HK1081971A - Binding molecules - Google Patents

Description

Binding molecules

no marking

During the pathogenesis of many diseases, the active concentrations of antibodies, enzymes and endocrine, paracrine or autocrine messengers change. These biologically active binding molecules may be proteins (e.g.insulin, growth factors, cytokines, secretion hormones, antibodies, enzymes, etc.), lipids (prostaglandins and similar fatty acid derivatives), steroids (e.g.glucocorticoids, mineralocorticoids). Hereinafter, the term cytokine will be interpreted as a biologically active binding molecule that participates in a binding process or a signaling process in a prototypic manner. Cytokines are biological messengers with a high degree of specificity and activity. They produce biological activity by binding to cytokine-specific receptors preferably located on the surface of target cells. When a messenger contacts a receptor, the latter typically initiates an intracellular signaling cascade that causes a biological effect within the cell or tissue. Typical effects triggered by these receptors are, for example, entry of the cell into the cell cycle with a consequent increase in the rate of proliferation, or triggering apoptosis, which is a form of cell death. In the course of inflammation, cytokines also play a very important role in the homeostatic balance of the respective biological organism in the immune system and in many local regulatory processes of the mammalian body. For many cytokines, such as most members of the interleukin family of cytokines, the receptors on the cell surface are composed of different proteins that aggregate by binding to the cytokine. Thus, many cytokines have specific binding sites that must bind to two or more receptor proteins simultaneously in order to elicit an effect with high efficiency. This suggests that in cytokine receptor interactions, precisely defined distances and spatial positioning of the binding regions in the cytokine molecule from each other are critical and necessary for full manifestation of the effect. In addition, for certain cytokines, this cytokine is composed of different proteins that need to be bound during receptor interactions (c.aul, w.schneider (Eds.), Biological Activities and Clinical effects, 1997, Springer Verlag Berlin). Recently, many cytokines have been recombinantly produced for use as drugs. For example, indications for which such drugs have been used are serious, life-threatening neoplastic diseases and immunodeficiency diseases.

However, recombinant formulations have the following disadvantages:

1. the preparation of recombinant proteins, such as cytokines, requires a high consumption during the "set-up" process. The cells used for expression must be guaranteed in a complex manner to produce precisely the protein in the exact "native" conformation. Many experiments are required to find suitable expression systems for cells, vectors and conditions to be suitable for high expression and stability in solution. In general, "assembly" conditions are not transferable to large-scale fermentations. Even if optimal conditions are found, expression systems tend to be unstable and prone to problems.

2. Since the cytokine is to be administered regularly, it must be ensured that it is purified after expression, which is usually carried out in bacteria, and is then absolutely free from bacterial contamination in order to avoid systemic allergies. This necessitates the use of complex methods, which have a large impact on pricing.

3. Many messengers, such as cytokines, have not only one receptor binding site, but other domains are present that are not in most cases fully understood by humans and may induce signaling pathways other than those intended. This may also explain to some extent the side effects frequently occurring when treating with these materials.

4. Recombinantly produced proteins often have a tertiary structure that is different from the native protein and are therefore recognized as "foreign" by the human body. The antibodies induced thereby neutralize the cytokine, resulting in loss of cytokine activity.

5. Recombinant proteins often exhibit significant instability towards proteolytic, i.e. proteolytic, enzymes. This necessitates frequent administration at higher concentrations, which on the one hand increases the stress on the patient and on the other hand makes the treatment expenditure very high.

An economical alternative to recombinant production of cytokines is the totally synthetic mimetic of biologically active proteins such as cytokines, which is feasible in organic chemistry.

For example, one approach taken here is to artificially design the catalytically active centers of known proteins, although this research is still in its infancy and to date only allows stabilization of the peptide structure (beta sheets, helices, turns) and in some cases targeted coordination of small molecules.

Another approach is computer-aided design of small, specific binding molecules with e.g. peptide properties. The sequence of such small specific binding molecules is typically less than 100 amino acids. Due to their size, such molecules cannot be used to bind receptor subunits that require full cytokine activity. In general, they cannot be designed to have high affinity for two or more binding sites on a large receptor molecule at the same time.

To date, peptides can be synthesized very simply by a variety of conventional methods, although separate synthetic methods must be established for each peptide. In general, solid phase synthesis of peptides is repeated by activating N-terminally protected amino acids, coupling, washing, deblocking, activation, etc., until the desired peptide is obtained. The product is removed from the solid phase, purified by HPLC and then transferred to other analyses, such as sequence identification and biological testing. Generally, the shorter the peptide, the simpler and more reliable the synthesis.

Studies with several peptide oligomers have shown that chemically synthesized and oligopeptides exhibit biological activity which is generally lower than that of the native protein, for example, in the case of erythropoietin (patent No. WO96/40772), but generally more potent than the monomer.

Up to now, the preparation of oligomers has also been carried out according to conventional methods described in the literature, using the most commercially available "crosslinkers". Often, these molecular structures, which do not exist in vivo, are biologically inert and unobtrusive in order to render a particular chemical component immune.

Peptides have a very short half-life in vivo, i.e. they are degraded and excreted very rapidly by endogenous enzymes. Various methods are used to metabolically stabilize peptides and proteins. On the one hand, unnatural amino acids which cannot be cleaved are used during synthesis, and on the other hand, proteins are modified by inert chemical groups to protect them from degradation. One example is known as the PEG intron (Schering-Plough), an interferon modified with polyethylene glycol.

Depending on the planned geometric arrangement of the peptides as specific binding molecules, different strategies must be used to prepare the possible support structures (support structures) to develop individual support structures for each specific problem. Until the present invention, there has been no suitable method to solve this problem synthetically by establishing a general strategy in which the number, type and mutual orientation of specific binding molecules is variable. The methods used previously for the preparation of suitable carrier structures are based either on the formation of disulfide bridges ((a) Nomiz, M., Utani, A., Shiraishi, N., Yamada, Y, Roller, P., "Synthesis and correlation of the synthetic linked-linkage of peptides"; Int J peptide Res (40) (1); 1992; 72-79) or on the use of lysine-based dendrimer structures ((a) receptors, M.D., Lerner, M.R.; Synthesis and conversion of bivalidational linked-to G-protein-linked-ligands "; Chem Bio 3 (7); Ser; 537; Syda, T., Oda, K., Synthesis, K., Moakkukuk-linked-linkage, K., K-linked-peptides, K., K, schleuder, D., Rauterberg, J., "Synthesis and folding of native collagen III models peptides"; biochemistry 38 (41); 1999; 13610-22) and thus these methods constitute a limit to the possibilities.

It is an object of the present invention to provide binding molecules with high physiological activity which do not have the above-mentioned disadvantages of the binding molecules of the prior art.

Surprisingly, the object forming the basis of the present invention is achieved by a binding molecule consisting of a carrier structure with at least one cyclic molecular subunit and at least two side chain subunits, wherein the side chain subunits are polypeptide chains synthesized from natural and/or unnatural D-and/or L-amino acids, wherein the side chain subunits are covalently bound to the carrier structure.

