CA2370298A1 - Peptide homodimers or peptide heterodimers derived from interleukin 12 - Google Patents
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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.
(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
Peptide Homodimers and Peptide Heterodimers Derived from Interleukin 12 The present invention relates to homodimers or heterodimers of peptide monomers which have the amino acid sequence KHYSCTAEDID (monomer I), PPVGEADPYRVKMQ (monomer II), AAVQNHNHQQIILDK (monomer III), IRDIIKPDPPKN (monomer IV), SLTFCVQVQGKSKR (monomer V) or RFTCWWLTTISTDLTF (monomer VI) or variants thereof, wherein the homodimers or heterodimers bind to the interleukin 12 (IL12) receptor and may optionally trigger a cellular signal. The present invention also relates to medicaments containing these homodimers or heterodimers. Furthermore, this invention concerns diagnostic compositions or diagnostic methods which make use of the homodimers or heterodimers according to the invention or antibodies directed thereagainst.
Tumor therapy is still based essentially on three main pillars: surgery, chemotherapy and radiotherapy. However, in the past few years the insight has generally been adopted that a primary (i.e. exclusive) or adjuvant (supporting) treatment using cytokines frequently extend~~ the life expectancy and often effects full recovery. Cytokines are endogenous messenger substances which are synthesized by different cells and subsequently secreted. The biological tasks of these cytokines are manifold and are only understood in part thus far. In any case, cyt:okines have chiefly fundamental immunoregulatory effects. One of these cytokines, i.e. interleukin 12 (IL12), is being used increasingly for the therapy of various tumors. IL12 is mainly produced in vivo by B cells and not so often by T
Tumor therapy is still based essentially on three main pillars: surgery, chemotherapy and radiotherapy. However, in the past few years the insight has generally been adopted that a primary (i.e. exclusive) or adjuvant (supporting) treatment using cytokines frequently extend~~ the life expectancy and often effects full recovery. Cytokines are endogenous messenger substances which are synthesized by different cells and subsequently secreted. The biological tasks of these cytokines are manifold and are only understood in part thus far. In any case, cyt:okines have chiefly fundamental immunoregulatory effects. One of these cytokines, i.e. interleukin 12 (IL12), is being used increasingly for the therapy of various tumors. IL12 is mainly produced in vivo by B cells and not so often by T
cells (D'Andrea et al., J. Exp. Med. ~ (1992), 1387-1398) and has a number of biological effects. Special attention should be paid to the following effects since they play a direct and important role for the therapeutic effect:
stimulation of the proliferation of human lymphoblasts (Gateley et al., J. Immunol. 147 (1991), 874), activation 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 aI . , J. Exp. Med. (1991) , 869) . Due to a synergistic effect with IL2 the dose of IL2 can be lowered drastically in the presence of IL12 in an adoptive immunot:herapy for producing lymphokine-activated killer cells, so that the serious side-effects of IL2 can markedly be reduced.
IL12 is a heterodimer which consists of a p40 <:hain and a p35 chain (Stern et al., Proc. Natl. Acad. Sci. U.S.A. $7 (1990), 6808). p40 has certain homologies to the extracellular domain of the I16 receptor (Gaearing and Cosman, Cell ~. (1991), 9) while p35 seems to b~e a homolog of IL6 (Mersberg et al . , Immunol. Today .1~. (1992) , 77) . The p35 chain is obviously responsible for the signal. triggering at the IL12 receptor while the p40 subunit is. likely to determine the species specificity. However, there are obviously also receptor isoforms which can be stimulated by the p40 chain alone (Presky et al., Proc. Natl. Acad. Sci.
U.S.A. ~. (1996), 14002). Since no x-ray structural analyses of both IL12 subunits exist thus far, only speculations can be made on the IL12 tertiary structure.
As in other cytokines or growth factors, the IL12 subunits also have certain short domains which interacts with the receptor. The administration of a short I112 domain which interacts with the receptor might be fully sufficient to achieve the desired cellular effects (e. g. immunostimulatory effect). However, no studies on IL12 exist thus far, carried out e.g. with synthesized peptides which could furnish information on suitable IL12 domains.
Since cytokines commonly only occur in extremely low concentrations in the serum, it is not possible to isolate therapeutically usable amounts from this medium. As a result, the therapeutically used cytokines have so far been produced recombinantly in bacteria or yeast. This. method is, however, accompanied by a number of serious drawbacks.
During the set-up, a recombinant production of cytokines is time-consuming and labor-intensive. For example, the following must be clarified: does the host cell express the protein accurately or in the exact native conformation, what is actually the best host cell or the best expre;~sion vector and under which conditions is the protein not degraded proteolytically. It should be noted that proteins generally produced by recombination have a tertiary structure differing from native protein and are therefore: recognized as "foreign" by the human immune system. The antibodies induced by this neutralize the protein, e.g. the cytokine, which results in a loss of its effectiveness.
The isolation and purification of recombinant proteins is difficult, since in principle always the same questions specific to every new product are posed: how can the proteolytic degradation of the protein and/or its precipitation following isolation be prevented. On the whole, recombinantly produced proteins show a striking instability towards proteases. This requires a frequent and/or large-dose administration which seriously strains the patient, on the one hand, and renders the therapy very expensive, on the other hand.
stimulation of the proliferation of human lymphoblasts (Gateley et al., J. Immunol. 147 (1991), 874), activation 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 aI . , J. Exp. Med. (1991) , 869) . Due to a synergistic effect with IL2 the dose of IL2 can be lowered drastically in the presence of IL12 in an adoptive immunot:herapy for producing lymphokine-activated killer cells, so that the serious side-effects of IL2 can markedly be reduced.
IL12 is a heterodimer which consists of a p40 <:hain and a p35 chain (Stern et al., Proc. Natl. Acad. Sci. U.S.A. $7 (1990), 6808). p40 has certain homologies to the extracellular domain of the I16 receptor (Gaearing and Cosman, Cell ~. (1991), 9) while p35 seems to b~e a homolog of IL6 (Mersberg et al . , Immunol. Today .1~. (1992) , 77) . The p35 chain is obviously responsible for the signal. triggering at the IL12 receptor while the p40 subunit is. likely to determine the species specificity. However, there are obviously also receptor isoforms which can be stimulated by the p40 chain alone (Presky et al., Proc. Natl. Acad. Sci.
U.S.A. ~. (1996), 14002). Since no x-ray structural analyses of both IL12 subunits exist thus far, only speculations can be made on the IL12 tertiary structure.
As in other cytokines or growth factors, the IL12 subunits also have certain short domains which interacts with the receptor. The administration of a short I112 domain which interacts with the receptor might be fully sufficient to achieve the desired cellular effects (e. g. immunostimulatory effect). However, no studies on IL12 exist thus far, carried out e.g. with synthesized peptides which could furnish information on suitable IL12 domains.
Since cytokines commonly only occur in extremely low concentrations in the serum, it is not possible to isolate therapeutically usable amounts from this medium. As a result, the therapeutically used cytokines have so far been produced recombinantly in bacteria or yeast. This. method is, however, accompanied by a number of serious drawbacks.
During the set-up, a recombinant production of cytokines is time-consuming and labor-intensive. For example, the following must be clarified: does the host cell express the protein accurately or in the exact native conformation, what is actually the best host cell or the best expre;~sion vector and under which conditions is the protein not degraded proteolytically. It should be noted that proteins generally produced by recombination have a tertiary structure differing from native protein and are therefore: recognized as "foreign" by the human immune system. The antibodies induced by this neutralize the protein, e.g. the cytokine, which results in a loss of its effectiveness.
