WO1990012870A1 - Monoclonal antibody to amyloid peptide - Google Patents
Monoclonal antibody to amyloid peptide Download PDFInfo
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- WO1990012870A1 WO1990012870A1 PCT/US1990/002002 US9002002W WO9012870A1 WO 1990012870 A1 WO1990012870 A1 WO 1990012870A1 US 9002002 W US9002002 W US 9002002W WO 9012870 A1 WO9012870 A1 WO 9012870A1
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- monoclonal antibody
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
Definitions
- the present invention relates to neurological disorders, and, more particularly, to monoclonal antibodies that are specific for peptides associated therewith.
- Alzheimer's disease is characterized by three major pathological markers. They are neuritic plaques, neurofibrillary tangles and cerebrovascular amyloidosis. Glenner, et al., Biochem. Biophys. Res. 5 Comm. , 120:885 (1984) and Wisniewski, et al., Int. Symp. Dementia and Amyloid "Neuropathology” , Suppl. .3:87 (1986). Alzheimer's cerebrovascular amyloid protein has been purified and a 24 amino acid residue sequence has been reported. Glenner, et al., 0 Biochem. Biophys. Res. Comm., 122:1131 (1984).
- Down's Syndrome is a disability characterized by the inheritance of an extra copy of chromosome 21 in each cell. Older persons afflicted with Down's Syndrome display dementia that resembles 0 Alzheimer's disease. The cerebral tissues of these individuals exhibit the same neuropathological findings of Alzheimer's disease, i.e., amyloid-containing neuritic plaques, neurofibrillary tangles and cerebrovascular amyloidosis. The amyloid 5 deposits of Down's Syndrome contain the same peptide as the amyloid deposits found in the brains of
- Alzheimer's disease victims See, Glenner et al., Biochem. Biophys. Res. Comm., 122:1131 (1984).
- Hybridoma technology is one of the most important biological tools for the analysis of complex antigens.
- monoclonal antibodies of the present invention which are specific to recently acquired amino acid sequences of beta - amyloid precursor proteins, it has become possible to study the beta-amyloid precursor protein which leads to significant deposits of vascular and neuritic amyloid plaques in Alzheimer's disease and Down's syndrome.
- These monoclonal antibodies provide the potential for non-invasive diagnosis thereof using body fluids such as spinal fluid, serum or urine.
- the invention provides a monoclonal antibody and antigen - binding fragment thereof which are specifically reactive with a peptide whose concentration level is elevated in individuals having Alzheimer's disease or Down's syndrome as compared to individuals of substantially the same age who are not so-afflicted and which do not react with other peptides of human origin.
- the monoclonal antibodies of the invention are primarily of subclass IgG, , while others are of a subclass selected from IgG_ , IgG 2b and IgM.
- Papain - digestion produced antigen - binding fragments e.g. Fab have also been shown to specifically recognize amyloid plaque.
- the monoclonal antibody is produced by a hybridoma formed by fusion of cells from a myeloma line, usually of mouse origin, and antibody-producing cells, also usually of mouse origin, previously immunized with a peptide whose concentration level is elevated in individuals having Alzheimer's disease or Down's syndrome as compared to individuals of substantially the same age who are not so-afflicted and which do not react with other peptides of human origin, such as one which forms a portion of a cerebrovascular amyloid plaque characteristic of Alzheimer's disease or Down's syndrome.
- a mouse NSO myeloma line is used, particularly with antibody-producing cells from a mouse previously immunized with a peptide having a C-terminal sequence comprising leu - val - phe - phe - ala - glu - asp - val.
- Examples are monoclonal antibody SCVAP-2F9 and monoclonal antibody SCVAP-4G8.
- the invention provides a hybridoma cell line capable of producing the monoclonal antibodies described above.
- the invention provides a composition for quantitatively determining a peptide whose concentration level is characteristic of Alzheimer's disease or Down's syndrome in an individual.
- the composition comprises a monoclonal antibody or antigen - binding fragment thereof in accordance with the invention and a detectable moiety which is directly or indirectly associated therewith.
- the monoclonal antibody is conjugated to an enzyme and the detectable moiety comprises a chromogenic redox substrate for the enzyme.
- the monoclonal antibody is conjugated to one partner of a specific binding pair and the other partner of the specific binding pair is conjugated to a detectable moiety or a substance capable of rendering the moiety detectable.
- the monoclonal antibody or antigen - binding fragment thereof additionally serves as an antigen of the specific binding pair.
- the partner for the monoclonal or fragment is an anti-immunoglobulin antibody, usually anti - IgG and usually from a species other than that of its partner, which is labeled, such as with a fluorophore like fluorescein isothiocyanate.
- one partner of the specific binding pair is selected from biotin and its binding analogs and the other partner is selected from avidin and its binding analogs.
- the detectable moiety is a chromophore, fluorophore or luminophore and the substance capable of rendering it detectable is an energy donor or catalyst therefor.
- the invention provides an immunoassay method. The method comprises contacting a sample, from the individual suspected of having Alzheimer's disease or Down's syndrome, with the composition of the invention and quantitatively observing any detectable response.
- Fig. 1 is a graph illustrating that the relative OD reading between concentrations of SCVAP in the range shown can be quantitated using the method in accordance with the invention.
- the ⁇ sensitivity of the method is in the range of 0.2 to 0.4 ng/100 ul of SCVAP in solution.
- mAB Monoclonal Antibody
- SCVAP Cerebrovascular Amyloid Peptide
- SCVAP cerebrovascular amyloid peptide
- the purified peptide (1 mg) was conjugated to keyhole-limpet hemocyanin (6 mg) with glutaraldehyde (3 ul) in 0 sodium phosphate (1.2 ml, 50 mM, pH 7.5) at 4°C for 2 hours to provide the SCVAP-KLH antigen.
