EP1171464A2

EP1171464A2 - Peptide homodimers or peptide heterodimers derived from interleukin 12

Info

Publication number: EP1171464A2
Application number: EP00938503A
Authority: EP
Inventors: Raimund J. Wieser
Original assignee: Individual
Current assignee: Individual
Priority date: 1999-04-27
Filing date: 2000-04-20
Publication date: 2002-01-16
Also published as: AU5388600A; WO2000064938A3; CA2370298A1; DE19919148A1; WO2000064938A2; JP2002543091A

Abstract

The invention relates to homodimers or heterodimers of peptide monomers which have the amino acid sequence KHYSCTAEDID (monomer I), PPVGEADPYRVKMQ (monomer II), AALQNHNHQQIILDK (monomer III), IRDIIKPDPPKN (monomer IV), SLTFCVQVQGKSKR (monomer V) or RFTCWWLTTISTDLTF (monomer VI) or variants thereof, whereby the homodimers or heterodimers can bind to the interleukin 12 (IL12) receptor and can optionally trigger a cellular signal. These dimers are suited for treating diseases of the immune system, diseases associated with increased or decreased cell proliferation, infectious or inflammable processes, and are suited for detecting diseases associated with, for example, an IL12 receptor which is modified or which is excessively or insufficiently expressed.

Description

Interleukin 12 derived peptide homodimers and peptide heterodimers

The present invention relates to homodimers or heterodimers from peptide monomers which have the amino acid sequence KHYSCTAEDID (Monomer I), PPVGEADPYRVKMQ (Monomer II), AALQNHNHQQIILDK (Monomer III), IRDIIKPDPPKN (Monomer IV), SLTFCRQQDOMWLLT or RFTTCVQDWLGLT (RFTQVDQWWLL) or SLTFCRQQDQWLGT (RFTQVDQWL) Variants thereof, the homodimers or heterodimers binding to the interleukin 12 (IL12) receptor and possibly being able to trigger a cellular signal. The present invention also relates to medicaments containing these homodimers or heterodimers. The present invention further relates to diagnostic compositions or diagnostic methods in which the homodimers or heterodimers according to the invention or antibodies directed against them are used.

Until now, tumor therapy has essentially been based on the three main pillars: surgery, chemotherapy and radiotherapy. In recent years, however, it has become known that primary (ie exclusive) or adjuvant (supportive) treatment with cytokines in many cases results in an extension of life expectancy, often even a complete cure. The cytokines are endogenous messenger substances that are synthesized by different cells and then secreted. The biological tasks of these cytokines are very diverse, but so far only partially understood. In any case, cytokines mainly have fundamental immunoregulatory effects. One of these cytokines, interleukin 12 (IL12), is increasingly used in the therapy of various tumors. IL12 is mainly produced in vivo by B cells, less by T cells (D'Andrea et al., J.Exp.Med. 176 (1992), 1387-1398) and has a variety of biological effects. The following effects are particularly noteworthy, since they play an important role in the therapeutic effect: stimulation of the proliferation of human lymphoblasts (Gateley et al., J. Immunol. 147 (1991), 874), Akti- crossing of NK cells (Manetti et al., J.Exp.Med. 177 (1999), 1199) and induction of the synthesis of IFN-gamma, IL2 and TNF (Chan et al., J.Exp.Med. 173 ( 1991), 869). Through a synergy effect with IL2, the dose of IL2 in the presence of IL12 can be drastically reduced in the case of adoptive immunotherapy for the generation of lymphokine-activated killer cells, so that the serious side effects of IL2 can be significantly reduced.

IL12 is a heterodimer consisting of a p40 and p35 chain (Stern et al., Proc. Natl. Acad. Sci. USA 87 (1990), 6808). p40 has certain homologies to the extracellular domain of the IL6 receptor (Gearing and Cosman, Cell ββ (1991), 9), while p35 appears to be a homologue of IL6 (Mersberg et al., Im unol. Today .13 (1992 ), 77). The p35 chain is apparently responsible for signaling at the IL12 receptor, while the p40 subunit is likely to determine species specificity. However, there are apparently also receptor isoforms that can be stimulated by the p40 chain alone (Presky et al., Proc. Natl. Acad. Sci. USA 9_3_ (1996), 14002). Since no X-ray structure analyzes of the two IL12 subunits have been available to date, one can only speculate about the IL-12 tertiary structure.

