DE19831429A1 - Preparation and identification of polytopic peptide, useful e.g. therapeutically and for detecting antigens or antibodies, by linking fragments of a protein and testing for specific binding - Google Patents

Abstract

A method (A) for the preparation and identification of a polytopic peptide (I) that forms a complex with the corresponding binding molecule (II) (antigen or ligand) via a (II)-binding site and comprises at least two peptide fragments (PF) attached together through a linker, is new. A method (A) for the preparation and identification of a polytopic peptide (I) that forms a complex with the corresponding binding molecule (II) (antigen or ligand) via a (II)-binding site and comprises at least two peptide fragments (PF) attached together through a linker, is new and comprises: (a) covalent linkage of two PF through a linker, optionally with additional PF covalently bonded to them in the same way; (b) treating the product with (II); (c) washing to remove non-complexed (II); and (d) identifying the (I)-(II) complex. The PF are partial amino acid (aa) sequences of proteins (III) (antibody or receptor) and are combined according to the systemic mixing principal in such a way that they are identifiable. The second PF may have the same sequence as the first but usually has a different sequence, also from (III). Independent claims are also included for the following: (1) a method (B) for optimizing (I) produced by (A); and (2) a kit for carrying out (A) and (B).

Description

The invention relates to a method for identification and chemical-synthetic Imitation of binding sites on proteins, especially antibodies or Receptors.

State of the art

In order to study the binding sites of antigen and antibody or ligand and receptor, various solutions were developed.

• (i) In the X-ray structure analysis, the complexes of antigen and Antibody or ligand and receptor analyzed three-dimensionally. [JONES and THORNTON (1996) Proc. Natl. Acad. Sci. USA, Vol. 93, pp 13-20] This method is only applicable if the amino acid sequence of the antibody or Receptors is known. The X-ray structure analysis is a very complicated one Process in which crystals of the complexes of antigen / antibody or Ligand / receptor must be present.
• (ii) Another method is to test overlapping peptides from an antibody sequence or receptor sequence for binding with the antigen or ligand. This process can also be reversed, with the antigen sequence or ligand sequence (if a protein or peptide is present) being tested with the antibody or the receptor. The peptides are synthesized in the solid phase and also analyzed there [FRANK et al. (1992) Tetrahedron, Vol. 48, pp 9217-9232] or synthesized and tested in the soluble phase. [HOUGHTON (1985) Proc. Natl. Acad. Sci, USA, Vol. 82, pp 5131-5135]
  It is disadvantageous that these peptides often have low binding capacities, since these peptide sequences usually have the high binding capacity together with at least one further peptide sequence arranged adjacently in the three-dimensional protein structure.
• (iii) It has also been described to use two peptide fragments derived from an antibody. The structure of the binding sites had been precisely determined by examining mutants of the antigen. The binding ability of the ligand and the peptide fragments was tested. [Bidart et al. (1990) Science, Vol. 248, pp 736-739]
  This method is limited solely to peptide sequences in which participation in the binding is already known by other methods.
• (iv) Antibodies have hypervariable regions that bind to the antigen. There are three peptide fragments each for the heavy and light chain (together 6 Chains), which are connected to one another by linkers (spacer peptide fragment). The antibody sequence is created randomly in the body's B cells. The selection of the correct antibody which forms a complex with the antigen occurs in the course of an immune response. The body of a vertebrate, especially one Mammal, represents the location of an essential step in this process. The Sequences of the amino acids of the antibodies can only be analyzed if monoclonal antibodies are present.

Task and solution

The task is to provide a manufacturing process and identification process for at least two peptide fragments of an antibody or receptor (= protein) offer, the peptide fragments with a known structure with respect to or unknown antigen or ligand (= binding molecule) form a complex and wherein the peptide fragments are from the sequence of an antibody or receptor come.

The object is achieved by a method for the production and identification of a polytop peptide,

• (a) which via an antigen or ligand binding site forms a complex with the corresponding antigen or ligand (binding molecule) forms,
• (b which comprises at least two peptide fragments,
  • (i) which are connected to each other by a linker,
  • (ii) the fragments of an amino acid sequence of an antibody or a receptor,
  • (iii) where the first fragment is combined with the partial amino acid sequence of the protein according to the systematic mixing principle with simultaneous identifiability with the second fragment with the same or - significantly more frequently - with a further partial amino acid sequence of the protein and
    if necessary, these two fragments are combined with one or more further fragments with the same or with at least one further partial amino acid sequence of the protein according to the systematic mixing principle,

comprising the following steps:

• - making a polytop peptide,
  • - wherein the first peptide fragment is covalently linked to the second peptide fragment via a linker and, if appropriate, this second peptide fragment is covalently linked via a further linker to at least one further peptide fragment,
• - Adding the binding molecule and washing off that which is not in the complex bound binding molecule,
• - identify the complexes of polytope peptide and binding molecule.

