RU2444528C2 - Il-31 monoclonal antibodies and methods of using - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is described is an IL-31 monoclonal antibody. What is offered is a method of treating a mammal suffering an inflammatory disease in which IL-31 in involved that consists in introducing the described antibody. There are offered methods for reducing, inhibiting and minimising itching in a mammal that consist in introducing the described antibody.

EFFECT: invention extends the range of anti-inflammatory agents.

39 cl, 4 tbl, 16 ex

Description

BACKGROUND OF THE INVENTION

The immune system includes a wide range of specific types of cells, including lymphocytes. Lymphocytes determine the specificity of the immune response in the body and include two classes: B-lymphocytes, which are the precursors of antibody-producing cells, and T-lymphocytes, which are necessary for certain regulatory functions, such as the development of a specific immune response.

Mature T cells can be activated, in particular, by an antigen or other stimuli and can produce, for example, cytokines, biochemical signaling molecules or receptors, which further affect the fate of the T cell population.

B-cell activation can occur through receptors on their cell surface, including the B-cell receptor, and other auxiliary molecules to perform auxiliary cellular functions, such as production of cytokines.

Activation of monocytes / macrophages and T cells can occur through receptors on their cell surface, and they play a central role in the immune response by presenting antigen to lymphocytes, and also act as auxiliary cells for lymphocytes through the secretion of a number of cytokines.

Natural killer (NK) cells share a precursor cell with T cells and B cells and are involved in immunological surveillance. NK cells, which make up up to 15% of blood lymphocytes, do not express receptors for antigens and, therefore, do not use MHC recognition as a necessary factor for binding of the target cell. NK cells are involved in the recognition and destruction of certain tumor cells and virus-infected cells. It is believed that in vivo NK cells require activation, but it has been shown that in vitro NK cells cause the death of some types of tumor cells in activation bases.

Interleukins are a family of cytokines that mediate the immunological response, including inflammation. Interleukins mediate a variety of inflammatory pathologies. The main role in the immune response is played by T cells, which produce many cytokines and exercise adaptive immunity against antigens. The cytokines produced by T cells are classified as type 1 and type 2 cytokines (Kelso, A. Immun. Cell Biol. 76: 300-317, 1998). Type 1 cytokines include IL-2, IFN-γ, LT-α, and they are involved in the inflammatory response, viral immunity, immunity against intracellular parasites and allograft rejection. Type 2 cytokines include IL-4, IL-5, IL-6, IL-10, and IL-13, and they are involved in the humoral response, helminth immunity, and allergic response. Common types 1 and 2 of cytokines include IL-3, GM-CSF, and TNF-α. There is some evidence that producing type 1 and type 2 T-cell populations predominantly migrate to various types of inflamed tissue.

The skin plays an important role in the immune system and consists of layers. The epidermis is a surface layer. Under the epidermis is the dermis, a layer of connective tissue. Under the dermis is the hypodermis, a layer of a large amount of adipose tissue. Circulating T-lymphocytes migrate to the skin under normal and inflammatory conditions. Cutaneous lymphocytic antigen (CLA) is considered a "homing" receptor for T cells tropic to the skin. Santamaria-Babi, L., Eur. J. Dermatol. 14: 13-18, 2004.

It is known that for certain skin diseases, high levels of CLA + T cells are detected, including atopic dermatitis, contact dermatitis, drug-induced allergic reactions, skin tropic viruses and the virus-associated itching, vitiligo, cutaneous T-cell lymphoma, focal alopecia, red blackheads, acne vulgaris, knotty pruritus and bullous pemphigoid. There is a need for the treatment of such cutaneous T-cell mediated diseases.

Demonstrated in vivo types of activity of the cytokine family illustrate the tremendous clinical potential and need for other cytokines, cytokine agonists and cytokine antagonists. IL-31 is a newly identified cytokine. When overexpressed in mice, IL-31 leads to dermatitis-like symptoms. The pathology of skin diseases in humans involves both T cells directed to the skin and epidermal keratinocytes. The present invention satisfies these needs by providing pro-inflammatory cytokine IL-31 antagonists. Such antagonists according to the invention, which can block, inhibit, reduce, antagonize or neutralize the activity of IL-31, include soluble IL-31RA receptors and neutralizing antibodies against IL-31. In addition, this invention relates to their use in inflammatory diseases, as well as to related compositions and methods.

