Dual specific antibodies, processes of their preparation and use

In addition, the invention provides a method of synthesizing a recombinant antibody of the invention by culturing a host cell of the invention in a suitable culture medium when the recombinant antibody of the invention is synthesized.

As an alternative to screening recombinant antibody libraries by phage display, other methods known in the art for screening large combination libraries can be used to identify dual-specific antibodies of the invention. One type of alternative expression system is a system in which a recombinant antibody library is expressed as an RNA-protein fusion described in PCT Publication No. WO 98/31700 by Szostak and Roberts and in Roberts, RW and Szostak, JW (1997) Proc. Nati. Acad. Sci. USA 94:12 297-1230. In this system, a covalent fusion is formed between the mRNA and its encoded peptide or protein, by translation in vitro of synthetic mRNAs that carry puromycin, the peptide acceptor of antibiotics at their 3 'end. Thus, a particular mRNA may be enriched from a complex mRNA mixture (eg, a combinatorial library) based on the properties of the encoded peptide or protein, e.g. an antibody or portion thereof, such as binding of the antibody or portion thereof to a dual specific antigen. Nucleic acid sequences encoding antibodies or portions thereof obtained by screening these libraries can be expressed in a recombinant manner as described above (e.g. in mammalian host cells) and in addition may be subject to further affinity maturation by either additional cycles of screening mRNA-peptide fusions in which mutations have been introduced into the originally selected sequence (s) or by other means of affinity maturation of recombinant antibodies in vitro as described above.

C. Combinational Approaches

The dual specificity antibodies of the invention may also be prepared using in vivo or in vitro combination approaches, in such a way that the dual specificity antigen is initially exposed to the antibody set in vivo in a host animal to stimulate production of antibodies that bind the dual specificity antigen, but wherein additional antibody selection and / or maturation (ie, improvement) is performed using one or more in vitro techniques.

In one embodiment, the combination approach comprises first immunizing a non-human animal (eg, mouse, rat, rabbit, goat or a transgenic version thereof or a chimeric mouse) with a dual specific antigen to stimulate an antibody response to the antigen, followed by preparation and screening of phage display antibody. libraries using immunoglobulin sequences from lymphocytes stimulated in vivo by exposure to dual cells

<img file="CZ20030291A3_D0001.tif" />

specific antigen. The first step of this combination process may be performed as described in section IIA, while the second step of this process may be performed as described in section IIB above. Preferred methods for hyperimmunization of non-human animals, followed by screening in vitro phage display libraries prepared from stimulated lymphocytes, include those described in BioSite Inc, see, e.g., PCT Publication WO 98/47 343, PCT Publication WO 91/17 271, U.S. Pat. 5 No. 427,908 and U.S. Patent No. 5,580,717.

In another embodiment, the combination procedure comprises first immunizing a non-human animal (eg, mouse, rat, rabbit, goat, or a knockout and / or transgenic version or chimeric mouse thereof) with a dual specific antigen to stimulate antibody response to antigen and lymphocyte selection, which produce antibodies with the desired dual specificity (eg, by screening hybridomas prepared from immunized animals). The rearranged antibody genes are then isolated from selected clones (by standard cloning methods such as reverse transcriptase - polymerase chain reaction) and are subject to affinity maturation in vitro, thereby enhancing the binding properties of the selected antibody or antibodies. The first step of the process may be performed as described in paragraph IIA above, while the second step of the process may be performed as described in paragraph IIB above, mainly using the in vitro maturation methods described in PCT Publication WO 97/29 131 and PCT WO 00/56 772.

In yet another combination method, recombinant antibodies are generated from individual, isolated lymphocytes, using a method known in the art as the selected lymphocyte antibody method (SLAM) described in US Patent No. 5,627,052, PCT Publication WO 92/02 551 and in Babcock, JS et al. (1996) Proc. Nati. Acad. Sci USA 93: 7843-7848. In this method, applied to the dual specific antibodies of the invention, first, a non-human animal (eg, mouse, rat, rabbit, goat, or a transgenic version thereof or a chimeric mouse) is immunized in vivo with a dual specific antigen to stimulate an antibody response against that antigen. and then individual cells secreting antibodies of interest, e.g., specific to a dual specificity antigen, are selected by an antigen-specific hemolytic plaque assay (e.g. the dual specific antigen itself or structurally related molecules of interest are attached to sheep red blood cells by means of a bridge such as biotin, thereby allowing the identification of individual cells that secrete antibodies of appropriate specificity by a hemolytic plaque assay). After identifying the cells secreting the antibodies of interest, the heavy and light chains of the cDNA variable regions are released from the cells by reverse transcriptase and PCR, and these variable regions can then be expressed in the context of the respective immunoglobulin constant regions (e.g., human constant regions). · Φ ······· · · Φ 9 9 9 9 9 9 9

9 9 9 9 9 9 9 9

999 999 9999 9 · 9 9 9 Φ φ ΦΦΦ φ hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitel hostitelφ φ These host cells, transfected with enhanced immunoglobulin sequences derived from in vivo selected lymphocytes, can then be further analyzed and selected in vitro, for example, by control of the transfected cells to isolate cells expressing antibodies with the desired dual specificity. These enhanced immunoglobulin sequences can be further influenced in vitro, such as in vitro affinity maturation, as described above.

In another embodiment, the combination method comprises the following steps to produce dual specific antibodies. First, the non-human animal is immunized with the first antigen and the second, non-human animal is immunized with the second, different antigen, wherein the second antigen is preferably structurally similar to the first antigen to stimulate the antibody response in vivo. A recombinant heavy chain library and a recombinant light chain library derived from a first non-human animal or a second non-human animal are constructed from antibody genes as described in Section IIB. The heavy chain library from the animal immunized with the first antigen is linked to the light chain library of the animal immunized with the second antigen to produce a library of antibodies X. Similarly, a heavy chain library from an animal immunized with a second antigen is linked to a light chain library from an animal immunized with a first antigen to create a library of antibody Y. In addition, libraries X and Y can be joined to form a library XY. Dual-specific antibodies that bind both the first and second antigens can be identified and isolated from X, Y and / or XY libraries.