- the purified peptide was also cleaved using trypsin into fragments containing residues 1-5, 6-16, 6-24 and 17-24 as follows.
- the peptide (1 mg) in 0.1 5 M H 4 HC0 3 (500 ul) was treated with TPCK-treated trypsin (Cooper Biomedical, Malvern, PA) overnight at 37°C.
- the peptide fragments were resolved by reverse-phase chromatography on a 4.6 x 50 mm C-8 column (J.T. Baker, Phillipsburg, NJ) eluted with a linear gradient of 0-60% acetonitrile in 0.1% trifluoroacetic acid for 40 minutes at 1 ml/minute.
- the fragments were identified by amino analysis and the sequences were determined to be as follows:
- Amino acids are designated by one-letter symbols in accordance with the convention described, inter alia, in Lehninger, Biochemistry (2nd Ed.) . Worth Publishers, Inc., New York, pg. 72 (1975). These fragments were used, as described below, to sublocalize the epitopes of the monoclonal antibodies prepared as described herein.
- mice were immunized , intraperitoneally with SCVAP-KLH antigen (25-50 ug) in Ribi adjuvant (200 ul) .
- SCVAP serum-derived neuropeptide
- the mice received intraperitoneal booster injections of SCVAP (200 ug) without adjuvant.
- Immune spleen cells were harvested as described in Galfre, et al., Methods Enzymol., 3_:73 (1981). A mouse with the highest titer after SCVAP-KLH antigen immunization was selected for hybrid clone production.
- NSO myeloma cells were obtained from Dr.
- the immune spleen cells were incubated with the NSO myeloma cells and fused at a ratio of
- hybridoma cells in 1.0 ml were added to each well of 96 well tissue culture plates. After 2 to 4 weeks of incubation, hybrid cell growth was detected in 300 of 420 wells.
- the supernatant fluid in 122 of hybrids from the 300 wells were found to contain SCVAP-specific antibody as determined by ELISA, as described below.
- u Hybrids that produced SCVAP specific antibody, as determined by the ELISA procedure were cloned three times by limiting dilutions to obtain pure clones.
- Normal BALB/CJ mouse macrophages 6,000 cells per well in 96 wells of a microtiter plate
- Fifty (50) stable cloned hybrid cell lines were established and are referred to here as purified antibodies. From these, thirty (30) well-adapted, good antibody producers were grown in tissue culture flasks and further characterized.
- Hybridoma cells were tested for the production of SCVAP antibodies by the ELISA technique 5 as described by Kim et al., J. Clin. Microbiol.,
- SCVAP antigen was coated onto Falcon 96 well, 3911 Micro Test 111TM flexible assay plates by adding 50 ul of SCVAP antigen (1 ul/ l) in carbonate buffer (ph 9.6) and incubating overnight at 0 4°C.
- the substrate used was 3,3",5,5'- tetramethylbenzidine (TMB) (1 ml of 10% stock solution of TMB dissolved in dimethyl sulfoxide, 100 ml of 0.1 M sodium acetate/citric acid buffer, pH 6.0, 148 ul of 30% hydrogen peroxide).
- TMB 3,3",5,5'- tetramethylbenzidine
- Substrate 5 solution was added to each well (100 ul/well) and incubated at room temperature for 30 minutes.
- the reaction was stopped by adding 2 M sulfuric acid (25 ul) to each well.
- the color change was measured with a Dynatech Micro Elisa Auto Reader MR580, using a 450 0 nm filter.
- Monoclonal antibodies produced as described above were recovered from culture fluids and characterized by Ouchterlony double diffusion tests with goat anti-mouse antibody specific to IgG, , IgG ' IgG 2b ' IgG 3 an ⁇ I 9 M - ⁇ l ⁇ e reagents were obtained from Meloy Laboratories, Inc., Springfield, VA. Most of the clones secreted the IgG, subclass of immunoglobulin; very few secreted IgG, . IqG 2 . and IgM subclasses and none secreted the IgG_ subclass.
- the SCVAP epitope for the monoclonal antibodies from the thirty (30) clones selected was determined as follows. To identify the amino acid sequence(s) in SCVAP which are immunogenic epitopes to monoclonal antibodies produced against SCVAP, SCVAP was trypsin digested as described above and fragments containing residues 1-5, 6-24, 6-16 and 17-24 were obtained. When each of the monoclonal antibodies were tested against these peptide fragments by ELISA, all of them reacted with peptides containing the amino acid sequences 1-24, 6-24 and 17-24 and none reacted with those containing the 6-16 or 1-5 amino acid residues.
- amino acid residues 17-24 are those which form the antigenic domain that induced production of the anti-SCVAP antibodies described above. It was surprising to find that this sequence contained the preferred epitope. This region is composed of the sequence leu - val - phe - phe - ala - glu - asp - val, in which six of the eight residues are hydrophobic and are probably restricted in a beta-helix. According to the rules that good epitopes should be hydrophilic and mobile, one would not predict that this sequence would be especially antigenic. Because these antibodies react as strongly with purified cerebrovascular amyloid protein and amyloid core proteins as they do with SCVAP, it is concluded that this epitope is equally exposed in the natural amyloids and the synthetic peptide.
- Example 2 Determination of Antigenic Determinant Commonality
- monoclonal antibodies purified from mouse ascites fluid were used in immunoassays in competition with homologous and heterologous antibody preparations to determine if they had epitopic sequences in common.
- ascites fluid was obtained by mtraperitoneal in f ection of 10 7 cells into BALB/CJ mice which had been pretreated with 0.25 ml of pristane (2,6,10,14-tetramethyl-pentadecane) 1 and 8 days before injection.