advantages

The advantage of the method according to the invention is that this method only requires that the amino acid sequence of the antibody or the Receptor (antibody or receptor = protein) is known. Their overall sequence is subdivided into individual peptide sequences which, if adjacent sequence parts are affected, overlap. For example, the total amino acid sequence of the Protein lysozyme in 41 overlapping peptides of 10 amino acids in length, each by 3 Amino acids on the primary structure are shifted, are divided. The polytope scan contains all possible combinations of any two of these 41 peptides. the ten-mer peptide fragments are linked with a linker made up of 2 β-alanine residues, so that each polytope peptide of the polytope scan consists of 22 amino acids. Of the Scan contains 41 × 41 = 1681 different polytope peptides, which in a 41 × 41 Matrix are constructed. Thus is a certain combination of two peptide fragments assigned to a specific, defined reaction site in which only this certain combination exists.

It is not necessary to know which amino acids des Protein at the binding of the antigen or ligand (antigen or ligand = Binding molecule) are involved.

Knowledge of the structure of the antigen or ligand is needed before the test not to be present.

If, for example, biological tests ensure that an antigen or a Ligand is present in a solution, this can be used in the identification process can be used. This is essential when you are looking for a mixture Antigens or ligands are present, which among other molecules also includes the desired molecule.

Several methods are possible for the identification of complexes: (i) One Antibody to one ligand is used in combination with a second for example enzyme-labeled or fluorescence-labeled antibodies are used. (ii) An enzyme label or a fluorescent label of the first antibody is used for testing. (iii) Radioactive, enzyme or fluorescence-labeled antigens or ligands are used.

It is advantageous to use the method according to the invention in the identification of polytop- Use peptides that act as an artificial antigen in the antibody determination Patients can be used in an ELISA. Otherwise the antibody corresponding antigen is replaced by the polytope peptides. So you can also Determine antibody titers that are not used as a test because of, for example, unstable antigens are to be established. Antigens that are expensive to produce can also be easily produced be replaced.

The polytope peptides which form a complex with the antigen or ligand can be used as a biochemical tool in research and development. With antigens can be detected in diagnostics.

The use of antibodies is sometimes desirable in therapy. Is disadvantageous their size. This disadvantage can be caused by the polytopic peptides, which according to the Processes according to the invention have been produced, are compensated. the Polytop peptides can have better pharmacokinetics than antibodies.

The method according to the invention is also to be used in order to obtain polytopic detergents Peptides manufacture the dyes and other dirt particles in place of large ones Can bind antibodies. First there is an antibody against the dye or that Produce dirt particles, which then with the invention Method is downsized to a polytop peptide.

In the food industry, antibodies against certain Minimize taste receptors to polytopic peptides. Then they can be used against the taste receptors directed polytop peptides as taste additives add to the food.

In biotechnology, substances are often afflicted with antibodies Columns cleaned. The inventive method allows these antibodies by to replace the corresponding polytop peptides. Such "minimalized" antibodies are significantly cheaper to manufacture. The polytop peptides are also chemical easier to handle than the antibodies.

What can be applied to pillars can also be used in environmental analysis use. Antibodies that bind to certain pollutants can become polytop- Peptides are made smaller, so that the latter in a simple test system can be used. However, this method is not only to be used in analysis, can also be used in the cleaning or recycling of liquids and gases use the "minimized" antibody or receptor.

It can be desirable to reduce enzymes to the catalytic center. The method according to the invention allows polytope peptides to be identified that can mimic the function of enzymes. A comparable approach took place, for example, with catalytic antibodies.

It is also conceivable to use these polytope peptides for their property as enzyme inhibitors to select. To this end, it is also promising to use sequences of proteins, which act as enzyme inhibitors for the identification of polytopic peptides use.

All pharmacologically interesting proteins can be reduced in size to a binding partner with respect to a binding site. The protein-protein interaction can be inhibited (antagonists) or stimulated (agonists). These proteins, which have been reduced in size according to the method according to the invention, can either be used directly therapeutically as polytop peptides or serve as a lead structure for the conversion of the peptides into peptide mimetics or small organic molecules. Examples of these proteins are:
Cytokines or growth factors and their receptors,
Cell adhesion proteins and their receptors,
Proteins of the signal transduction pathways and their binding partners,
cytosolic receptors, steroid receptors,
Blood clotting proteins,
Neurotransmitters and their receptors,
Proteins in metabolic pathways,
Proteins in replication, transcription and translation,
Proteins that are introduced into the organism by pathogens (bacteria, viruses, eukaryotic single cells and parasites) and interact with each other or with proteins of the host.

Other embodiments Linker and N- and C-terminal building blocks

A polytop peptide can also comprise only two peptide fragments, in which case the Term duotope peptide used for this special form of the polytop peptide will.

