EP1082339A2 - Peptides con us de maniere rationnelle, leur production et leur utilisation - Google Patents
Peptides con us de maniere rationnelle, leur production et leur utilisationInfo
- Publication number
- EP1082339A2 EP1082339A2 EP99936345A EP99936345A EP1082339A2 EP 1082339 A2 EP1082339 A2 EP 1082339A2 EP 99936345 A EP99936345 A EP 99936345A EP 99936345 A EP99936345 A EP 99936345A EP 1082339 A2 EP1082339 A2 EP 1082339A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- peptides
- peptide
- autoantibodies
- binding
- activity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
- C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
Definitions
- the invention relates to rationally designed peptides, their preparation and their use. Areas of application of the invention are medicine and the pharmaceutical industry.
- DCM Dilated cardiomyopathy
- *% AK + proportion of antibody-positive patients with the respective clinical diagnosis.
- the procedure known as immunapheresis, removes all of the immunoglobulin from the body or the bloodstream.
- target-specific autoantibodies only small portions of the body's immunoglobulin, so-called target-specific autoantibodies, could often be attributed to the cause of the disease.
- DCM dilated cardiomyopathy
- the pathological autoantibodies are detected in a microscopic biological test on cultivated, neonatal, spontaneously contracting rat cardiac muscle cells. Their beat frequency increases in the presence of certain ß1 receptor-specific autoantibodies.
- the aim of the invention is to provide new peptides suitable for the binding and removal of pathogenic autoantibodies. It is based on the task of developing an effective method for generating such peptides.
- the invention is implemented according to the claims, the subclaims are preferred variants.
- the method according to the invention for generating peptides which are suitable for binding autoantibodies is characterized by the following steps:
- the physicochemical space which is the size of the number of amino acids of the peptide, represents not only the sequences "around the seed peptide", but the entire sequence space.
- DCM dilated cardiomyopathy
- the amino acid sequences of the epitopes recognized by autoantibodies on the corresponding receptors are known in detail at DCM.
- the natural sequences of the relevant receptor areas are used in order to use them after the solid phase coupling for the elimination of the autoantibodies from the blood plasma.
- the natural amino acid sequence (10 amino acids; pos. 107-116: ARRC YNDPKC) or partial sequences thereof from Loop2 of the ⁇ 1-adrenergic receptor was selected as the original or seed peptide.
- This epitope contains the autoimmunologically relevant structure. It is known to bind autoantibodies and if this peptide is added to an appropriate biological test system, the autoantibodies present in the test system can be neutralized. This peptide is also coupled in a solid phase, ie it recognizes and binds the corresponding ß1-adrenergic autoantibodies from immunoglobulin fractions from patients.
- the natural peptide does not fully fulfill this task and has a low neutralizing capacity of the autoantibodies in the biological test.
- the peptide from the Loop2 region of the ⁇ 1-adrenergic human receptor was the starting point for the search strategy to find a peptide with higher antibody binding efficiency.
- an amino acid sequence was identified that does not occur in nature or has never been described in the corresponding databases.
- variants of peptides were developed with the help of a special computer-aided computation method, which obey certain physicochemical laws and accordingly have different binding properties towards autoantibodies and immunoglobulins.
- the peptide DRFGDKDIAF was identified, which no longer has any sequence similarity to the natural epitope (Loop2) and efficiently neutralizes the effect of the autoantibodies on the pulsation frequency of the heart muscle cells in the cell biological test used. With the help of this peptide, the binding and elimination of the Loop2-specific, ßl-adrenergic autoantibodies succeed.
- a certain number of sequence variants (DCM project: 90 variants) is generated using a defined algorithm: each amino acid residue is described with suitable physicochemical parameters.
- DCM seed peptide these were the properties 'volume' (1) and 'hydrophobicity' (libophobia) (2).
- the variants of the seed peptide to be produced are now preferably taken from the closer environment of the seed peptide in the chemical space, but also include some peptides far from the seed peptide.