- Immunoglobulins of the IgG subclasses were purified from the ascites fluid with the Affi-Gel Protein A MAPS system (Bio-Rad Laboratories, Richmond, CA) .
- the purified IgG fractions from the Protein A MAPS column were combined and dialyzed against several changes of PBS and stored at -70°C until used. Monoclonal antibodies obtained in this manner are referred to herein as purified antibody.
- HRP was conjugated to anti-SCVAP monoclonal antibodies from each of the purified preparations by the periodate method described in Wilson and Nakane, In Knapp et al(eds.), Immunofluorescence and Related Techniques, Elsevier Sci. Publ. Co., Amsterdam, pp. 215-225 (1978).
- the conjugate solution was stored at 70°C in small aliquots after adding 1% BSA, 50% glycerol and 0.01% Thimerosal.
- HRP-conjugated antibody (1:5,000 dilution of stock conjugate) was mixed with various amounts (1, 10, 100 ug/ml) of either unlabelled homologous or one of the other (heterologous) monoclonal antibodies and added to ELISA plate wells coated with limiting concentrations of SCVAP antigen.
- culture supernatants of 44 initial clones which exceeded 1.5 OD units in ELISA on SCVAP-coated wells were screened for im unocytochemical staining of cerebral amyloid.
- tissue sections were dephosphorylated prior to application of the antibodies using 43 ug/ml calf intestinal alkaline phosphatase type VII (Sigma B.ochemicals, St. Louis, MO).
- 43 ug/ml calf intestinal alkaline phosphatase type VII Sigma B.ochemicals, St. Louis, MO.
- Grundke-Iqbal et al. Proc. Nat. Acad. Sci. USA, 12:4913 (1986) and Grundke-Iqbal et al., J. Biol. Che ., 12:6084 (1986).
- tau-1 In the case of the tangles, access of the monoclonal antibody tau-1 to microtubule associated polypeptides , tau-1 is known to be blocked by phosphorylation , as reported in Grundke-Iqbal, P.N.A.S. USA, s E-ta. Therefore, tissue sections were also treated with alkaline phosphatase before immunostaining with the anti-SCVAP antibodies. ° However, no change in immunostaining pattern or intensity was observed when the tissue sections or tangles containing neurons were dephosphorylated prior to application of immunocytochemically reactive or unreactive antibodies. 5
- Cerebrovascular amyloid material was obtained from autopsy-derived meninges as described by Glenner and Wong, Biochem. Biophys. Res. Commun., 120:885 (1984). Cerebrovascular amyloid peptides 0 were isolated by size exclusion chromatography and reverse-phase chromatography as follows. Cerebrovascular amyloid was dissolved in 6 M guanidine HC1-50 mM Tris HC1, pH 7.5. The solution was dialyzed and lyophilized, and the residue of 5 amyloid peptides was dissolved in 70% formic acid, 0.1% acetonitrile. This preparation was chromatographed on a 1 x 30 cm Pharmacia HR-12 size exclusion column equilibrated with the preceding solvent. The fractions containing cerebrovascular 0 amyloid peptide were dried and further fractionated on a J.T. Baker C-8 column 0.4 x 10 cm in a solvent containing 0-60% acetonitrile in 40% formic acid.
- Neuritic plaque amyloid cores were prepared as described by Bobin, et al., Acta. Neuropathol., 74:313 (1987). Briefly, autopsy material shown to be rich in neuritic plaque cores was homogenized in 5 phosphate-buffered saline (PBS), pH 7.0, and centrifuged through 10 M sucrose to remove lipids. The pellet was digested with pepsin and collagenase and sedimented through Percoll.
- PBS phosphate-buffered saline
- the layer containing the amyloid cores was further washed with 6 M guanidine HC1, 8 M urea, and 10% sodium deoxycholate to remove adherent lipids and cerebrovascular amyloid, Neuritic plaque amyloid core peptides were purified by the same procedure as used for the purification of the cerebrovascular amyloid peptides, except that the cores were initially dissolved in 90% .formic acid.
- Anti-SCVAP Fragment 4 mAB to Amyloid Core and Peptide Monoclonal antibodies specific for amyloid 0 residues (Fragment 4) selectively stained both plaque core amyloid and cerebrovascular amyloid in brain tissue from patients with histologically confirmed Alzheimer's disease. These monoclonal antibodies were further tested for their immunoreactivity by ELISA using 96 well plastic plates coated with the purified neuritic plague core amyloid peptides and cerebrovascular amyloid peptides prepared as described above. All of these monoclonal antibodies reacted well with peptides from both amyloid core and cerebrovascular plaque by ELISA.
- Example 5 Quantitative Diagnostic Alzheimer ELISA Method A quantitative ELISA procedure for the 5 detection of SCVAP in solution using the monoclonal antibodies of the present invention was developed as follows.
- SCVAP 4G8-HRP conjugate a specific monoclonal antibody reactive to residues 17-24 of Alzheimer amyloid peptide conjugated to horseradish peroxidase
- SCVAP 4G8-HRP conjugate a specific monoclonal antibody reactive to residues 17-24 of Alzheimer amyloid peptide conjugated to horseradish peroxidase
- the relative OD reading between concentrations of SCVAP in this range can be quantitated using this method.
- the sensitivity of the method is in the range of 0.2 to 0.4 ng/100 ul of SCVAP in solution.
Abstract
Disclosed is a monoclonal antibody and specific-binding fragment thereof which is specifically reactive with a peptide whose concentration level is elevated in individuals having Down's syndrome or Alzheimer's disease as compared to individuals of substantially the same age who are not so-afflicted and which monoclonal antibody and specific-binding fragment thereof does not react with other peptides of human origin. Also disclosed is a hybridoma cell line capable of producing the monoclonal antibody, a reagent composition which incorporates the monoclonal antibody or specific-binding fragments thereof and an immunoassay method for their use.