A process according to the invention in which the linker is oligomeric is preferred can be synthesized from building blocks by the following exemplary bonds are linked to one another: (i) amide, (ii) ester, (iii) ether, (iv) urea, (v) -C-C and (vi) sulfonamide linkages. Such building blocks can be, for example, the following oligomerizable monomers: (i) Amino acids (L- and D-forms; genetically encoded and synthetic amino acids) (ii) Peptoids, (iii) PNAs, (iv) ribose phosphate elements, (v) ethylene glycol and (vi) Acrylic acid. A linker with a length of 0 to 16 building blocks, more, is preferred preferably from 1 to 10, even more preferably from 2 to 7, most preferably from 2 up to 4 modules.

A linker consisting of amino acids is more preferred. A linker is preferred from 0 to 16 amino acids in length, more preferably from 1 to 10, even more preferably from 2 to 7, most preferably from 2 to 4 amino acids. Even if the Linker does not consist of amino acids, the term duotope peptide or polytopic Peptide can be used, which thus also includes linkers that do not contain amino acids or have at least one polymer building block that is not an amino acid.

Frame building blocks

It can be advantageous if the duotope peptides have at least one further building block outside the N-terminal and / or C-terminal end in the form of at least have a further frame building block, the frame building blocks expanded polytope peptides essentially have the function that the polytope Peptide without a framework. The expression frame building block is analogous to the term polymer building block (see above) defines only its position in the polytope is different. Preferred framework building blocks are framework amino acids.

The sequence of the polytop peptide can be at the N-terminal and / or C-terminal end instead of a protective group connected to further frame building block sequences be. These additional frame building block sequences are used to bind the polytop Peptides are not essential, but they can carry other functions, like that for example, include enzymatic functions. It is also possible to use polytop Coupling peptides one behind the other, with frame building block sequences between the Individual sequences are arranged.

In order to decide in individual cases whether a polytope peptide with at least one framework building block sequence is part of the subject matter of the invention, a comparison between

• a) this polytope peptide with framework building block sequence and
• b) the same polytope peptides without a frame building block sequence

to employ. Both molecules should essentially perform the same functions the binding to the corresponding protein.

Protecting groups and their variants

It is advantageous if the polytop peptides depending on the end or amino protective groups Have carboxyl protecting groups or their variants.

The protective group or its variants for the N-terminus can consist of:
Alkyl, aryl, alkylaryl, aralkyl, alkylcarbonyl or arylcarbonyl groups with 1 to 10 carbon atoms, preferred are naphthoyl, naphthylacetyl, naphthylpropionyl, benzoyl groups or an acyl group with 1 to 7 carbon atoms.

The protective group or its variants for the C-terminus can consist of:
An alkoxy or aryloxy group having 1 to 10 carbon atoms or an amino group.

Further protective groups can be composed of building blocks, where the Building blocks amines, natural substances such as sugar, nucleic acids, phosphate (deoxy) riboses, Include PNAs, steroids, and lipids.

Further protective groups or their variants are given in Houben-Weyl (1974) Georg Thieme Verlag, 4th edition. The description of the protecting groups in the cited literature is part of the disclosure.

In order to decide in individual cases whether a polytope peptide with at least one protective group is part of the subject matter of the invention, a comparison between

• a) this polytope peptides with protective group and
• b) the same polytope peptide without a protective group

to employ. Both molecules should essentially perform the same functions the binding to the corresponding protein.

Amino acid derivatives

A method according to the invention in which the amino acids are natural is preferred or artificial amino acids.

The amino acids come from

• a) from the group of natural amino acids in L or D form or
• b) from the group of derivatives of amino acids.

Mixed forms are also possible within a peptide sequence. Derivatives of Amino acids are usually characterized by the fact that the side chains are substituted are. They can also include β-amino acids, γ-amino acids, and ω-amino acid. In addition to the twenty L-amino acids, there is a large number of amino acid derivatives, for example in the catalog of BACHEM, Bubendorf, Basellandschaft, Switzerland. The artificial amino acids are in Houben-Weyl (1974) Georg Thieme Verlag, 4th edition described. Such peptides that changed Containing amino acids often have different pharmacokinetics Day. They behave differently in the organism than the peptides, which consist exclusively of natural amino acids. They're more stable to proteases, they sometimes penetrate the cell membranes poorly, which makes it a drug The synthetic peptide used is more suitable for action in the extracellular area as a sequence of L-amino acids.

A better membrane transfer is also conceivable and in many cases (cytosolic Receptors) desired.

Usually peptides are N-C linked. However, the Partial amino acid sequences of the polytope peptide via the C-termini or via the N- Terms are linked.