The cyclic molecular subunits are preferably cyclic polypeptides synthesized from natural or unnatural D-and/or L-amino acids, aromatic rings, aromatic ring systems, polylactones, polylactams, benzenetriamides (benzanetriamides), trihydroxybenzoic acids, trilactones or trilactams. In certain preferred embodiments where just two side chains are attached, the cyclic carrier structure may also be minimized in structure and replaced with a linear symmetric structure.

The amino acids forming the cyclic polypeptide are advantageously linked by peptide bonds. If the cyclic structure is formed by the formation of disulfide bridges, cyclic molecular subunits in the sense of the present invention are not present. This type of cyclic peptide structure is an integral part of many naturally occurring proteins. Due to their instability, are unsuitable as central cyclic molecular subunits of the vector structure in the sense of the present invention. Of course, this does not exclude that the binding molecules of the invention comprise a disulfide bridge in addition to the more stable cyclic molecular subunits in the carrier structure. Such additional disulfide bonds are preferably located at sites remote from the carrier structure, which introduction will stabilize a particular spatial configuration within and/or between two or more side chains.

If the cyclic molecular subunit is a cyclic polypeptide, in a preferred embodiment the at least two side chain subunits of the binding molecule are bound to the cyclic molecular subunit via amino acid side chain moieties of amino acids such as lysine, serine, threonine, glutamic acid, asparagine and/or aspartic acid in the cyclic molecular subunit.

The cyclic polypeptide preferably consists of 2 to 10 amino acids, in particular 2, 3, 4 or 6 amino acids.

In a preferred embodiment, the cyclic molecular subunit has one of the following structures:

a) benzene triamides

b) Cyclic hexapeptides

Or a cyclohexapeptide with 1-3 side chain peptides Seq and a sequence of DKDDK,

c) a trilactone:

d) a trilactam:

e) trihydroxybenzoic acid carrier structure:

f) cyclic dipeptide: loop-L-Ala-L-Lys, derivatized with Seq,

wherein Seq or Pep represents a side chain subunit or-OH. According to the invention, the molecule comprises at least two side chain subunits, which may be identical or different from each other.

If the at least two side chain subunits in the binding molecule of the invention are polypeptide chains, they may have the same amino acid sequence or different sequences.

The polypeptide chain of the side chain subunit preferably consists of 5 to 60 amino acids, preferably 10 to 50 amino acids, in particular 12 to 40 amino acids. The molecular weight of such polypeptide chains is preferably no more than 10kDa, in particular less than 8kDa or less than 5 kDa.

The spatial position of two or more side chains of a circular carrier structure or a linear linker molecule end can be fixed by introducing a disulfide bond between two sterically suitable adjacent residues (e.g. cysteine or homocysteine) of each side chain during the whole molecule synthesis process, which is also considered part of the present invention. For this purpose, the amino acid sequence of the side chain may be modified or optimized to contain one or more suitable SH-containing residues at the appropriate positions.

Another object of the invention is a binding molecule wherein the side chain subunits are oligomeric or polymeric nucleic acids consisting of natural and non-natural nucleic acid units. Thus, the nucleotides may be linked in a natural manner or be present as Peptide Nucleic Acids (PNAs). The molecular weight of such side chain subunits is preferably not more than 10kDa, in particular less than 8kDa, or less than 5 kDa. For example, such binding molecules of the invention may be used to bind a DNA binding protein or protein complex having multiple DNA binding sites.

In a preferred embodiment, the side chain subunits are linked to the carrier structure by spacer molecule subunits (spacers), which in particular consist of natural and non-natural carbohydrates or nucleic acids.

Preferably, the free ends of the side chain subunits are covalently bound to a suitable protecting group to prevent degradation by exopeptidases. Depending on the type of bond between the side chain subunit and the support structure, the N-or C-terminus may be free. Preferred protecting groups are D-amino acids, preferably di-or tripeptides. In addition, protective groups within the scope of solid phase synthesis are generally used, and monomeric and polymeric carbohydrates can be used.

To prevent exopeptidases from cleaving the side chain peptide, the internal amino acids may also be covalently bound to a protecting group. Preferred protecting groups are carbohydrates, preferably selected from glucose, mannose, N-acetylglucosamine or N-acetylgalactosamine or sialic acid.

In a preferred embodiment, the unnatural amino acid that can be a constituent of a binding molecule of the invention is of the formula NH₂(CH₂)_nCOOH, wherein n is 2-8; in particular NH₂(CH₂)₂COOH，NH₂(CH₂)₃COOH or NH₂(CH₂)₄COOH。

The present invention enables the development and preparation of fully synthetic physiologically binding molecular mimetics that overcome the above-described disadvantages of the prior art. The present invention designs a carrier structure with the orientation of the binding molecules on its backbone enabling the simulation of the binding characteristics of the bioactive binding molecules. One of the great advantages of the present invention is that the synthetic preparation of the binding molecules of the present invention is simple. Compared to the recombinant production of similar protein molecules, organic chemical synthesis can be performed more cheaply and with better control. In addition, it is theoretically possible to use unnatural amino acids, such as D-amino acids or other suitable monomeric units, for which better compatibility or immunological tolerance is desired.

Among other things, the invention described herein overcomes the disadvantages of the prior art from the following aspects:

a) it describes an easily prepared cyclic carrier structure, which is generally easy to prepare and obtain, and which can be used to localize specific binding molecules.

b) The cyclic structure of the carrier structure enables the specific binding molecules to be positioned, in particular in any desired spatial orientation, by varying the ring size and the distance between the sites to which the side chain subunits are bound, and optionally by introducing suitable spacer molecules. The binding molecules of the present invention differ from the binding molecules of the prior art in that only random cross-linking of the peptide to the synthetic molecule is performed, wherein a defined spatial orientation of the molecular components is not possible.

c) By selecting appropriate monomer units, the cyclic support structure can be synthesized in a certain sequence on a solid phase, and the specific binding molecules can be combined or synthesized as side chain subunits. In the case of amino acids as monomer units, this enables solid-phase synthesis of peptides oriented in a parallel or antiparallel manner to be accomplished on a support structure.

d) The structure of the carrier may vary depending on the number, type and length of specific binding molecules bound as side chain subunits.

e) According to the invention, amino acid sequences oriented in a parallel or antiparallel manner can be coupled to a support structure in one synthesis step in the solid phase, optionally in one synthesis step. The latter is advantageous with regard to the yield and purity of the product and the production costs. Here, parallel orientation means that all side chain subunits are bound to the support structure in the same direction via the N-or C-terminus. By antiparallel orientation is meant that at least one side chain subunit is attached to the N-terminus of the support structure and at least one is attached to the C-terminus of the support structure.

By using unnatural amino acids and D-amino acids, the flexibility of planning the localization of specific binding molecules can be increased. The use of unnatural amino acids and D-amino acids or lactones or lactams increases the stability against proteolytic degradation. Due to the variability of the support structure, other functional groups can be introduced that increase stability and half-life. For example, this applies to glycosylation, acetylation, formation of disulfide bridges or other chemical derivatization methods known to the skilled person from protecting group chemistry.