- sequences were chosen so that their distances from the seed peptide in chemical space approximated a Gaussian distribution (or other localized distribution).
- the rational approach here is that peptides with a similar or even higher activity (effect, property) can be expected in the immediate vicinity of the seed peptide in the chemical space (3).
- a Gaussian distribution of peptides is very important for the next computer-aided design step, the development of sequence-activity relationships.
- the new peptides generated in step 1 are examined for their activity (action, desired property) using a suitable test method.
- Figure 1 shows the result: As the distance (Euclidean distance measure) from the seed peptide increases, the average activity of the peptide variants decreases. Two peculiarities can be found: on the one hand the average activity of the peptides closely adjacent to the seed peptide is above that of the seed peptide, and on the other hand there are significant activities far away from the seed peptide. This observation confirms the knowledge-based approach and the method for the systematic generation of peptide libraries: Several non-natural peptides with an increased ability to bind antibodies compared to the seed peptide were found.
- sequence-activity relationships are modeled.
- artificial neural networks are used for this.
- the peptides generated in step 1 and tested in step 2 together with the seed peptide are numerically described with suitable physicochemical parameters (see step 1).
- suitable physicochemical parameters see step 1.
- a mathematical connection is established between the description of the properties of the peptides and their measured activity. This is an optimization process that is described in detail in the literature (4,5).
- the consideration of both peptides with high activity and inactive peptides in this process is necessary for a meaningful modeling.
- the defined selection of peptides in step 1 fulfills this requirement.
- the neural network optimized in step 3 is used as a quality function for the computer-aided systematic search in the sequence space (amount of all possible peptides of a given one Length).
- An evolutionary algorithm is used as a search strategy (this method is described in the literature (6)). The principle is as follows: In an iterative process, new variants of a parent peptide are generated by the computer program, then all variants are evaluated with regard to their ability to bind antibodies using the neural network (see step 3), and the best peptide variant (s) ) are selected as parent peptides for the next cycle.
- the DCM project we received the sequence DRFGDKDIAF as the output of the computer program. The neural network calculated the highest activity for this peptide.
- the sequence has an identity with the original seed peptide (see step 1) in only two positions (arginine-2, aspartic acid-7). 73% of all potentially strongly antibody-binding peptides have the Asp-7 residue according to the prediction of the neural network, 17% have an asparagine in position 7.
- the system can also be used to generate peptides that do not have a certain property, therefore do not bind antibodies.
- the sequence FVRRTYYPER was proposed as such by the computer system.
- Test systems especially bioassays:
- the peptides designed by the computer in step 4 are experimentally examined for their actual biochemical biological effect.
- this test consisted of measuring the beat rate of embryonic rat myocytes with and without adding peptides to the medium.
- the normal rate of increase of human antibodies against the ⁇ 1-adrenergic receptor was in the medium.
- the exact test procedure for the DCM project is described in the literature (7).
- the result corresponded to the expectations and predictions of the computer program:
- the peptide DRFGDKDIAF was active, ie the rate of heart muscle cells increased by the pathogenic antibodies was depressed to the normal frequency, and the peptide FVRRTYYPER, which was designed as inactive, had no measurable effect (Figure 2) .
- the peptide DRFGDKDIAF had more pronounced effects than the natural antigen of the ⁇ 1-adrenergic receptor at all peptide concentrations used (10 ⁇ g / ml, 1 ⁇ g / ml, 0.1 ⁇ g / ml).
- the natural antigen had only a very weak effect (only 10% of the normal activity of the myocardial cells was observed), while the peptide designed according to the invention was able to restore 50% of the normal activity of the myocardial cells.