III. Characteristics of dual specific antibodies

The present invention provides dual specific antibodies, as well as portions thereof, that can be prepared by the method of the invention. These antibodies, or portions thereof, are preferably isolated antibodies. These antibodies, or portions thereof, are preferably neutralizing antibodies. The antibodies of the invention are also monoclonal and recombinant antibodies, or portions thereof. In various embodiments, the antibodies or portions thereof may comprise amino acid sequences derived entirely from individual animal species, such as antibodies or portions thereof of entirely human or entirely murine origin. In other embodiments, the antibodies or portions thereof may be chimeric antibodies, CDR implanted antibodies, or other forms of human antibodies.

The term "antibody" as used herein refers to immunoglobulin molecules composed of four polypeptide chains, two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.

♦ ·······«

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This heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (referred to herein as LCVR or LV) and a light chain constant region. The light chain constant region is a single domain, CL. The VH and LH regions can be further subdivided into hypervariability regions, which are called complementarity determining regions (CDRs), which are separated by regions that are more conservative and are called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the N-terminus to the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The term "antigen-binding portion" of an antibody (or simply a "portion of an antibody") as used herein refers to one or more fragments of a dual specificity antibody that retain the ability to specifically bind to two different but structurally related antigens. It has been found that the antigen-binding function of an antibody may be a fragment of the entire antibody length. Examples of binding fragments included within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, which is a monovalent fragment comprising the VL, VH, CL and CH1 domains; (ii) F (ab ')₂ a fragment, which is a bivalent fragment comprising two Fab fragments bound by a disulfide bridge at the post region; (iii) a Fd fragment comprising the VH and CH1 domains; (iv) a FV fragment comprising the VL and VH domains of one arm of an antibody; (v) a dAB fragment (Ward et al., (1989) Nature 341: 544-546) that comprises a VH domain; and vi) an isolated complementarity determining region (CDR). In addition, although the two domains of the Fv fragment, VH and VL, are encoded by separate genes, they can be linked using a synthetic bridge using recombinant methods, allowing them to be produced as a single protein chain in which the VL and VH regions form monovalent molecules ( known as single chain Fv (scFv), see, eg, Bird et al. (1988) Science 242: 423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). These single chain antibodies are also understood to mean the "antigen-binding portions" of the antibody. Other forms of chain antibodies, such as diabodies, are also included within the term. Diabodies are bivalent, bispecific antibodies whose VH and VL domains are expressed in one polypeptide chain, but due to a bridge that is too short to allow pairing between the two domains within one chain, these domains pair with the complementary domains of the other chains, and thus forming two antigen binding sites (see, eg, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1121- 1123.

The antibody or antigen-binding portion thereof may be part of a larger immunoadhesive molecule, formed by covalent or non-covalent association of the antibody or portion thereof with one or more other proteins or peptides. Examples of such immunoadhesive molecules include

<img file="CZ20030291A3_D0002.tif" />

The use of the streptavidin nuclear region for the formation of a tetrameric scFv molecule (Kyprianov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93 to 101) and the use of a cysteine ​​residue, a marker peptide and a C-terminal polyhistidine tag for generating bivalent and biotinylated scFv molecules (Kyprianov, SM et al. (1994) Mol. Immunol. 31: 1047-1058). Antibody portions, such as Fab and F (ab ')₂ fragments, can be prepared from whole antibodies in a conventional manner, such as papain or pepsin digestion of whole antibodies. In addition, antibodies, antibody portions, and immunoadhesive molecules can be obtained by standard recombinant techniques.

The term "isolated dual specificity antibody" as used herein refers to a dual specificity antibody that essentially contains no other antibodies having different antigen specificities (eg, an isolated antibody that specifically binds two different but structurally related antigens but is essentially free of antibodies that specifically bind other unrelated antigens). In addition, the isolated dual specificity antibody may be substantially free of other cellular material and / or other chemicals.

The term "neutralizing antibody" as used herein refers to an antibody whose binding to a particular antigen results in inhibition of the biological activity of that antigen. This inhibition of the biological activity of an antigen can be estimated by measuring one or more indicators of the biological activity of the antigen by means of a suitable in vitro or in vivo assay.

The term "monoclonal antibody" as used herein refers to a hybridoma derived antibody (eg, an antibody secreted by a hybridoma, prepared by hybridoma technology, such as the standard hybridoma technology of Kohler and Milstein). Thus, a dual specificity hybridoma-derived antibody of the invention is still referred to as a monoclonal antibody, although it has antigen specificity against more than one individual antigen.

The phrase "recombinant antibody" refers to antibodies that are prepared, expressed, generated or isolated in a recombinant manner, such as antibodies expressed using a recombinant expression vector transfected into a host cell, an antibody isolated from a recombinant combination antibody library isolated from an animal (e.g. mice) that is transgenic to human immunoglogulin genes (see, e.g., Taylor, LD, et al. (1992) Nucl. AddsRes. 20: 6287-6295), or an antibody that is prepared, expressed, engineered, or isolated by another method that comprises linking the respective immunoglobulin gene sequences (such as human immunoglobulin gene sequences) to other DNA sequences. Examples of recombinant antibodies include chimeric, CDR implanted and humanized antibodies.

• fc ··· · fcfc

The term "human antibody" refers to antibodies having variable and constant regions corresponding to or derived from immunoglobulin human germline sequences as described, eg, in Kabat et al. (see Kabat, et al. (1991) Sequeunces of Proteins of Immunological Interest, Fifth Edition, US Department of Gealth and Human Services, NIH Publication No. 91-3242). However, in some embodiments, such recombinant human antibodies are subject to in vitro mutagenesis or, when an animal transgenic to human Ig sequences is used, in vivo somatic mutagenesis, and the amino acid sequences of the VH and LH regions of recombinant antibodies are sequences that, when derived and related from human germline VH and λ / L sequences, cannot exist within the natural set of germline human antibodies in vivo. In certain embodiments, however, these recombinant antibodies are the result of selective mutagenesis, backmutation, or both.

The term "backmutation" refers to a process in which some or all of the somatically mutated amino acids are replaced by the corresponding germline residues from the homologous germline antibody sequence sequences. The heavy and light chain sequences of the human antibody of the invention are separately aligned with the germline sequences of the VBASE database to identify sequences with the highest homology. The differences in the human antibody of the invention are corrected according to germline sequences by mutating defined nucleotide positions encoding these different amino acids. The role of each amino acid identified as a candidate for back mutation should be tested for its direct or indirect role in antigen binding, and any amino acid that would alter the desired human antibody characteristics after mutation should not be included in the resulting human antibody to minimize the number amino acids that are back mutated for those amino acid positions that were found to be different from the nearest germline sequence, but identical to the corresponding amino acid in the second germline, and which may remain, provided that the second germline sequence is identical and collinear to the human antibody sequence of the invention in at least 10 , preferably 12, amino acids on both sides of the amino acid in question. Backmutations can occur at any stage of antibody optimization.