If the specific binding molecules to be positioned on the carrier structure of the invention are peptides, they can be oriented and synthesized in a parallel and antiparallel manner on the carrier backbone.

A preferred embodiment of the carrier structure of the present invention is a cyclic peptide. Suitable prior art protecting group strategies are applicable to cyclic peptides. Because cyclic peptides are only cleaved by endopeptidases, they degrade more slowly in vivo. In addition, according to the methods of the present invention, cyclic peptides with increased proteolytic tolerance can be synthesized by introducing unnatural amino acids, such as D-amino acids. In the case of the attachment of two functional side chains, the support structure can be minimized to a linear structure.

Interleukin-2 receptor specific binding molecules

In one embodiment of the invention, synthetic binding molecules are provided that, like interleukin-2 (IL-2), have the ability to bind with high affinity to IL-2 specific receptors. IL2 is a cytokine for tumor therapy. It is a protein consisting of 133 amino acids, with a molecular weight of 15.4 kDa. IL2 binds to a specific receptor (IL2R), thereby triggering IL 2-specific intracellular signaling. High affinity receptors are receptor complexes consisting of subunits a, b, and g (also designated as p55, p75, and p64, respectively). Stimulation of the receptor complex consisting of p75 and p64 is sufficient to induce the full activity of IL 2.

IL2 has stimulatory effects on T lymphocytes and B lymphocytes, activating cytotoxic and cytolytic NK cells. Thus, it is of critical importance in the regulation of immune responses. Thus, IL2 is important in the immune response to tumor and inflammatory responses. An important mechanism for the defense of tumors with IL2 might be the induction of LAK ("lymphokine-activated killer cells"). These cells are capable of destroying tumor cells.

In recent years, various attempts have been made to treat tumors using IL 2. To date, a recombinant preparation for the treatment of metastatic renal cell carcinoma and metastatic myeloma has been marketed (Aldesleukin, Proleukin byChiron). Recombinant products have high development, approval, and production costs, resulting in correspondingly high market prices. In addition, reconstituted products are highly prone to problems. Only about 30% of treated patients respond to IL 2. The extremely short half-life (less than 10min) and extensive toxicity in humans hamper the widespread clinical use of IL2, with a treatment-related mortality rate of 4% observed in the study.

In addition to nausea, vomiting and vertigo, the above IL2 treatment is accompanied by organ dysfunction and neuropsychiatric effects. The toxicity is so high that only 10% of the doses determined to be optimal in animal studies can be used in humans. Thus, the therapeutic potential of this cytokine cannot be fully exploited.

Occasionally, administration of a short IL2 domain that interacts with a receptor is sufficient to enable at least some cellular effects (e.g., immunostimulatory effects) to be achieved. One study may show that peptides having the amino acid sequence 1-30 IL2 have similar effects only at significantly higher concentrations (Eckenberg, R., Xu, D., Moreau, J., Bossus, M., Mazie, J., Tartar, A., Liu, X., Alzari, P., Bertoglo, J., Th ze, J. (1997) Analysis of human IL-2/IL-receptor beta mechanisms: Monoclonal antibody H2-8 and new IL-2Mutants the critical role of alpha Helix-A of IL-z. Cytokine 9: 488-498). This is probably due to the fact that the peptide binds only to the beta-chain of the receptor complex, activating only the moderate affinity receptor complex. The same applies to The other sequences described from another IL2 region (Eckenberg, R., Rose, T., Moreau, J. -L., Weil, R., Gesbert, F., Dubois, S., Tello, D., Bossus, M., Gras, H., Tartar, A., Bertoglo, J., Chouiab, S., Goldberg, M., Jacques, Y., alzari, PM., Th ze, J. (2000) The first "-Helix of Interleukin (IL) -2 Folds as a Homotetramer, actas an Agonite of The IL-2 Receptor. beta. Chain, and Induce-activated kinase. Kinect. J., Australin. ex, Wallace, 2. Ex. J., J. and J. repair. 2. repair. binder. J. and sample. repair. 9. J. 2. repair. J. of The IL-2 Receptor. beta. Chassis, and repair. J. 9. insert. 2. repair. J. insert, W. sub., W. 23. repair. insert. 2. insert. repair. 2. insert. 2. insert. repair. in, 2. repair. insert. 2. insert. 2. insert. in, 2. insert. However, none of these peptides are synthesized bound to a carrier structure that is characteristic of the present invention. Thus, they are unable to dimerize physiological receptors via medium to low affinity receptor subunits and act exclusively. Therefore, its activity is weak. Furthermore, these peptides cannot be protected against proteolytic degradation by introducing protecting groups. Therefore, they degrade rapidly in vivo.

The present invention enables the provision of synthetic IL2R specific binding molecules with high activity, which are able to avoid the disadvantages associated with the preparation and use of IL-2 or IL-2 peptide fragments.

Here, the side chain subunit preferably has at least one of the following sequences:

·APTSSSTKKT₁ QLQLEHX¹X²X³X⁴X⁵QMILNGINN or

·TIVX⁶FLNRWIT FX⁷QSX⁸ISTLT₁，

Wherein

X¹Is selected from the group consisting of I or L,

X²is selected from the group consisting of I or L,

X³is selected from the group consisting of V, L or M,

X⁴is selected from the group consisting of E, D or K,

X⁵is selected from the group consisting of L or F,

X⁶is selected from the group consisting of E or D,

X⁷is selected from the group consisting of A, G or C,

X⁸is selected from A or I, and

at T₁The protecting group according to the prior art, preferably a carbohydrate moiety, preferably selected from glucose, mannose, N-acetylglucosamine or N-acetylgalactosamine, is covalently bound to threonine in a suitable manner, preventing proteolytic degradation. According to the invention, it is also possible to use binding molecules whose side chain subunits have at least 80%, preferably 90% or 95%, sequence homology to the above-mentioned sequences. In this case, the sequence differences are mainly conservative amino acid exchanges.

Preferred binding molecules have the following structure:

· APTSSSTKKT₁QLQLEHX¹X²X³X⁴X⁵QMILNGINN-support structure

TIVX⁶FLNRWTT FX⁷QSX⁸ISTLT₁；

·TIVX⁶FLNRWITFX⁷QSX⁸ISTLT₁-support structure-APTSSSTKKT₁

QLQLEHX¹X²X³X⁴ X⁵QMILNGINN；

·APTSSSTKKT₁QLQLEHX¹X²X³X⁴X⁵QMILNGINN-support structure

APTSSSTKKT₁QLQLEHX⁰¹X⁰²X⁰³X⁰⁴X⁰⁵QMILNGINN

Or TIVX⁶FLNRWIT FX⁷QSX⁸ISTLT₁-support structure-TIVX⁰⁶FLNRWIT

FX⁰⁷QSX⁰⁸ISTLT₁

Wherein amino acid X⁰¹、X⁰²、X⁰³、X⁰⁴、X⁰⁵Selection of (A) and X¹、X²、X³、X⁴And X⁵The same is true. According to the invention, it is also possible to use binding molecules whose side chain subunits have at least 80%, preferably 90% or 95%, sequence homology to the above-mentioned sequences. In this case, the sequence differences are mainly conservative amino acid exchanges.