- Literature :
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
L'invention concerne des peptides conçus de manière rationnelle, leur production et leur utilisation à des fins biotechnologiques, médicales, diagnostiques et thérapeutiques. Selon l'invention, le procédé de production de peptides adaptés à la liaison d'auto-anticorps est caractérisé par les étapes suivantes: identification d'un peptide de semence présentant une activité appropriée; production d'une sélection restreinte de variantes de peptides à partir de la zone physico-chimique autour du peptide de semence, au moyen d'une méthode de calcul assistée par ordinateur; synthèse et test de l'activité de ces variantes de peptides; modélisation d'un rapport quantitatif séquence/activité au moyen d'un réseau neuronal artificiel; et production de peptides par évolution simulée dans la zone de séquence au moyen du réseau neuronal ayant subi un apprentissage. L'invention concerne en particulier le peptide DRFGDKDIAF.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19824641 | 1998-06-02 | ||
DE19824641 | 1998-06-02 | ||
DE19826442A DE19826442A1 (de) | 1998-06-02 | 1998-06-13 | Rational designte Peptide, ihre Herstellung und ihre Verwendung |
DE19826442 | 1998-06-13 | ||
PCT/DE1999/001575 WO1999062933A2 (fr) | 1998-06-02 | 1999-05-28 | Peptides conçus de maniere rationnelle, leur production et leur utilisation |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1082339A2 true EP1082339A2 (fr) | 2001-03-14 |
Family
ID=26046590
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP99936345A Withdrawn EP1082339A2 (fr) | 1998-06-02 | 1999-05-28 | Peptides con us de maniere rationnelle, leur production et leur utilisation |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP1082339A2 (fr) |
AU (1) | AU5150499A (fr) |
WO (1) | WO1999062933A2 (fr) |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10041560A1 (de) * | 2000-08-24 | 2002-03-07 | Max Delbrueck Centrum | Peptide des alpha1-adrenergen Rezeptors und ihre Verwendung bei Psoriasis |
WO2002020564A2 (fr) * | 2000-09-05 | 2002-03-14 | Callistogen Ag | Procede servant a identifier des sequences de peptides possedant une fonctionnalite specifique |
EP1832600A1 (fr) | 2006-03-09 | 2007-09-12 | Max-Delbrück-Centrum Für Molekulare Medizin | Peptides contre autoanticorps associees du glaucome et leur utilisation. |
EP2172481B1 (fr) | 2008-10-06 | 2014-10-29 | Novoplant GmbH | Formats d'anticorps stables de manière protéolytique |
EP2199305A1 (fr) | 2008-12-18 | 2010-06-23 | Max-Delbrück-Centrum | Peptides contre les anticorps associés au CRPS et utilisation de ces peptides |
MX2012001741A (es) | 2009-08-19 | 2012-03-21 | Merck Patent Gmbh | Anticuerpos para la deteccion de complejos de integrina en material incrustado en parafina fijado con formalina (ffpe). |
RU2014104690A (ru) | 2011-08-12 | 2015-09-20 | Е.Р.Д.Е.-ААК-Диагностик ГмбХ | АГОНИСТИЧЕСКИЕ АУТОАНТИТЕЛА К α1- И β2-АДРЕНЕРГИЧЕСКИМ РЕЦЕПТОРАМ ПРИ БОЛЕЗНИ АЛЬЦГЕЙМЕРА И СОСУДИСТОЙ ДЕМЕНЦИИ |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU7410998A (en) * | 1996-11-26 | 1998-06-22 | Johns Hopkins University, The | Ligand detection system and methods of use thereof |
US5933819C1 (en) * | 1997-05-23 | 2001-11-13 | Scripps Research Inst | Prediction of relative binding motifs of biologically active peptides and peptide mimetics |
-
1999
- 1999-05-28 EP EP99936345A patent/EP1082339A2/fr not_active Withdrawn
- 1999-05-28 WO PCT/DE1999/001575 patent/WO1999062933A2/fr not_active Application Discontinuation
- 1999-05-28 AU AU51504/99A patent/AU5150499A/en not_active Abandoned
Non-Patent Citations (1)
Title |
---|
See references of WO9962933A3 * |
Also Published As
Publication number | Publication date |
---|---|
WO1999062933A3 (fr) | 2000-04-06 |
AU5150499A (en) | 1999-12-20 |
WO1999062933A2 (fr) | 1999-12-09 |
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