Optimization procedure

An optimization method is preferred in which a polytope peptide which has been produced according to the method according to the invention is optimized in a further method by following the steps of the method according to the invention

• a) at least one amino acid in the sequence of the polytope peptide through a natural or unnatural amino acid is substituted,
• b) after each substitution the modified polytope peptide on the Binding function tested against the binding molecule and the am the best binding, modified polytope peptides are selected,
• c) the best binding, modified polytope peptides, if applicable at least one further cycle according to points (a) and (b) of the Go through the optimization process.

It is preferred if the polytope peptide is cyclized.

The result of this optimization can be a polytope peptide which is linked to the originally found sequence only has parts in common. the Binding capacity can be increased considerably, as can be seen in the example of Invention emerges.

The starting peptide and all peptides that differ from this starting peptide in a Differentiate amino acids are synthesized. Usually this will be Libraries divided into groups. Within a group, a position of the Starting peptide exchanged for all amino acids required for optimization should be used. (L-, D- and non-genetically encoded amino acids). One complete optimization library consists of as many groups as Amino acid positions are present in the peptide.

Two positions can also be optimized depending on one another. Will for example, all 20 L-amino acids used for optimization result in 20 Subgroups with 20 different peptides each. According to the combinatorial Chemistry, the two positions of the peptide to be substituted are systematic exchanged for the 20 L-amino acids, so that 20 × 20 approaches are formed. Corresponding procedures are also interdependent for 3 or more optimizing positions applicable.

This procedure is further illustrated by the examples. A routine editing of sequences corresponding to polytopic peptides is easily possible with this method. The degrees of order that occur can also be done without computer technology with the help of cellulose as a carrier material deal with.

Test reagents

The invention further comprises a set of test reagents for the production and identification of a polytop peptide,

• (a) which via an antigen or ligand binding site forms a complex with forms the corresponding binding molecule,
• (b) which comprises at least two peptide fragments,
  • (i) which are connected to each other by a linker,
  • (ii) are the fragments of an amino acid sequence of a binding molecule,
  • (iii) where the first fragment is combined with the partial amino acid sequence of the protein according to the systematic mixing principle with simultaneous identifiability with the second fragment with the same or significantly more frequently with a further partial amino acid sequence of the protein and
    optionally these two fragments are combined with one or more further fragments with the same or with at least one further partial amino acid sequence of the protein according to the systematic mixing principle,

comprising the following parts:

• - A carrier with reaction sites in which polytope peptides are synthesized and / or can be saved,
• - various polytop peptides on or in the reaction sites of the carrier,
• - Identification reagents for the complexes.

Use of a set of test reagents according to the invention for Optimization of polytope peptides, which by the method according to the invention have been produced and identified, with the modified polytop peptides with substituted amino acids are arranged.

Explanation of terms Abbreviations

The abbreviations used in the text are determined by the rules adopted by the IUPAC-IUB Commission for Biochemical Nomenclature have been established (Biochemistry 11: 1726 (1972) and Biochem. J. 219: 345 (1984)). The following usual Abbreviations are used: Ala = A = alanine; Arg = R = arginine; Asn = N = Asparagine; Cys = C = cysteine; Gln = Q = glutamine; Glu = E = glutamic acid; Gly = G = Glycine; His = H = histidine; Ile = I = isoleucine; Leu = L = leucine; Lys = K = lysine; Mead = M = methionine; Phe = F = phenylalanine; Pro = P = proline; Ser = S = serine; Thr = T = Threonine; Trp = W = tryptophan; Tyr = Y = tyrosine and Val = V = valine.

antibody

The term antibody includes all fragments of an antibody which is still has at least one weakened binding function. This includes the Example Fab fragments, the light chain, the heavy chain, the hypervariables Areas with framework amino acids (framework) of antibodies.

receptor

The term receptor includes all the glycosylated or unglycosylated proteins and Protein complexes that can bind a ligand. The receptors can be in the Cytoplasm, inserted into membranes or located in the extracellular matrix. the Receptors can also include coenzymes. Also the T cell receptors and antigen-binding fragments thereof are to be grouped under the term antibodies. Receptors often have a quaternary structure. The receptor can also act towards you Antibodies can be an antigen.

antigen

The term antigen includes all natural and artificial products which are produced by an antibody or a fragment thereof can be recognized.

According to the systematic mixing principle with simultaneous identifiability:
The peptide fragments, which are part of the amino acid sequences of the protein, are mixed. The entire sequence of the protein can be divided into partially overlapping partial amino acid sequences, that is to say into the peptide fragments.

• (i) These peptide fragments are now mixed with one another, so that due to the Reaction sites must be identified exactly, at which reaction site which one Peptide fragment with which further peptide fragment is reacted. Thus, the localization of the mixture and belongs to every mixing the identifiability of the mixing partners.
• (ii) Alternatively, spherical carrier material can also be used which the peptides are synthesized, possible. (K.S. LAM et al. (1991) Nature, Vol 354, 82). In our example 41 approaches are run, one approach per part Amino acid sequence. After the successive synthesis steps to produce the entire partial amino acid sequence, the balls are shuffled and re-inserted into 41 Reaction vessels divided. When selecting the positive balls determined and the sequence of the peptides located on it determined. The positive ones Spheres carry the sequences that bind the binding molecule. Disadvantage of this The method is the cumbersome handling after the synthesis.