IL2R specific binding molecules may overcome the basic disadvantages of the prior art. The binding molecules can be produced easily and economically. There is no high production cost and high physiological instability of recombinant molecules. The insufficient receptor stimulation observed with peptides does not occur because the binding molecules of the invention bind to the receptor complex in a functionally synergistic manner in a spatial arrangement with a carrier structure having no affinity for the receptor. The receptor molecules of the invention also offer the possibility of increased activity and minimized side effects by purposeful sequence optimization.

The synthetic IL 2R-specific binding molecules described above are suitable for the treatment of immune system diseases, such as inflammatory and arthritic processes, or immunodeficiency syndromes of all types and origins; diseases associated with increased cell proliferation, such as cancer diseases (carcinoses), e.g., carcinomas, sarcomas, lymphomas, and leukemias; or an infectious process. The combination of interleukin 2 and interleukin 12 is known to have a positive effect in the treatment of tumors. In addition, these agents may also be used in combination with the agents of the present invention as preferred combination agents for inducing apoptosis in target cells, particularly tumor cells. In this case, the expected positive effect of therapeutic success is achieved by local apoptosis in the tumor and ubiquitous immune stimulation caused by the inventive drug resulting in increased local efficacy. Here, it is particularly preferable to use TRAIL (TNF-related apoptosis-inducing ligand) receptor-specific binding molecules and effector molecules.

Although the above sequences and binding molecules were designed to act optimally on the human IL2 receptor, the entire concept can be applied to any species of IL2 receptor for which homologous sequences are known.

Each human sequence can be replaced with its corresponding part in the animal, combined in a manner unique to the present invention to optimize the effect in the animal for optimal veterinary use.

The following arrangement shows the sequences of cats and dogs homologous to the human sequence. These sequences are characterized by the ability to bind the respective receptor subunit in a cat or dog. The present invention includes all of the common modifications and combinations of these side chains as outlined by the above-mentioned human sequences. In preferred embodiments, these sequences can be modified by introducing conservative exchanges of amino acids in the sequence, SH-containing residues and disulfide bridges, and other chemical modifications that optimize the spatial design and stability of the resulting molecule. As side chains, they may be combined on the support structure according to the invention to become able to bind to the receptor dimer.

TRAIL receptor-specific binding molecules

TRAIL (TNF-related apoptosis-inducing ligand) is a protein belonging to the TNF family that selectively triggers programmed cell death (apoptosis) in tumor cells. TRAIL-specific binding molecules and effector molecules synthesized here, preferably based on suitable circular carrier structures, bind specifically to TRAIL receptors expressed by tumor cells and trigger apoptosis. Apoptosis-inducing proteins such as TNF (tumor necrosis factor) or Fas and AOP1 have been disclosed for a long time. Initially, the cytokine TNF was identified as a protein with a strong tumor killing effect in mice. However, due to its strong systemic toxicity, it cannot be used therapeutically in humans. The same applies to Fas and APO 1. TRAIL was first described in 1999 (Walczak, H.et al, Nature Medicine 5(1999) 157-163). The protein is composed of 281 amino acids, is II type membrane protein, and is converted into soluble molecules composed of amino acid 114-281 by cutting. Following spontaneous trimerization, it binds to a specific receptor, thereby inducing trimerization of the receptor. Thus, it triggers an intracellular cell death program mediated by caspases. To date, four different receptors for TRAIL have been reported: TRAIL-R1 and TRAIL-R2, both of which are involved in signaling; and the receptors TRAIL R3 and TRAIL R4, which do not have a so-called "death domain" and therefore are unable to trigger a signal after binding TRAIL. Both TRAIL itself and the above receptors are expressed in most human tissues and organs; the effect of selective killing of tumors is probably due to the fact that normal cells have more "trapped receptors" 3 and 4, which prevent efficient signal initiation by inhibiting trimerization of active receptors.

In the amino acid sequence of natural or recombinantly produced TRAIL, there are several domains responsible for binding to the receptor and thus for its biological activity, these domains being referred to as domains AA131-163, AA201-205, AA214-220, AA237-240, AA 258-283. Spatially adjacent these short domains are cooperatively responsible for interaction with TRAIL receptors.

The present invention can provide binding molecules that specifically bind to TRAIL receptors with high affinity. In the binding molecules of the invention, similar domains as described above are coupled to a cyclic carrier structure in a suitable synergistic arrangement to obtain a biological effect.

In a preferred embodiment, the TRAILR binding molecule of the invention is present as a trimer. Due to their covalent binding to cyclic molecular subunits, they have a high biological half-life, and due to trimerization it is possible to obtain both maximum biological activity and minimal side effects. These peptides are the minimal RAIL structures necessary for their biological activity. This enables the highest specificity and minimal side effects to be achieved. In preferred embodiments, the trimer may be prepared in the homotrivalent form, or by a cyclic support structure, or by a benzenetriamide linker.

Advantageously, TRAIL peptides are prepared in a sugar-modified form so that they have a high biological half-life. This can reduce the frequency and amount of medication and, in turn, reduce stress on the patient. By purposeful amino acid exchanges, the peptides can be converted on the one hand into a form with a higher biological activity, or into a form with a higher biological half-life, or on the other hand into an antagonistic form, i.e. into an inhibitor form.

In one embodiment, at least one side chain subunit of the TRAILR-specific binding molecule has the following sequence:

SKNEKALGRKINSWESSRSGHSFLS

in a specific embodiment, the binding molecule having affinity for a TRAIL receptor has at least one side chain subunit having the sequence:

SKNEKALGRKIX₁X₂X₃X₄SSRX₅GHSFLS, wherein

X₁Either N or L is added to the reaction mixture,

X₂either S or Q or L or E,

X₃w or L or R or a or Y,

X₄e or N or a or D or H,

X₅either S or a.

Another object of the invention are binding molecules having a side chain subunit with at least 80%, preferably 90% or 95% sequence homology to the above sequences. Here, the sequence differences are mainly amino acid exchanges.

Particular embodiments are molecules having the following structure:

according to the invention, it is also possible to use binding molecules whose side chain subunits have at least 80%, preferably 90% or 95%, sequence homology to the above-mentioned sequences. Here, the sequence differences are mainly conservative amino acid exchanges.

Antibodies and anti-idiotypic (anti-idiotype) thereof

Another important class of biologically active substances relevant to the present invention are antibodies. Antibodies are produced in vivo and bind specifically to substances known as antigens, which are usually heterologous to humans. Antibodies are important in the defense against pathogens and for the prevention of pathogen infection. Antibodies with relevant binding capacity for the proposed indications are used or developed in various therapeutic and diagnostic applications. Thus, the antibodies are used to specifically enrich the tumor for cytostatic or other tumor-damaging agents, or they are added with control agents to account for specific parts of the body or diseased areas during imaging. Antibodies can be isolated from the human body as natural antibodies. So-called monoclonal antibodies are generally antibodies that are immortalized by immunization followed by fusion of antibody producing cells with myeloma cells, and that are grown in laboratory rodents by isolation for monoclonality. Furthermore, antibodies can also be monoclonal by recombinant techniques that transfer the encoded genetic information into an appropriate expression system, often a bacterial or yeast based expression system. Recombinantly produced antibodies can be obtained from all species with a collection of immunoglobulin genes that are well sequenced and are well known. Herein, naturally occurring, monoclonal or recombinantly produced antibodies may be used in accordance with the above description.

