Peptides having binding affinity to an antibody which recognizes an epitope on an α1 loop 2 or β2 loop 1 of an adrenoreceptor

Abstract

A peptide having an ED.sub.50 of less than 500 nm, in particular 10 nM to an antibody which recognizes an epitope on an extracellular 1 loop 2 or 2 loop1 of a human adrenoceptor wherein the antibody's binding to the epitope results in an increase of -secretase activity, an increased release of -amyolid molecules and/or cellular dysfunction of cerebral blood vessel cells, glia cells or neurons, wherein the ED.sub.50 value is measured by bioassay.

Claims

1. A peptide consisting of an amino acid sequence having at least 70% homology to the amino acid sequence X1-APEDET-X9 (SEQ ID NO: 1), wherein X1=amino group, amide, acetyl group, biotin group, heterologous spacer, heterologous linker, C, GKK, SGKK (SEQ ID NO: 6), or deletion and X9=amide, hydrazide, azide, carbamate, heterologous spacer, heterologous linker, C, GKK, SGKK (SEQ ID NO: 6), or deletion, wherein X1 and X9 cannot both be a deletion.

2. The peptide of claim 1 wherein neither X1 nor X9 can be a deletion.

3. The peptide of claim 1 consisting of an amino acid sequence having at least 80% homology to the amino acid sequence X1-APEDET-X9 (SEQ ID NO: 1).

4. The peptide of claim 1 consisting of an amino acid sequence having at least 90% homology to the amino acid sequence X1-APEDET-X9 (SEQ ID NO: 1).

5. The peptide according to claim 1, characterized in that the peptide is immobilized and/or fixed to magnetic, paramagnetic and/or non magnetic nanoparticles.

6. The peptide according to claim 1, characterized in that the peptide is bound to a solid phase.

7. A pharmaceutical composition comprising the peptide of claim 1 in combination with a pharmaceutically tolerable carrier.

8. A kit comprising the peptide of claim 1, or a solid phase on which the peptide is immobilized, together with instructions for combining contents of the kit and/or providing a formulation.

Description

EXAMPLES, MATERIALS AND METHODS

(1) Isolation and cultivation of neonatal heart cells were performed as described previously (Wallukat, G. and Wollenberger, A. 1987. Effects of the serum gammaglobulin fraction of patients with allergic asthma and dilated cardiomyopathy on chronotropic -adrenoceptor function in cultured rat heart myocytes. Biomed. Biochim. Acta. 78:634-639)

(2) Cell culture. Briefly, single cells were dissociated from the minced ventricles of Wistar rats (1-2 days old) with a 0.25% solution of crude trypsin and were cultured as monolayers with a density of 800 cells/mm2 in Halle SM 20-I medium equilibrated with humidified air. The medium contained 10% heat-inactivated FCS and 2 mol/1 fluorodeoxyuridine (Serva, Heidelberg, Germany) the latter to prevent proliferation of nonmuscle cells.

(3) On the third or fourth days, the cells were incubated for 2 h in 2 ml fresh serum-containing medium. Seven to 10 selected cells or synchronously contracting cell clusters per flask were counted for 15 s. The change of the pulsation rate were expressed as beats per minute. This procedure was repeated twice in different cultures to yield results representing a total of up to 30 cells for each sample.

(4) Immunoglobulin fractions, agonist and antagonist drugs, peptides etc. were added singly or cumulatively as indicated. The basal contraction rate of the spontaneously beating cardiomyocytes was 1624 min.

(5) Preparation of the immunoglobulin fraction. The immunoglobulin fraction was isolated from 1- to 2-ml serum samples by ammonium sulfate precipitation at a saturation of 40%. The precipitates were washed and dissolved in dialysis buffer (154 mmol/l NaCl, 10 mmol/l sodium phosphate; pH 7.2). The procedure of precipitating, washing, and dissolving was repeated twice.

(6) Finally, the immunoglobulins were taken up in 1 ml PBS (pH 7.2) and dialyzed at 4 C. for 30 hours against 11 of dialysis buffer. The buffer was changed five times during dialysis. For detection of autoantibodies, the immunoglobulin fractions were added at a dilution of 1:20 or 1:40 to the flasks.

(7) After the dialysis, the IgG fractions feature a concentration of 10-15 mg/ml, a maximum of 10 g/ml of which are specific receptor antibodies. 50 l of this IgG solution are pre-incubated with the peptides with the following concentration of peptides: 5 l of a buffered solution of 100 g/ml per 50 l IgG solution. This mixture is pre-incubated at room temperature for 30 minutes and subsequently filled into the cell culture vials, after the former nutrient solution has been removed by pipet. The amount of the medium is 2 ml. The final concentration therefore is of 0.5-0.75 mg/ml for the IgG and 0.5 g for the peptides. If the molecular weight of the peptide (10 mer) is set with 1 KD and that of the IgG is set with 150 KD, there is an excess of agAAB.