As with other cytokines or growth factors, the IL12 subunits also have certain short domains that interact with the receptor. The administration of a short IL12 domain that interacts with the receptor could possibly be sufficient to achieve the desired cellular effects (e.g. immunostimulatory effect). To date, however, there have been no studies on IL12, for example with synthesized peptides, which could provide information about suitable IL12 domains.

Since cytokines are generally only present in extremely low concentrations in the serum, it is not possible to isolate therapeutically usable amounts from this medium. Therefore, the therapeutically used cytokines have been recombinantly used in Bacteria or yeast. However, this procedure has a number of serious disadvantages.

The recombinant production of cytokines requires a lot of effort in the "set-up". For example, it must be clarified whether the host cell expresses the protein exactly or in the exact native conformation, which is the best host cell or the best expression vector and under which conditions the protein is not proteolytically degraded. It should be noted here that generally recombinantly produced proteins are from the native protein. have a different tertiary structure and are therefore recognized as "foreign" by the human immune system. The antibodies induced thereby neutralize the protein, for example the cytokine, and thus lead to a loss of its effectiveness.

The isolation and purification of recombinant proteins is difficult because, in principle, the same questions that are specific to each new product always arise, namely how can the protein be prevented from being proteolytically degraded and / or failing after isolation. In general, recombinantly produced proteins show a striking lability towards proteases. This requires frequent and / or high-dose administration, which on the one hand is very stressful for the patient, but on the other hand also makes the therapy very expensive.

Since the cytokines normally have to be administered in a regular sequence during therapy, it must be ensured that they are absolutely free of contamination, i.e. especially components of the host cell. As a result, complex procedures must be used that have an unfavorable effect on pricing.

In the meantime, there has been widespread recognition that messenger substances (e.g. cytokines) not only have a binding site for the receptor, but that other - currently. yet domains that are not fully understood may exist that may induce signaling pathways other than those that are intended. Among other things, this could explain the frequently occurring side effects of the cytokines used to date, which in some cases make treatment with these impossible.

Thus, the invention is essentially based on the technical problem of providing compounds with a biological activity of interleukin 12 (IL12) in such a way that the previous disadvantages of the prior art are avoided, i.e. when administered as a medicinal product, fewer side effects, a longer half-life and high biological activity are obtained.

This technical problem has been solved by providing the embodiments characterized in the patent claims.

One embodiment of the present invention relates to a synthetic homodimer or heterodimer composed of peptide monomers which have the amino acid sequence KHYSCTAEDID (Monomer I), PPVGEADPYRVKMQ (Monomer II), AALQNHNHQQIILDK (Monomer III), IRDIIKPDPPKN (Monomer IV), VGTLKTQMDQTLKTQVQQWLTLKTQQQQWLLKTQVQQQWLLKTQVQQQWLLKLQQQWLQWLKVQQQQWLQWLWLQWLVLQWLVLQVLQWLQWLQWLVWLQWLKVLQLQLQLQQQQQQWLQKVQQQVLQKVLQKVQQLQKVLQKVQQQVLQKVLQKVLQKVLQKVLQKVLQKVKV (Clerox) VI), where the homodimer or heterodimer binds to the interleukin 12 (IL12) receptor and can possibly trigger a cellular signal. These peptides according to the invention represent the binding regions of the IL12 subunits p40 and p35 and take advantage of the knowledge that the peptides from p35 and p40 must be used in a dimerized form for a biological effect, since the receptors must also lie together twice in order to Triggering the intracellular signals to be able to. Monomers I-III comprise 3 loops which are located at the point of contact of p35 with the interleukin 12 receptor, while analogously the monomers IV-VI represent the contact points for p40 with the receptor. The dimers according to the invention have the following advantages: They are easy to use using standardized methods synthesize and purify them and they can be used immediately for the necessary tests (e.g. structural examinations, biological tests, preclinical and clinical studies). These peptides represent the minimal structures of the respective cytokine that are required for its biological effectiveness. This ensures the highest specificity and high biological activity combined with the least side effects. Due to the small size of the peptides, they can be microcapsulated, which results in depot forms with different duration of action - depending on the pore size of the capsules. The depot form can be applied locally, so that the highest concentration of the active ingredient is guaranteed over a long period of time at the desired location (eg cancer focus).

According to the invention, all combinations of the monomers I - VI with one another are capable of forming dimers, and the dimers formed achieve the object.