Antibodies are larger molecules whose specific binding region usually contains 6 highly variable regions (based on amino acid sequence). The binding characteristics of an antibody are defined by the so-called CDRs ("complementarity determining regions"). However, in many cases not all 6 participate in the binding synergistically, and a single or multiple regions are not necessary for binding in a critical (critical) manner. Both monoclonal antibodies and recombinantly produced antibodies can be used therapeutically or diagnostically in a variety of indications. However, the therapeutic use of antibodies has several disadvantages. On the one hand, antibody production by both methods (monoclonal and recombinant) is complex and very expensive. Antibodies, on the other hand, are themselves natural molecules with which the patient's immune system reacts. The development of patient autoantibodies (anti-therapeutic antibodies) or allergic reactions can greatly hamper such treatment. To overcome these problems, the process of preparing therapeutic antibodies must be modified, for example, the introduction of parietal monoclonal antibodies into recombinant production must be carried out in a molecular biological, time-consuming, complex process, and simultaneously humanized in such a way that the therapeutic use is optimized by the introduction of human sequences.

According to the prior art, it has now been possible to isolate anti-idiotypic material from libraries, such as libraries of recombinant peptides. Generally, peptides isolated in this way bind to antibodies only with low affinity and cannot or only marginally competitively hinder the formation of pathogenic antibodies.

The present invention solves the above problems by providing synthetic binding molecules that mimic the binding characteristics of therapeutically relevant antibodies. The carrier structure of the present invention overcomes the above-mentioned problems of the prior art and allows the development and design of fully synthetic mimetics of each antibody. Organic chemical synthesis can be performed more economically and with greater control than recombinant preparation of similar molecules. In addition, unnatural amino acids or other suitable monomeric units that are desirable for better compatibility, half-life, or immune tolerance can also be used in general.

However, antibodies are not only important in therapy, but also can be pathogenic agents. Thus, antibodies are pathogenic if they attack autologous tissues, thereby eliciting so-called autoimmune diseases, or if they are at least associated with autoimmune diseases. Another variant of the pathogenic effect of antibodies or other substances of the immunoglobulin superfamily consists in the excitation of allergic substances in patients allergic to the respective substance. These diseases are also false reactions between immune components, the initiation of which is initiated by the binding of the respective allergens. In all these cases, it is necessary to react the pathogenicity-related antibodies with a so-called anti-idiotypic substance to neutralize the pathogenicity-related antibodies. For anti-idiotypes, it must be understood that the substance that specifically binds to the antibody binding cavity is like the actual antigen. This prevents pathogenic binding to autologous antigens.

Another object of the invention is a method for the preparation of binding molecules, said method being carried out as a solid phase synthesis method, wherein the peptides of the side chain subunits bound to the solid phase are successively extended by the respective amino acid, optionally coupled to a support structure in a final step.

Another object of the invention is a peptide having the following sequence:

·APTSSSTKKT₁QLQLEHX¹X²X³X⁴X⁵QMILNGINN, or

·TIVX⁶FLNRWIT FX⁷QSX⁸ISTLT₁Wherein

X₁Is selected from the group consisting of I or L,

X₂is selected from the group consisting of I or L,

X₃is selected from the group consisting of V, L or M,

X₄is selected from the group consisting of E, D or K,

X₅is selected from the group consisting of L or F,

X₆is selected from the group consisting of E or D,

X₇is selected from the group consisting of A, G or C,

X₈is selected from A or I.

Another object of the invention are peptides having a side chain subunit with at least 80%, preferably 90% or 95% sequence homology to the above sequences which are not completely homologous to a TRAIL sequence fragment.

Another object of the invention are pharmaceutical and diagnostic reagents comprising the binding molecules or peptides of the invention.

The binding molecules of the invention are preferably used for the preparation of a medicament or diagnostic agent for the treatment or identification of diseases of the immune system, in particular inflammation, the progression of arthritis, immunodeficiency syndrome, autoimmune syndrome and immunodeficiency syndrome, fertility disorders, contraception or antiviral prophylaxis, diseases associated with increased cell proliferation, in particular tumour diseases, cancer diseases, sarcomas, lymphomas and leukemias, in humans or mammals; and/or the progress of infection.

The medicaments of the invention are in particular provided in the form of microcapsules, liposome formulations or depot formulations comprising suitable additives, carriers, adjuvants and/or at least one additional active substance. Optionally, the agents of the invention are combined with a common suitable pharmaceutical carrier. For example, suitable carriers include buffered sodium chloride solutions, water, emulsions such as oil/water emulsions, wetting agents, disinfectant solutions, and the like. The medicament of the present invention may be provided in the form of injection, tablet, ointment, suspension, emulsion, suppository, aerosol, etc., and administered in a long-acting form (microcapsule, zinc salt, liposome, etc.). The peptides can be microencapsulated, due to their small size, to produce long-acting forms with different effect times, depending on the size of the respective pore of the capsule. The long acting form may be administered locally, with the highest concentration being ensured over a prolonged period in the desired area (e.g. the precancerous area). Wherein the mode of administration of the medicament depends on the form in which the active substance is present; it can be administered orally or parenterally. Several methods are known to the skilled person. The appropriate dosage will be determined by the attending physician and will depend on several factors, such as the age, sex, weight, nature and stage of the disease, mode of administration, and the like of the patient.

In a preferred embodiment, the use of the binding molecules of the invention takes place by ex vivo preparation of human LAK (lymphokine-activated killer cells), in particular in a manner specific for IL2, using said molecules. In this ex vivo treatment, cells are taken from the patient, treated with the active substance, activated in a culture dish and subsequently re-infused into the patient.

In a particular embodiment of the invention, interleukin 12, an interleukin 12 receptor specific binding molecule and an effector molecule or recombinant IL12 are used as additional active substances.

In another embodiment TRAIL (TNF-related apoptosis-inducing ligand), a TRAIL receptor-specific binding molecule and an effector molecule or recombinantly produced TRAIL is used as the additional active substance.

In a particular embodiment, a viral growth inhibitor, in particular a viral growth inhibitor capable of blocking or blocking the entry of viral particles, in particular HIV particles, into target cells, in particular T-lymphocytes, is added as an additional active substance to the medicament of the invention.

Another object of the invention is a binding molecule wherein the carrier structure consists of a linear peptide of 1-10 amino acids, wherein said amino acids are selected from the group consisting of G, beta-D, L-alanine or NH₂(CH₂)_xCOOH(x＝3-8)。

Example 1:

peptide multimerization principle based on cyclic peptides

A: antiparallel dimers:

the cyclic peptide is produced and used as the starting point for solid phase synthesis. First, a first peptide chain is synthesized in solid phase, then a cyclic peptide is coupled thereto, and subsequently a second peptide chain or a previously purified peptide B is gradually synthesized on a loaded resin.