Description
ϋ MONOCLONAL ANTIBODY TO AMYLOID PEPTIDE
BACKGROUND OF THE INVENTION
Field Of The Invention 5 The present invention relates to neurological disorders, and, more particularly, to monoclonal antibodies that are specific for peptides associated therewith.
0 Brief Description Of The Prior Art
Alzheimer's disease is characterized by three major pathological markers. They are neuritic plaques, neurofibrillary tangles and cerebrovascular amyloidosis. Glenner, et al., Biochem. Biophys. Res. 5 Comm. , 120:885 (1984) and Wisniewski, et al., Int. Symp. Dementia and Amyloid "Neuropathology" , Suppl. .3:87 (1986). Alzheimer's cerebrovascular amyloid protein has been purified and a 24 amino acid residue sequence has been reported. Glenner, et al., 0 Biochem. Biophys. Res. Comm., 122:1131 (1984). It has also been observed that the sequenced portion of the cerebrovascular amyloid peptide is part of the sequence of a precursor protein. Robakis, et al., Proc. Nat. Acad. Sci. USA, £4:4190 (1987) and 5 Robakis, et al., The Lancet, 1:384 (1987).
Down's Syndrome is a disability characterized by the inheritance of an extra copy of chromosome 21 in each cell. Older persons afflicted with Down's Syndrome display dementia that resembles 0 Alzheimer's disease. The cerebral tissues of these individuals exhibit the same neuropathological findings of Alzheimer's disease, i.e., amyloid-containing neuritic plaques, neurofibrillary tangles and cerebrovascular amyloidosis. The amyloid 5 deposits of Down's Syndrome contain the same peptide
as the amyloid deposits found in the brains of
Alzheimer's disease victims. See, Glenner et al., Biochem. Biophys. Res. Comm., 122:1131 (1984).
As applied to other peptides and antigens, including many associated with various diseases, monoclonal antibody products of hybridoma technology have often proven valuable in studying factors associated with these diseases and in diagnosis of the disease in clinical settings. Kim, et al., Neuroscience Research Communications, 2:121 (1988) discloses production and characterization of monoclonal antibodies specific for the synthetic cerebrovascular amyloid protein described in Glenner et al., Biochem. Biophys. Res. Comm., 122:1131 (1984), supra.
SUMMARY OF THE INVENTION Hybridoma technology is one of the most important biological tools for the analysis of complex antigens. Using the monoclonal antibodies of the present invention, which are specific to recently acquired amino acid sequences of beta - amyloid precursor proteins, it has become possible to study the beta-amyloid precursor protein which leads to significant deposits of vascular and neuritic amyloid plaques in Alzheimer's disease and Down's syndrome.
These monoclonal antibodies provide the potential for non-invasive diagnosis thereof using body fluids such as spinal fluid, serum or urine.
In one aspect, the invention provides a monoclonal antibody and antigen - binding fragment thereof which are specifically reactive with a peptide whose concentration level is elevated in individuals having Alzheimer's disease or Down's syndrome as compared to individuals of substantially the same age who are not so-afflicted and which do
not react with other peptides of human origin. The monoclonal antibodies of the invention are primarily of subclass IgG, , while others are of a subclass selected from IgG_ , IgG2b and IgM. Papain - digestion produced antigen - binding fragments (e.g. Fab) have also been shown to specifically recognize amyloid plaque.
The monoclonal antibody is produced by a hybridoma formed by fusion of cells from a myeloma line, usually of mouse origin, and antibody-producing cells, also usually of mouse origin, previously immunized with a peptide whose concentration level is elevated in individuals having Alzheimer's disease or Down's syndrome as compared to individuals of substantially the same age who are not so-afflicted and which do not react with other peptides of human origin, such as one which forms a portion of a cerebrovascular amyloid plaque characteristic of Alzheimer's disease or Down's syndrome. Preferably, a mouse NSO myeloma line is used, particularly with antibody-producing cells from a mouse previously immunized with a peptide having a C-terminal sequence comprising leu - val - phe - phe - ala - glu - asp - val. Examples are monoclonal antibody SCVAP-2F9 and monoclonal antibody SCVAP-4G8. Further in this regard, the invention provides a hybridoma cell line capable of producing the monoclonal antibodies described above.
In another aspect, the invention provides a composition for quantitatively determining a peptide whose concentration level is characteristic of Alzheimer's disease or Down's syndrome in an individual. The composition comprises a monoclonal antibody or antigen - binding fragment thereof in accordance with the invention and a detectable moiety which is directly or indirectly associated
therewith. In one embodiment, the monoclonal antibody is conjugated to an enzyme and the detectable moiety comprises a chromogenic redox substrate for the enzyme. In another embodiment, the monoclonal antibody is conjugated to one partner of a specific binding pair and the other partner of the specific binding pair is conjugated to a detectable moiety or a substance capable of rendering the moiety detectable. One example of this embodiment is where the monoclonal antibody or antigen - binding fragment thereof additionally serves as an antigen of the specific binding pair. The partner for the monoclonal or fragment is an anti-immunoglobulin antibody, usually anti - IgG and usually from a species other than that of its partner, which is labeled, such as with a fluorophore like fluorescein isothiocyanate. Another example is where one partner of the specific binding pair is selected from biotin and its binding analogs and the other partner is selected from avidin and its binding analogs. Usually, the detectable moiety is a chromophore, fluorophore or luminophore and the substance capable of rendering it detectable is an energy donor or catalyst therefor. * Further in this regard, the invention provides an immunoassay method. The method comprises contacting a sample, from the individual suspected of having Alzheimer's disease or Down's syndrome, with the composition of the invention and quantitatively observing any detectable response.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a graph illustrating that the relative OD reading between concentrations of SCVAP in the range shown can be quantitated using the method in accordance with the invention. The
ϋ sensitivity of the method is in the range of 0.2 to 0.4 ng/100 ul of SCVAP in solution.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
5 Example 1
Monoclonal Antibody (mAB) To Synthetic Cerebrovascular Amyloid Peptide (SCVAP) and Fragments In the experiments reported by this example, 0 a synthetic polypeptide, an antigenic conjugate thereof and tryptic fragments of the polypeptide were used to prepare and characterize the monoclonal antibodies of the invention.