It is possible to split the entire sequence of a protein into partial amino acid sequences split up or just particularly interesting sequence sections. So can Example an interesting part of a particularly conservative passage in the overall Be the amino acid sequence of the protein that is shared by a family of genes. A mix is identifiable when each specific mix that is in their Composition is known, a specific reaction site is to be assigned.

Ligand

The ligands include all natural and synthetically produced substances, which can bind to a receptor or from a T cell receptor or fragment be recognized by it. The ligand can also be directed against the receptor Be antibodies.

Identification of the complexes

Several methods are possible for the identification of complexes: (i) One Antibody to one ligand is used in combination with a second for example enzyme-labeled or fluorescence-labeled antibodies are used. (ii) An enzyme label or a fluorescent label of the first antibody is used for testing. (iii) radioactively, enzyme or fluorescently labeled Antigens or ligands are used.

Solid phase synthesis

Solid phase synthesis is described in detail in Solid Phase Synthesis, E. ATHERTON and R. C. SHEPPARD (1989) IRL Press, ISBN 1-85221-133-4 and Amino Acid and Peptide Syntheses, J. JONES, Oxford Science Publication (1992) ISBN 0-19- 855668-3.

Liquid phase synthesis

The liquid phase synthesis or solution technology is in methods of organic Chemistry (HOUBEN / WEYL), Vol. 15 / No. 1 and 2, E. WÜNSCH (editor), Thieme Verlag Stuttgart, 1974 illustrated.

Carrier materials

A wide variety of carrier materials are described in the standard literature. Particularly suitable in this process are all essentially planar supports which allow simple application of the reactive amino acids. Cellulose and polypropylene work particularly well. Materials from WHATMAN, type:
Whatman paper 540 or 50 (hardened cellulose) found very useful. Processes in which the carrier material consists of cellulose are preferred. It has the advantage of having a support which is more useful for the synthesis and on which the amino acids can easily be applied. The flat surface and the high absorbency make handling easier.

Polystyrene beads are also suitable as carrier material, on which the solid phase synthesis of the peptide library can also be carried out. The simultaneous synthesis of individual components (for example, in the case of a hexapeptide with the sequence AA _{1 .AA 2} .AA ₃ .AA ₄ .AA ₅ .AA ₆ (AA = specific amino acid) of the peptide library) can be carried out well on a multiple peptide synthesis machine (for example from the company Abimed AMS 422) 48 batches can be synthesized simultaneously with a device mentioned above, using this method larger amounts of free peptides can be obtained, which can be used for cell tests, for example.

Methods are advantageous in which the polytope peptides via alanine residues with the Support material are connected. The peptides are on or in the carrier material coupled to β-alanine residues which are covalently linked to the carrier material. β- Alanine is linked to the cellulose material via an ester bond.

Another suitable peptide coupling amino acid is glycine, others amino-carbon acids (except β-alanine), polyethylene glycol (RAPP et al. in Peptides 1988; Ed. G. JUNG, E. BAYER, Walter deGRUYTER, Berlin 1989, pages 199-201) and all Groups for peptide synthesis that are commercially available and of which after the synthesis, the peptide can be cleaved off again (cf. catalog from NOVABIOCHEM 93).

Reaction site

A reaction space is closed under reaction location (which can also be referred to as application location) understand in which identical reaction conditions prevail. It is important that the Reaction site makes it possible to understand at which point on a carrier or also in a reaction vessel which reactions have taken place.

A reaction site can thus be a part of a cellulose carrier or a separate one Be a reaction vessel with soluble substances. On the cellulose carrier pro Reaction site brought the same amino acids to reaction.

In order to make the experiment meaningful, the one or two-dimensional or also possibly three-dimensional definition of places on or in the Carrier material required to avoid that the amino acids on the corresponding reaction sites uncontrolled with the amino acids of others Can mix reaction sites.

If the reaction sites are reaction vessels, the reaction can take place as Solid phase synthesis or liquid phase synthesis (solution technology).

Process for the production of the peptides, polytop peptides Chemicals for peptide synthesis

The Fmoc-protected amino acids are from Novablochem (Switzerland) and Bachem (Bubendorf; Switzerland) available. Their production is state of the art. In the Synthesis of peptides on polystyrene resin are the amino acids in situ using PyBOP (Benzotriatol-1-yl-oxy-tripyrrolidinophasphonium hexaffuophosphate) and NMM (N- Methyl morpholine) activated.