The isolation and purification of recombinant proteins is difficult, since in principle always the same questions specific to every new product are posed: how can the proteolytic degradation of the protein and/or its precipitation following isolation be prevented. On the whole, recombinantly produced proteins show a striking instability towards proteases. This requires a frequent and/or large-dose administration which seriously strains the patient, on the one hand, and renders the therapy very expensive, on the other hand.
Since in a therapy the cytokines must usually be administered at regular intervals, it must be ensured that they are absolutely free from contaminations, i.e. above all from host cell constituents. Because of this complex methods must be used which have an unfavorable effect on :pricing.
In the meantime, the insight has largely been adopted that messenger substances (e.g. cytokines) do not only have a binding site for the receptor but that further - presently not yet fully understood - domains exist which might induce signal paths other than those actually intended. This could explain inter alia the frequently occurring side-effects of the cytokines used thus far, which sometimes render treatment therewith impossible.
Thus, the technical problem of this invention is chiefly to provide compounds having a biological acaivity of interleukin 12 (IL12) so as to avoid the former drawbacks of the prior art, i.e. fewer side-effects, a longer half-life and strong biological activity are obtained upon administration as medicament.
This technical problem was solved by providing the embodiments characterized in the claims.
An embodiment of the present invention relates to a synthetic homodimer or heterodimer 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), wherein the homodimer or heterodimer can bind to the interleukin 12 (IL:L2) receptor and optionally trigger a cellular signal. These peptides according to the invention represent the binding regions of the IL12 subunits p40 and p35 and make use of t:he insight that for a biological effect the peptides from p35 and p40 must be used in dimerized form, since the receptors must also lie together in twos to be able to trigger the intracellular signals. Monomers I-III comprise 3 loops which lie at the point where p35 and the interleukin :12 receptor contact while analogously monomers IV-VI represent the contact points for p40 with the receptor. The dimers according to the invention have the following advantages:
They can be synthesized and purified easily by standardized methods and can be used directly for the required tests (e.g. structural studies, biological tests, preclinical and clinical studies). These peptides represent the minimum structures of the respective cytokine which are required for its biological effectiveness. This ensures utmost specificity and high biological activity in combination with minimum side-effects. Because the peptides have such a small size, they can be microcapsulated so as to yield depot forms which have differently long duration of effect - depending on the respective pore size of the capsules. The depot form can be applied locally so as to ensure the maximum concentration of the active substance at the desired site over a long period of time.
According to the invention all combinations of monomers I -VI among one another can form dimers, and the dimers forming solve the problem.
The peptides according to the invention are usually synthesized according to known solid-phase synthesis methods, as described in below Example 1, for instance. The biological activity (binding to the receptor, triggering a cellular signal and proliferation-stimulating effect) is studied according to methods with which a person skilled in the art is familiar, e.g. according to Lewis, ~T. Immunol.
Methods X85 (1995), 9-17; Grander et al., Eur. J: Cancer 28A
(1992), 815-818 or also according to the methods described in the below examples.
In a preferred embodiment according to the invention the above described peptides are homodimers or hete:rodimers in which the monomers are linked via their C termini or N
termini or the N terminus of one monomer is linked with the C terminus of the other monomer. Preferably, the C terminus of monomer I is linked with the N or C terminus of monomer II or the N terminus of monomer I is linked with the N or C
terminus of monomer II. By way of example, the desired N-terminus peptide is thus activated in a side chain function-protected form after removing the proteci~ing group (preferably Fmoc) by treatment with e.g. piperidine with a cross-linker and the product is purified via HPLC. Another previously synthesized monomer is also provided (at either the C terminus or the N terminus - depending on the desired dimer) in a side chain function-protected form with a group which reacts exclusively with the N-terminus group of the bound peptide. After adding this monomer to the activated monomer, the dimers which differ markedly from the monomer as regards the elution behavior are purified a.nd isolated after removing the side chain protection groups according to standard methods by reversed-phase HPLC. If enabled by the amino acid composition, the activation groups and cross-linkers can also be chosen such that dimerization at free monomers can be carried out directly in a solution after removing the side chain protecting groups. Alternatively, the synthesis strategy can be performed at the HYCHRON
anchor as described in the example so as to maintain the side chain protecting groups and carry out dimerization in aqueous solution.
This linkage can be carried out e.g. by means of the method described in the below examples or by other standard methods, attention having to be paid to the fact that dimerization takes place such that effective binding to the receptor pair is still possible effectively. For example, dimerization can be carried out with a lysine residue as the branching point (Wrighton et al., Nature Biotechnology 15 (1997), 1261-1265). The homomers of the dimers according to the invention are preferably linked covalently with one another via linkers, such as polyethylene glycol, peptide(s), activated benzodiazepines, oxazolones, azalactones, aminimides, diketopiperazines or (a) monosaccharide(s). The chemical reactions to be carried out correspondingly are known to the person skilled in the art and working in this field (e. g. Hermannson, Hioconjugate Techniques (1996), Academic Press, San Diego; Peeters et al., J. Immunol. Meth. 120, (1989), 133-144; Inman et al., Bioconjugate Chem. 2 (1991), 458-463).
The present invention also relates to homodimers or heterodimers which are characterized in that t:he monomers have deletions, additions or substitutions of one or more amino acids and/or (one) modified amino acid(~~) over the corresponding starting monomers I to VI. In this connection, homodimers or heterodimers are obtained which (a) as compared to the original forms bind to the IL12 receptor in a similar or better way and/or can trigger a cellular signal, or (b) although they can still bind too the IL12 receptor they have an antagonistic effect, i.e. upon administration the biologically active form is displaced by this form which no longer triggers a cellular :signal after binding to the receptor. It can be studied by means of the methods described in the below examples or the above described methods to what extent these modified homodimers or heterodimers have the desired biological properties. It is preferred to introduce natural amino acids in the amino acid substitutions or additions, modified amino acids being not excluded. The preferred modifications comprise glycosylations with monosaccharides or disacc:harides of serine, threonine and asparagine, farnesylation and palmitoylation of cysteine, phosphorylation of threonine, serine and tyrosine, the modifications which relate to the central amino acids usually resulting in antagonistic forms.
The deletions, additions, substitutions and/or modifications of the above described homodimers or heterodimers modified according to the invention relate to at most 10, preferably at most 7, more preferably at most 3 and most preferably at most 1 amino acids) per monomer. The homodimers or heterodimers according to the invention which consist of modified monomers contain in addition to the domain responsible for receptor binding no further domains which are characteristic of the natural cytokine.
The dimer according to the invention and/or the dimer consisting of modified monomers are preferably the following homodimers: [AALQNHNHQQIILDK]2 or [AALQNHNKQQIILDK]2, which even have a markedly higher specific activity over IL12.
These two monomers have both C-C and C-N linkage.
The homodimers or heterodimers according to the invention may be available as such but can also be linked with other compounds, e.g. (poly)peptides. For examples, carrier proteins, e.g. transferrin or albumin which are not recognized to be foreign by the body, are counted among these (poly)peptides. The homodimers or heterodimers according to the invention can also be fused with other (poly)peptides, e.g. 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 penetratrin from Drosophila.
In a further embodiment, the present invention relates to the above homodimers or heterodimers which a.re further characterized in that one or more amino acids a:re modified covalently by fatty acids, monosaccharides and/or oligosaccharides. This may be done by generally known methods, e.g. during the synthesis of the monomers using amino acids which already carry the above modifications. The modifications may also be made subsequently. For example, a higher resistance to proteolytic degradation and thus an even longer biological half-life can be achieved by these modifications.
The present invention also relates to an antibody or a fragment thereof against the above described dimers according to the invention. These antibodies may also be used in diagnostic assays, for example.