The peptides according to the invention are usually synthesized by known solid-phase synthesis processes, for example as described in Example 1 below. The biological activity (binding to the receptor, triggering a cellular signal and proliferation-stimulating effect) is investigated by methods known to the person skilled in the art, e.g. according to Lewis, J. Immuno1. Methods 185 (1995), 9-17; Grander et al., Eur. J. Cancer 28A (1992), 815-818 or, for example, also according to the methods described in the examples below.

In a preferred embodiment according to the invention, the peptides described above are homodimers or heterodimers in which the monomers are linked via their C-terminus or N-termini or the N-terminus of one monomer is linked to the C-terminus of the other monomer is linked. Preferably, the C-terminus of monomer I is linked to the N- or C-terminus of monomer II, or the N-terminus of monomer I is linked to the N- or C-terminus of monomer II. For example, the desired N-terminal peptide in a side-function-protected form is activated after removal of the protective group (preferably Fmoc) by treatment with, for example, piperidine with a crosslinker, and the product is purified by HPLC. Another previously synthesized monomer is also provided (either at the C- or N-terminus - depending on the desired dimer) in its enfunction-protected form with a group which only reacts with the N-terminal group of the bound peptide. After this monomer has been added to the activated monomer, the dimers, which differ significantly from the monomers in terms of elution behavior, are purified and isolated after removal of the side protecting groups by standard methods using reverse phase HPLC. If the amino acid addition permits, the activation groups and crosslinkers can also be selected in such a way that dimerization of free monomers can be carried out directly in solution after removal of the side chain protective groups. Alternatively, the synthesis strategy can be carried out on the HYCHRON anchor as described in the example, so that the side chain protective groups are retained and dimerization can be carried out in aqueous solution.

This linkage can be carried out, for example, using the method described in the examples below or other standard methods, it being necessary to ensure that the dimerization takes place in such a way that binding to the receptor pair is still possible effectively. For example, the dimerization can be carried out on a lysine residue as a branch point (Wrighton et al., Nature Biotechnology 15 (1997), 1261-1265). The homomers of the dimers according to the invention are preferably covalently linked to one another via linkers, such as polyethylene glycol, peptide (s), activated benzodiazepines, oxazolones, azalactones, amine imides, diketopiperazines, or (a) monosaccharide (s). The corresponding chemical reactions to be carried out are known to the person skilled in the art in this field (for example Hermannson, Bioconjugate Techniques (1996), Academic Press, San Diego; Peeters et al., J. Immunol. Meth. 120, (1989), 133-144; Inman et al., Biocon- j ugate Chem. 2: 458-463 (1991)).

The present invention also relates to homodimers or heterodimers which are characterized in that the monomers have deletions, additions or substitutions of one or more amino acids and / or (a) modified amino acid (s) with respect to the corresponding starting monomers I to VI. Homo- or heterodimers are obtained, which (a) compared to the original. Can bind forms similar or better to the IL12 receptor and / or can trigger a cellular signal, or (b) can still bind to the IL12 receptor, but have an antagonistic effect, ie after administration, the biologically active form becomes this form, the no longer leads to the triggering of a cellular signal after binding to the receptor. The extent to which these modified homo- or heterodimers have the desired biological properties can be investigated using the methods described in the examples below or the methods described above. Natural amino acids are preferably introduced in the case of the amino acid substitutions or additions, with modified amino acids not being excluded. The preferred modifications include glycosylations with mono- or disaccharides of serine, threonine and asparagine, farnesylation and palmitoylation of cysteine, phosphorylation of threonine, serine and tyrosine, the modifications which affect the central amino acids mostly leading to antagonistic forms. In the case of the modified homodimers or heterodimers according to the invention described above, the deletions, additions, substitutions and / or modifications relate to at most 10, preferably at most 7, more preferably at most 3 and most preferably at most 1 amino acid (s) per monomer. The inventive, from modified monomers Existing homo- or heterodimers contain, in addition to the domain responsible for receptor binding, no other domains which are characteristic of the natural cytokine. The dimer according to the invention or consisting of modified monomers is preferably the following homodimers: [AALQNHNHQQIILDK] ₂ or [AALQNHNKQQIILDK] ₂ , which even have a significantly higher specific activity than IL12. Both monomers are linked to both CC and CN.

The homodimers or heterodimers according to the invention can be present as such, but can also be linked to other further compounds, for example (poly) peptides. These (poly) peptides include, for example, carrier proteins, e.g. Transferin or albumin, which the body does not recognize as foreign. The homodimers or heterodimers according to the invention can also be fused with other (poly) peptides, for example a leader peptide which enables or supports the penetration of the peptide according to the invention into the cell. An example of such a leader peptide is Penetrin from Drosophila.