Coupling of lysine cyclic peptide and aspartase in solution

0.2ml NMM and 1mmol CDMT were added to a solution of 1mmol aspartic acid in 10ml dichloromethane, cooled to 0 ℃ under argon and the mixture was stirred at 0 ℃ for 2 h. Then, an additional 0.2ml of NMM and 1 molar equivalent of cyclic peptide dissolved in 10ml of pure DMF were added. The mixture is stirred at 0 ℃ for 2h, at room temperature overnight, 50ml of ice-water are added and extracted twice with 100ml of ethyl acetate each time. The combined organic phases were extracted by shaking with saturated sodium bicarbonate solution and 5% sodium bisulfate solution. After drying over sodium sulfate, the remaining solution was concentrated in a rotary evaporator, the residue was suspended in dichloromethane and the product was in the form of a white solid.

Batch size:

Boc-L-Asp(OBz) 162mg 0.5mmol

Cyclo-L-Ala-L-Lys 100mg 0.5mmol

CDMT 89mg

NMM 0.2ml

Dichloromethane 5ml

DMF 5ml

Yield of

Theory: 0.25g 100%

Actually: 0.18g 72%

B: parallel dimers

The cyclohexapeptide of sequence D-K-D-K-D-K was produced according to the protocol described below. By appropriate choice of the protecting group, the cyclic peptide can be bound to the polymeric support via the side chain of one of the aspartic acids, optionally via a spacer molecule, and then the three peptide chains can be synthesized in parallel on a resin, or the purified peptide can be coupled to a solid phase.

A ═ DB ═ KX, Y ═ spacer molecules

PG1/PG2 (orthogonal protecting group)

PG3/PG4 (orthogonal protecting group)

Example 2

According to example 1B an embodiment of peptide a in the form of a specific binding molecule for IL 2R:

the peptides were synthesized as follows:

synthesis of: peptides with variable sequences were prepared automatically in a synthesizer (Sophas-3, Zinsser, Analytik) using solid phase synthesis (100 mg Wang resin (0.4mmol/g) for each experiment). Fmoc-derived amino acids (c ═ 0.5mol/l) with other protecting groups suitable in the prior art were used as monomer units. The amino acid units and coupling reagents (HOBt, c ═ 0.766 g/ml; PyBOP, c ═ 0.2602g/ml) were provided in DMF. The coupling steps were each performed for 2X 45min, with DMF washing of the resin between steps. Cleavage of Fmoc was performed by treatment with 50% (v/v) piperidine in DMF for 20 min. Subsequent cleavage from the resin was performed in the presence of a mixture of 95% trifluoroacetic acid, 2.5% water, 2.5% diisopropylsilane for 20 minutes. The cleaved peptide was precipitated by addition of tert-butyl methyl ether, centrifuged, combined with tert-butyl methyl ether again and centrifuged. The peptides were then lyophilized, purified by reverse phase HPLC, and characterized by LC/ms (thermoquest lcqduo). The purified peptides were examined in cell culture for their cytotoxicity increasing effects.

Peptides having the following structure were prepared and tested:

sequence 1: APTSSSTKKT QLQLEHLLLK LQMILNGINN

Sequence 2: APTSSSTKKT QLQLEHFLLD FQMILNGINN

And (3) sequence: APTSSSTKKT QLQLEHFLMK FQMILNGINN

Cytotoxicity assays：

For cytotoxicity assays, NK cells that specifically kill tumor cells as effector cells and tumor cells as target cells are incubated in the presence of the test peptide. Cytotoxicity assays were performed according to the conventional protocol using the cytotoxicity test kit (Promega, Germany) to measure the release of Lactate Dehydrogenase (LDH) in a microplate reader. YT cells (DSMZ, Germany) were used as NK cells (E). Daudi cells (DSMZ, Germany) were used as target cells (T). YT cells and Daudi cells were seeded in a 1: 10 ratio in a microplate and incubated in the presence of 0.3, 3, 30: M test peptide for 16 h. Human IL-2(Sigma) at a concentration of 5nM was used as a positive control.

The following day, 10: 1 lysis buffer was added to the cells and the cells were incubated for an additional 2 h. The 5: 1 reagent was then transferred to a second microplate and combined with the 50: 1 substrate buffer. After incubation for 30min, the reaction was stopped with 50: 1 stop solution and the light absorption of the MTT coloured complex formed was determined in a microplate reader.

Table 1 shows that the cytotoxicity inducing effect of the tested peptides was as high as that of human IL-2 at a concentration of 0.3: M.

Table 1: summary of cytotoxicity test results

Material (series #)	Cytotoxicity
		Control	10％
IL-2	60％
		Sequence of1 30∶M	30％
Sequence 13: M	30％
		Sequence 10.3: M	30％
Sequence 230: M	10％
		Sequence 23: M	80％
Sequence 20.3: M	30％
		Sequence 330: M	0％
Sequence 33: M	20％
		Sequence 30.3: M	0％

Example 3:

synthesis of trihydroxybenzoic acid carrier structure

Accurate mass 1065

Molecular formula ═ C₆₂H₅₅N₃O₁₄

And (3) analysis:

170mol, 4mol, 0.68g of 2, 4, 6-trihydroxybenzoic acid

Fmoc-Ala-OPfp 477.4g/mol 16mmol 7.16g

HOBt 135.1g/mol 13mmol 1.76g

DMF 100ml

4mmol of trihydroxybenzoic acid are dissolved in as little aqueous sodium carbonate solution as possible (pH 9) and a suspension of 16mmol of pentafluorophenol ester suspended in 130ml of acetone is added. The mixture was stirred at rt overnight and acidified to pH 1 with 5% HCl at 0 ℃. The solution was concentrated to half the original volume in a rotary evaporator and extracted by shaking with 2X 150ml of ethyl acetate. The combined organic phases are extracted by shaking with 100ml of water, 5X concentrated sodium hydrogensulfate solution and dried over sodium sulfate. After removal of the volatile components in a rotary evaporator, the residue is suspended in methanol, filtered with suction and washed again with ice-cold methanol. If the product does not precipitate, it is completely rotary evaporated.

Example 3：

Synthesis of Interleukin-2 immunomers (immomers)

Chemical formula of the material to be synthesized:

x ═ D-alanine

beta-Ala ═ beta-alanine

Acronym material

DMF N, N-dimethylformamide (peptide synthesis grade)

DCM dichloromethane (peptide synthesis grade)

HOBT 1-hydroxybenzotriazole (Anhydrous)

DBU 1, 8-azobicyclo [5, 4, 0] undec-7-ene 98%

PyBOP benzotriazol-1-yl-oxytripyrrolidinylphosphine hexafluorophosphate

DIPEA N-ethyldiisopropylamine 98 +%

TIS triisopropylsilane 99%

MeOH methanol HPLC (grade)

TFE 2, 2, 2-trifluoroethanol 99.8%

EDT ethanedithiol

General strategy:

synthesis was carried out on 2-chlorotrityl chloride resin (2-chlorotrityl chloride resin, 200-400 mesh) at a substitution rate of 0.2 mmol/g. The first 7 amino acids were coupled as fragments. The following amino acid combinations were achieved by single, double or triple coupling with a PyBOP/HOBT/DIPEA coupling additive and a 10-fold excess of amino acid. N-terminal protection of the growing peptide chain was achieved by double treatment with piperidine/DMF (1/3). In difficult cases, triple treatments were performed with DBU/piperidine/DMF (2/2/96). The number of coupling and deprotection cycles used is shown in the following scheme (monitoring the different coupling/deprotection information obtained for each extension by HPLC-MS).