The term "chimeric antibody" refers to antibodies that comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, as well as antibodies having mouse heavy and light chain variable regions linked to human constant regions.

The term &amp; quot; CDR-implanted antibody &amp; quot; refers to antibodies that comprise heavy and light chain variable region sequences of one species in which:

<img file="CZ20030291A3_D0003.tif" />

However, the sequences of one or more of the VH and / or VL CDRs are replaced by CDR sequences from other species, such as antibodies having mouse heavy and light chain variable regions in which it is located. one or more murine CDRs (eg, CDR3), replaced with a human CDR sequence.

The term "humanized antibody" refers to antibodies that comprise heavy and light chain variable region sequences of non-human species (eg, murine) but in which at least a portion of the VH and / or VL sequence is changed to more "human", ie, more similar human germline variable sequences. One type of humanized antibody is an antibody with an implanted CDR in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences.

One way of measuring antibody binding kinetics is surface plasmon resonance. The term "surface plasmon resonance" as used herein refers to an optical phenomenon that allows real-time analysis of biospecific interactions by detecting changes in protein concentration in the biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscaraway, NY). For further description, see Jonsson, U. Et al. (1993) Ann. Biol. Clin. 51:19 to 26; Jonson, U. et al. (1991) Biotechniques 11: 620-622; Johnsson, B., et al. (1995) J. Mol. Recognit. 8: 125-131 and Johnnson, B., et al. (1991) Anal. Biiochem. 198: 268-277.

The term “K_Pff ”, as used herein, refers to the decay rate constant of antibody dissociation from the antibody / antigen complex.

The term "K d" as used herein refers to the dissociation constant of an individual antibody-antigen interaction.

The dual specific antibodies of the invention are prepared using a variety of antibody preparation methods as described in paragraph II above. The dual-specific antibodies of the invention can be directed against essentially any structurally related antigens, although preferred dual-specific antibodies of the invention are those that specifically bind IL-Ια and IL-Ιβ, which can be prepared using dual-specific antigens, such as the antigens described in Examples 1-4. Other structurally related antigens that can be used in the present invention include, but are not limited to, caspase family members, cytokine family members such as IL-1 family members (eg, IL1 / IL-18), TNF family members (eg. . TNFα / TNFP), IL-6 family members, interferons, TGFP family members, EGF family members, FGF family members, PDGF family members, VEGF family members, angiopoietin family members, bone morphogenic proteins, secreted proteases (metallo-proteinases) and • · Tititi9 ♦ titi ti ti ti ti ti ti ti ti * titititi «·· ♦♦ ti cytokine receptor family, such as IIL-1 receptor family members, TNF receptor family members, TGFP receptor family members, EGF receptor family members, receptor family members FGF, members of the PDGF receptor family, members of the VEGF receptor family, and members of the angiopoietin receptor family.

The dual specific antibodies of the invention may exhibit the same binding activity against two different but structurally related antigens to which the dual specific antibody binds, or alternatively may bind more preferably to one of the two antigens, but still has specificity against the two related antigens as compared to with unrelated antigens. The binding activity of a dual specificity antibody against structurally related antigens, as well as against unrelated antigens, can be estimated using standard in vitro assays such as ELISA or BIAcore analyzes. It is preferred that the ratio K a of the antibody against structurally unrelated antigens to the K d of the antibody against the structurally related antigens is at least 3, more preferably the ratio is at least 5 or more preferably the ratio is at least 10 or more preferably the ratio is at least 50,

100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000.

In quantitative terms, one measure is the difference between background binding and dual specificity. For example, background binding is at a low level, eg, less than 5%, more preferably less than 3%, and most preferably 0.1 to 1%, while the specific cross-reactivity or dual-specific binding is at a higher level, eg, greater than 1%, more preferably greater than 3%, even more preferably greater than 5% and even more preferably greater than 10%. In addition, the IC50 of the dual specificity antibody to the target antigen is close to the ED50 value of the antigens in a given bioassay.

The dual specificity antibody or antigen-binding portion thereof of the invention is preferably selected to have the desired binding kinetics (e.g., high affinity, low dissociation, slow disintegration rate, strong neutralizing activity) against one, preferably both, antigens to which it specifically binds. binds. For example, a dual specificity antibody or portion thereof may bind one or preferably both structurally related antigens with a rate constant koff of 0.1 s'¹ or less, more preferably 1 x 10 '² with'¹ or less, more preferably 1 x 10 '³ with'¹ or less, more preferably 1 x 10⁴ with'¹ or less, more preferably 1 x 10 '⁵ with¹ or less as determined by surface plasmon resonance. Additionally or otherwise, a dual specificity antibody or portion thereof may inhibit the activity of one, preferably both, structurally related antigens having an IC 50 value of 1 x 10 M or less, more preferably an IC 50 value of 1x10 µM or less, even more preferably an IC 50 value of 1 x 10 '⁸M or less, more preferably with an IC 50 of 1 x 10 '⁹M or less, more preferably with an IC 50 of 1 x 10 '^I0M or less, more preferably IC₅₀ 1 x 10 '¹¹ M or less. Preferably, the IC 50 is measured by

44 4» 4 • · · 4 4 · · · ♦ ··· *44 4

4 4 · 44 444 • 4 4444 sensitive bioassay in which the IC50 values ​​should be close to the ED50 of the antigens in this assay.

The invention also provides pharmaceutical compositions comprising a dual specificity antibody or antigen-binding portion thereof of the invention and a pharmaceutically acceptable carrier. The pharmaceutical composition of the invention may further comprise at least one additional therapeutic agent, eg, one or more additional therapeutic agents for the treatment of a disease, wherein the use of a dual specificity antibody is useful for ameliorating the disease. For example, if a dual specificity antibody specifically binds IL-1β and IL-1β, the pharmaceutical composition may further comprise one or more therapeutic agents for the treatment of diseases in which IL-1 activity is deleteriously involved.