In a further embodiment, the present invention relates to the above homodimers or heterodimers, which are further characterized in that one or more amino acids are covalently modified by fatty acids, mono- and / or oligosaccharides. This can be done by generally known methods, for example during the synthesis of the monomers through the use of amino acids which already carry the above modifications. However, the modifications can also be made retrospectively. These modifications can, for example, achieve a higher resistance to proteolytic degradation and thus an even longer biological half-life.

The present invention further relates to an antibody or a fragment thereof against the dimers according to the invention described above. These antibodies can also be used in diagnostic assays, for example. These antibodies can be monoclonal, polyclonal or synthetic antibodies or fragments thereof. In this Context, the term "fragment" means all parts of the monoclonal antibody (for example Fab, Fv or "single chain Fv" fragments) which have the same epitope specificity as the complete antibody. The production of such fragments is known to the person skilled in the art. The antibodies according to the invention are preferably monoclonal antibodies. The antibodies according to the invention can be produced according to standard methods, the homodimers or heterodimers according to the invention serving as antigen. Methods for obtaining monoclonal antibodies are known to the person skilled in the art.

In a particularly preferred embodiment, the monoclonal antibody according to the invention is an antibody derived from an animal (for example a mouse), a humanized antibody or a chimeric antibody or a fragment thereof. Chimeric, human antibody-like or humanized antibodies have a reduced potential antigenicity, but their affinity for the target is not reduced. The production of chimeras and humanized antibodies or of antibodies similar to human antibodies has been described in detail (see, for example, Queen et al., Proc. Natl. Acad. Sci. USA 86 (1989), 10029, and Verhoeyan et al., Science 239 (1988), 1534). Humanized immunoglobulins have variable scaffold areas, which essentially come from a human immunoglobulin (called acceptor immunoglobulin) and the complementarity of the determining areas, which essentially come from a non-human immunoglobulin (e.g. from the mouse) (called donor) Immunoglobulin). The constant region (s), if any, also originate essentially from a human immunoglobulin. When administered to human patients, humanized (as well as human) antibodies offer a number of advantages over antibodies from mice or other species: (a) the human immune system should not recognize the framework or constant region of the humanized antibody as foreign, and therefore should Antibody response against one injected antibodies are lower than against a completely foreign mouse antibody or a partially foreign chimeric antibody; (b) since the effector area of the humanized antibody is human, it interacts better with other parts of the human immune system, and (c) injected humanized antibodies have a half-life that is substantially equivalent to that of naturally occurring human antibodies, which allows to administer smaller and less frequent doses compared to antibodies from other species. The present invention also relates to a hybridoma that produces the monoclonal antibody described above.

Furthermore, the present invention relates to the medicaments containing homodimers or heterodimers according to the invention and their use for the treatment of diseases of the immune system, of diseases which are associated with decreased cell proliferation, or of infectious or inflammatory processes (for example HIV, leishmaniasis): Mountford et al., J. Immuol. 156 (996), pp. 4739-4745). Certain modified forms of the dimers according to the invention can also have an antagonistic effect. The present invention thus also relates to the medicaments containing the homodimers or heterodimers according to the invention and their use for the treatment of diseases which are associated with increased cell proliferation, e.g. Doors, arthritic processes, allergic reactions or certain forms of skin diseases such as psoriasis (Wigginton et al., J. Natl. Cancer Inst. 88 (1996), 38-43; Brunda et al., J. Exp. Med 178: 1223-1230 (1993). Finally, the present invention also relates to medicaments containing the dimers according to the invention and their use for reducing the IL2 side effects in the case of therapy with IL2, for example immunotherapy.

The medicament according to the invention is optionally in combination with a suitable pharmaceutical carrier. Suitable carriers and the formulation of such drugs are known to the person skilled in the art. Suitable carriers include, for example, phosphate-buffered saline solutions, water, emulsions, for example oil / water emulsions, wetting agents, sterile solutions, etc. The medicament according to the invention can be in the form of a solution for injection, tablet, ointment, suspension, emulsion, suppository, etc. It can also be administered in the form of depots (microcapsules, zinc salts, liposomes, etc.). The mode of administration of the drug depends, among other things, on the form in which the active ingredient is present, and can be administered orally or parenterally. Methods for parenteral administration include topical, intra-arterial (e.g. directly to a tumor), intramuscular, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, transdermal or transmucosal (nasal, vaginal, rectal, sublingual) administration. The appropriate dosage is determined by the attending physician and depends on various factors, for example the age, gender, weight of the patient, the type and stage of the disease, the type of administration, etc.