The antibodies may be monoclonal, polyclonal or synthetic antibodies or fragments thereof. In this connection, the term "fragment" means all parts of the monoclonal antibody (e.g. Fab, Fv or single chain Fv fragments) which have an epitope specificity the same as that of the complete antibody. The person skilled in the art is familiar with the production of such fragments. 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 an antigen. Methods of 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 (e.g. a mouse), a humanized antibody or a chimeric antibody or a fragment thereof. Antibodies similar to chimeric human antibodies or humanized antibodies have a reduced potential antigenicity. However, their affinity for the target is not lowered. The production of chimeric and humanized antibodies or of antibodies similar- to human antibodies was described in detail (see e.g. Queen et al., Proc. Natl. Acad. Sci. U.S.A. $~ (1989), 10029, and Verhoeyan et al., Science 239 (1988), 1534). Humanized immunoglobulins have variable framework regions which are derived substantially from a human immunoglobulin (designated acceptor immunoglobulin) and the complementarity of the determining regions which are derived substantially from a non-human immunoglobulin (e. g. from a mouse) (designated donor immunoglobulin). The constant regions) also originate(s), if present, substantially from a human immunoglobulin. Upon administration to human patients humanized (and the human) antibodies offer a number of advantages over antibodies of mice or other species: (a) the human immune system should not recognize the framework or the constant region of the humanized antibody as being foreign, and the antibody response to such an injected antibody should therefore be less than that to a fully foreign mouse antibody or a partially foreign chimeric antibody (b) since the effector region 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 which is substantially equivalent to that of naturally occurring human antibodies, which permits the administration of doses smaller and less frequent as compared to antibodies of other species. The present invention also relates to a hybridoma which produces the above described monoclonal antibody.
The present invention also relates to medicaments containing the homodimers and/or heterodimers according to the invention and to their use for treating diseases of the immune system, diseases associated with a reduced cell proliferation or infectious or inflammatory processes (e. g.
HIV, leishmaniasis: Mountford et al., J. Immunol. 156 (996).
pp. 4739-4745). Certain modified forms of the dimers according to the invention can also have an antagonistic effect. Thus, the present invention also relates to medicaments containing the homodimers or heterodimers according to the invention and their use for treating diseases which are associated with an increased cell proliferation, e.g. tumors, 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 (7.993), 1223-1230). Finally, the present invention also relates to medicaments containing the dimers according to the invention and their use for reducing of the IL2 side-effects in a therapy with IL2, e.g. in an immunotherapy.
The medicament according to the invention is optionally available in combination with a suitable ph<~rmaceutical carrier. Suitable carriers and the formulation of such medicaments are known to the person skilled _Ln the art.
Suitable carriers are e.g. phosphate-buffered common salt solutions, water, emulsions, e.g. oil/water emulsions, wetting agents, sterile solutions, etc. The medicament according to the invention may be available in the form of an injection solution, tablet, ointment, :suspension, emulsion, suppository, etc. It may also be administered in the form of depots (microcapsules, zinc salts, liposomes, etc.). The kind of administration of the medicament depends inter alia on the form of active substance which :is present;
it can be made orally or parenterally. The methods for the parenteral administration comprise the topical, intra-arterial (e. g. directly to a tumor), int:ramuscular, intramedullary, intrathekal, intraventricular, intravenous, intraperitoneal, transdermal or transmucosa:l (nasal, vaginal, rectal, sublingual) administration. The suitable dosage is determined by the attending physician and depends on various factors, e.g. on the age, sex and weight of the patient, the kind and stage of the disease, t:he kind of administration, etc.
The present invention also 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 for the diagnosis of diseases which are associated with a modified I112 receptor or one expressed expressively or insufficiently anct with an excessively high or low concentration of IL12. In this connection, the homodimers or heterodimers according to the invention may be available in generally common assay formats for diagnostic detection. This detection comprises e.g. (a) the collection of a cell sample from a patient, (b) the contacting of the resulting cell sample with a homodimer or heterodimer or antibody according to the invention as a probe under conditions which permit the specific binding to the target, and (c) the detection of the binding to the target. This detection can be carried out using standard techniques known to the person skilled in the art. In this connection, the compounds according to the invention can be bound e.g. in liquid phase or to a solid carrier and can be labeled in different ways. Suitable markers and labeling methods are known to the person skilled in the art. He is also familiar with cell breakdown methods permitting that IL12 or the receptor can be contacted specif ical7_y with the antibody or homodimer or heterodimer according to the invention.
Finally, the present invention relates to a diagnostic kit for carrying out the above described diagnostic method, which contains a dimer according to the invention or the antibody according to the invention or the fragment thereof.
Depending on the development of the diagnostic kit, the dimer or the antibody or the fragment thereof can be immobilized.
The following examples explain the invention.
Monomers I-VI were produced according to a standard solid-phase synthesis (Seitz et al., Angew. Chem. 107 (1995), 901). The synthesis was made at a HYCHRON anchor with a Fmoc-protected amino acid and was started via the C
terminus. The carboxyl groups of glutamic acid and asparagic acid were protected in the form of tert-butyl esters. The protecting groups of the side chains of Tyr comprised tetrahydropyranyl, tert-butyl(Boc) and trityl groups. The side chain protecting groups of serine and threonine comprised acetyl, benzoyl and benzyloxycarbonyl (=Cbz) groups. The protecting groups for the side chain of arginine comprised Cbz, mesitylene-2-sulfonyl (=Mts) or tert-butyloxycarbonyl (=Boc). The side chain of lysine was protected by tosyl (=Tos) or Boc. After removing the a-amino protective group and corresponding wash steps, followed by activation of the carboxyl group of the next amino acid, the latter was linked to the preceding amino acid. T:he complete monomers were synthesized in this order. The prepared monomers were removed from the column by palladium(0)catalyzed allyl transfer, the protecting groups at the side chains being maintained. Thereafter, the Fmoc group was removed by morpholine, and the rest of the protecting groups was cleaved by treatment with TFA.
Monomers I were dimerized .by dissolving 25 mg activated bifunctional polyethylene glycol (PEG ~~uccinimidyl propionate, molecular weight about 3,400; Sigma, Taufkirchen, Germany) in 4 ml PBS, pH value 7.5, whereupon a threefold molar excess of monomers, dissolved in 1 ml 0.1 trifluoroacetic acid, were added. After 3 hours of incubation on ice, lyophilized monomers were added again so that there was a final ratio of 3.5 mol monomer to 1 mol PEG. The mixture was incubated on ice for another 17 hours.
PEG was inactivated by the addition of 1 M Tris/HCl, pH
value 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).
onomer II
Monomers II were dimerized by dissolving 25 mg of activated bifunctional polyethylene glycol (PEG succinimidyl propionate, molecular weight about 3,400; Sigma, Taufkirchen) in 4 ml PBS, pH value 7.5, whereupon a threefold molar excess of monomers, dissolved in 1 ml 0.1 %
trifluoroacetic acid were added. Following 3 hours of incubation on ice lyophilized monomers were added again, so that there was a final ratio of 3.5 mol monomer to 1 mol PEG. The mixture was incubated on ice for another 17 hours.
PEG was inactivated by the addition of 1 M Tris/HC1, pH
value 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 dimerized by dissolving 25 mc~ activated bifunctional polyethylene glycol (PEG-succinimidyl propionate, molecular weight about 3,400; Sigma, Taufkirchen) in 4 ml PBS, pH value 7.5. Thereafter, a threefold molar excess of monomers, dissolved in 1 ml 0.1 trifluoroacetic acid was added. After 3 hours of incubation on ice lyophilized monomers were added again, so that there was a final ratio of 3.5 mol monomer to 1 mol PEG. The mixture was incubated on ice for another 17 hours. PEG was inactivated by adding 1 M Tris/HCl, pH value 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 made via reversed-phase HPLC, since both dimers markedly differ as regards their elution behavior (depending on the composition there is a difference between 4 and ~3 minutes in the elution peak) and can thus be separated from one another.