5 Preparation of SCVAP and Fragments
Preparation of SCVAP and its tryptic fragments was as follows. A 24-residue synthetic cerebrovascular amyloid peptide (SCVAP) corresponding to the published sequence of the Alzheimer's 0 cerebrovascular amyloid peptide was obtained from Peninsula Laboratories, Belmont, CA. The sequence mentioned was published in Glenner, et al., Biochem. Biophys. Res. Comm., 120:885 (1984). The preparation as received was purified by reverse phase liquid 5 chromatography and the sequence was confirmed by amino-terminal sequencing as described in Bobin, et al., Acta Neuropathol. , 4-.313 (1987). The purified peptide (1 mg) was conjugated to keyhole-limpet hemocyanin (6 mg) with glutaraldehyde (3 ul) in 0 sodium phosphate (1.2 ml, 50 mM, pH 7.5) at 4°C for 2 hours to provide the SCVAP-KLH antigen.
The purified peptide was also cleaved using trypsin into fragments containing residues 1-5, 6-16, 6-24 and 17-24 as follows. The peptide (1 mg) in 0.1 5 M H4HC03 (500 ul) was treated with TPCK-treated
trypsin (Cooper Biomedical, Malvern, PA) overnight at 37°C. The peptide fragments were resolved by reverse-phase chromatography on a 4.6 x 50 mm C-8 column (J.T. Baker, Phillipsburg, NJ) eluted with a linear gradient of 0-60% acetonitrile in 0.1% trifluoroacetic acid for 40 minutes at 1 ml/minute. The fragments were identified by amino analysis and the sequences were determined to be as follows:
Fragment Residues Sequence 1 1-5 DAEFR
2 6-16 HDSGYQVHHQK
3 6-24 HDSGYQVHHQKLVFFAEDV
4 17-24 LVFFAEDV
Amino acids are designated by one-letter symbols in accordance with the convention described, inter alia, in Lehninger, Biochemistry (2nd Ed.) . Worth Publishers, Inc., New York, pg. 72 (1975). These fragments were used, as described below, to sublocalize the epitopes of the monoclonal antibodies prepared as described herein.
Preparation of Hybrid Cell Lines
Procedures for producing antibody-secreting hybrid cell lines as described in Togashi, et al., Arch. Virol., £2:149 (1981) and modified as described in Kim, et al., J. Clin. Microbiol., 1S.:331 (1983) were applied as follows.
BALB/CJ female mice (Jackson Laboratories, Bar Harbor, ME) were immunized via the back foot pads with Ribi adjuvant (200 ul) (Ribi Immunochem
Research, Inc., Hamilton, MT) containing SCVAP-KLH antigen (25-50 ug) . At 21 days after the initial injection, these mice were immunized , intraperitoneally with SCVAP-KLH antigen (25-50 ug) in Ribi adjuvant (200 ul) . Four days prior to
fusion, at 6 weeks after the last immunization, the mice received intraperitoneal booster injections of SCVAP (200 ug) without adjuvant. Immune spleen cells were harvested as described in Galfre, et al., Methods Enzymol., 3_:73 (1981). A mouse with the highest titer after SCVAP-KLH antigen immunization was selected for hybrid clone production.
NSO myeloma cells were obtained from Dr.
Choi, Kyung Hee of Albert Einstein Medical School,
New York, NY. The immune spleen cells were incubated with the NSO myeloma cells and fused at a ratio of
5:1 in the presence of polyethyleneglycol 1500
(Boehringer Mannheim, Indianapolis, IN) as the fusing agent. The cells were maintained in Iscoves modified
Dulbecco minimum essential medium containing 15%
4 fetal bovine serum, 10- M hypoxanthme, 1.6 x
10- 5 M thymidine and 4.0 x 10-7 M aminopteπn for
14 days. Subsequent feedings with medium containing only hypoxanthine and thymidine were performed on days 18, 22 and 25. After day 25, cells were fed with medium that did not include hypoxanthine, thymidine and aminopterin. Normal BALB/CJ mouse macrophages (6,000 cells per well in 96 wells of a microtiter plate) as a feeder layer were used for the initial hybridoma production.
Establishment of Hybrid Clones Secreting Anti-SCVAP mABs
After the initial hybridoma production, as 4 described above, 5 x 10 hybridoma cells in 1.0 ml were added to each well of 96 well tissue culture plates. After 2 to 4 weeks of incubation, hybrid cell growth was detected in 300 of 420 wells. The supernatant fluid in 122 of hybrids from the 300 wells were found to contain SCVAP-specific antibody as determined by ELISA, as described below.
u Hybrids that produced SCVAP specific antibody, as determined by the ELISA procedure, were cloned three times by limiting dilutions to obtain pure clones. Normal BALB/CJ mouse macrophages (6,000 cells per well in 96 wells of a microtiter plate) as a feeder layer were used here also. Fifty (50) stable cloned hybrid cell lines were established and are referred to here as purified antibodies. From these, thirty (30) well-adapted, good antibody producers were grown in tissue culture flasks and further characterized.