DETAILED DESCRIPTION OF THE INVENTION

Before a detailed presentation of this invention, for its understanding, it may be appropriate to define the following terms:

As used herein, the term “antibodies” includes polyclonal antibodies, affinity purified polyclonal antibodies, monoclonal antibodies and antigen binding fragments, such as F (ab ′) ₂ and Fab proteolytic fragments. The definition also includes whole antibodies or fragments obtained by genetic engineering methods, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen binding peptides and polypeptides. Non-human antibodies can be humanized by transplanting non-human CDRs into the framework region and constant regions of a person, or by embedding whole non-human variable domains (optionally "masking" them like a human surface by replacing the exposed residues, resulting in a "hyperchimerized" antibodies). In some cases, non-human residues can be stored in humanized antibodies in the framework domains of the human variable region to enhance the necessary binding properties. By humanizing antibodies, the biological half-life can be increased, and when administered to humans, the possibility of adverse immune reactions is reduced.

The term "chimeric antibody" or "chimeric antibodies" refers to antibodies whose light and heavy chain genes were constructed primarily by genetic engineering from the variable and constant region genes of immunoglobulins belonging to another species. For example, variable gene segments from a monoclonal mouse antibody can be attached to human constant segments, such as gamma 1 and gamma 3. Thus, a typical therapeutic chimeric antibody is a fusion protein consisting of a variable or antigen binding domain of a mouse antibody and a constant domain of human antibodies, although other mammalian species may be used.

As used herein, the term “immunoglobulin” refers to a protein consisting of one or more polypeptides, mainly encoded by immunoglobulin genes. One form of immunoglobulin is the main structural element of an antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, where each pair has one light and one heavy chain. In each pair, the variable regions of the light and heavy chains are jointly responsible for binding to the antigen, and the constant regions are responsible for the effector functions of the antibody.

The full-length immunoglobulin light chains (approximately 25 kDa or 214 amino acids) are encoded by the variable region gene at the NH2 end (approximately 110 amino acids) and the kappa or lambda constant region genome at the COOH end. The full-length immunoglobulin heavy chains (approximately 50 kDa or 446 amino acids) are likewise encoded by the variable region gene (approximately 116 amino acids) and one of the other constant region genes mentioned above (approximately 330 amino acids). Heavy chains are classified as gamma, mu, alpha, delta or epsilon, and they determine the antibody isotype IgG, IgM, IgA, IgD and IgE, respectively. In the light and heavy chains, the variable and constant regions are connected by a “J” region of about 12 or more amino acids, and the heavy chain also includes a “D” region of about 10 or more amino acids. (See mainly Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7 (incorporated by reference in its entirety for any purpose).

The variable region of the light or heavy chain of an immunoglobulin consists of a “frame” region interrupted by three hypervariable regions. Thus, the term “hypervariable region” refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region contains amino acid residues from the “complementarity determining region” or “CDR” (ie, residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the variable domain of the heavy chain (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md . (1991)) and / or these residues from the "hypervariable loop" (ie, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the variable domain of the light chain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable domain of the heavy chain; Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917) (both of which are included in the present description by reference.) The framework region or FR residues are those variable domain residues that differ from the hypervariable region residues as defined herein. The frame region sequences of various light and heavy chains are relatively conserved within the species . Thus, a "human framework region" is a framework region that is substantially identical (approximately 85% or more, typically 90-95% or more) of the framework region of a naturally occurring human immunoglobulin. The frame region of the antibody, which is a combined frame region of its light and heavy chains, serves to localize and align the CDR. CDRs are primarily responsible for binding to an antigen epitope.

Thus, the term “humanized” immunoglobulin refers to an immunoglobulin containing a human framework region and one or more CDRs of a non-human immunoglobulin (typically a mouse or rat). A non-human immunoglobulin providing a CDR is called a “donor”, and a human immunoglobulin providing a framework region is called an “acceptor”. There is no need for constant regions, however, if present, they should be substantially identical to the constant regions of human immunoglobulin, i.e. at least about 85-90%, preferably about 95% or more identical. Thus, all parts of the humanized immunoglobulin, with the possible exception of CDR, are essentially identical to the corresponding parts of the natural sequences of human immunoglobulin. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody does not include a typical chimeric antibody as defined above, for example, due to the fact that the entire variable region of the chimeric antibody is non-human.