The formation of the dimers described in the above examples was checked by analytical reversed-phase HPLC. T'he analysis was carried out by means of a Vydac-C18 protein peptide column (0.46 x 25 cm) (The Separation Group, U.S.A.), a BioRad HPLC system and a two-wavelength detector from Perkin-Elmer. The column was equilibrated using 0.1 % TFA in distilled water, and 10 minutes after injecting the sample (as a rule 6 ml), there was a 45-minute linear gradient (0-100) of acetonitrile with 0.1 % TFA. The flow rate was kept continuously at 1 ml/min. Under these conditions,. the cross-linker appeared in the flow. The reaction product eluted after 37 minutes, while the unconjugated monomer was already eluted after 32 minutes and could thus be separated clearly from the dimer. The dimer was further purified on a preparative reversed-HPLC column (2.2 x 25 cm) with the same HPLC system. The column was equilibrated with a constant flow rate of 8 ml/min. using distilled water . acetonitrile of 80:20 (both contained 0.1 % TFA). 20 minutes after injecting the sample (6 ml) there was a 60-minute linear gradient with 100 o acetonitrile/0.1 % TFA. The main peak was collected and lyophilized. A gentler gradient (20-800 acetonitrile/0.1 % TFA above 60 %) was used for monomer II
so as to reach better separation. The isolated dimers and the native monomers were used in binding studies (see Example 7) and proliferation tests (see Example 8).
~~ amn~e 6~ radioactive labeling of the dimers The dimers were iodized as described below. 20 ~.1 chloramine T ( 0 . 5 mg/ml ) were added to 2 0 ~l Nalasl ( 2 mC:i , Amersham Buckler, Braunschweig, Germany), the mixture was added to 50 ~.1 dimer (5 ~g in PBS) 2 minutes later and incubated for 15 seconds. In order to complete the reaction, 30 ~,l sodium metabisulfite (1 mg/ml in PBS) and, after 30 seconds, 50 ~,1 KI ( 10 mg/ml in PBS ) were added . Ge lat in ( 5 0 ~.l , 1 mg/ml in distilled water) was added as carrier and the solution was applied immediately onto a BioSil SEC 125-5 column (BioRad, ml) which was equilibrated with 0.25 % gel;atin/0.02 %
sodium azide in PBS. The fractions which contained the radioactive product were combined and further purified and isolated on the reversed-phase HPLC column as described in Example 5. The purified dimers had a specific radioactivity of 50-100 ~,Ci/~.g, yielded in tricin-SDS-PAGE a single band and 95-100 % of the radioactivity could be precipitated with the chloroform/methanol method (Wessel et al., Anal.
Biochem. 138 (1984), 141-143). These results show that the radioactively labeled product consisted of a pure dimer and that the radioactivity could be ascribed exclusively to the dimer.
These studies show whether the dimers according to the invention bind specifically to cells which express the IL12 receptor. This example uses the homodimer consisting of monomers III. The experiment was carried out: with PHA-stimulated human lymphoblasts according to a modified protocol of Chizzonite et al., J. Immunol. ],~$., (1992), 3117-3124.
The stimulated cells (7.5 x 105 cells /ml RPMI medium) were incubated for 90 minutes with radioactively labeled dimer at varying concentrations (0.01 to 1 ng) and thereafter washed in PBS by means of 3 centrifugation steps, and the cell-bound radioactivity was measured in a liquid scintillation counter. The extent of unspecific binding was determined in the presence of an IL12 concentration which was 100 times as high, labeled homodimer and IL12 having been added to the cells at the same time. The results are shown in Table I.
Test substance Binding o maximum binding) Interleukin 12 100 AALQNHNHQQIILDK
[AALQNHNHQQIILDK 2 66 These studies should show whether similar to native IL12 the dimers according to the invention have a proliferation-stimulating effect. They were carried out again with PHA-stimulated human lymphoblasts in a 48-hour assay according to Gately and Chizzonite, Curr. Protocols in Immunology Voll (1992), pages 6.16 - 6.16.8, using monomer I:CI. In this connection, the cells were seeded in 200 ~.1 culture medium in a cell number of 5 x 104 and cultured in the presence of various dimer concentrations (0.01 - 1 ng) for 48 hours.
Having added tritiated thymidine (0.25 ~Ci/ml:) the cells were cultured for another 4 hours and then isolated via a cell harvester. The introduced radioactivity was measured in a liquid scintillation counter. The values a:re shown in Table II as specific activity in units x 10-'/mg IL12.
Test substance Specific ac-tiv ty interleukin 12 5-3 AALQNHNHQQIILDK 0.03 _A_AT~QNHNHQQIILDK 2 2.4 [AALQNHNKQQIILDK 2 10.5 The heterodimer produced in Example 4 was subjected to 18 SDS polyacrylamide gel electrophoresis. After staining the gel using 4 M sodium acetate, an about 6.5 k.B band was excised out of the gel and incubated in phosphate-buffered common salt solution. The dimer is eluted by means of an electroeluter (BioRad) from the gel and lyophilized after a kB dialysis overnight. 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/0.02 mol carrier in a total of 2 ml PBS. 2 ml glutardialdehyde (0..2 °s in PBS) are added dropwise to the mixture with continuous stirring and incubated at room temperature for 1 hour. The. mixture is then dialyzed for 48 hours with 12-hour buffer cycle in a 20 kB dialysis hose against PBS and subsequently freeze-dried.
The animals are immunized with the mixture consisting of dimer BSA complexes, BSA-BSA complexes and dime:r complexes with 100 ~.g (rabbit and chicken) or with 30 ug (rnouse) .
- Immunization protocol for polyclonal antibodies in rabbits 100 ~.g of gel-purified heterodimer in 0.7 ml PBS and 0.7 ml of complete or incomplete Freund's adjuvant were used per immunization:
Day 0: lgt immunization (complete Freund's adju.vant) Day 14: 2nd immunization (incomplete Freund's adjuvant;
icFA) Day 28: 3rd immunization (icFA) Day 56: 4th immunization (icFA) Day 80: bleeding to death.
The rabbit serum was tested in an immunoblot. For this purpose, a heterodimer of Example 4 according to the invention was subjected to SDS polyacry:Lamide gel electrophoresis and transferred to a nitrocellulose filter (cf. Khyse-Andersen, J., J. Biochem. Biophys. Meth. 10 (1984) , 203-209) . The Western blot analysis was carried out as described in Bock, C.-T. et al., Virus Genes. 8, (1994), 215-229. For this purpose, the nitrocellulose filter was incubated with a first antibody at 37°C for one: hour. This antibody was the rabbit serum (1:10000 in PBS). After several wash steps using PBS, the nitrocellulose filter was incubated with a second antibody. This antibody was an alkaline phosphatase-coupled monoclonal goat anti-rabbit IgG
antibody (Dianova company) (1:5000) in PBS. 30 minutes of incubation at 37°C were followed by several wash steps using PBS and subsequently by the alkaline phosphatase detection reaction with developer solution (36 ~,M 5'-bromo-4-chloro-3-indolylphosphate, 400 ~M nitro blue tetrazol:ium, 100 mM
Tris-HC1, pH 9.5, 100 mM NaCl, 5 mM MgCl2) at room temperature until bands became visible.
It showed that polyclonal antibodies according to the invention can be prepared.
- Immunization protocol for polyclonal antibodies in chickens 100 ~g of gel-purified heterodimer in 0.8 ml PBS and 0.8 ml of complete or incomplete Freund's adjuvant wez-e used per immunization.
Day 0: 18t immunization (complete Freund's adjuvant) Day 28: 2nd immunization (incomplete Freund'~; 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 were detected.