Preparation of the peptides or peptide derivatives and the carrier Synthesis of Soluble Peptides

The synthesis of soluble peptide mixtures and peptides or peptide derivatives is on a multiple peptide synthesizer (MPS) AMS 422 from ABIMED (Langenfeld) executable (H. GAUSEPOHL et al. (1992) Peptide Research 5/6: 315-320).

Synthesis of peptides on solid support

For the peptide synthesis, hardened cellulose (Whatman 540; Catalog number 1540917) from Whatman (Maidstone, Great Britain) and Tenta Gel SRAM polystyrene resin (capacity 0.25 meq / g) from Rapp Polymers (Tübingen, Germany) used.

The synthesis of peptides on cellulose is described by Frank and Döring. (R. FRANK (1992) Tetrahedron 48: 9217-9232 and R. FRANK and R. DÖRING (1988) Tetrahedron 44: 6031-6040)

Modification of cellulose

The cellulose membrane is chemically modified to provide suitable anchor functions for to attach the subsequent peptide synthesis. These anchor functions also serve as a spacer to ensure free access to the immobilized peptides guarantee. In a first step, the entire membrane is covered with a for 3 hours 0.2 M Fmoc-β-alanine solution (activated with 0.24 M DIC and 0.4 M NMI in DMF) incubated so that an esterification with the OH groups of the cellulose uniform distribution of β-alanine results. After washing three times with DMF the Fmoc protecting groups of the esterified β-alanine are removed by incubation with 20% Cleaved piperidine in DMF (20 minutes). The membrane is five times with DMF and washed twice with methanol and dried.

In a second step, a 0.3 M Fmoc-β-alanine solution (activated with 0.3 M TBTU, 1: 1 in NMP) on defined points of the membrane with the help of a drawn out Glass capillary dripped on (approx. 0.5 µl per application site) so that a second β- Alanine is coupled (15 minutes incubation). The points are previously under Use a stencil with a pencil on the back of the membrane marked. This process can also be carried out with a peptide synthesis robot (basic device from AMS 422 from Abimed) can be automated. All remaining amino functions of the membrane by acetylation (twice for 3 minutes 2% acetic anhydride in DMF, once for 30 Minutes 2% acetic anhydride plus 1% DIPA in DMF). After five times Washing with DMS is the Fmoc protecting group with 20% piperidine in DMF (15 Minutes) split. The free amino groups are now by dyeing with 0.01% (w / v) BPB visualized in DMF and appear as blue distinct Application sites. The membrane is then washed twice with methanol and then dried.

Synthesis of the peptides

The solid phase peptide synthesis on cellulose is described in detail in Achim KRAMER and Jens SCHNEIDER-MERGENER (1998) Sytheses and Screening of Peptide Libraries on Continuous Cellulose Membrane Supports, Methods in Molecular Biology, Vol 87, Combinatorial Peptide Library Protocols pp 25-39.

Another peptide synthesis in which the peptide or peptide derivative has an anchor group is fixed and is only split off at the end of the synthesis, takes place on Polystyrene resin (batch size for example 50 µmol). The solvent used is DMF and DCM used. Double couplings are always carried out. Of the Synthesis cycle of the automat consists of the cleavage of the Fmoc protective group with 20% piperidine (twice 15 minutes) and then six times Washing procedure with DMF before the amino acids are coupled (twice 15th Minutes). Before the next Fmoc cleavage is again six times with DMF washed.

During the reaction, the automat activates 200 µmol amino acid in 400 µl DMF Addition of 200 µmol PyBOP in 220 µl DMF and 400 µmol NMM in 100 µl DMF.

The N-terminus of the peptides or peptide derivatives is made by adding DMF / acetic anhydride / DIPA in a volume ratio of 7: 2: 1 acetylated (stir for 30 minutes).

For splitting off the peptide or peptide derivative from the resin and for splitting off the Side protecting groups is 2 to 3 ml of 90% TFA, 5% triisobytylsiolane, 5% water and 5% phenol used. After five hours of incubation, the peptides or Peptide derivatives precipitated with approx. 35 ml of ice-cold ether. The precipitation will centrifuged off, the pellet (sediment) resuspended with 15 to 20 ml of ice-cold ether and washed. The centrifugation and washing is repeated five times before the Peptides or peptide derivatives in the smallest possible volume of 5% acetic acid be solved. Then the peptides or peptide derivatives freeze dried. The peptides or peptide derivatives are after the cleavage C- terminal as an amide.

