of the different forms of A(1-42) in the concentration range from 100 pmol/l to 0.01 pmol/l in PBS+0.2 mg/ml BSA were made. Of each sample, 1 l was applied to a nitrocellulose membrane. Detection was performed with suitable mouse plasma samples (diluted 1:400). Staining was done with anti-mouse-lgG conjugated alkaline phosphatase and addition of the staining reagent NBT/BCIP
(190) A-Standards for Dot-Blot
(191) 1. A(1-42) Globulomer
(192) The preparation of the A(1-42) globulomer is described in example 1a.
(193) 2. HFIP Pretreated A(1-42) Monomer in Pluronic F68
(194) 3 mg A(1-42), (Bachem Inc.; cat. no. H-1368) were dissolved in 0.5 ml HFIP (6 mg/ml suspension) in an 1.7 ml Eppendorff tube and was shaken (Eppendorff Thermo mixer, 1400 rpm) for 1.5 h at 37 C. till a clear solution was obtained. The sample was dried in a SpeedVac concentrator (1.5 h) and resuspended in 13.2 l DMSO, shaken for 10 sec., followed by sonification (20 sec), and shaking (e.g. in Eppendorff Thermo mixer, 1400 rpm) for 10 min. 6 ml 20 mM NaH.sub.2PO.sub.4; 140 mM NaCl; 0.1% Pluronic F68; pH 7.4 was added and stirred for 1 h at room temperature. The sample was centrifuged for 20 min at 3000 g. The supernatant was discarded and the precipitate solved in 0.6 ml 20 mM NaH.sub.2PO.sub.4; 140 mM NaCl; 1% Pluronic F68, pH 7.4 3.4 ml H.sub.2O was added and stirred for 1 h at room temperature followed by 20 min centrifugation at 3000 g. Eight aliquots of each 0.5 ml of the supernatant were stored at 20 for further use.
(195) 3. A(20-42) Globulomer
(196) The preparation of the A(20-42) globulomer is described in example 1c.
(197) 4. A(12-42) Globulomer
(198) The preparation of the A(1-42) globulomer is described in example 1d.
(199) 5. A(1-40) Monomer, HFIP Pretreated, 5 mM in DMSO
(200) 1 mg A(1-40), (Bachem Inc, cat. no. H-1194) were suspended in 0.25 ml HFIP (4 mg/ml suspension) in an Eppendorff tube. The tube was shaken (e.g. In Eppendorff Thermo mixer, 1400 rpm) for 1.5 h at 37 C. to get a clear solution and afterwards dried in a SpeedVac concentrator (for 1.5 h). The sample was redissolved in 46 l DMSO (21.7 mg/ml solution=5 mM), shaken for 10 sec and subsequently sonicated for 20 sec. After shaking (e.g. In Eppendorff Thermo mixer, 1400 rpm) for 10 min the sample is stored at 20 C. for further use.
(201) 6. A(1-42) monomer, 0.1% NH.sub.4OH
(202) 1 mg A(1-42) (Bachem Inc., cat no. H-1368) were dissolved in 0.5 ml 0.1% NH.sub.4OH in H.sub.2O (freshly prepared) (=2 mg/ml) and immediately shaken for 30 sec. at room temperature to get a clear solution. The sample was stored at 20 C. for further use.
(203) 7. A(1-42) Fibrils
(204) 1 mg A(1-42) (Bachem Inc. Catalog Nr.: H-1368) were solved in 500 l aqueous 0.1% NH.sub.4OH (Eppendorff tube) and the sample was stirred for 1 min at room temperature. 100 l of this freshly prepared A(1-42) solution were neutralized with 300 l 20 mM NaH.sub.2PO.sub.4; 140 mM NaCl, pH 7.4. The pH was adjusted to pH 7.4 with 1% HCl. The sample was incubated for 24 h at 37 C. and centrifuged (10 min at 10000 g). The supernatant was discarded and the fibril pellet resuspended with 400 l 20 mM NaH.sub.2PO.sub.4; 140 mM NaCl, pH 7.4 by vortexing for 1 min.