- Immunization protocol for monoclonal antibodies in mice 30 ~g of gel-purified fusion protein in 0.25 ml 3?BS and 0.25 ml of complete or incomplete Freund's adjuvant were used per immunization. The fusion protein was dissolved. in 0.5 ml (without adjuvant) in the 4th immunization.
Day0: 1St immunization (complete Freund's adjuvant) Day28: 2nd immunization (incomplete Freund's adjuvant;
icFA) Day56: 3rd immunization (icFA) Day84: 4th immunization (PBS) Day87: fusion.
Supernatants of hybridomas were tested in a Western blot.
Monoclonal antibodies according to the invention were identified.
In the meantime, the insight has largely been adopted that messenger substances (e.g. cytokines) do not only have a binding site for the receptor but that further - presently not yet fully understood - domains exist which might induce signal paths other than those actually intended. This could explain inter alia the frequently occurring side-effects of the cytokines used thus far, which sometimes render treatment therewith impossible.
Thus, the technical problem of this invention is chiefly to provide compounds having a biological acaivity of interleukin 12 (IL12) so as to avoid the former drawbacks of the prior art, i.e. fewer side-effects, a longer half-life and strong biological activity are obtained upon administration as medicament.
This technical problem was solved by providing the embodiments characterized in the claims.
An embodiment of the present invention relates to a synthetic homodimer or heterodimer 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), wherein the homodimer or heterodimer can bind to the interleukin 12 (IL:L2) receptor and optionally trigger a cellular signal. These peptides according to the invention represent the binding regions of the IL12 subunits p40 and p35 and make use of t:he insight that for a biological effect the peptides from p35 and p40 must be used in dimerized form, since the receptors must also lie together in twos to be able to trigger the intracellular signals. Monomers I-III comprise 3 loops which lie at the point where p35 and the interleukin :12 receptor contact while analogously monomers IV-VI represent the contact points for p40 with the receptor. The dimers according to the invention have the following advantages:
They can be synthesized and purified easily by standardized methods and can be used directly for the required tests (e.g. structural studies, biological tests, preclinical and clinical studies). These peptides represent the minimum structures of the respective cytokine which are required for its biological effectiveness. This ensures utmost specificity and high biological activity in combination with minimum side-effects. Because the peptides have such a small size, they can be microcapsulated so as to yield depot forms which have differently long duration of effect - depending on the respective pore size of the capsules. The depot form can be applied locally so as to ensure the maximum concentration of the active substance at the desired site over a long period of time.
According to the invention all combinations of monomers I -VI among one another can form dimers, and the dimers forming solve the problem.
The peptides according to the invention are usually synthesized according to known solid-phase synthesis methods, as described in below Example 1, for instance. The biological activity (binding to the receptor, triggering a cellular signal and proliferation-stimulating effect) is studied according to methods with which a person skilled in the art is familiar, e.g. according to Lewis, ~T. Immunol.
Methods X85 (1995), 9-17; Grander et al., Eur. J: Cancer 28A
(1992), 815-818 or also according to the methods described in the below examples.
In a preferred embodiment according to the invention the above described peptides are homodimers or hete:rodimers in which the monomers are linked via their C termini or N
termini or the N terminus of one monomer is linked with the C terminus of the other monomer. Preferably, the C terminus of monomer I is linked with the N or C terminus of monomer II or the N terminus of monomer I is linked with the N or C
terminus of monomer II. By way of example, the desired N-terminus peptide is thus activated in a side chain function-protected form after removing the proteci~ing group (preferably Fmoc) by treatment with e.g. piperidine with a cross-linker and the product is purified via HPLC. Another previously synthesized monomer is also provided (at either the C terminus or the N terminus - depending on the desired dimer) in a side chain function-protected form with a group which reacts exclusively with the N-terminus group of the bound peptide. After adding this monomer to the activated monomer, the dimers which differ markedly from the monomer as regards the elution behavior are purified a.nd isolated after removing the side chain protection groups according to standard methods by reversed-phase HPLC. If enabled by the amino acid composition, the activation groups and cross-linkers can also be chosen such that dimerization at free monomers can be carried out directly in a solution after removing the side chain protecting groups. Alternatively, the synthesis strategy can be performed at the HYCHRON
anchor as described in the example so as to maintain the side chain protecting groups and carry out dimerization in aqueous solution.
This linkage can be carried out e.g. by means of the method described in the below examples or by other standard methods, attention having to be paid to the fact that dimerization takes place such that effective binding to the receptor pair is still possible effectively. For example, dimerization can be carried out with a lysine residue as the branching point (Wrighton et al., Nature Biotechnology 15 (1997), 1261-1265). The homomers of the dimers according to the invention are preferably linked covalently with one another via linkers, such as polyethylene glycol, peptide(s), activated benzodiazepines, oxazolones, azalactones, aminimides, diketopiperazines or (a) monosaccharide(s). The chemical reactions to be carried out correspondingly are known to the person skilled in the art and working in this field (e. g. Hermannson, Hioconjugate Techniques (1996), Academic Press, San Diego; Peeters et al., J. Immunol. Meth. 120, (1989), 133-144; Inman et al., Bioconjugate Chem. 2 (1991), 458-463).
The present invention also relates to homodimers or heterodimers which are characterized in that t:he monomers have deletions, additions or substitutions of one or more amino acids and/or (one) modified amino acid(~~) over the corresponding starting monomers I to VI. In this connection, homodimers or heterodimers are obtained which (a) as compared to the original forms bind to the IL12 receptor in a similar or better way and/or can trigger a cellular signal, or (b) although they can still bind too the IL12 receptor they have an antagonistic effect, i.e. upon administration the biologically active form is displaced by this form which no longer triggers a cellular :signal after binding to the receptor. It can be studied by means of the methods described in the below examples or the above described methods to what extent these modified homodimers or heterodimers have the desired biological properties. It is preferred to introduce natural amino acids in the amino acid substitutions or additions, modified amino acids being not excluded. The preferred modifications comprise glycosylations with monosaccharides or disacc:harides of serine, threonine and asparagine, farnesylation and palmitoylation of cysteine, phosphorylation of threonine, serine and tyrosine, the modifications which relate to the central amino acids usually resulting in antagonistic forms.
The deletions, additions, substitutions and/or modifications of the above described homodimers or heterodimers modified according to the invention relate to at most 10, preferably at most 7, more preferably at most 3 and most preferably at most 1 amino acids) per monomer. The homodimers or heterodimers according to the invention which consist of modified monomers contain in addition to the domain responsible for receptor binding no further domains which are characteristic of the natural cytokine.
The dimer according to the invention and/or the dimer consisting of modified monomers are preferably the following homodimers: [AALQNHNHQQIILDK]2 or [AALQNHNKQQIILDK]2, which even have a markedly higher specific activity over IL12.
These two monomers have both C-C and C-N linkage.
The homodimers or heterodimers according to the invention may be available as such but can also be linked with other compounds, e.g. (poly)peptides. For examples, carrier proteins, e.g. transferrin or albumin which are not recognized to be foreign by the body, are counted among these (poly)peptides. The homodimers or heterodimers according to the invention can also be fused with other (poly)peptides, e.g. 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 penetratrin from Drosophila.
In a further embodiment, the present invention relates to the above homodimers or heterodimers which a.re further characterized in that one or more amino acids a:re modified covalently by fatty acids, monosaccharides and/or oligosaccharides. This may be done by generally known methods, e.g. during the synthesis of the monomers using amino acids which already carry the above modifications. The modifications may also be made subsequently. For example, a higher resistance to proteolytic degradation and thus an even longer biological half-life can be achieved by these modifications.
The present invention also relates to an antibody or a fragment thereof against the above described dimers according to the invention. These antibodies may also be used in diagnostic assays, for example.