The antibodies and portions thereof of the invention may be incorporated into pharmaceutical compositions suitable for administration to a subject. These pharmaceutical compositions typically comprise an antibody or portion thereof of the invention and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, antibacterial and antithungal agents, isotonic sensors, and absorption retardants and the like, as long as they are physiologically acceptable. Examples of pharmaceutically acceptable carriers include one or more such as water, saline, phosphate saline, glucose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it is preferred that the isotonic agents include, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. Pharmaceutically acceptable carriers may further comprise minor amounts of excipients such as wetting or emulsifying agents, buffer preservatives that increase the half-life of the antibody or antibody portion.

The antibodies and antibody portions of the invention may be incorporated into pharmaceutical compositions suitable for parenteral administration. Preferably, the antibody or antibody portions are prepared as an injectable solution containing 0.1-250 mg / ml antibody. This solution for injection may contain either a liquid or lyophilized dosage form in quartz or brown glass vials, ampoules or pre-filled syringes. The buffer may be L-histidine (1 to 50 mM), optimally 5 to 50 mM, at pH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride may be used to alter the toxicity of the solution at a concentration of 0 to 300 mM (optimally 150 mM for the liquid dosage form). In the lyophilized dosage form, cryoprotectants may be present, in particular 0 to 10% sucrose (optimally 0.5 to 1.0%). Other suitable cryoprotectants are trehalose and lactose.

• · · · · • · ···· ··

Fillers may be included in the lyophilized dosage form, especially 1 to 10% mannitol (optimally 2 to 4%). Stabilizers, in particular 1 to 50 mM L-methionine (optimally 5-10 mM) may be present in both liquid and lyophilized dosage forms. Other suitable fillers are glycine, arginine, and an amount of 0 to 0.05% may contain polysorbate-80 (optimally 0.005 to 0.01%). Additional surfactants may include, but are not limited to, polysorbate 20 and BRIJ.

The compositions of the invention may be in various forms. Such forms are, for example, liquid, semi-solid and solid dosage forms such as liquid solutions (eg, injection and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories.

The preferred dosage form depends on the intended route of administration and therapeutic application. Typical preferred compositions are in the form of solutions for injection or infusion, such as those similar to those used in passive immunization of humans with other antibodies. A preferred route of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous injection or infusion.

In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

Therapeutic compositions must be sterile and stable under the conditions of manufacture and storage. The composition must be formulated as a solution, microemulsion, dispersion, liposome or other desired structure suitable for high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (ie, antibody or antibody portion) in the required amount in a suitable solvent with one or more of the above ingredients, followed by sterile filtration. Dispersions are generally prepared by incorporating the active compound into a sterile solvent that contains a basic dispersion medium and other necessary ingredients within the above. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and spray drying, which provide a powder of the active substance and any additional desired ingredient from their pre-sterile filtered solutions. The intrinsic fluidity of the solution can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of wetting agents. Prolonged absorption of injectable compositions may be brought about by the inclusion of such agent in a composition that retards absorption, for example, monostearate salts and gelatin.

The antibodies and antibody portions of the present invention can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route / mode of administration is subcutaneous injection, intravenous injection or infusion. One skilled in the art will readily understand that the route and / or mode of administration will vary depending on the desired results. In certain embodiments, the 9 &amp; quot; &amp; quot;

9 9 9 9 9 99 9 9 9

9 9 9 9 9 9 9 9

999 9 9 9 9999 9 • · 9 · 9 · · 9 ·

9999 The active compound is formulated with a carrier that protects the compound from rapid release, as is the case with a controlled release formulation, including implants, transdermal patches and microencapsulation delivery methods. Biodegradable and compatible polymers such as ethylene (vinyl) acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Many methods for preparing these formulations are patented or generally known to those skilled in the art. See, eg, Sustained and Controlled Release Drug Delivery Systems, JR Fobilnson, eds., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, the antibodies or portions thereof can be administered orally with, for example, an inert solvent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be contained in a hard or soft gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compound may be included in a filler and used in the form of tablets, nasal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, and the like. For administration of a compound of the invention by other than parenteral administration, it may be necessary to coat or administer the compound with a material that will prevent the compound from inactivating.

Supplementary active compounds may also be incorporated into the compositions.

In certain embodiments, an antibody of the invention, or a portion thereof, may be formulated together and / or administered with one or more additional therapeutic agents that are useful for treating diseases in which IL-1 activity is negatively involved. For example, dual-specific antibodies to IL-loc / IL-β or parts thereof may be co-formulated and / or administered with one or more antibodies that bind to other targets (e.g. antibodies that bind other cytokines or that bind molecules on the cell surface). In addition, one or more of the antibodies of the invention may be used in combination with one or more of the foregoing therapeutic agents. Such combination therapies may advantageously utilize low doses of the administered therapeutic agents and thus eliminate undesirable possible toxicities or complications associated with various monotherapies.

IV. Use of dual specific antibodies

Because of their ability to bind to two different but structurally related antigens, the dual specific antibodies of the invention or portions thereof are used to detect either one or both antigens (eg, in biological samples such as serum or plasma) using conventional immunological an assay such as enzyme linked immunosorbent assay (ELIS A), radioimmunoassay (RIA), or tissue immunohistochemistry. · · ♦ · · · · «4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 44 9 4

4 4 4 4 4 4 4 4

9444 The invention provides a method of detecting an antigen in a biological sample comprising contacting the biological sample with a dual specificity antibody of the invention or a portion thereof that specifically recognizes the antigen and detecting either the antibody (or antibody portion) bound to the antigen or unbound antibody (or a portion thereof) thereby detecting the antigen in the biological sample. The antibody is directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine influorescein, dansyl chloride or phycoerythrin; an example of a luminescent material may be luminol, and examples of suitable radioactive materials are¹²⁵AND, ¹³¹AND,³⁵S or 3^h.

Alternatively to the labeling of the antibody, the antigen (s) can be tested in biological fluids by competition in a radioimmunoassay using antigenic standards labeled with a detectable substance and an unlabeled dual specificity antibody specific to that antigen (s). In this assay, the biological sample, the labeled antigen standards and the dual specificity antibody are mixed and the amount of labeled antigen standard that is bound to the unlabeled antibody is determined. This amount of antigen in the biological sample is inversely proportional to the amount of labeled antigen standard bound to the unlabeled antibody.