ELISA Screening for Anti-SCVAP mABs
Hybridoma cells were tested for the production of SCVAP antibodies by the ELISA technique 5 as described by Kim et al., J. Clin. Microbiol.,
1£:331 (1983). SCVAP antigen was coated onto Falcon 96 well, 3911 Micro Test 111TM flexible assay plates by adding 50 ul of SCVAP antigen (1 ul/ l) in carbonate buffer (ph 9.6) and incubating overnight at 0 4°C. The substrate used was 3,3",5,5'- tetramethylbenzidine (TMB) (1 ml of 10% stock solution of TMB dissolved in dimethyl sulfoxide, 100 ml of 0.1 M sodium acetate/citric acid buffer, pH 6.0, 148 ul of 30% hydrogen peroxide). Substrate 5 solution was added to each well (100 ul/well) and incubated at room temperature for 30 minutes. The reaction was stopped by adding 2 M sulfuric acid (25 ul) to each well. The color change was measured with a Dynatech Micro Elisa Auto Reader MR580, using a 450 0 nm filter.
The antibody titers as determined by ELISA for tissue culture supernatant and ascites fluids were mostly around 1:20,000 and 1:1,000,000, respectively. 5
Characterization of Immunoglobulin Subclasses
Monoclonal antibodies produced as described above were recovered from culture fluids and characterized by Ouchterlony double diffusion tests with goat anti-mouse antibody specific to IgG, , IgG ' IgG 2b' IgG 3 an^ I9M- τlιe reagents were obtained from Meloy Laboratories, Inc., Springfield, VA. Most of the clones secreted the IgG, subclass of immunoglobulin; very few secreted IgG, . IqG2. and IgM subclasses and none secreted the IgG_ subclass.
Characterization of SCVAP mAB-Inducing Epitope
The SCVAP epitope for the monoclonal antibodies from the thirty (30) clones selected was determined as follows. To identify the amino acid sequence(s) in SCVAP which are immunogenic epitopes to monoclonal antibodies produced against SCVAP, SCVAP was trypsin digested as described above and fragments containing residues 1-5, 6-24, 6-16 and 17-24 were obtained. When each of the monoclonal antibodies were tested against these peptide fragments by ELISA, all of them reacted with peptides containing the amino acid sequences 1-24, 6-24 and 17-24 and none reacted with those containing the 6-16 or 1-5 amino acid residues.
These data demonstrate that amino acid residues 17-24 are those which form the antigenic domain that induced production of the anti-SCVAP antibodies described above. It was surprising to find that this sequence contained the preferred epitope. This region is composed of the sequence leu - val - phe - phe - ala - glu - asp - val, in which six of the eight residues are hydrophobic and are probably restricted in a beta-helix. According to the rules that good epitopes should be hydrophilic
and mobile, one would not predict that this sequence would be especially antigenic. Because these antibodies react as strongly with purified cerebrovascular amyloid protein and amyloid core proteins as they do with SCVAP, it is concluded that this epitope is equally exposed in the natural amyloids and the synthetic peptide.
Example 2 Determination of Antigenic Determinant Commonality In the experiments reported by this example, monoclonal antibodies purified from mouse ascites fluid were used in immunoassays in competition with homologous and heterologous antibody preparations to determine if they had epitopic sequences in common.
Preparation of Purified mABs from Ascites Fluid
From the cloned hybrid cell lines, prepared as described in Example 1, that continued to produce antibody for 3 months, ascites fluid was obtained by mtraperitoneal infection of 10 7 cells into BALB/CJ mice which had been pretreated with 0.25 ml of pristane (2,6,10,14-tetramethyl-pentadecane) 1 and 8 days before injection.
Immunoglobulins of the IgG subclasses were purified from the ascites fluid with the Affi-Gel Protein A MAPS system (Bio-Rad Laboratories, Richmond, CA) . The purified IgG fractions from the Protein A MAPS column were combined and dialyzed against several changes of PBS and stored at -70°C until used. Monoclonal antibodies obtained in this manner are referred to herein as purified antibody.
Anti-SCVAP AB - Horseradish Peroxidase (HRP) Conjugate
HRP was conjugated to anti-SCVAP monoclonal antibodies from each of the purified preparations by the periodate method described in Wilson and Nakane, In Knapp et al(eds.), Immunofluorescence and Related Techniques, Elsevier Sci. Publ. Co., Amsterdam, pp. 215-225 (1978). The conjugate solution was stored at 70°C in small aliquots after adding 1% BSA, 50% glycerol and 0.01% Thimerosal.
Identification of Antigenic Peterminants
For each monoclonal antibody a fixed amount of HRP-conjugated antibody (1:5,000 dilution of stock conjugate) was mixed with various amounts (1, 10, 100 ug/ml) of either unlabelled homologous or one of the other (heterologous) monoclonal antibodies and added to ELISA plate wells coated with limiting concentrations of SCVAP antigen.
All of the monoclonal antibodies reactive to residues 17-24 (Fragment 4) effectively competed with each other and, therefore, appear to be directed either against the same epitope, or overlapping epitopes present in the amino acid sequence of residues 17-24.
Example 3 Immunocytochemistrv of Alzheimer-Specific mABs
In this example, culture supernatants of 44 initial clones which exceeded 1.5 OD units in ELISA on SCVAP-coated wells were screened for im unocytochemical staining of cerebral amyloid.