The antibodies may be monoclonal, polyclonal or synthetic antibodies or fragments thereof. In this connection, the term "fragment" means all parts of the monoclonal antibody (e.g. Fab, Fv or single chain Fv fragments) which have an epitope specificity the same as that of the complete antibody. The person skilled in the art is familiar with the production of such fragments. 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 an antigen. Methods of 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 (e.g. a mouse), a humanized antibody or a chimeric antibody or a fragment thereof. Antibodies similar to chimeric human antibodies or humanized antibodies have a reduced potential antigenicity. However, their affinity for the target is not lowered. The production of chimeric and humanized antibodies or of antibodies similar- to human antibodies was described in detail (see e.g. Queen et al., Proc. Natl. Acad. Sci. U.S.A. $~ (1989), 10029, and Verhoeyan et al., Science 239 (1988), 1534). Humanized immunoglobulins have variable framework regions which are derived substantially from a human immunoglobulin (designated acceptor immunoglobulin) and the complementarity of the determining regions which are derived substantially from a non-human immunoglobulin (e. g. from a mouse) (designated donor immunoglobulin). The constant regions) also originate(s), if present, substantially from a human immunoglobulin. Upon administration to human patients humanized (and the human) antibodies offer a number of advantages over antibodies of mice or other species: (a) the human immune system should not recognize the framework or the constant region of the humanized antibody as being foreign, and the antibody response to such an injected antibody should therefore be less than that to a fully foreign mouse antibody or a partially foreign chimeric antibody (b) since the effector region 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 which is substantially equivalent to that of naturally occurring human antibodies, which permits the administration of doses smaller and less frequent as compared to antibodies of other species. The present invention also relates to a hybridoma which produces the above described monoclonal antibody.
The present invention also relates to medicaments containing the homodimers and/or heterodimers according to the invention and to their use for treating diseases of the immune system, diseases associated with a reduced cell proliferation or infectious or inflammatory processes (e. g.
HIV, leishmaniasis: Mountford et al., J. Immunol. 156 (996).
pp. 4739-4745). Certain modified forms of the dimers according to the invention can also have an antagonistic effect. Thus, the present invention also relates to medicaments containing the homodimers or heterodimers according to the invention and their use for treating diseases which are associated with an increased cell proliferation, e.g. tumors, 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 (7.993), 1223-1230). Finally, the present invention also relates to medicaments containing the dimers according to the invention and their use for reducing of the IL2 side-effects in a therapy with IL2, e.g. in an immunotherapy.
The medicament according to the invention is optionally available in combination with a suitable ph<~rmaceutical carrier. Suitable carriers and the formulation of such medicaments are known to the person skilled _Ln the art.
Suitable carriers are e.g. phosphate-buffered common salt solutions, water, emulsions, e.g. oil/water emulsions, wetting agents, sterile solutions, etc. The medicament according to the invention may be available in the form of an injection solution, tablet, ointment, :suspension, emulsion, suppository, etc. It may also be administered in the form of depots (microcapsules, zinc salts, liposomes, etc.). The kind of administration of the medicament depends inter alia on the form of active substance which :is present;
it can be made orally or parenterally. The methods for the parenteral administration comprise the topical, intra-arterial (e. g. directly to a tumor), int:ramuscular, intramedullary, intrathekal, intraventricular, intravenous, intraperitoneal, transdermal or transmucosa:l (nasal, vaginal, rectal, sublingual) administration. The suitable dosage is determined by the attending physician and depends on various factors, e.g. on the age, sex and weight of the patient, the kind and stage of the disease, t:he kind of administration, etc.
The present invention also 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 for the diagnosis of diseases which are associated with a modified I112 receptor or one expressed expressively or insufficiently anct with an excessively high or low concentration of IL12. In this connection, the homodimers or heterodimers according to the invention may be available in generally common assay formats for diagnostic detection. This detection comprises e.g. (a) the collection of a cell sample from a patient, (b) the contacting of the resulting cell sample with a homodimer or heterodimer or antibody according to the invention as a probe under conditions which permit the specific binding to the target, and (c) the detection of the binding to the target. This detection can be carried out using standard techniques known to the person skilled in the art. In this connection, the compounds according to the invention can be bound e.g. in liquid phase or to a solid carrier and can be labeled in different ways. Suitable markers and labeling methods are known to the person skilled in the art. He is also familiar with cell breakdown methods permitting that IL12 or the receptor can be contacted specif ical7_y with the antibody or homodimer or heterodimer according to the invention.
Finally, the present invention relates to a diagnostic kit for carrying out the above described diagnostic method, which contains a dimer according to the invention or the antibody according to the invention or the fragment thereof.
Depending on the development of the diagnostic kit, the dimer or the antibody or the fragment thereof can be immobilized.
The following examples explain the invention.
Monomers I-VI were produced according to a standard solid-phase synthesis (Seitz et al., Angew. Chem. 107 (1995), 901). The synthesis was made at a HYCHRON anchor with a Fmoc-protected amino acid and was started via the C
terminus. The carboxyl groups of glutamic acid and asparagic acid were protected in the form of tert-butyl esters. The protecting groups of the side chains of Tyr comprised tetrahydropyranyl, tert-butyl(Boc) and trityl groups. The side chain protecting groups of serine and threonine comprised acetyl, benzoyl and benzyloxycarbonyl (=Cbz) groups. The protecting groups for the side chain of arginine comprised Cbz, mesitylene-2-sulfonyl (=Mts) or tert-butyloxycarbonyl (=Boc). The side chain of lysine was protected by tosyl (=Tos) or Boc. After removing the a-amino protective group and corresponding wash steps, followed by activation of the carboxyl group of the next amino acid, the latter was linked to the preceding amino acid. T:he complete monomers were synthesized in this order. The prepared monomers were removed from the column by palladium(0)catalyzed allyl transfer, the protecting groups at the side chains being maintained. Thereafter, the Fmoc group was removed by morpholine, and the rest of the protecting groups was cleaved by treatment with TFA.
Monomers I were dimerized .by dissolving 25 mg activated bifunctional polyethylene glycol (PEG ~~uccinimidyl propionate, molecular weight about 3,400; Sigma, Taufkirchen, Germany) in 4 ml PBS, pH value 7.5, whereupon a threefold molar excess of monomers, dissolved in 1 ml 0.1 trifluoroacetic acid, were added. After 3 hours of incubation on ice, lyophilized monomers were added again so that there was a final ratio of 3.5 mol monomer to 1 mol PEG. The mixture was incubated on ice for another 17 hours.
PEG was inactivated by the addition of 1 M Tris/HCl, pH
value 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).
onomer II
Monomers II were dimerized by dissolving 25 mg of activated bifunctional polyethylene glycol (PEG succinimidyl propionate, molecular weight about 3,400; Sigma, Taufkirchen) in 4 ml PBS, pH value 7.5, whereupon a threefold molar excess of monomers, dissolved in 1 ml 0.1 %
trifluoroacetic acid were added. Following 3 hours of incubation on ice lyophilized monomers were added again, so that there was a final ratio of 3.5 mol monomer to 1 mol PEG. The mixture was incubated on ice for another 17 hours.
PEG was inactivated by the addition of 1 M Tris/HC1, pH
value 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 dimerized by dissolving 25 mc~ activated bifunctional polyethylene glycol (PEG-succinimidyl propionate, molecular weight about 3,400; Sigma, Taufkirchen) in 4 ml PBS, pH value 7.5. Thereafter, a threefold molar excess of monomers, dissolved in 1 ml 0.1 trifluoroacetic acid was added. After 3 hours of incubation on ice lyophilized monomers were added again, so that there was a final ratio of 3.5 mol monomer to 1 mol PEG. The mixture was incubated on ice for another 17 hours. PEG was inactivated by adding 1 M Tris/HCl, pH value 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 made via reversed-phase HPLC, since both dimers markedly differ as regards their elution behavior (depending on the composition there is a difference between 4 and ~3 minutes in the elution peak) and can thus be separated from one another.