(8) For the neutralization experiments, synthetic peptides corresponding to the sequence of the three extracellular loops of the human 2 AR or 1A-AR was added in excess (0.5 g in 50 l) to 50 l of the immunoglobulin fraction. The mixtures were shaken and incubated at room temperature for 1 h. The 100-l samples were then added to neonatal rat heart muscle cells cultured in 2 ml medium to a final dilution of 1:40. The beating rate was counted for 15 s, about 60 min after the addition of the peptide/immunoglobulin mixture.

(9) For identifying the autoantibody neutralizing epitopes synthetic overlapping peptides corresponding to the sequence of the first extracellular loop of the human 2-AR or second extracellular loop of 1A-AR was added to the immunoglobulins. After 1 h incubation at room temperature the samples were used as described above.

(10) ED50 calculation. For the estimation of the 50% effective dosage of agAAB to stimulate neonatale rat cardiomyocytes the affinity chromatographic purified antibodies which bind to the epitopes in loop1 of the human 2 adrenoceptor or loop2 of the human al adrenocepotor (sub type A) the antibodies were given into the tissue culture containing neonatal rat cardiomyocytes sequencially with increasing of antibody concentration until the maximum of the increase of the beating rate was observed. The ED50 was calculated by a computer program.

(11) Patients. Sera of 14 patients with dementia of the Alzheimer type were recruited from the Clinic of Psychiatry of the Hospital Luedenscheid. The controls are immunoglobulin fraction from the blood serum of healthy donors from the Max Delbrck Centre of Moleculare Medicine, Berlin.

(12) For immunoglobulin preparation, the samples were centrifuged at 4,000 g for 30 min and stored at 20 C. Reagents was purchased from Sigma-Aldrich Chemie (Deisenhofen, Germany). All other chemicals were of analytical grade.

(13) Results

(14) The figures show the following:

(15) FIG. 1: Antibodies of patients (n=11-14) with Morbus Alzheimer stimulate rat cardiomyocytes via 2 adrenergic receptor and al adrenergic receptor.

(16) The 2 adrenergic antagonist ICI 118,551 blocks specifically but partially the stimulating effects of the agonistic antibodies. A more effective inhibition of cellular activity of contraction is observed if additional the adrenergic antagonist prazosin was given into the cell culture. 11 from 14 serum samples or immunoglobulins activate the cardiac myocytes.

(17) FIG. 2: Antibodies from patients (n=6) with Morbus Alzheimer bind to the first extracellular loop of the 2 Adrenoceptor. After coincubation of the extracellular loop peptides 1, 2 or 3 with the immunoglobulins of the patients only the loop 1 neutralise the antibody mediated increase of the cellular beating rate.

(18) FIG. 3: Antibodies from patients (n=6) with Morbus Alzheimer bind to a peptide epitope embedded in the first extracellular loop of the 2-AR. After co-incubation of the immunoglobulins with overlapping epitopes (2 amino acids) of the loop 1 peptides a neutralisation of antibody mediated increase of the beating rate was observed with the epitope FGNFWCE (SEQ ID NO: 5).

(19) FIG. 4: Antibodies of patients (n=6) with Morbus Alzheimer and directed to the 2 Adrenoceptor belong to the immunoglobulin G subclass 1. After co-incubation of the immunoglobulins with monoclonal antibodies specific for the subclasses 1-4 only with the precipitate performed with the monoclonal antibody against IgG1 the antibody mediated activity disappeared.

(20) FIG. 5: Antibodies from patients (n=6) with Morbus Alzheimer bind to the second extracellular loop of the al Adrenoceptor. After co-incubation of the loops 1, 2 or 3 with the immunoglobulins of the patients only the loop 2 neutralise the antibody mediated increase of the cellular beating rate.

(21) FIG. 6: Antibodies from patients (n=5) with Morbus Alzheimer are neutralised by a peptide embedded in the second extracellular loop of the 1A Adrenoceptor. Overlapping peptides (one or two aminoacids) were co-incubated with the immunoglobulins. The peptide with the amino acid sequence APEDET SEQ ID NO: 1) neutralises the cellular activity of the immunoglobulins.

(22) FIG. 7: Antibodies of patients (n=5) with Morbus Alzheimer and directed to the 1A Adrenoceptor belong to the immunoglobulin G subclass 1. After co-incubation of the immunoglobulins with monoclonal antibodies specific for the subclasses 1-4 only with the precipitate performed with the monoclonal antibody against IgG1 the antibody mediated activity disappeared.