Immunostaining Procedure
All supernatants were diluted with equal volumes of PBS containing 20 mg/ml BSA and applied on
6 urn sections of paraffin-embedded, formalin fixed Alzheimer cortex. Bound antibodies were visualized using the avidin biotin complex technique (ABC Kit, Vector Labs, Burlingame, CA) as described in Wang et al., Acta Neuropathol. (Berl. ) , £2:268 (1984). Selected supernatants were also tested on sections of Alzheimer hippocampus, a non-demented aged subject and tangles-containing neuronal cell bodies isolated from Alzheimer cortex as described in Iqbal et al., Acta Neuropathol. (Berl.) , .62:167 (1984). In some instances tissue sections were dephosphorylated prior to application of the antibodies using 43 ug/ml calf intestinal alkaline phosphatase type VII (Sigma B.ochemicals, St. Louis, MO). In this regard, see Grundke-Iqbal et al., Proc. Nat. Acad. Sci. USA, 12:4913 (1986) and Grundke-Iqbal et al., J. Biol. Che ., 12:6084 (1986).
Immunostaining of Cerebral Amyloid - Results Fifty (50) percent of the culture supernatants reactive with the 17-24 amino acid sequence of SCVAP also strongly reacted with amyloid present in neuritic plaques and cortical and meningeal vessels both in brain sections of Alzheimer patients and non-demented controls. No staining of Alzheimer neurofibrillary tangles or axons/neurofilaments either in tissue sections or in isolated neurons was observed with any of the supernatants.
In the case of the tangles, access of the monoclonal antibody tau-1 to microtubule associated polypeptides , tau-1 is known to be blocked by phosphorylation , as reported in Grundke-Iqbal, P.N.A.S. USA, s E-ta. Therefore, tissue sections were also treated with alkaline phosphatase before immunostaining with the anti-SCVAP antibodies.
° However, no change in immunostaining pattern or intensity was observed when the tissue sections or tangles containing neurons were dephosphorylated prior to application of immunocytochemically reactive or unreactive antibodies. 5
Example 4 Immunohistology Using Alzheimer-Specific mABs
In the experiments reported by this example, purified cerebrovascular amyloid peptides and 0 purified neuritic plaque amyloid core peptides were used for testing of recognition by monoclonal antibody specific for Fragment 4, described in Example 1.
5 Preparation of Cerebrovascular A yloid Peptides
Cerebrovascular amyloid material was obtained from autopsy-derived meninges as described by Glenner and Wong, Biochem. Biophys. Res. Commun., 120:885 (1984). Cerebrovascular amyloid peptides 0 were isolated by size exclusion chromatography and reverse-phase chromatography as follows. Cerebrovascular amyloid was dissolved in 6 M guanidine HC1-50 mM Tris HC1, pH 7.5. The solution was dialyzed and lyophilized, and the residue of 5 amyloid peptides was dissolved in 70% formic acid, 0.1% acetonitrile. This preparation was chromatographed on a 1 x 30 cm Pharmacia HR-12 size exclusion column equilibrated with the preceding solvent. The fractions containing cerebrovascular 0 amyloid peptide were dried and further fractionated on a J.T. Baker C-8 column 0.4 x 10 cm in a solvent containing 0-60% acetonitrile in 40% formic acid.
5
° Preparation of Neuritic Plaque Core Peptides
Neuritic plaque amyloid cores were prepared as described by Bobin, et al., Acta. Neuropathol., 74:313 (1987). Briefly, autopsy material shown to be rich in neuritic plaque cores was homogenized in 5 phosphate-buffered saline (PBS), pH 7.0, and centrifuged through 10 M sucrose to remove lipids. The pellet was digested with pepsin and collagenase and sedimented through Percoll. The layer containing the amyloid cores was further washed with 6 M guanidine HC1, 8 M urea, and 10% sodium deoxycholate to remove adherent lipids and cerebrovascular amyloid, Neuritic plaque amyloid core peptides were purified by the same procedure as used for the purification of the cerebrovascular amyloid peptides, except that the cores were initially dissolved in 90% .formic acid.
Anti-SCVAP Fragment 4 mAB to Amyloid Core and Peptide Monoclonal antibodies specific for amyloid 0 residues (Fragment 4) selectively stained both plaque core amyloid and cerebrovascular amyloid in brain tissue from patients with histologically confirmed Alzheimer's disease. These monoclonal antibodies were further tested for their immunoreactivity by ELISA using 96 well plastic plates coated with the purified neuritic plague core amyloid peptides and cerebrovascular amyloid peptides prepared as described above. All of these monoclonal antibodies reacted well with peptides from both amyloid core and cerebrovascular plaque by ELISA.
Example 5 Quantitative Diagnostic Alzheimer ELISA Method A quantitative ELISA procedure for the 5 detection of SCVAP in solution using the monoclonal
antibodies of the present invention was developed as follows.
Fifty (50) ul of various dilutions of monoclonal antibody SCVAP 4G8-HRP conjugate (a specific monoclonal antibody reactive to residues 17-24 of Alzheimer amyloid peptide conjugated to horseradish peroxidase) were titrated by ELISA on wells coated with the maximum amount of SCVAP antigen (5 ug/ml) .
The dilution of monoclonal antibody SCVAP 4G8-HRP conjugate that gave a relative OD reading close to 1.0 when incubated with TMB for 30 minutes as described above was determined. This concentration of monoclonal antibody SCVAP 4G8-HRP conjugate was mixed with an equal volume of SCVAP solution containing varying concentrations of SCVAP and incubated overnight at 4°C. The amount of unadsorbed monoclonal antibody SCVAP 4G8-HRP conjugate was then measured by titrating the mixture on ELISA 96 well plates coated with 5 ug/ml of SCVAP. After 4 hours of incubation at room temperature the ELISA OD was determined by using TMB as the substrate.
As shown in Fig. 1, the relative OD reading between concentrations of SCVAP in this range can be quantitated using this method. The sensitivity of the method is in the range of 0.2 to 0.4 ng/100 ul of SCVAP in solution.