The formation of the dimers described in the above examples was checked by analytical reversed-phase HPLC. T'he analysis was carried out by means of a Vydac-C18 protein peptide column (0.46 x 25 cm) (The Separation Group, U.S.A.), a BioRad HPLC system and a two-wavelength detector from Perkin-Elmer. The column was equilibrated using 0.1 % TFA in distilled water, and 10 minutes after injecting the sample (as a rule 6 ml), there was a 45-minute linear gradient (0-100) of acetonitrile with 0.1 % TFA. The flow rate was kept continuously at 1 ml/min. Under these conditions,. the cross-linker appeared in the flow. The reaction product eluted after 37 minutes, while the unconjugated monomer was already eluted after 32 minutes and could thus be separated clearly from the dimer. The dimer was further purified on a preparative reversed-HPLC column (2.2 x 25 cm) with the same HPLC system. The column was equilibrated with a constant flow rate of 8 ml/min. using distilled water . acetonitrile of 80:20 (both contained 0.1 % TFA). 20 minutes after injecting the sample (6 ml) there was a 60-minute linear gradient with 100 o acetonitrile/0.1 % TFA. The main peak was collected and lyophilized. A gentler gradient (20-800 acetonitrile/0.1 % TFA above 60 %) was used for monomer II
so as to reach better separation. The isolated dimers and the native monomers were used in binding studies (see Example 7) and proliferation tests (see Example 8).
~~ amn~e 6~ radioactive labeling of the dimers The dimers were iodized as described below. 20 ~.1 chloramine T ( 0 . 5 mg/ml ) were added to 2 0 ~l Nalasl ( 2 mC:i , Amersham Buckler, Braunschweig, Germany), the mixture was added to 50 ~.1 dimer (5 ~g in PBS) 2 minutes later and incubated for 15 seconds. In order to complete the reaction, 30 ~,l sodium metabisulfite (1 mg/ml in PBS) and, after 30 seconds, 50 ~,1 KI ( 10 mg/ml in PBS ) were added . Ge lat in ( 5 0 ~.l , 1 mg/ml in distilled water) was added as carrier and the solution was applied immediately onto a BioSil SEC 125-5 column (BioRad, ml) which was equilibrated with 0.25 % gel;atin/0.02 %
sodium azide in PBS. The fractions which contained the radioactive product were combined and further purified and isolated on the reversed-phase HPLC column as described in Example 5. The purified dimers had a specific radioactivity of 50-100 ~,Ci/~.g, yielded in tricin-SDS-PAGE a single band and 95-100 % of the radioactivity could be precipitated with the chloroform/methanol method (Wessel et al., Anal.
Biochem. 138 (1984), 141-143). These results show that the radioactively labeled product consisted of a pure dimer and that the radioactivity could be ascribed exclusively to the dimer.
These studies show whether the dimers according to the invention bind specifically to cells which express the IL12 receptor. This example uses the homodimer consisting of monomers III. The experiment was carried out: with PHA-stimulated human lymphoblasts according to a modified protocol of Chizzonite et al., J. Immunol. ],~$., (1992), 3117-3124.
The stimulated cells (7.5 x 105 cells /ml RPMI medium) were incubated for 90 minutes with radioactively labeled dimer at varying concentrations (0.01 to 1 ng) and thereafter washed in PBS by means of 3 centrifugation steps, and the cell-bound radioactivity was measured in a liquid scintillation counter. The extent of unspecific binding was determined in the presence of an IL12 concentration which was 100 times as high, labeled homodimer and IL12 having been added to the cells at the same time. The results are shown in Table I.
Test substance Binding o maximum binding) Interleukin 12 100 AALQNHNHQQIILDK
[AALQNHNHQQIILDK 2 66 These studies should show whether similar to native IL12 the dimers according to the invention have a proliferation-stimulating effect. They were carried out again with PHA-stimulated human lymphoblasts in a 48-hour assay according to Gately and Chizzonite, Curr. Protocols in Immunology Voll (1992), pages 6.16 - 6.16.8, using monomer I:CI. In this connection, the cells were seeded in 200 ~.1 culture medium in a cell number of 5 x 104 and cultured in the presence of various dimer concentrations (0.01 - 1 ng) for 48 hours.
Having added tritiated thymidine (0.25 ~Ci/ml:) the cells were cultured for another 4 hours and then isolated via a cell harvester. The introduced radioactivity was measured in a liquid scintillation counter. The values a:re shown in Table II as specific activity in units x 10-'/mg IL12.
Test substance Specific ac-tiv ty interleukin 12 5-3 AALQNHNHQQIILDK 0.03 _A_AT~QNHNHQQIILDK 2 2.4 [AALQNHNKQQIILDK 2 10.5 The heterodimer produced in Example 4 was subjected to 18 SDS polyacrylamide gel electrophoresis. After staining the gel using 4 M sodium acetate, an about 6.5 k.B band was excised out of the gel and incubated in phosphate-buffered common salt solution. The dimer is eluted by means of an electroeluter (BioRad) from the gel and lyophilized after a kB dialysis overnight. 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/0.02 mol carrier in a total of 2 ml PBS. 2 ml glutardialdehyde (0..2 °s in PBS) are added dropwise to the mixture with continuous stirring and incubated at room temperature for 1 hour. The. mixture is then dialyzed for 48 hours with 12-hour buffer cycle in a 20 kB dialysis hose against PBS and subsequently freeze-dried.
The animals are immunized with the mixture consisting of dimer BSA complexes, BSA-BSA complexes and dime:r complexes with 100 ~.g (rabbit and chicken) or with 30 ug (rnouse) .
- Immunization protocol for polyclonal antibodies in rabbits 100 ~.g of gel-purified heterodimer in 0.7 ml PBS and 0.7 ml of complete or incomplete Freund's adjuvant were used per immunization:
Day 0: lgt immunization (complete Freund's adju.vant) Day 14: 2nd immunization (incomplete Freund's adjuvant;
icFA) Day 28: 3rd immunization (icFA) Day 56: 4th immunization (icFA) Day 80: bleeding to death.
The rabbit serum was tested in an immunoblot. For this purpose, a heterodimer of Example 4 according to the invention was subjected to SDS polyacry:Lamide gel electrophoresis and transferred to a nitrocellulose filter (cf. Khyse-Andersen, J., J. Biochem. Biophys. Meth. 10 (1984) , 203-209) . The Western blot analysis was carried out as described in Bock, C.-T. et al., Virus Genes. 8, (1994), 215-229. For this purpose, the nitrocellulose filter was incubated with a first antibody at 37°C for one: hour. This antibody was the rabbit serum (1:10000 in PBS). After several wash steps using PBS, the nitrocellulose filter was incubated with a second antibody. This antibody was an alkaline phosphatase-coupled monoclonal goat anti-rabbit IgG
antibody (Dianova company) (1:5000) in PBS. 30 minutes of incubation at 37°C were followed by several wash steps using PBS and subsequently by the alkaline phosphatase detection reaction with developer solution (36 ~,M 5'-bromo-4-chloro-3-indolylphosphate, 400 ~M nitro blue tetrazol:ium, 100 mM
Tris-HC1, pH 9.5, 100 mM NaCl, 5 mM MgCl2) at room temperature until bands became visible.
It showed that polyclonal antibodies according to the invention can be prepared.
- Immunization protocol for polyclonal antibodies in chickens 100 ~g of gel-purified heterodimer in 0.8 ml PBS and 0.8 ml of complete or incomplete Freund's adjuvant wez-e used per immunization.
Day 0: 18t immunization (complete Freund's adjuvant) Day 28: 2nd immunization (incomplete Freund'~; 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 were detected.