(205) 8. sAPP
(206) Supplied from Sigma (cat.no. S9564; 25 g in 20 mM NaH.sub.2PO.sub.4; 140 mM NaCl; pH 7.4). The sAPP was diluted with 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4, 0.2 mg/ml BSA to 0.1 mg/ml (=1 pmol/l).
(207) Materials for Dot Blot:
(208) A-Standards:
(209) Serial dilution of A-antigens in 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4+0.2 mg/ml BSA 1) 100 pmol/l 2) 10 pmol/l 3) 1 pmol/l 4) 0.1 pmol/l 5) 0.01 pmol/l
Nitrocellulose: Trans-Blot Transfer medium, Pure Nitrocellulose Membrane (0.45 m); BIO-RAD
Anti-Mouse-AP: AQ330A (Chemicon)
Detection Reagent: NBT/BCIP Tablets (Roche)
Bovine Serum Albumin, (BSA): A-7888 (Fa. SIGMA)
Blocking Reagent: 5% low fat milk in TBS
Buffer Solutions: TBS 25 mM Tris/HCl-buffer pH 7.5 +150 mM NaCl TTBS 25 mM Tris/HCl-buffer pH 7.5 +150 mM NaCl +0.05% Tween 20 PBS+0.2 mg/ml BSA 20 mM NaH.sub.2PO.sub.4 buffer pH 7.4 +140 mM NaCl +0.2 mg/ml BSA
Antibody Solution I: Plasma of the TG2576 mice actively immunized 1/400 diluted in 20 ml 1% low fat milk in TBS
Antibody Solution II: 1:5000 dilution Anti-Mouse-AP in 1% low fat milk in TBS
Dot Blot Procedure: 1) 1 l each of the different A standards (in their 5 serial dilutions) were dotted onto the nitrocellulose membrane in approximately 1 cm distance from each other. 2) The A standards dots are allowed to dry on the nitrocellulose membrane on air for at least 10 min at room temperature (RT) (=dot blot) 3) Blocking: The dot blot is incubated with 30 ml 5% low fat milk in TBS for 1.5 h at RT. 4) Washing: The blocking solution is discarded and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT. 5) Antibody solution 1: The washing buffer is discarded and the dot blot incubated with antibody solution I overnight at RT 6) Washing: The antibody solution I is discarded and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT. The washing solution is discarded and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT. The washing solution is discarded and the dot blot incubated under shaking with 20 ml TBS for 10 min at RT. 7) Antibody solution II: The washing buffer is discarded and the dot blot incubated with antibody solution II for 1 h at RT. 8) Washing: The antibody solution II is discarded and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT. The washing solution is discarded and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT. The washing solution is discarded and the dot blot incubated under shaking with 20 ml TBS for 10 min at RT. 9) Development: The washing solution is discarded. 1 tablet NBT/BCIP is dissolved in 20 ml H.sub.2O and the dot blot is incubated for 5 min with this solution. The development is stopped by intensive washing with H.sub.2O.
(210) Results are shown in
(211) Sera of different immunization groups: a) A(20-42) globulomers; b) A(12-42) globulomers; c) A(1-42) monomer, 0.1% NH.sub.4OH; d) vehicle control were tested against different A forms in a dot blot for differing antibody profiles.
(212) 1. A(1-42) globulomer
(213) 2. A(1-42) monomer, HFIP pretreated, in 0.1% Pluronic F68
(214) 3. A(20-42) globulomer
(215) 4. A(12-42) globulomer
(216) 5. A(1-40) monomer, HFIP pretreated, 5 mM in DMSO
(217) 6. A(1-42) monomer, dissolved in 0.1% NH.sub.4OH
(218) 7. A(1-42) fibril preparation
(219) 8. sAPP (Sigma); (first dot: 1 pmol)
(220) In contrast to the active immunizations with either vehicle as control or A(1-42) monomer the immunization with A(20-42) globulomer or A(12-42) globulomer exhibits even after approximately one year of the last immunization a high titer of antibodies. These antibodies are selective for the A(20-42) globulomer in the case of the A(20-42) globulomer immunization or A(20-42) globulomer and A(12-42) globulomer selective in the case of the A(12-42) globulomer immunization. This shows that the truncated A(20-42) globulomer and A(12-42) globulomer represent a very good antigen and that antibodies directed against them persist very long in vivo.