Furthermore, the present invention relates to a diagnostic composition which contains the homodimer or heterodimer according to the invention or the antibody according to the invention and can be used to diagnose diseases which are associated with an altered or too high or too low expressed IL12 receptor or with a too high or low concentration of IL12. The homodimers or heterodimers according to the invention can be present in generally customary assay formats for diagnostic detection. This detection includes, for example, (a) obtaining a cell sample from the patient, (b) contacting the cell sample thus obtained with the homo- or. Heterodimer or antibody as a probe under conditions that allow specific binding to the target and (c) detection of binding to the target. This detection can be performed using standard techniques known to those skilled in the art. The compounds according to the invention can be bound, for example, in the liquid phase or to a solid support. they will be marked in different ways. Suitable markers and marking methods are known to the person skilled in the art. These are also known cell disruption methods which allow IL12 or the receptor to be brought into specific contact with the antibody or homo- or heterodimer according to the invention.

Finally, the present invention relates to a diagnostic kit for carrying out the diagnostic method described above, which contains a dimer according to the invention or the antibody according to the invention or the fragment thereof. Depending on the configuration of the diagnostic kit, the dimer or the antibody or the fragment thereof can be immobilized.

The following examples illustrate the invention.

Example 1: Solid phase synthesis of KHYSCTAEDID (Monomer I),

PPVGEADPYRVKMQ (Monomer II), AALQNHNHQQIILDK (Monomer III), IRDIIKPDPPKN (Monomer IV). SLTFCVOVOGKSKR (Monomer V) and RFTCWWLTTISTDLTF (Monomer VI)

The monomers I-VI were prepared according to a standard solid phase synthesis (Seitz et al., Angew. Chem. 107 (1995), 901). The synthesis was carried out on a HYCHRON anchor with an Fmoc-protected amino acid starting from the C-terminus. The carboxyl groups of glutamic acid and aspartic acid were protected in the form of tert-butyl esters. The protecting groups of the side chains of Tyr included tetrahydropyranyl, tert-butyl (Boc) and trityl groups. The side chain protecting groups of serine and threonine included acetyl, benzoyl and benzyloxycarbonyl (= Cbz) groups. The protecting groups for the side chain of arginine included Cbz, mesitylene-2-sulfonyl (= Mts) or tert-butyloxycarbonyl (= Boc). The side chain of lysine was protected by tosyl (= tos) or boc. After removal of the cc-amino protecting group and corresponding washing steps, followed by activation of the carboxyl group of next amino acid, this was coupled to the previous amino acid. The complete monomers were synthesized in this order. The finished monomers were separated from the column by a palladium (0) catalyzed allyl transfer, the protecting groups on the side chains being retained. The Fmoc group was then replaced by morpholine, the remaining protective groups were removed by treatment with TFA.

Example 2: Polyethylene glycol dimerization of monomer I.

The dimerization of the monomers I was carried out by dissolving 25 mg of activated bifunctional polyethylene glycol (PEG-succinimidyl propionate, MW approx. 3400; Sigma, Taufkirchen) in 4 ml of PBS, pH 7.5, after which a triple molar excess of monomers was dissolved in 1 ml of 0.1% trifluoroacetic acid. After 3 hours of incubation on ice, lyophilized monomers were again added, so that at the end there was a ratio of 3.5 mol of monomer to 1 mol of PEG. The mixture was incubated on ice for an additional 17 hours. PEG was inactivated by adding 1 M Tris / HCl, pH 7.5 (final concentration: 50 mM Tris) (incubation: 1 hour on ice). The mixture was subjected to both analytical and preparative HPLC (see Example 5).

Example 3: Polväthylenqlvkol dimerization of Monomer II

The dimerization of the monomers II was carried out by dissolving 25 mg of activated bifunctional polyethylene glycol (PEG-succinimidyl propionate, MW approx. 3400; Sigma, Taufkirchen) in 4 ml of PBS, pH 7.5, after which a triple molar excess of monomers was dissolved in 1 ml of 0.1% trifluoroacetic acid. After 3 hours of incubation on ice, lyophilized monomers were again added, so that at the end there was a ratio of 3.5 mol of monomer to 1 mol of PEG. The mixture was incubated on ice for an additional 17 hours. PEG was inactivated by adding 1 M Tris / HCl, pH 7.5 (final concentration: 50 mM Tris) (incubation: 1 hour on ice). The mixture was subjected to both analytical and preparative HPLC (see Example 5).