- Immunization protocol for monoclonal antibodies in mice 30 ~g of gel-purified fusion protein in 0.25 ml 3?BS and 0.25 ml of complete or incomplete Freund's adjuvant were used per immunization. The fusion protein was dissolved. in 0.5 ml (without adjuvant) in the 4th immunization.
Day0: 1St immunization (complete Freund's adjuvant) Day28: 2nd immunization (incomplete Freund's adjuvant;
icFA) Day56: 3rd immunization (icFA) Day84: 4th immunization (PBS) Day87: fusion.
Supernatants of hybridomas were tested in a Western blot.
Monoclonal antibodies according to the invention were identified.
Claims (10)
1. Synthetic homodimer or heterodimer of peptide monomers which have the amino acid sequences KHYSCTAEDID
(monomer I), PPVGEADPYRVKMQ (monomer II), AALQNHNHQQIILDK (monomer III), IRDIIKPDPPKN (monomer IV), SLTFCVQVQGKSKR (monomer V) or RFTCWWLTTISTDLTF
(monomer VI), wherein the homodimer or heterodimer binds to the interleukin 12 (IL12) receptor.
(monomer I), PPVGEADPYRVKMQ (monomer II), AALQNHNHQQIILDK (monomer III), IRDIIKPDPPKN (monomer IV), SLTFCVQVQGKSKR (monomer V) or RFTCWWLTTISTDLTF
(monomer VI), wherein the homodimer or heterodimer binds to the interleukin 12 (IL12) receptor.
2. The homodimer or heterdimer 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 with the C terminus of the other monomer.
3. The heterodimer according to claim 2, characterized in that the C terminus of monomer I is linked with the N
or C terminus of monomer II, or the N terminus of monomer I is linked with the N or C terminus of monomer II.
or C terminus of monomer II, or the N terminus of monomer I is linked with the N or C terminus of monomer II.
4. The homodimer or heterodimer according to any of claims 1 to 3, characterized in that the monomers are covalently bonded with one another via polyethylene glycol, peptide(s), activated benzodiazepines, oxazolones, azalactones, aminimides, diketopiperazines, or (a) monosaccharide(s).
5. The homodimer or heterodimer according to any of claims 1 to 4, characterized in that the monomers have deletions, additions or substitutions or at most 3 11. Use of the homodimer or heterodimer according to any of claims 1 to 4, 7 and 8 or the homodimer or heterodimer produced according to the method as defined in claim 9 for treating diseases of the immune system or diseases associated with increased cell proliferation.
12. Use according to claim 11, wherein the disease is a cancerous disease.
13. Use of the homodimer or heterodimer according to any of claims 1 to 8 or of the homodimer or heterodimer produced according to the method as defined in claim 9 for the diagnosis of diseases which are associated with a modified or excessively or insufficiently expressed interleukin 12 (Il12) receptor or an excessively high or low interleukin 12 concentration.
14. Kit for carrying out the diagnostic method as defined in claim 13, containing the homodimer or the heterodimer according to any of claims 1 to 8, the homodimer or heterodimer produced according to the method as defined in claim 9.
amino acids per monomer and/or at most 3 modified amino acids per monomer, as compared to the original forms the homodimers or heterodimers (a) binding to the interleukin 12 (IL12) receptor and/or (b) triggering a cellular signal in a similar or better way.
12. Use according to claim 11, wherein the disease is a cancerous disease.
13. Use of the homodimer or heterodimer according to any of claims 1 to 8 or of the homodimer or heterodimer produced according to the method as defined in claim 9 for the diagnosis of diseases which are associated with a modified or excessively or insufficiently expressed interleukin 12 (Il12) receptor or an excessively high or low interleukin 12 concentration.
14. Kit for carrying out the diagnostic method as defined in claim 13, containing the homodimer or the heterodimer according to any of claims 1 to 8, the homodimer or heterodimer produced according to the method as defined in claim 9.
amino acids per monomer and/or at most 3 modified amino acids per monomer, as compared to the original forms the homodimers or heterodimers (a) binding to the interleukin 12 (IL12) receptor and/or (b) triggering a cellular signal in a similar or better way.
6. The homodimer according to claim 5, which is the homodimer [AALQNHNHQQIILDK]2 or [AALQNHNKQQIILDK]2.
7. The homodimer or heterodimer according to any of claims 1 to 4, characterized in that the monomers have deletions, additions or substitutions of at most 3 amino acids per monomer and/or at most 3 modified amino acids per monomer, wherein the homodimers or heterodimers can bind to the interleukin 12 (IL12) receptor and can have an antagonistic effect.
8. The homodimer or heterodimer according to claim 5, 6 or 7, characterized in that the at most 3 amino acids per monomer are covalently modified by fatty acids, monosaccharides and/or oligosaccharides.
9. A method of producing the homodimer or heterodimer as defined in claims 1 to 8, which comprises a solid-phase synthesis of the monomers and subsequent dimerization.
10. Use of the homodimer or heterodimer according to any of claims 1 to 6 and 8 or the homodimer or heterodimer produced according to the method as defined in claim 9 for treating diseases of the immune system, diseases associated with reduced cell proliferation, infectious or inflammatory processes or for reducing the IL2 side effects in a therapy carried out using IL2.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19919148A DE19919148A1 (en) | 1999-04-27 | 1999-04-27 | Interleukin 12-derived peptide homodimers and peptide heterodimers |
| DE19919148.4 | 1999-04-27 | ||
| PCT/DE2000/001260 WO2000064938A2 (en) | 1999-04-27 | 2000-04-20 | Peptide homodimers or peptide heterodimers derived from interleukin 12 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2370298A1 true CA2370298A1 (en) | 2000-11-02 |
Family
ID=7906047
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002370298A Abandoned CA2370298A1 (en) | 1999-04-27 | 2000-04-20 | Peptide homodimers or peptide heterodimers derived from interleukin 12 |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP1171464A2 (en) |
| JP (1) | JP2002543091A (en) |
| AU (1) | AU5388600A (en) |
| CA (1) | CA2370298A1 (en) |
| DE (1) | DE19919148A1 (en) |
| WO (1) | WO2000064938A2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010503687A (en) * | 2006-09-15 | 2010-02-04 | ザ バーナム インスティチュート | High affinity EphB receptor binding compounds and methods of use thereof |
| AU2012228769B2 (en) * | 2011-03-14 | 2016-09-08 | Phlogo Aps | Antagonists of the interleukin- 1 receptor |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2125763C (en) * | 1993-07-02 | 2007-08-28 | Maurice Kent Gately | P40 homodimer of interleukin-12 |
| US5891680A (en) * | 1995-02-08 | 1999-04-06 | Whitehead Institute For Biomedical Research | Bioactive fusion proteins comprising the p35 and p40 subunits of IL-12 |
-
1999
- 1999-04-27 DE DE19919148A patent/DE19919148A1/en not_active Withdrawn
-
2000
- 2000-04-20 JP JP2000614287A patent/JP2002543091A/en active Pending
- 2000-04-20 CA CA002370298A patent/CA2370298A1/en not_active Abandoned
- 2000-04-20 EP EP00938503A patent/EP1171464A2/en not_active Withdrawn
- 2000-04-20 AU AU53886/00A patent/AU5388600A/en not_active Abandoned
- 2000-04-20 WO PCT/DE2000/001260 patent/WO2000064938A2/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| DE19919148A1 (en) | 2000-11-16 |
| JP2002543091A (en) | 2002-12-17 |
| WO2000064938A3 (en) | 2001-04-12 |
| EP1171464A2 (en) | 2002-01-16 |
| AU5388600A (en) | 2000-11-10 |
| WO2000064938A2 (en) | 2000-11-02 |
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