The term "genetically modified antibodies" means antibodies, where the amino acid sequence differs from the sequence of a natural antibody. Due to the importance of recombinant DNA methods in producing antibodies, there is no need to limit the amino acid sequences found in natural antibodies; antibodies can be redesigned to obtain the desired characteristics. Possible changes are numerous and vary from a change in only one or several amino acids to a complete redesign, for example, a variable or constant region. Changes in the constant region, as a rule, are carried out in order to improve or change characteristics, such as complement fixation, interaction with membranes and other effector functions. Changes in the variable region are carried out in order to improve the characteristics relating to antigen binding.

In addition to antibodies, immunoglobulins can exist in many other forms, including, for example, single chain antibodies or Fv, Fab and (Fab ') ₂ , as well as dimers, linear antibodies, polyvalent or multispecific hybrid antibodies (as described above and in more detail in: Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in the form of individual chains (e.g., Huston et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883 (1988), and Bird et al., Science, 242, 423-426 (1988), which are incorporated herein by reference). (See mainly Hood et al., "Immunology", Benjamin, NY, 2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporated herein by reference )

As used herein, the terms “single chain Fv”, “single chain antibodies”, “Fv” or “scFv” refer to antibody fragments that contain variable regions from both the heavy and light chains, but lack constant regions but within the same polypeptide chain. Typically, a single chain antibody between the VH and VL domains further comprises a polypeptide linker, which allows them to form the desired structure, which allows antigen binding. Single chain antibodies are described in detail by Pluckthun in The Pharmacology of Monoclonal Antobodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994); see also International Patent Application Publication No. WO 88/01649 and US Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference for any purpose. In specific embodiments, single chain antibodies may also be bispecific and / or humanized.

A "Fab fragment" contains one light chain and C.sub.H1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

The "Fab'fragment" contains one light chain and one heavy chain, which between the domains C _H1 and C _H2 contains a large part of the constant region, so that between the two chains an interchain disulfide bond can form to form the F (ab ') ₂ molecule.

The "F (ab ') ₂ fragment" contains two light chains and two heavy chains containing a portion of the constant region between the C _H1 and C _H2 domains, so that an interchain disulfide bond forms between the two heavy chains.

It is understood that the molecular weights and lengths of polymers determined by inaccurate analytical methods (e.g. gel electrophoresis) are approximate values. When such a value is expressed as “approximately” X or “approximately” X, it should be understood that the indicated value of X is ± 10% accurate.

All references cited herein are incorporated by reference in their entirety.

The present invention is partially based on the discovery associated with the use of antibodies as antagonists of IL-31, thus inhibiting inflammation in general and the symptoms of dermatitis and pruritic diseases. This invention relates to the use of monoclonal antibodies to inhibit, reduce, prevent or minimize the effects of dermatitis and pruritic diseases, as described later in the present description. In one embodiment, the dermatitis is atopic dermatitis. In another embodiment, dermatitis is nodular pruritus. In another embodiment, dermatitis is eczema. IL-31 is a recently discovered T-cell cytokine that, when overexpressed in mice, leads to dermatitis-like symptoms. See also Dillon, et al., Nature Immunol. 5: 752-760, 2004. Both skin-directed T cells and epidermal keratinocytes are involved in the pathology of skin diseases in humans. The expression of mRNA and IL-31 protein in both patients with atopic dermatitis (AD) and healthy individuals is limited to the skin-directed population of CLA + T cells, while receptor analysis for IL-31, IL-31RA, by immunohistochemistry ( IHC) confirms slightly higher levels of IL-31RA expression on skin keratinocytes in the skin biopsies of patients with acute and chronic AD compared with healthy individuals.