Example 4: Polväthylenqlvkol-Dimerisierunq of the monomers I and

II

The monomers were dimerized by dissolving 25 mg of activated bifunctional polyethylene glycol (PEG-succinimidyl propionate, MW approx. 3400; Sigma, Taufkirchen) in 4 ml of PBS, pH 7.5, after which a triple molar excess of monomers, dissolved in 1 ml of 0.1% trifluoroacetic acid was added. After 3 hours of incubation on ice, lyophilized monomers were again added, so that at the end there was a ratio of 3.5 mol of monomer to 1 mol of PEG. The mixture was incubated on ice for an additional 17 hours. PEG was inactivated by adding 1 M Tris / HCl, pH 7.5 (final concentration: 50 mM Tris) (incubation: 1 hour on ice). The mixture was subjected to both analytical and preparative HPLC (see Example 5).

The monomers were used in a ratio of 1: 1, the separation was carried out using reverse phase HPLC, since the two dimers differ significantly in their elution behavior (depending on the composition, a difference in the elution peak of between 4 and 9 minutes) and can therefore be separated from one another.

Example 5: Analytical and preparative HPLC

The formation of the dimers described in the previous examples was checked by analytical reverse phase HPLC. The analysis was performed using a Vydac-C18 protein peptide column (0.46 x 25 cm) (The Separation Group, USA), a BioRad HPLC system and a dual-wavelength detector from Perkin-Elmer. The column was washed with 0.1% TFA in dist. Water equilibrated, and 10 min. after the sample was injected (usually 6 ml), a 45-minute linear gradient (0-100) of acetonitrile was run with 0.1% TFA. The flow rate became continuous at 1 ml / min. held. Under these conditions the cross-linker appeared in the flow. The reaction product eluted after 37 min. , while the unconjugated monomer already after 32 min. was eluted and thus could be clearly separated from the dimer. The dimer was further purified on a preparative reverse HPLC column (2.2 × 25 cm) on the same HPLC system. The column was run at a constant flow rate of 8 ml / min. with dist. Water: 80:20 acetonitrile (both contained 0.1% TFA) equilibrated. 20 min. after injection of the sample (6 ml), a 60-minute linear gradient was run on 100% acetonitrile / 0.1% TFA. The main peak was collected and lyophilized. A flatter gradient (20-80% acetonitrile / 0.1% TFA over 60%) was run for the monomer II in order to achieve a better separation. The isolated dimers and the native monomers were used in binding studies (see Example 7) and proliferation tests (see Example 8).

Example 6: Radioactive labeling of the dimers

The dimers were iodinated as described below. 20 ul chloramine T (0.5 mg / ml) were added to 20 ul Na ^{1 5} I (2 mCi, Amersham Buchler, Braunschweig), the mixture was 2 min. later mixed to 50 μl dimer (5 μg in PBS) and 15 sec. incubated. To terminate the reaction, 30 μl sodium metabulfite (1 mg / ml in PBS) and after 30 sec. 50 μl KI (10 mg / ml in PBS) were added. Gelatin (50 μl, 1 mg / ml. In distilled water) was added as carrier and the solution was immediately applied to a BioSil SEC 125-5 column (BioRad, 10 ml), which was filled with 0.25% gelatin / 0 , 02% sodium azide in PBS was equilibrated. The fractions containing the radioactive product were pooled and further purified and isolated on the reverse phase HPLC column as described in Example 5. The purified dimers had a specific radioactivity of 50-100 μCi / μg, gave a single band in the tricin-SDS-PAGE and the radioactivity could be 95-100% with the chloroform / methanol method (Wessel et al., Anal Biochem., 138: 141-143 (1984). These results show that the radioactively labeled product consisted of a pure dimer and that the radioactivity was exclusively attributable to the dimer.

Example 7: Cell binding studies

These studies provide information as to whether the dimers according to the invention bind specifically to cells which express the IL12 receptor. In this example, the homodimer consisting of the monomers III was used. The experiment was carried out on PHA-stimulated human lymphoblasts according to a modified protocol according to Chizzonite et al. , J. Immuno1. 148: 3117-3124 (1992).