BPB Bromophenol blue DCM Dichloromethane DIC Diisopropyl darbodiimide DIPA Diisopropylethylamine DMF Dimethylformamide Fmoc o-fluorenylmethoxycarbonyl PyBOP Benzotriatol-1-yl-oxy-tripyrrolidinophosphonium hexafluorophosphate NMI N-methylimidazole NMM N-methyl morpholine NMP N-methylpyrrolidone TBTU 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate TFA Trifluoroacetic acid

General manufacture of peptides

Furthermore, the peptides found with the method according to the invention are or peptide derivatives easily produced. Such peptides or peptide derivatives can be manufactured using a technique known to those skilled in the art Peptide synthesis is known. A summary of many of these techniques can be used in J. M. STEWART and J.D. YOUNG, San Francisco, 1969; and J. MEIERRHOFER, Hormonal Proteins and Peptides, Vol. 2 p 46, Academic Press (New York), 1973 for the solid phase method and E. SCHRODER and K. LUBKE, The Peptides, Vol. 1, Academic Press (New York) 1965 for the liquid phase method will. The synthesis steps are described in EP-A 0 097 031. the general procedural steps from the European publications can be analogously to the synthesis of the peptides according to the invention described here or Transfer peptide derivatives. Further literature on solid phase synthesis are: Solid Phase Synthesis, E. ATHERTON and R. C. SHEPPARD (1989) IRL Press, LSBN 1- 85221-133-4 and Amino Acid and Peptide Synthesis, J. JONES, Oxdford Science Publication (1992) ISBN 0-19-855668-3.

Examples Polytopic production Identification and characterization of a polytope derived from the sequence of the Lysozyme derived from chicken egg white, and from monoclonal antibody D1.3 is tied

An antigen was given (lysozyme from chicken protein, here lysozyme for short) that was produced by the monoclonal antibody D1.3 is bound. This model system serves as a Proof of the practicality of the process described in this application. The model system has been examined in detail and a spatial structure of the Complex is available. (Bhat et al. (1994)) Proc. Natl. Acad. Sci. USA, Vol. 91, pp 1089-1093] A peptide consisting of the lysozyme sequence should be identified is derived and binds to the antibody D1.3. This was done using a duotop scan according to the invention with the help of spot synthesis (carrier-bound protein synthesis on the smallest reaction site) on cellulose. The principle of synthesis is described in Frank (1992) Tetrahedron, Vol 48, pp 9217-9323. Lysozyme possesses 129 amino acids. This sequence was found in 41 overlapping peptides of 10 Amino acids length, each shifted by 3 amino acids on the primary structure, divided up. The Doutop-Scan contains all possible combinations of two of these 41 peptides. The ten-mer peptide fragments have a linker from FIG β-alanine residues connected so that each peptide of the duotope scan consists of 22 amino acids consists. The scan contains 41 × 41 = 1681 peptides in a 41 × 41 matrix are constructed.

The cellulose-bound duotop scan was incubated with the antibody D1.3 (0.4 μg / ml, in TBS buffer with pH 8.0 at 4 ° C. overnight) TBS = Tris buffered cream. The peptide-linked antibody was detected with a secondary peroxidase-labeled antibody and a chemiluminescent substrate. The strongest signal gave the peptide:
Gly Thr Asp Val Gln Ala Trp Ile Arg Gly β-Ala β-Ala Asp Asn Tyr Arg Gly Tyr Ser Leu Gly Asn (see also SEQ ID NO: 5)
The two peptide fragments correspond exactly to the two binding regions described in the X-ray crystal structure analysis of the complex. They are separated from one another in the primary structure of the lysozyme 90 AS, but form a coherent surface in the three-dimensional lysozyme structure. The dissociation constant of the complex of peptide and antibody D1.3 was determined according to Kriguet [FRIGUET et al. (1985) J. Immunol. Meth., Vol. 77, pp 305-319) determined to be 27 μM.

Optimization of a polytope from the interleukin-10 sequence was derived and attached to the monoclonal antibody CB / RS / 1 binds

The antibody CB / RS / 1 binds the cytokine Interieukin-10 (IL-10). The CBIRS / 1-binding peptide His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg
- Gly Gly Gly Gly Ser
- Ser Thr His Phe Pro Gly Asn Leu Pro Asn
(SEQ ID NO: 1)
identified. A secondary peroxidase-labeled antibody was used in conjunction with a chemiluminescent substrate for detection. The peptide consists of two IL-10-derived peptide fragments that are linked by a linker made up of four glycine and one serine residue. A substitution analysis, in which old amino acids were replaced one after the other by all other L-amino acids, resulted in higher signal intensities for many substitutions compared to the starting peptide. Five of these substitutions were used for a new peptide:
His Asp Asn Gln Leu Trp Glu Ala Leu Lys Gln Leu Arg Leu Arg Leu Arg
- Gly Gly Gly Gly Ser
- Ser Thr His Phe Pro Gly Asn Leu Pro Asn,
(SEQ ID NO: 2)
(the substitutions are underlined) The aforementioned peptide has a dissociation constant for the peptide-CB / RS / 1 complex of 6.8 µM. This peptide was again optimized with the aid of a substitution analysis. The resulting peptide
His Asp Asn Gln Leu Leu Glu Thr Leu Lys Gln Abs Arg Leu Arg Asn Arg
- Arg Gly Asn Gly Ser
- Ser Thr His Phe Glu Gly Asn Leu Pro Asn,
(SEQ ID NO: 3)
has a dissociation constant of 200 nM. Cyclizations of this peptide were tested with the aid of two cysteine residues which had been oxidized to a disulphide bridge. All possible distributions of two cysteine residues over the whole peptide (466 variants) were synthesized on cellulose, oxidized and tested for binding to the antibody CB / RS / 1. The dissociation constant for the complex of the best of these 466 peptides
His Asp Asn Gln Leu Leu Glu Thr Cys Lys Gln Asp Arg Leu Arg Asn Arg
- Arg Gly Asn Gly Ser
- Ser Thr His Phe Glu Gly Asn Leu Pro Cys
(SEQ ID NO: 4)
with CB / RS / 1 is 35 nM. With the same half-maximal concentration, this peptide also inhibits the interaction between CB / RS / 1 and IL-10.