(221) (Note that on some dot-blots an unspecific staining signal is observed which is most likely a cross reaction of murine antibodies to the BSA used in the serial dilutions of the A peptides.)
Example 11
Brain-Levels of A(20-42) Globulomer Epitopes in Alzheimer's Disease Patients
(222) SDS-DTT-Brain Extract:
(223) AD brain samples: RZ 16; RZ 52 und RZ 55 (obtained from Brain Net, Munich) Control sample: RZ 92 (obtained from Brain Net, Munich)
(224) One tablet of Complete Protease Inhibitor (Roche, Cat. No. 1697 498) is dissolved in 1 ml H.sub.2O (=protease inhibitor solution). 100 mg of AD brain sample are homogenized in 2.5 ml NaH.sub.2PO.sub.4, 140 mM NaCl, 0.05% Tween 20, 0.5% BSA (supplemented with 25 l protease inhibitor solution) with 20 strokes in a glass potter. The suspension is sonified for 30 sec on ice, then incubated at 37 C. for 16 h. The suspension is centrifuged at 100,000 g and 8 C. for one hour, then the supernatant is collected. The residue is dissolved in 5 mM NaH.sub.2PO.sub.4, 35 mM NaCl, pH 7.4 and homogenized with 10 strokes in a glass potter. 75 l of 10% SDS and 125 l of 0.16 mg/ml DTT are added and stirred for 20 minutes at ambient temperature. The sample was centrifuged for 10 minutes at 10,000 g, and the supernatant is stored overnight at 20 C. Before use the supernatant is thawed and centrifuged for another 10 min at 10,000 g. The supernatant (=SDS/DTT brain extract) is used for ELISA.
(225) a) Sandwich-ELISA for A(20-42) Globulomer Epitope
(226) Reagent List:
(227) 1. F96 Cert. Maxisorp NUNC-Immuno Plate (Cat.No.:439454) 2. Binding antibody: 5F7, 7C6, 10F11 3. Coupling buffer: 100 mM sodium hydrogen carbonate, pH9.6 4. Blocking reagent for ELISA (Roche Diagnostics GmbH Cat.No.: 1112589) 5. PBST buffer: 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, 0.05% Tween 20, pH 7.4 6. A(20-42) calibration standard 7. Primary antibody: anti-A pRAb BA199; affinity purified (by A(1-42) globulomer-Sepharose) IgG solution in PBS; Konz.: 0.22 mg/ml 8. Secondary antibody: anti-rabbit-POD conjugate; (Jackson ImmunoResearch, Cat.No.: 111-036-045) 9. Development: TMB; (Roche Diagnostics GmbH Cat.No.: 92817060) 42 mM in DMSO 3% H.sub.2O.sub.2 in H.sub.2O 100 mM sodium acetate, pH4.9 Stop solution: 2M sulfuric acid
Preparation of Reagents: 1. Binding Antibody: The individual binding antibodies 5F7, 7C6 and 10F11 are diluted to a final concentration of 0.7 g/ml in coupling buffer. 2. Blocking reagent For preparation of the blocking stock solution the blocking reagent is dissolved in 100 ml H.sub.2O and stored at 20 C. in aliquots of 10 ml each. 3 ml of the blocking stock solution are diluted with 27 ml H.sub.2O for blocking one ELISA plate. 3. A(20-42) calibration standard (CS 1)
(228) The preparation of the A(1-42) globulomer is described in example 1a.