In a preferred embodiment, the dual specificity antibody recognizes IL-β and IL-β and the foregoing detection methods are used to detect IL-β and / or IL-β. Accordingly, the present invention provides a method for detecting IL-Ια or IL-Ιβ in a biological sample or tissue comprising contacting the biological sample or tissue expected to contain IL-Ια or IL-Ιβ with a dual specific antibody of the invention, or a portion thereof and detecting IL-lot or IL-β in the biological tissue or sample. For example, the biological sample may be an in vitro sample such as a sample of cells, tissues, or body fluids (e.g., blood, plasma, urine, wet, etc.). in addition, tissues that are placed in vivo in a subject, eg, tissues visualized in vivo by imaging tissue (eg, using a labeled antibody), can be detected.

The dual specific antibodies of the invention can also be used for diagnostic purposes. In one embodiment, an antibody of the invention is used

<td> ·· ·*</td><td> ··</td><td> •</td><td> ··</td><td></td><td> ····</td>

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in an in vitro diagnostic test, such as a laboratory test for the detection of antigen of interest (s) or, if necessary, a test of detection of the antigen of interest (s). Examples of well established in vitro assays include ELISA, RIA, Western blotting and the like. In another embodiment, an antibody of the invention is used in an in vivo laboratory assay, such as an in vivo imaging assay. For example, the antibody may be labeled with a detectable substance capable of in vivo detection, the labeled antibody may be administered to a subject, and the labeled antibody may be detected in vivo, thereby allowing in vivo imaging.

The dual specific antibodies of the invention that specifically recognize IL-Ια and IL-Ιβ can be used in diagnostic assays to detect IL-Ια and IL-Ιβ for diagnostic purposes, for example, in various inflammatory diseases and disorders, as well as spontaneous fetal resorption . With respect to specific types of diseases and disorders, dual-specific IL-β and IL-β antibodies of the invention can be used for diagnostic purposes in any of the diseases / disorders described herein with respect to the therapeutic uses of these antibodies (see below), such as disorders in which IL-1 activity is detrimental, as discussed below.

The dual specificity antibodies of the invention and portions thereof are preferably capable of neutralizing, both in vivo and in vitro, the activity of the antigen to which they bind. Accordingly, the antibodies of the invention and portions thereof may be used to inhibit antigen activity, eg, in cell cultures containing antigens or in human subjects or other mammalian objects having antigens with which a dual specificity antibody of the invention reacts . In one embodiment, the invention provides a method of inhibiting antigen activity, comprising contacting the antigen with a dual specificity antibody of the invention or a portion thereof, such that the antigen activity is inhibited. In a preferred embodiment, the dual specificity antibody EL-1α and IL-1β bind and the method is a method of inhibiting IL-1α and / or IL-1β activity by contacting IL-1α and / or IL-1β with a dual specificity antibody or a portion thereof. The activity of IL-1α and / or IL-β may be inhibited, for example, in vitro. For example, in the case of cell cultures that contain or are expected to have IL-Ια and / or IL-Ιβ, the antibody of the invention or a portion thereof may be added to the culture medium to inhibit IL-1α and / or IL activity. -Ιβ in culture. IL-1 and / or IL-1β activity can be inhibited in vivo in a subject.

In another embodiment, the invention provides a method of inhibiting antigen activity in a subject suffering from a disease in which antigen activity is detrimentally involved. The invention provides methods for inhibiting antigen activity in a subject suffering from such a disease, and the method comprises administering a dual specificity antibody of the invention or a portion thereof of the invention. · · · · · · · · · · · · · · · · · · · · · · · · · · · Φ · antigen inhibited in this subject. Preferably, the antigen is a human antigen and the subject is a human subject. The antibody of the invention may be administered to a human subject for therapeutic purposes. In addition, the antibody of the invention may be administered to a non-human mammal expressing the antigen with which the antibody binds, for veterinary purposes or as an animal model of human disease. In view of the latter, such animal models may be useful for evaluating the therapeutic efficacy of the antibodies of the invention (e.g. dose testing and timing of administration).

Preferably, the dual specificity antibody binds EL-1α and IL-1β and the method of inhibiting antigen activity in a subject is a method of inhibiting IL-1 activity in a subject, for example, a subject suffering from a disease to which IL-1 activity is detrimental. As used herein, the term "disease in which IL-1 activity is harmful" refers to diseases and other disorders in which the presence of IL-1 (which contains IL-1 and IL-1β) in a subject suffering from the disease is found. as responsible or suspected of being responsible for the pathophysiology of the disease or contributing to the worsening of the disease. Accordingly, a disease in which IL-1 activity is deleterious is a disease in which inhibition of IL-1 activity (ie, either or both of IL-1 and IL-1β) is expected to ameliorate symptoms and / or development of the disease. For example, evidence of such diseases is an increase in the concentration of EL-1 in the biological fluid of a subject suffering from the disease (eg, an increase in IL-1 concentration in serum, plasma, synovial fluid, etc. of the subject) that can be detected, eg using anti-IL -1 antibodies as described above.

Interleukin 1 has been implicated in the pathology of many diseases associated with immune and inflammatory elements. These diseases include, but are not limited to, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathies, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory colitis, diabetes, asthma, allergic diseases, psoriasis, dermatitis scleroderma, host transplant reaction, organ transplant rejection, acute chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, scattered intravascular coagulation, Kawasaki's disease, Graves' disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schoenlein hepatitis, uveitis seitis, microscopic active , toxic shock syndrome, septic syndrome, cachexia, infectious diseases, parasitic diseases, AIDS, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignant diseases, cardiac

<img file="CZ20030291A3_D0004.tif" />

failure, myocardial infarction, Addison's disease, sporadic, polyglandular type I deficiency and polyglandular type II deficiency, Schmidt's syndrome, adult (acute) respiratory pain syndrome, alopecia, alopecia areata, seronegative arthopathia, arthropathies, ulcerative arthritis, psoriasis, colitis, psoriasis, colitis , enteropathic synovitis, arthropathies associated with chlamydia, yersinia and salmonella, spondyloarthropathies, atheromatous disease / arteriosclerosis, atopic allergies, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs positive haemolytic anaemia, acquired pernicious anaemia, chronic arterial illness, perennial mycosis, , primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, AIDS, AIDS related diseases, hepatitis C, general variable immunodeficiency (general variable hypogammaglobulinaemia), dilated cardiomyopathy, female infertility, ovarian disorders, premature ovarian disorders, fibrotic lung disease, cryptogenic fibrosis alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonia, interstitial pneumonia, tonsillitis tissues associated with interstitial lung disease, systemic sclerosis associated with lung disease, rheumatoid arthritis associated with lung disease, systemic lupus erythematosus associated with interstitial lung disease, dermatomyositis / polymyositis associated with lung disease, Sjögren's disease associated with lung disease, ankylosing spondylitis associated with pulmonary disease, pulmonary disease, vascular disease, vascular disease , drug-induced interstitial lung disease, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, Lymphocytic lung infiltration disease, post-infectious interstitial lung disease, gout arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis (classic autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune-mediated insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ transplantation, osteoarthrosis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leukopaenia, autoimmune neutropaenia, renal disease NOS, glomerulonephritis, microscopic renal vasculitis, Lyme disease, discoid lupus erythematosus, male idiopathic infertility or NOS, spermal autoimmunity, sperm autoimmunity) pulmonary hypertension secondary to connective tissue disease, Goodpasture syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjorgren's syndrome, Takayasu's disease / arteritis, autoimmune • gr ······ · *