The stimulated cells (7.5 x 10 ⁵ cells / ml RPMI medium) were incubated for 90 minutes with radiolabelled dimer in various concentrations (0.01 to 1 ng) and then washed by means of 3 centrifugation steps in PBS and the cell-bound radioactivity measured in the liquid scintillation counter. The extent of non-specific binding was determined in the presence of a 100-fold higher concentration of IL12, with labeled homodimer and IL12 being added to the cells at the same time. The results are shown in Table I.

Table I test substance binding% maximum binding)

Interleukin 12 100

AALQNHNHQQIILDK

[AALQNHNHQQIILDK]: ° ό

Example 8: Proliferation studies

These studies should show whether the dimers of the invention similar to native IL12 have proliferation stimulating effects. They were again tested on PHA-stimulated human lymphoblasts in a 48 hour assay according to Gately and Chizzonite, Curr. Protocols in Immunology Voll (1992), p.6.16-6.16.8, with Monomer III. The cells were sown in 200 μl of culture medium in a cell count of 5 × 10 ⁴ and cultivated in the presence of different concentrations (0.01-1 ng) of the dimers for 48 hours. After the addition of tritiated thymidine (0.25 μCi / ml), the cells were cultivated for a further 4 hours and then isolated using a cell harvester. The built-in radioactivity was measured in the liquid scintillation counter. The values are shown in Table II as specific activity in units x 10 ^"7 / mg IL12.

Table II Test substance Specific .activity

Interleukin 12

AALQNHNHQQIILDK: 0.03

[AALQNHNHQQIILDK] 2

[AALQNHNKQQIILDK] 2 i 10.5

Example 9: Production and detection of an antibody according to the invention

The heterodimer prepared in Example 4 was subjected to 18% SDS-polyacrylamide gel electrophoresis. After staining the gel with 4 M sodium acetate, an approximately 6.5 kB band was cut out of the gel and boiled in phosphate-buffered Incubated saline. The dimer is eluted from the gel using an electroeluctor (BioRad) and lyophilized overnight after kB dialysis. The peptide is taken up in a concentration of 5 mg / ml PBS and mixed with the carrier protein albumin in a ratio of 1 mol peptide / p. 02 mol carrier in a total of 2 ml PBS. 2 ml of glutaraldehyde (0.2% in PBS) are added dropwise to the mixture with continuous stirring and incubated for 1 hour at room temperature. The mixture is then dialyzed against PBS for 48 hours with a 12-hour buffer change in a 20 kB dialysis tube and then freeze-dried. The animals are immunized with the mixture consisting of dimer-BSA complexes, BSA-BSA complexes and dimer complexes with 100 μg (rabbit and chicken) or with 30 μg (mouse):

Immunization protocol for polyclonal antibodies in rabbits

100 ug of gel-purified heterodimer in 0.7 ml of PBS and 0.7 ml of complete or incomplete Freund's adjuvant were used for each immunization.

Day 0: 1st immunization (complete Freund's adjuvant) Day 14: 2nd immunization (incomplete Freund's adjuvant; icFA)

Day 28 3rd Immunization (icFA) Day 56 4th Immunization (icFA) Day 80 Bleed

The rabbit's serum was tested in the immunoblot. For this purpose, a heterodimer according to the invention from Example 4 was subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter (cf. Khyse-Andersen, J., J. Biochem. Biophys. Meth. 10, (1984), 203-209). Western blot analysis was performed as in Bock, C.-T. et al. , Virus Genes 8, (1994), 215-229. For this purpose, the nitrocellulose filter was incubated for one hour at 37 ° C. with a first antibody. This antibody was the serum of the Rabbit (1: 10,000 in PBS). After several washes with PBS, the nitrocellulose filter was incubated with a second antibody. This antibody was an alkaline phosphatase-coupled goat monoclonal anti-rabbit IgG antibody (Dianova) (1: 5000) in PBS. After 30 minutes of incubation at 37 ° C, several washing steps with PBS followed and then the alkaline phosphatase detection reaction with developer solution (36μM 5 'Bronio-4-ch.loro--3-indolylphosph.at, 400μM nitroblue tetrazolium, 100mm Tris HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl ₂ ) at room temperature until bands were visible.

It was found that polyclonal antibodies according to the invention can be produced.

Immunization protocol for chicken polyclonal antibodies

For each immunization, 100 μg gel-purified heterodimer in 0.8 ml PBS and 0.8 ml complete or incomplete Freund's adjuvant were used.