(229) The A(20-42) globulomer protein concentration was determined (6.81 mg/ml) after Bradford (BioRad). 14.68 l A(20-42) globulomer (6.81 mg/ml) are diluted in 10 ml 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, 0.05% Tween20, pH 7.4, 0.5% BSA (=10 g/ml). 10 l of the 10 g/ml solution are further diluted in 10 ml 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, 0.05% Tween20, pH 7.4, 0.5% BSA (=10 ng/ml=CS1)
(230) Calibration Standards for A(20-42):
(231) TABLE-US-00007 final volume of concentration Calibration calibration A(20-42) standard standard PBST + 0.5% BSA (pg/ml) CS1.1 1 ml of CS1 0 ml 10000 CS1.2 0.316 ml of CS1.1 0.684 ml 3160 CS1.3 0.316 ml of CS1.2 0.684 ml 1000 CS1.4 0.316 ml of CS1.3 0.684 ml 316 CS1.5 0.316 ml of CS1.4 0.684 ml 100 CS1.6 0.316 ml of CS1.5 0.684 ml 31.6 CS1.7 0.316 ml of CS1.6 0.684 ml 10 CS1.8 0.0 1.0 ml 0.0
SDS/DTT-Brain Extracts:
(232) SDS/DTT-brain extracts=E#
(233) (#represents the 4 human brain samples (1) RZ 16; (2) RZ 52; (3) RZ 55; (4) RZ 92)
(234) TABLE-US-00008 extraction PBST + 0.5% dilution sample volume of extraction sample BSA factor E#. 1 1 ml of E# 0.0 ml direct E#. 2 0.316 ml of E#. 1 0.684 ml 1:3.16 E#. 3 0.316 ml of E#. 1 0.684 ml 1:10 E#. 4 0.316 ml of E#. 1 0.684 ml 1:31.6 4. Primary Antibody: The anti-A pRAb stock solution is diluted to 0.05 g/ml in PBST+0.5% BSA. The antibody solution is used immediately. 5. Secondary Antibody: Lyophilized anti-rabbit-POD conjugate is dissolved in 0.5 ml H.sub.2O and mixed with 500 l glycerol. The antibody concentrate is then stored at 20 C. in aliquots of 100 l. The concentrate is diluted 1:10,000 in PBST buffer. The antibody solution is used immediately. 6. TMB solution: 20 ml of 100 mM sodium acetate, pH 4.9, are mixed with 200 l TMB solution and 29.5 l of 3% hydrogen peroxide. This solution is used immediately. ELISA-Plate for A(20-42): Calibration standards (CS1.1-CS1.8) and SDS/DTT-brain extracts of the 4 human brain samples (1) RZ 16; (2) RZ 52; (3) RZ 55; (4) RZ 92 (=E1-E4 in their 4 serial dilutions E#.1-E#.4) are determined in double:
(235) TABLE-US-00009 1 2 3 4 5 6 7 8 9 10 11 12 A CS1.1 CS1.1 E1.1 E1.1 E3.1 E3.1 B CS1.2 CS1.2 E1.2 E1.2 E3.2 E3.2 C CS1.3 CS1.3 E1.3 E1.3 E3.3 E3.3 D CS1.4 CS1.4 E1.4 E1.4 E3.4 E3.4 E CS1.5 CS1.5 E2.1 E2.1 E4.1 E4.1 F CS1.6 CS1.6 E2.2 E2.2 E4.2 E4.2 G CS1.7 CS1.7 E2.3 E2.3 E4.3 E4.3 H CS1.8 CS1.8 E2.4 E2.4 E4.4 E4.4 ELISA is performed with each of the binding monoclonal antibodies 5F7, 7C6, 10F11.
Procedure 1. Add 100 l mAb solution per well. Incubate the ELISA plate overnight at +6 C. (fridge). 2. Decant the antibody solution and wash wells three times with 250 l PBST buffer each. 3. Add 250 l/well of blocking solution. Incubate for 2 hours at ambient temperature. 4. Decant the blocking solution and wash wells three times with 250 l PBST buffer each. 5. Add 100 l/well each of calibration standards and SDS/DTT brain extracts. Incubate plate for 2 hours at ambient temperature, then overnight at 6 C. 6. Decant the calibration standards and SDS/DTT brain extracts solution and wash wells three times with 250 l PBST buffer each. 7. Add 200 l/well of primary antibody solution and incubate for 1 hour at ambient temperature. 8. Decant the primary antibody solution and wash wells three times with 250 l PBST buffer each. 9. Add 200 l/well of secondary antibody solution and incubate for 1 hour at ambient temperature. 10. Decant the secondary antibody solution and wash wells three times with 250 l PBST buffer each. 11. Add 100 l/well of TMB solution. 12. Monitor plate colour during development (5-15 min at ambient temperature) and terminate reaction by adding 50 l/well of stop solution when an appropriate colour has developed. 13. Measure extinction at 450 nm. 14. Calculate results using calibration. 15. Evaluation: If the extinctions of the samples are beyond the linear calibration range, dilute them again and repeat.