JO thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid disease, hyperthyroidism, gout autoimmune hypothyroidism (Hashimoto's) disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo, diseases of the central nervous system (eg, depression, schizophrenia, Alzheimer, Parkinson, etc.), acute and chronic pain, and lipid imbalance. The human antibodies of the invention and parts thereof can be used to treat people suffering from autoimmune diseases, especially those associated with inflammation, including rheumatoid spondylitis, allergies, autoimmune diabetes, and autoimmune uveitis.

The EL-1T / IL-β dual-specific antibodies of the invention or antigen-binding portions thereof are preferably used for the treatment of rheumatoid arthritis, Crohn's disease, multiple sclerosis, insulin-dependent diabetes and psoriasis.

The IL-1α / IL-ββ dual specificity antibody of the invention or a portion thereof may also be administered with one or more additional therapeutic agents useful in the treatment of autoimmune and inflammatory diseases.

The antibodies of the invention or antigen-binding portions thereof can be used alone or in combination for the treatment of these diseases. It will be appreciated that the antibodies of the invention or antigen-binding portions thereof may be used alone or in combination with an adjunct agent, eg, a therapeutic agent, when said adjunct agent is selected by a trained person according to the intended use. For example, the adjunctive agent may be a therapeutic agent expertly determined to be useful for treating a disease or condition that is treated with an antibody of the present invention. The additional agent may also be an agent that imparts a beneficial property to the therapeutic composition, e.g., an agent that affects the viscosity of the composition.

It will also be appreciated that those combinations encompassed by the present invention are combinations useful for their intended use. The following agents are intended to be illustrative and not intended to be limiting. Combinations of the invention may be the antibodies of the present invention and at least one additional agent, eg, two or three additional agents, provided that the combination in the composition thus formed can fulfill the intended purpose.

Preferred combinations are non-steroidal anti-inflammatory drugs (medicaments), also referred to as non-steroidal anti-inflammatory drug (s), including ibuprofen-like drugs, and COX-2 inhibitors. Other preferred combinations are corticosteroids, including prednisolone; The well-known side effects of steroid use can be reduced or eliminated by reducing the steroid dose required to treat the patient in

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combination with anti-IL-1 antibodies of the invention. Non-limiting examples of therapeutic agents against rheumatoid arthritis with which an antibody of the invention or a portion thereof may be combined are as follows: cytokine suppressive anti-inflammatory drug (s) (CSAIDs) antibodies or antagonists against human cytokines or growth factors, e.g. IL-2, IL-6, IL-7, IL-8, IL-12, IL-15, IL-16, IL-18, EMAP-II, GM-GSF, FGF, and PGDF. The antibodies of the invention or antigen-binding portions thereof may be combined with antibodies against cell surface molecules such as CD2, CD3, CD4, D8, CD25,

CD28, CD30, CD40, CD45, CD69, CD80 (B7, 1), CD86 (B7.2), CD90 or ligands thereof, including CD 154 (gp39 or CD40L).

Preferred combinations of therapeutic agents may interfere at various sites in the autoimmune and subsequently inflammatory cascades; preferred examples are including TNF antagonists such as chimeric, humanized or human TNF antibodies, D2E7, (PCT publication no. WO 97/29 131), CA2 (Remicade ™), CDP 571, CDP 870, Thalimide and soluble p55 or p75 TNF receptors, derivatives thereof (p75TNFRgG (Enbrel ™) or p55TNFR1gG (Lenercept) as well as TNFα converting enzyme inhibitors (TACE) similarly, IL-1 inhibitors (interleukin-1-converting enzyme inhibitors, IL-1RA, etc.) may be effective for the same reason. Other important participants in the autoimmune response that can act in parallel are other preferred combinations depending on or consistent with IL-1 function; particularly preferred are IL-12 and / or IL-18 antagonists, including IL-12 and / or IL-18 antibodies or soluble IL-121 and / or IL-18 receptors or IL-12 and / or IL-18 binding proteins. It has been found that IL-12 and IL-18 have overlapping, but different functions and combinations of their antagonists may be most effective. Yet another preferred combination is unused anti-VD4 inhibitors. Still other preferred inhibitors are costimulatory pathway (B7, 1) or CD86 (B7.2) antagonists, including antibodies, soluble receptors or antagonist ligands.

The antibodies of the invention or antigen-binding portions thereof may also be combined with agents such as methotrexate, 6-MP, azathioprine sulfasalazine, mesalazine, olsalazine chloroquinine / hydroxychloroquine, penicillamine, aurothiomalate (intramuscular or oral), azathioprine, corticosteroid, by inhalation and local injection), β-2 adrenoreceptor agonists (salbutamol, terbutaline, salmeteral), xanthines (theophylline, aminophylline), cromoglycate, nedocromil, ketofmen, ipraropium and oxitropium, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs such as ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adensonin agonists, antithrombotic agents, complement inhibitors, interferences, adrenergic agents Cytokines such as TNFα or IL-1 (e.g. IRAK, NIK, IKK, p38n or MAP kinase inhibitors), EL-1β converting enzyme inhibitors, TNFα converting enzyme inhibitors (TACE), T-cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulsalazine, azathioprine, 6-mercaptopurines , angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g. soluble p55 or p75 TNF receptors and derivatives p75TNFRIgG (Enbrel ™) and p55TNFRIgG (Lenercept)) sIL-1RI, sIL-1RII, sIL-6R) and anti-inflammatory cytokines (eg, IL-4, IL-10, IIL-11, IL Preferred combinations include methotrexate or leflunomide and, in mild or severe cases, rheumatoid arthritis, cyclosporine.