Sequence listing

Claims (12)

1. Process for the preparation and identification of a polytopic peptide,

• (a) which forms a complex with the corresponding antigen or ligand (binding molecule) via an antigen or ligand binding site,
• (b) which comprises at least two peptide fragments,
  • (i) which are connected to each other by a linker,
  • (ii) the fragments of an amino acid sequence of an antibody or a receptor (= protein) are,
  • (iii) where the first fragment is combined with the partial amino acid sequence of the protein according to the systematic mixing principle with simultaneous identifiability with the second fragment with the same or - much more frequently - with a further partial amino acid sequence of the protein and optionally these two fragments according to the systematic mixing principle with one or more further fragments is combined with the same or with at least one further partial amino acid sequence of the protein,

comprising the following steps:

• - making a polytop peptide,
  wherein the first peptide fragment is covalently linked to the second peptide fragment via a linker and, if appropriate, this first or second is covalently linked via a further linker to at least one further peptide fragment,
• - adding the binding molecule and washing off the binding molecule not bound in the complex,
• - Identify the complexes of polytope peptide and binding molecule.

2. The method of claim 1, wherein the linker synthesize oligomerically leaves 3. The method of claim 2, wherein the linker is an amide, ester, ether, Includes urea, -C-C and sulfonamide linkages. 4. The method according to claim 3, wherein the linker from building blocks is preferred Amino acids, peptoids, PNAs, ribose phosphate elements, ethylene glycols and acrylic acids. 5. The method of claim 4, wherein the linker is from 0 to 16 in length Is amino acids. 6. The method according to any one of the preceding claims, wherein the polytope peptides at least one other building block outside of the N-terminal and I or C- terminal end in the form of at least one further frame module have, with the expanded polytope peptides in the framework essentially have the function that the polytope peptide without Frame module owns. 7. The method according to any one of the preceding claims, wherein the polytope peptides each have amino protective groups or carboxyl protective groups after the end, wherein the polytope peptides extended by the protective group essentially have the function possessed by the polytope peptide without a protective group. 8. The method according to any one of the preceding claims, wherein the amino acids are natural or artificial amino acids. 9. Optimization method in which a polytope peptide which has been produced by a method according to one of the preceding claims is optimized in a further method by following the steps of the method according to the preceding claims

• (a) at least one amino acid in the sequence of the polytop peptide is substituted by a natural or unnatural amino acid,
• (b) after each substitution, the modified polytope peptide is tested for its binding function with respect to the binding molecule and the modified polytope peptides which bind best are selected,
• (c) the modified polytope peptides which bind best, if appropriate, run through at least one further cycle according to points (a) and (b) of the optimization process.

10. Set of test reagents for the production and identification of a polytop peptide,

• (a) which forms a complex with the corresponding binding molecule via an antigen or ligand binding site,
• (b) which comprises at least two peptide fragments,
  • a) which are connected to each other by a linker,
  • b) the fragments of an amino acid sequence of a protein are,
  • c) wherein the first fragment is combined with the partial amino acid sequence of the protein according to the systematic mixing principle with simultaneous identifiability with the second fragment with the same or with a further partial amino acid sequence of the protein and
    where appropriate, these two fragments are combined with one or more other fragments with the same or with at least one further partial amino acid sequence of the protein according to the systematic mixing principle,

comprising the following parts:

• 1. a carrier with reaction sites in which polytope peptides can be synthesized and / or stored,
• 2. different polytop peptides on or in the reaction sites of the carrier,
• 3. Identification reagents for the complexes.

11. Use of a set of test reagents according to claim 10 for Optimization of polytop peptides, which according to the method according to one of the previous claims 1 to 9 have been produced and identified, the modified polytope peptides on a carrier in reaction sites are arranged with substituted amino acids.