RES-XXNNIGN-LCMQLDLLLHELQLQTKKTBGGB-TLTSIISQAFTIWRNCSEVITXXHHHH

COUPLING： 22221211111111122222222-223333333333333333333333333

DEPROTECT： 22222222222222322222222-223333333333333333333333333

1. 2, 3 ═ number of coupling or deprotection cycles

X ═ D-alanine

Beta-alanine

Scheme 1: synthesis of the first peptide fragment

2mmol of the first amino acid and 4mmol of DIPEA were dissolved in 10ml of anhydrous DCM. The solution was added to 1.0g of anhydrous 2-chlorotrityl chloride resin (200-400 mesh) and the mixture was reacted for 60 minutes on a vortex apparatus. At the end of the reaction, the resin was reacted twice with 20ml DCM/MeOH/DIPEA for 3 min, washed twice with 20ml DCM and twice with DMF. The resin was then treated twice with 20ml piperidine/DMF (1: 3) for 3/20 minutes and washed 6 times with 20ml DMF. The addition of amino acids 2 to 7 is effected by the following steps: 5mmol of amino acid, 7.5mmol of HOBT and 10mmol of DIPEA are dissolved in 15ml of DMF. After 5min, 5mmol PyBOP and 10mmol DIPEA were added and the solution was poured onto the resin. After 60 minutes on a vortex apparatus, the resin was washed 6 times with 20ml DMF, treated twice with 20ml piperidine/DMF (1: 3) for 20/20 minutes and washed 6 times with 20ml DMF. In the case of the N-terminal amino acid, the resin was not treated with piperidine/DMF.

Scheme 2: cleavage of peptide fragments

After incorporation of the last amino acid, the resin was washed 6 times with 20ml DMF and twice with 20ml DCM and reacted with 50ml TFE/DCM (2/8) for 60 min. The resin was filtered off, the solvent was removed in vacuo, and the crude peptide fragment was used without further purification.

Scheme 3: re-conjugation of peptide fragments

1mmol of the fragment and 2mmol of DIPEA were dissolved in 50ml of anhydrous DCM. This solution was added to 5.0g of anhydrous 2-chlorotrityl chloride resin (200-400 mesh) and the mixture was reacted on a vortex apparatus for 12 hours. At the end of the reaction, the resin was reacted twice with 50ml DCM/MeOH/DIPEA for 3 min, washed twice with 50ml DCM and twice with 50ml DMF.

Scheme 4: coupling of amino acids 8 to 57

The dried resin was swollen in 50ml piperidine/DMF (1/3) for 30min, treated with 30ml piperidine/DMF (1/3) for 20 min, treated with 30ml DBU/piperidine/DMF (2/2/96) for 20 min and washed 6 times with 30ml DMF. 10mmol of amino acid, 15mmol of HOBT and 20mmol of DIPEA are dissolved in 30ml of DMF. After 5min, 10mmol PyBOP and 20mmol DIPEA were added and the solution was poured onto the resin. After 60 minutes on a vortexer, the resin was washed twice with 30ml DMF and the coupling was repeated 1 or (in different cases) twice for 60 minutes. The resin was then washed 6 times with 30ml DMF. The resin can be used directly for coupling the next amino acid, or washed 2 times with 30ml DCM, dried in vacuo and stored at-80 ℃.

Scheme 5: cleavage and deprotection

After the last amino acid coupling, the N-terminal protecting group was removed by a double treatment with 30ml piperidine/DMF (1/3) for 20 minutes and DBU/piperidine/DMF (2/2/96) for 20 minutes. The resin was washed 6 times with 30ml DMF, twice with 30ml DCM and treated with 50ml TFE/DCM (2/8) for 180 min. After filtration, the solvent was removed in vacuo and the protecting group was removed by treatment with 30ml TFA/TIS/EDT/water (94/1/2.5/2.5) under inert gas for 180 min. The solution was poured into 300ml of cold ether, the precipitate was dissolved in methanol, the peptide was purified by RP-HPLC (Kromasil 100C 410 μm, 250X 4.6mm), and the collected fractions were used directly in the unfolding step.

Scheme 6: unfolding step

Each elution fraction was diluted to a final volume of 20ml using the same solvent present in the elution fraction. This solution of purified product was incubated in a beaker with 10ml Ni-NTA Superflow (Qiagen) for 30 minutes at room temperature with slight agitation. Superflow particles were loaded onto an empty FPLC column and attached to an FPLC instrument. Using the chromatography procedure of the instrument, the solvent was exchanged for water in a 10min. gradient, and then another 10min. gradient was used to exchange the solvent for a mixture of water/trifluoroethanol (1/1). During the 24 hour period, the reservoir was charged with oxygen and the solvent was oxidized using oxygen as the gas atmosphere in the bottle to close the disulfide bridge. During the oxidation, a constant flow of 1ml/min was passed along the column for 24h, while the eluate was constantly refluxed into the reservoir bottle.

At the end of the 24h process, the unfolded and blocked end product by disulfide bridges was eluted using phosphate buffer containing 150mM imidazole (PBS, pH7.2) or using 0.1M acetate buffer (pH 4.5).

The binding molecules are capable of binding to β/γ interleukin 2 receptor heterodimers, activating them and inducing signaling.

Claims (25)

1. A binding molecule consisting of a carrier structure having at least one cyclic molecular subunit and at least two side chain subunits, characterized in that the side chain subunits are polypeptide chains and/or polynucleotide chains consisting of natural and/or unnatural D-and/or L-amino acids and said side chain subunits are covalently bound to the carrier structure.

2. The binding molecule according to claim 1, characterized in that said cyclic molecular subunit is a cyclic polypeptide, an aromatic ring system, a polylactone, a polylactam, a benzenetriamide, a trilactone or a trilactam, synthesized from natural or non-natural D-and/or L-amino acids.

3. The binding molecule of claim 2, characterized in that the amino acids forming the cyclic polypeptide are linked by polypeptide bonds.

4. A binding molecule according to claim 2 or 3, characterized in that the cyclic molecular subunit is a cyclic polypeptide and the at least two side chain subunits are linked to the cyclic molecular subunit via the amino acid side chain part of an amino acid of the cyclic molecular subunit, such as lysine, serine, threonine, glutamic acid, asparagine and/or aspartic acid.

5. Binding molecule according to at least one of claims 2 to 4, characterized in that the cyclic polypeptide consists of 2 to 10 amino acids, preferably 2, 3, 4 or 6 amino acids.