(236) Results are shown in
(237) Brain levels of A(20-42) globulomer epitopes in brain extracts from AD patients and control subjects
(238) A sandwich ELISA was used to assess brain extracts for their truncated A(20-42) globulomer epitope content. ELISAs with the respective antibodies against the A(20-42) globulomer were used for calibration.
(239) Extraction of Alzheimer's disease brain tissue shows that the A(20-42) globulomer epitope content is significantly elevated compared to a control patient. This shows that Indeed the A(20-42) globulomer epitope is a relevant Au-species in human Alzheimer's disease brain and not only relevant for Alzheimer's disease animal models. Antibodies directed against the A(20-42) globulomer epitope therefore are highly desirable for the treatment of Alzheimer's disease.
Example 12
Development of Anti-A(20-42) Globulomer Hybridoma Cell Lines
(240) Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references Incorporated herein by reference in their entireties). The term monoclonal antibody as used herein is not limited to antibodies produced through hybridoma technology. The term monoclonal antibody refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
(241) The particular protocol used to produce the antibodies described herein is as follows:
(242) Immunization of mice: Balb/c and A/J mice (6-8 week old) were immunized subcutaneously with 50 ug of antigen in CFA. Animals were boosted every three weeks with 50 ug of antigen in Immuneasy (Qiagen) for a total of three boosts. Four days prior to fusion, mice were boosted with 10 ug of antigen Intravenously.
(243) Cell fusion and hybridoma screening: Spleen cells from Immunized animals were fused with SP2/0-Ag14 myeloma cells at a ratio of 5:1 using standard techniques. Seven to ten days post fusion, when macroscopic colonies were observed, SN were tested by ELISA for antibody to A(20-42) globulomer Cells from ELISA positive wells were scaled up and cloned by limiting dilution.
(244) Antibody isotype determination: The isotype of the anti-A(20-42) globulomer mAbs was determined using the Zymed EIA isotyping kit.
(245) Scale up and purification of monoclonal antibodies: Hybridomas were expanded into media containing 5% Low IgG. Fetal bovine serum (Hyclone). Supernatant was harvested and concentrated. mAb was purified using Protein A chromatography and dialyzed into PBS.
(246) Serum titers: Ten mice were immunized with the A(20-42) globulomer. All mice seroconverted with ELISA titers ( Max OD 450 nm) of 1:5000-10,000.
DESIGNATIONS OF HYBRIDOMAS PRODUCING MONOCLONAL ANTIBODIES
(247) Internal designations of Abbott Laboratories used for the deposits.
(248) Deposited Cell Lines:
(249) 1) ML 13-7C6.1D4.4A9.5G8 (also referred to herein as 7C6)
(250) 2) ML15-5F7.5B10 (also referred to herein as 5F7)
(251) 3) ML15-10F11.3D9 (also referred to herein as 10F11)
(252) 4) ML15-4B7.3A6 (also referred to herein as 4B7)
(253) 5) ML15-2F2.3E12 (also referred to herein as 2F2)
(254) 6) ML15-6A2.4B10 (also referred to herein as 6A2)
(255) 7) ML13-4D10.3F3 (also referred to herein as 4D10)
(256) 8) ML15-7E5.5E12 (also referred to herein as 7E5)
(257) 9) ML15-10C1.5C6.3H4 (also referred to herein as 10C1)
(258) 10) ML15-3B10.2D5.3F1 (also referred to herein as 3B10)