IL-31 also has the name HUGO, which was assigned to the cytokine, which was previously described as Zcyto17rlig in published US patent application (see publication number 20030224487, Sprecher, Cindy et al., 2003, incorporated by reference). See also Dillon, et al., Nature Immunol., Supra. A heterodimeric receptor for IL-31 is also described in 20030224487 as zcytor17 (name HUGO, IL-31RA), which form a heterodimer with oncostatin M beta receptor (OSMRbeta). IL-31 was isolated from a cDNA library obtained from activated human peripheral blood cells (hPBC), among which selection was performed on CD3. CD3 is a cell surface marker for cells of exclusively lymphoid origin, in particular T cells. The polynucleotide and polypeptide sequences of human IL-31 are presented in SEQ ID NO: 1 and 2, respectively. The polynucleotide and polypeptide sequences of mouse IL-31 are presented in SEQ ID NO: 10 and 11, respectively. As used in the present description, the term IL-31 means IL-31, as used in US patent publication number 20030224487, as shown above. The IL-31 secretory signal sequence contains amino acid residues from 1 (Met) to 23 (Ala), and the mature polypeptide contains amino acid residues from 24 (Ser) to 164 (Thr) (as shown in SEQ ID NO: 2). Additional analysis with N-terminal sequencing of purified IL-31 from T-cells 293 showed the localization of the N-terminal at position 27 (Leu), as shown in SEQ ID NO: 2, and the mature polypeptide contained amino acid residues from 27 (Leu) to 164 (Thr) (as shown in SEQ ID NO: 2).

The IL-31RA polypeptide sequence (receptor for IL-31) is shown in SEQ ID NO: 5, and the oncostatin M beta receptor polypeptide sequence (OSMRbeta) is shown in SEQ ID NO: 7.

The IL-31RA and OSMRbeta receptors belong to class I of the cytokine receptor subfamily, which includes, but is not limited to, receptors for IL-2, IL-4, IL-7, Lif, IL-12, IL-15, EPO, TPO , GM-CSF and G-CSF (for a review, see Cosman, "The Hematopoietin Receptor Superfamily" in Cytokine 5 (2): 95-106, 1993). The IL-31RA subunit is fully described in PCT Joint Patent Application No. US01 / 20484 (WIPO Publication No. WO 02/00721). Analysis of the distribution of IL-31RA subunit mRNA in tissues revealed expression of CD4 + and CD8 + subtypes, CD14 + monocytes in activated T cells, and weaker expression in CD19 + B cells. Moreover, mRNA was present in both resting and activated monocytic cell lines THP-1 (ATCC No. TIB-202), U937 (ATCC No. CRL-1593.2) and HL60 (ATCC No. CCL-240).

Inhibition, neutralization, and blocking of signal transduction by the IL-31 antagonists described herein can be determined using a number of assay methods known to one skilled in the art. For example, assays to determine a decrease in proliferation include dye reduction assays such as AlamarBlue ™ (AccuMed International, Inc. Westlake, Ohio), 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide ( Mosman, J. Immunol. Meth. 65: 55-63, 1983); 3, (4,5-dimethylthiazol-2-yl) -5-3-carboxymethoxyphenyl-2H-tetrazolium; 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl) -5 - [(phenylamino) carbonyl] -2H-tetrazolium hydroxide; and cyanoditholyltetrazolium chloride (which are commercially available from Polysciences, Inc., Warrington, PA); mitogenesis assays, such as measurement of ³ H-thymidine incorporation; dye displacement assays using, for example, naphthalene black or trypan blue; capture of the dye using diacetylfluorescein; and chromium release. See mainly Freshney, Culture of Animal Cells: A Manual of Basic Technique, 3rd ed., Wiley-Liss, 1994, which is incorporated herein by reference. In addition to the above, see US Published Patent Publication No. 20030224487 (Sprecher, Cindy et al., 2003) for an example of BaF3 cells expressing IL-31RA and full-length OSMRbeta.

Typically, cytokines are thought to have a structure of four alpha helices, where helices A, C, and D are most important for ligand-receptor interactions and the most highly conserved among family members. An indication of the amino acid sequence of human IL-31 presented in SEQ ID NO: 2, the alignment of human IL-31, human IL-3, and the amino acid sequences of human cytokines suggests that the A helix in IL-31 is determined by amino acid residues 38-52; helix B is determined by amino acid residues 83-98; helix C is determined by amino acid residues 104-117; and the D helix is defined by amino acid residues 137-152, as presented in SEQ ID NO: 2. Structural analysis suggests that the A / B loop is long, the B / C loop is short, and the C / D loop is long. This structure of the loops leads to the organization of the spiral up-up-down-down. Based on the structure of the 4-helix globule, the cysteine residues in IL-31, which are conserved, correspond to amino acid residues 72, 133, and 147 in SEQ ID NO: 2; and 74, 137, and 151 in SEQ ID NO: 11, as described herein. The appropriate arrangement of cysteine is an additional confirmation of the structure of the four-helix globule. Also in IL-31, the Glu residue is highly conserved, as described in SEQ ID NO: 2, at position 43. These IL-31 helices can be specific targets for inhibiting, decreasing, or neutralizing with the antibodies described herein to block effects of IL-31 signal transmission through a receptor recognizing it.