Day O. 1st immunization (Freund's complete adjuvant) Day 28: 2nd immunization (Freund's incomplete adjuvant; icFA) Day 50: 3rd immunization (icFA)

Antibodies were extracted from egg yolk and tested in a Western blot. Polyclonal antibodies according to the invention have been detected.

Immunization protocol for mouse monoclonal antibodies

30 μg of gel-purified heterodimer in 0.25 ml of PBS and 0.25 ml of complete or incomplete Freund's adjuvant were used per immunization; at the 4th immunization, the fusion protein dissolved in 0.5 ml (without adjuvant).

Day 0. 1. Immunization (Freund's complete adjuvant) Day 28 2. Immunization (Freund's incomplete adjuvant; icFA)

Day 56 3. Immunization (icFA) Day 84 4. Immunization (PBS) Day 87 Fusion

Supernatants from hybridomas were tested in a Western blot. Monoclonal antibodies according to the invention have been detected

Claims

claims

1. Synthetic homodimer or heterodimer from peptide monomers which have the amino acid sequences KHYSCTAEDID (Monomer I), PPVGEADPYRVKMQ (Monomer II), AALQNHNHQQIILDK (Monomer III), IRDIIKPDPPKN (Monomer IV), SLTKRVQVWWTLMT () or variants thereof, wherein the homodimer or heterodimer binds to the interleukin 12 (IL12) receptor.

2. Homodimer or heterodimer according to claim 1, wherein the monomers are linked via their C-termini or N-termini or the N-terminus of one monomer is linked to the C-terminus of the other monomer.

3. Heterodimer according to claim 2, characterized in that the C-terminus of monomer I is linked to the N- or C-terminus of monomer II, or the N-terminus of monomer I is linked to the N- or C-terminus of monomer II is.

4. Homodimer or heterodimer according to one of claims 1 to

3, characterized in that the monomers are covalently linked to one another via polyethylene glycol, peptide (s), activated benzodiazepines, oxazolones, azalactones, aminimides, diketopiperazines, or (a) monosaccharide (s).

5. homodimer or heterodimer according to any one of claims 1 to

4, characterized in that the monomers have deletions, additions or substitutions of one or more amino acids and / or (a) modified amino acid (s), the homodimers or heterodimers being similar or better (a) to the Interleukin 12 (IL12) receptor bind and / or (b) can trigger a cellular signal.

6. Homodimer according to claim 5, which is the homodimer [AALQNHNHQQIILDK] ₂ or [AALQNHNKQQIILDK] ₂

7. Homodimer or heterodimer according to one of claims 1 to 4, characterized in that the monomers have deletions, additions or substitutions of one or more amino acids and / or (a) modified amino acid (s), the homodimers or heterodimers on the interleukin 12 (IL12) receptor can bind, but have an antagonistic effect.

8. Homodimer or heterodimer according to one of claims 1 to 7, characterized in that one or more amino acids are covalently modified by fatty acids, mono- and / or oligosaccharides.

9. Antibody or fragment thereof that specifically binds to the homodimer or heterodimer according to any one of claims 1 to 8.

10. The antibody of claim 9 which is a monoclonal antibody or a fragment thereof.

11. A process for the preparation of the homodimer or heterodimer defined in claims 1 to 8, comprising a solid phase synthesis of the monomers and subsequent dimerization.

12. Use of the homodimer or heterodimer according to one of claims 1 to 6 and 8, or of the homodimer or heterodimer produced by the process according to claim 11 for the treatment of diseases of the immune system, of diseases which are associated with a reduced cell proliferation, of infectious or inflammatory processes or to reduce the side effects of IL2 during therapy with IL2.

13. Use of the homodimer or heterodimer according to one of claims 1 to 4, 7 and 8, or of the homodimer or heterodimer produced by the process according to claim 11 for the treatment of diseases of the immune system or diseases associated with increased cell proliferation.

14. Use according to claim 13, wherein the disease is a cancer.

15. Use of the homodimer or heterodimer according to one of claims 1 to 8 or of the homodimer or heterodimer produced by the method according to claim 11 or of the antibody according to claim 9 or 10 or the fragment thereof for the diagnosis of diseases which are altered or too high or interleukin 12 (IL12) receptor which is expressed too low or an interleukin 12 concentration which is too high or too low.

16. Kit for carrying out the diagnostic method defined in claim 15, containing the homodimer or heterodimer according to one of claims 1 to 8, the homodimer or heterodimer produced by the method according to claim 11 or the antibody according to claim 9 or 10 or the fragment thereof .