Claims
1. A monoclonal antibody or antigen - binding fragment thereof which is specifically reactive with a peptide whose concentration level is elevated in individuals having Down's syndrome or Alzheimer's disease as compared to individuals of substantially the same age who are not so-afflicted and which does not react with other peptides of human origin.
2. The monoclonal antibody or binding fragment of claim 1 which is specifically reactive with a cerebrovascular amyloid protein whose elevated levels are characteristic of Down's syndrome and Alzheimer's disease.
3. The monoclonal antibody or binding fragment of claim 1 which is of subclass IgG,. i
4. The monoclonal antibody or binding fragment of claim 1 which is of a subclass selected from the group consisting of IgG_ , IgG_, and IgM.
5. The monoclonal antibody or binding fragment of claim 1 which is produced by a hybridoma formed by fusion of cells from a mouse myeloma line and antibody-producing cells from a mouse previously immunized with a peptide whose concentration level is elevated in individuals having Down's syndrome or Alzheimer's disease as compared to individuals of substantially the same age who are not so-afflicted and which does not react with other peptides of human origin.
6. The monoclonal antibody or binding fragment of claim 1 which is produced by a hybridoma formed by fusion of cells from a mouse myeloma line and ° antibody-producing cells from a mouse previously immunized with a cerebrovascular amyloid protein whose elevated levels are characteristic of Alzheimer's disease or Down's syndrome.
5 7. The monoclonal antibody or binding fragment of claim 1 which is produced by a hybridoma formed by fusion of cells from a mouse NSO myeloma line and antibody-producing cells from a mouse previously immunized with a peptide whose concentration level is 0 elevated in individuals having Down's syndrome or Alzheimer's disease as compared to individuals of substantially the same age who are not so-afflicted.
8. The monoclonal antibody or binding fragment of 5 claim 1 which is produced by a hybridoma formed by fusion of cells from a mouse NSO myeloma line and antibody-producing cells from a mouse previously immunized with a cerebrovascular amyloid protein whose elevated levels are characteristic of 0 Alzheimer's disease or Down's syndrome.
9. A monoclonal antibody which is produced by a hybridoma formed by fusion of cells from a mouse NSO myeloma line and antibody-producing cells from a 5 mouse previously immunized with a peptide having a
C-terminal sequence comprising leu - val - phe - phe - ala - glu - asp - val, or an antigen - binding fragment of said monoclonal antibody.
0 10. A monoclonal antibody selected from the group consisting of monoclonal antibody SCVAP 2F9 and monoclonal antibody SCVAP 4G8, or an antigen - binding fragment of said monoclonal antibody.
5
11. A hybridoma cell line capable of producing the monoclonal antibody of claim 1.
12. The hybridoma cell line of claim 11 which is formed by fusion of cells from a myeloma line and antibody-producing cells previously immunized with a peptide whose concentration level is elevated in individuals having Down's syndrome or Alzheimer's disease as compared to individuals of substantially the same age who are not so-afflicted.
13. The hybridoma cell line of claim 11 which is formed by fusion of cells from a mouse myeloma line and antibody-producing cells from a mouse previously immunized with a peptide whose concentration level is elevated in individuals having Down's syndrome or Alzheimer's disease as compared to individuals of substantially the same age who are not so-afflicted.
14. The hybridoma cell line of claim 11 which is formed by fusion of cells from a mouse NSO myeloma line and antibody-producing cells from a mouse previously immunized with a peptide whose concentration level is elevated in individuals having Down's syndrome or Alzheimer's disease as compared to individuals of substantially the same age who are not so-afflicted.
15. The hybridoma cell line of claim 11 which is formed by fusion of cells from a mouse NSO myeloma line and antibody-producing cells from a mouse previously immunized with a peptide having a C-terminal sequence comprising leu - val - phe - phe - ala - glu - asp - val. ° 16. A composition for quantitatively determining a peptide whose elevated levels are characteristic of Alzheimer's disease or Down's syndrome in an individual, which composition comprises the monoclonal antibody or specific - binding fragment of
5 claim 1 and a detectable moiety which is directly or indirectly associated therewith.
17. The composition of claim 16 wherein said monoclonal antibody or specific - binding fragment is 0 conjugated to an enzyme and said detectable moiety comprises a chromogenic redox substrate for said enzyme.
18. The composition of claim 16 wherein said 5 monoclonal antibody or specific - binding fragment is specifically bindable with a substance attached to said detectable moiety.
19. The composition of claim 18 wherein the substance 0 attached to said detectable moiety is an antibody specifically bindable with an immunoglobulin.
20. The composition of claim 16 wherein said monoclonal antibody is conjugated to one partner of a 5 specific binding pair and which further comprises the other partner of said specific binding pair conjugated to said detectable moiety or a substance capable of rendering said moiety detectable.
0 21. The composition of claim 20 wherein one partner of said specific binding pair is selected from the group of biotin and its binding analogs and the other partner of said binding pair is selected from the group of avidin and its binding analogs. 5
° 22. The composition of either of claims 20 or 21 wherein said detectable moiety is a chrornophore, fluorophore or luminophore and said substance capable of rendering said moiety detectable is an energy donor or catalyst therefor. 5
23. A method for quantitatively determining a peptide whose elevated levels are characteristic of Alzheimer's disease or Down's syndrome in an individual, which method comprises contacting a 0 sample from said individual with the composition of any of claims 16-20 and quantitatively observing any detectable response.
5
0
5
0
5
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US33830289A | 1989-04-14 | 1989-04-14 | |
US338,302 | 1989-04-14 |
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WO1990012870A1 true WO1990012870A1 (en) | 1990-11-01 |
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PCT/US1990/002002 WO1990012870A1 (en) | 1989-04-14 | 1990-04-13 | Monoclonal antibody to amyloid peptide |
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