Non-limiting examples of therapeutic agents for intestinal inflammatory disease with which an antibody of the invention or a portion thereof may be combined are as follows: budenoside; epidermal growth factor; corticosteroids; cyclosporine, sulfalazine;

aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; EL-1 receptor antagonists; anti-IL-β monoclonal antibodies; anti-IL-6 monoclonal antibodies; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; antibodies or antagonists against other human cytokines or growth factors; for example, TNF, LT, IL-2, IL-6, IL-7, IL-8, EL-12, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PGDF. Antibodies of the invention may be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90, or ligands thereof. The antibodies of the invention or antigen-binding portions thereof may also be combined with agents such as methotrexate, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAEDs, for example ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adensonin agonists, complement, adrenergic agents, agents that interfere with pro-inflammatory cytokine signaling, such as TNFα or IL-1 (e.g. IRAK, NIK, IKK, p38n or MAP kinase inhibitors), IL1β converting enzyme inhibitors, TNFα converting enzyme (TACE) inhibitors, T-cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulsalazine, azathioprine, 6-mercaptopurines, angiotensin inhibitors converting enzyme, soluble cytokine receptors and derivatives thereof (e.g. soluble p55 or p75 TNF receptors and derivatives of p75TNFRIgG (Enbrel ™) and p55TNFRIgG (Lenercept)), sIL-1RI, sIL-1RII, sIL-6R) and anti-inflammatory cytokines (eg, IL-4, IL-10, IIL-11, IL-13 and β.

Preferred examples of therapeutic agents against Crohn's disease in which an antibody or antigen-binding portion thereof may be combined include the following: TNF

<img file="CZ20030291A3_D0008.tif" />

antagonists such as anti-TNF antibodies, D2E7 (PCT Publication No. WO 97/29 131), CA2 (Remicade ™), CDP 571, TNFR-Ig constructs, (p75NFRIgG (Enbrel ™) and p55TNFRIgG (Lenercept)) inhibitors and PDE4 inhibitors . The antibodies of the invention or antigen-binding portions thereof may be combined with corticosteroids such as budenoside and dexamethasone. Antibodies of the invention or antigen-binding portions thereof may also be combined with agents such as sulfalasin, 5-aminosalicylic acid and olsalasin and agents that interfere with the synthesis or effect of pro-inflammatory cytokines such as IL-1, for example, EL-β-converting inhibitors and EL-Ira. The antibodies of the invention or antigen-binding portion thereof may also be used with inhibitors of T-cell signaling, for example, 6-mercaptopurines, tyrosine kinase inhibitors. The antibodies of the invention or antigen-binding portions thereof may be combined with IL-11.

Non-limiting examples of multiple sclerosis therapeutic agents that may be combined include the following: corticosteroids; prednisolone; methylprednisolone; azathioprine; cyclophosphamide; cyclosporin; methotrexate; 4-aminopyridine; tizanidine; interferon-pia (Avonex; Biogen); interferon-pib (Betaseron; Chiron / Berlex); Copolymer 1 (Cop-1;

Copaxone; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous immunoglobulin; klabrinin; antibodies or antagonists of other human cytokines or growth factors such as TNF, LT, IL-2, IL-6, IL-7, EL-8, IL-12, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PGDF. Antibodies of the invention or antigen-binding portions thereof may be combined with antibodies against cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or ligands thereof. Antibodies or antigen-binding portions thereof may be combined with agents such as methotrexate, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs for example ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adensonin agonists, antithrombotic agents, antithrombotic agents agents that interfere with pro-inflammatory cytokine signaling, such as TNFα or IL-1 (e.g. BRAK, NIK, IKK, p38n or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TNFα converting enzyme (TACE) inhibitors, T-cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulsalazine, azathioprine, 6-mercaptopurines , angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g. soluble p55 or p75 TNF receptors and derivatives of p75TNFRIgG (Enbrel ™) and p55TNFRIgG (Lenercept)), sEL-1RI, sIL-1RII, sIL-6R) and anti-inflammatory cytokines (eg, IL-4, IL-10, IIL-11, IL-13 and TGFβ.

• Φ · 4 4 4 4 4 9 9 9 9 9 9 9 9 9 9 4 4 9 9 9 9 9 9 9 9 9 9 9 9

Preferred examples of anti-multiple sclerosis therapeutic agents in which an antibody or antigen-binding portion thereof may be combined include interferon-β such as ΙΕΝβΙδ and IF-1α; copaxone; corticosteroids; EL-1 inhibitors, TNF inhibitors, and antibodies against CD40 and CD80 ligands.

The pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody of the invention or a portion thereof. "Therapeutically effective amount" means an amount that is effective at the dosages and time periods necessary to achieve the desired therapeutic result. The therapeutically effective amount of the antibody or portion thereof may vary depending on factors such as the disease state, age, sex and weight of the individual and the ability of the antibody or portion thereof to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the deleterious or toxic effects of an antibody or portion thereof are outweighed by therapeutically beneficial effects. "Prophylactically effective amount" means an amount effective at the dosages and time periods necessary to achieve the desired prophylactic effect. Because the prophylactic dose is used in subjects prior to or earlier stages of the disease, the prophylactic effective amount is usually less than the therapeutically effective amount.

Dosage regimens should be adjusted to achieve the optimum desired response (eg, a therapeutic or prophylactic response). For example, a single dose may be administered, several divided doses over time, or the dose may be proportionally reduced or increased as the therapeutic situation appears to be necessary. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. By dosage unit is meant discrete units suitable as unitary amounts for the mammalian subject to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required therapeutic carrier. The specifications for dosage unit forms of the invention are dictated and directly dependent upon the unique characteristics of the active compound and the principal therapeutic or prophylactic effect to be achieved and the limitations contained in the technique of using such an active compound to treat sensitivity in individuals.

An exemplary, non-limiting range of a therapeutically or prophylactically effective amount of an antibody of the invention or a portion thereof is 0.1 to 20 mg / kg, more preferably 1 to 10 mg / kg. It should be noted that dosage values ​​may vary with the type and severity of the condition to be improved. It is also to be further understood that for any individual subject, the dosage regimen should be adjusted to a time appropriate to the individual need and professional judgment of the person administering or supervising the administration of the compositions, and that the dosage regimens herein are exemplary and not intended to be limiting. or the use of the claimed claims.