6. The binding molecule of claim 1, characterized in that it has one of the following structures:

a) benzene triamides

b) Cyclic hexapeptides

Or a cyclohexapeptide with 1-3 side chain peptides Seq and a sequence of DKDDK,

c) a trilactone:

d) a trilactam:

e) trihydroxybenzoic acid carrier structure:

f) cyclic dipeptide: loop-L-Ala-L-Lys, derivatized with Seq,

wherein Seq or Pep represents a side chain subunit or-OH.

7. Binding molecule according to at least one of claims 1 to 6, characterized in that the polypeptide chains have the same amino acid sequence.

8. Binding molecule according to at least one of claims 1 to 7, characterized in that the polypeptide chain consists of 5 to 60 amino acids, preferably 10 to 50 amino acids, in particular 12 to 40 amino acids.

9. The binding molecule according to at least one of claims 1 to 8, characterized in that the side chain subunits are bound to the carrier structure by spacer molecule subunits (spacers), which in particular consist of carbohydrates or nucleic acids.

10. Binding molecule according to at least one of claims 1 to 9, characterized in that the unnatural amino acid has the formula NH₂(CH₂)_nCOOH, wherein n is 2-8; preferably NH₂(CH₂)₂COOH，NH₂(CH₂)₃COOH or NH₂(CH₂)₄COOH。

11. Binding molecule according to at least one of claims 1 to 10, characterized in that the side chain subunits bound to the carrier structure have at least one of the following sequences:

·APTSSSTKKT₁ QLQLEHX¹X²X³X⁴X⁵QMILNGINN (SC1) or

·TIVX⁶FLNRWIT FX⁷QSX⁸ISTLT₁(SC2) or

TKETQQQLEQLLLDLRLLLNGVNNPE (SC3) or

TKETEQQMEQLLLDLQLLLNGVNNYE (SC4) or

NYDDETATIVEFLNKWITFAQSIFSTLT (SC5) or

·EYDDETATITEFLNKWITFAQSIFSTLT (SC6)，

Wherein

X¹Is selected from the group consisting of I or L,

X²is selected from the group consisting of I or L,

X³is selected from the group consisting of V, L or M,

X⁴is selected from the group consisting of E, D or K,

X⁵is selected from the group consisting of L or F,

X⁶is selected from the group consisting of E or D,

X⁷is selected from the group consisting of A, G or C,

X⁸is selected from the group consisting of A or I,

at T₁In position, a protecting group according to the prior art, preferably a carbohydrate moiety, preferably selected from glucose, mannose, N-acetylglucosamine or N-acetylgalactosamine, to prevent proteolytic degradation, optionally covalently binds threonine in a suitable manner;

optionally protection against exopeptidases is achieved by binding of two D-amino acid residues at the free C-and/or N-terminus;

after binding to the support structure, the sequence is optionally modified by replacing a single residue with a residue having an SH group to generate a disulfide bridge between the polypeptide side chains;

the side chain subunits in the binding molecule have at least 80% homology with the above sequence;

and fragments of the above sequences are shortened by up to 6 amino acids from the N-or C-terminus.

12. The binding molecule of claim 11, having the structure

SCX-Carrier Structure-SCY

Wherein SCX (side chain X) may be any one of the 6 side chains (SC1-6) of claim 11 and SCY (side chain Y) may be any one of the side chains (SC1-6) of claim 11.

13. The binding molecule of at least one of claims 1 to 11, wherein at least one side chain subunit has the following sequence: SKNEKALGRKIX₁X₂X₃X₄SSRX₅GHSFLS, wherein

X₁Either N or L is added to the reaction mixture,

X₂either S or Q or L or E,

X₃w or L or R or a or Y,

X₄e or N or a or D or H,

X₅either of the groups S or A are,

and the side chain subunits in the binding molecule have at least 80% homology with the above sequence.

14. The binding molecule of claim 1, characterized in that it has one of the following structural formulae:

or

Pep＝SKNEKALGRKINSWESSRSGHSFLS

H₂N- -COOH

Or

Or

And the side chain subunits in the binding molecule have at least 80% homology with the above sequence.

15. A method for the preparation of a binding molecule according to at least one of claims 1 to 14, wherein the method is performed using a solid phase synthesis method, wherein the peptide of the side chain subunit is bound to a solid phase, is extended continuously from each amino acid, and is optionally coupled to a support structure in a final step.

16. A peptide having the structure:

·APTSSSTKKT₁ QLQLEHX¹X²X³X⁴ X⁵QMILNGINN or

·TIVX⁶FLNRWIT FX⁷QSX⁸ISTLT₁Wherein

X¹Is selected from the group consisting of I or L,

X²is selected from the group consisting of I or L,

X³is selected from the group consisting of V, L or M,

X⁴is selected from the group consisting of E, D or K,

X⁵is selected from the group consisting of L or F,

X⁶is selected from the group consisting of E or D,

X⁷is selected from the group consisting of A, G or C,

X⁸is selected from the group consisting of A or I,

and the side chain subunits in the binding molecule have at least 80% homology with the above sequence.

17. A medicament containing a binding molecule according to any one of claims 1 to 14 and/or a peptide according to claim 16.

18. Diagnostic agent comprising a binding molecule according to any one of claims 1 to 14 and/or a peptide according to claim 16.

19. Use of a binding molecule according to any one of claims 1 to 14 and/or a peptide according to claim 16 for the preparation of a medicament or diagnostic agent for the treatment or detection of immune system diseases, in particular inflammation, arthritis progression, immunodeficiency syndrome, autoimmune syndrome and immunodeficiency syndrome, fertility disorders, contraception or antiviral prophylaxis, diseases associated with increased cell proliferation, in particular tumour diseases, cancer diseases, sarcomas, lymphomas and leukemias, in humans or mammals; and/or the progress of infection.

20. The medicament according to claim 17, in the form of microcapsules, liposome formulations or depot formulations containing suitable additives, carriers, adjuvants and/or at least one additional active substance.

21. Pharmaceutical according to claim 17, characterized in that interleukin 12, an interleukin 12 receptor specific binding molecule and an effector molecule or recombinant IL12 are used as additional active substances.

22. Medicament according to claim 17, characterized in that TRAIL (TNF-related apoptosis-inducing ligand), TRAIL receptor-specific binding molecules and effector molecules or recombinantly produced TRAIL is used as additional active substance.

23. Pharmaceutical according to claim 17, characterized in that a viral growth inhibitor, in particular a viral growth inhibitor capable of blocking or blocking the entry of viral particles, in particular HIV particles, into target cells, in particular T-lymphocytes, is used as additional active substance.

24. The binding molecule according to claims 1 and 12, characterized in that exactly two side chains are bound to said support structure, said support structure being minimized to a linear peptide consisting of 2-10 amino acids comprising a peptide selected from the group consisting of G, P, β -alanine and NH₂(CH₂)_xAt least two different amino acids of COOH (x-3-8).

25. The binding molecule of claim 24, defined by the formula:

x ═ D-alanine

beta-Ala ═ beta-alanine