The present invention is further illustrated by the following examples, which are not intended to be limiting. The contents of all references cited, including literature references, published patents and published patent applications cited in this application, are hereby expressly incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Construction of dual-specific antigens based on the interface of topological identity

In this example, the largest interface topology of identity between two different but structurally related proteins, EL-Ια and IL-Ιβ, was determined as the basis for constructing dual-specific antigens to grow dual-specific antibodies to IL-1 and IL-1β. A BLAST algorithm was used to compare the two proteins, which made it possible to measure the tendency of one residue to replace another in structurally or functionally similar regions. This analysis allowed the identification of the largest contact topological region of identity between IL-1 and and EL-β and extended this region with any similarity range to produce a linear peptide that serves as a dual specific antigen. The peptide that best matches the criteria has the following amino acid sequence:

NEAQNITDF (SEQ ID NO: 1)

An asterisk (*) indicates identical residues in both proteins. The other residues are very similar, according to the BLAST algorithm. For example, lysine will often replace arginine in homologous proteins, but not phenylalanine. The peptide of SEQ ID NO: 1 is a hybrid formed from two different sections of the structure that run in the opposite direction, so that the corresponding representation of this epitope is:

dNdEdAdQNITDF, (where the prefix "d" indicates that the amino acid residue is a D amino acid residue).

The amino acid L form of this peptide, as well as its version, which is partially substituted by D &amp; apos; amino acid residues, are synthesized by standard chemical methods. The peptide is then conjugated to a proteinaceous carrier (eg, KLH or albumin) and the conjugated peptide is used to select antibodies in vitro or in vivo.

Example 2: Construction of a dual specific antigen based on a cyclic peptide that mimics a loop of a common native structure

In this example, it was a cyclic peptide that structurally mimics the main loop of the common native structure of two different but structurally related proteins. IL-Ία and IL-1 β, designed for use as dual-specific antigen for the production of dual-specific antibodies against EL-Ια and IL-Ιβ. This selected loop represents residues 168-184 of IL-1β and residues 160-176 of IL-β. The agreed sequence is:

Cyclo-MAFLRANQNNGKISVAL (PG) * cbcccccccc ** c * b * (SEQ ID NO: 2)

The asterisk (*) indicates identical residues between IL-Ία and IA-Ιβ, c indicates the agreed residues, ie residues similar to IL-Ία and IL-Ιβ, but which are actually not present in this position in either protein, and b denotes that there is no agreed residue as long as the sequence identity of IL-β is maintained. This linear peptide is synthesized by standard chemical methods. Proline and glycine residues are added to cyclize the peptide. This cyclic peptide can be synthesized using standard high dilution synthesis conditions in N, N- (dimethyl) formamide (1 mg / ml). Prototype reactions proceed at room temperature using an excess of a coupling reagent such as benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexaflurophosphate (PyBOP; 2 moles) and sodium bicarbonate (10 moles). This peptide is conjugated to a carrier protein. (e.g. KLH or albumin) and the conjugated peptide are used to select antibodies by in vitro or in vivo methods.

Example 3: Construction of a dual-specific hybrid peptide-based antibody

In this example, a hybrid peptide was constructed for use as a dual specificity antigen for raising dual specific anti-IL-β and IL-β antibodies, containing alternating or overlapping sequences of two different but structurally related proteins, IL-β and IL-β. In order to produce this hybrid peptide, alternating and overlapping IL--α and EL-Ιβ sequences were identified and linked to form the following peptide:

• φ «r · · ·«

<img file="CZ20030291A3_D0009.tif" />

TKGGQDITDFQELENQ (SEQ ID Ν 0: 3) bbbbbbbbbb aaaaaaaaaa

The letters a and b indicate which protein is the source of the residue (a = IL-Ια; b = IL-1 β). The TIDF motif, common to both proteins, was contained in the hybrid protein. In addition, this hybrid peptide targets sequences from the C-terminus of both proteins, which are also known in both proteins for their antigenicity to neutralizing antibodies. This hybrid peptide is synthesized by standard chemical synthetic methods. The peptide is then conjugated to a protein carrier (e.g. KLH or albumin) and the conjugated peptide are used to select antibodies by in vitro or in vivo methods.

Example 4: Generation of Dual-Specific Antibodies Against EL-Ια and IL-1β

NEAQNITDF (SEQ ID NO: 1)

Cyclo-MAFLEANQNNGKISVAL (PG) (SEQ ID NO: 2)

TKGGQDITDFQILENQ (SEQ ID NO: 3)

The peptides of SEQ ID NOs: 1, 2 and 3 were conjugated to KLH and individual rabbits were immunized. Antisera of immunized rabbits against each of the three peptides showed a good antibody response against the peptide used as the antigen. However, only the rabbit antiserum immunized with the peptide of SEQ ID NO: 3 was able to bind to both IL-loc and IL-β protein.

Five mice (BA119 to BA123) were immunized once every three weeks with a peptide of SEQ ID NO: 3 conjugated to KLH and Freund's incomplete adjuvant (FIA), three times in total, followed by two challenging intravenous doses of the peptide of SEQ ID NO: 3 with KLH. Blood was drawn from each mouse 10 days after each immunization and the antibody titer was determined by ELISA. Spleen cells from BA119 and BA123 mice, respectively, were fused to the myeloma cell line P3X36Ag8,653 as described in section IIA, and the resulting fused cells were seeded one at a time into wells in several 96-well plates using limited dilution. Those hybridoma clones that grew were first tested for IgG and IgM production by standard ELISA to identify clones producing antibody. A total of 945 clones were isolated from fusion # BA123. In 335 clones tested, ELISA supernatants showed antigen binding activity to IL-1α, IL-1β, or both.

<td>clone</td><td>antigen specificity (against full-length EL-lot and / or IL-Ιβ)</td><td>Isotype</td>

<td> 249</td><td>IL-1α only</td><td>IgG</td>

<td> 19</td><td>EL-la only</td><td>IgM</td>

<td> 15</td><td>Only IL-1β</td><td>IgG</td>

<td> 2</td><td>IL-β only</td><td>IgM</td>

<td> 57</td><td>IL-1α and β</td><td>IgG</td>

<td> 13</td><td>EL-1a and β</td><td>IgM</td>

It will be clear to those skilled in the art that even when using routine experiments there are many equivalents to specific embodiments of the present invention. It is intended that these equivalents be included in the following claims.

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