Based on a comparison of human and mouse IL-31 sequences, conserved residues were found in regions presumably encoding alpha helices C and D. Corresponding polynucleotides encoding polypeptide regions, domains, motifs, residues, and sequences of human IL-31 described in the present description, represented in SEQ ID NO: 1. These IL-31 helices can be specific targets for inhibiting, reducing, or neutralizing with the antibodies described herein to block the effects of IL-31 signaling through its recognizing receptor.

While helix D is relatively conserved in human and mouse IL-31, helix C is the most conserved. Although both species are dominated by acidic amino acids in this region, their differences can cause species specificity in the interaction between IL-31 and its receptor, IL-31RA, which includes monomeric, heterodimeric, or multimeric receptors. Loop A / B and helix B of IL-31 are less conserved, and helix C with loop C / D and helix D are the most conservative between species; conservatism in this area suggests that it is functionally significant. Human and mouse D-helices IL-31 are also conserved. IL-31RA receptor antagonists can be constructed through mutations in the IL-31 D-helix. These may include shortening the protein from the Thr156 residue (SEQ ID NO: 2), or retaining residues that allow the ligand to bind to the receptor but reduce signal transmission activity.

Methods for producing polynucleotides encoding antibodies described herein (including DNA and RNA) are well known in the art. Total RNA can be obtained using extraction with guanidinium isothiocyanate, followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18: 52-94, 1979). Poly- (A) ⁺ RNA is obtained from total RNA using the Aviv and Leder method (Proc. Natl. Acad. Sci. USA 69: 1408-12, 1972). Complementary DNA (cDNA) is obtained from poly (A) ⁺ RNA using known methods. Alternatively, genomic DNA can be isolated. Polynucleotides encoding anti-IL-31 antibodies are then identified and isolated, for example, by hybridization or PCR.

The polynucleotide sequence of the mouse IL-31 ortholog was identified and presented in SEQ ID NO: 3. The mature mouse IL-31 sequence is thought to begin with Met ₁ ; as presented in SEQ ID NO: 4, which corresponds to Met ₁ , as represented in SEQ ID NO: 2, in the human sequence. Tissue analysis revealed that mouse IL-31 expression is found in the testes, brain, CD90 + cells, prostate cells, salivary glands and skin. Additional N-terminal sequencing analysis of purified IL-31 from T-cells 293 showed the location of the N-terminus at residue 31 (Ala) as shown in SEQ ID NO: 4, and the mature polypeptide contains amino acid residues from 31 (Ala) to 163 (Cys) (as presented in SEQ ID NO: 4).

You can get the Hopp / Woods hydrophilicity profile for the protein sequence of IL-31, as presented in SEQ ID NO: 2 (Hopp et al., Proc. Natl. Acad. Sci. 78: 3824-3828, 1981; Hopp, J. Immun. Meth. 88: 1-18, 1986 and Triquier et al., Protein Engineering JU: 153-169, 1998). The profile is based on a moving window of six residues. In-depth residues G, S, and T and exposed residues H, Y, and W are not taken into account. For example, in human IL-31, hydrophobic sites include amino acid residues 54-59 in SEQ ID NO: 2, amino acid residues 129-134 in SEQ ID NO: 2, amino acid residues 53-58 in SEQ ID NO: 2, amino acid residues 35-40 in SEQ ID NO: 2 and amino acid residues 33-38 in SEQ ID NO: 2. For example, in IL-31 mice, hydrophobic regions include amino acid residues 34-39 in SEQ ID NO: 11, amino acid residues 46-51 in SEQ ID NO: 11, amino acid residues 131-136 in SEQ ID NO: 11, amino acid residues 158-163 in SEQ ID NO: 11 and amino acid residues 157-162 in SEQ ID NO: 11.

Specialists in this field will understand that hydrophilicity or hydrophobicity will be taken into account when developing modifications in the amino acid sequence of the IL-31 polypeptide so as not to violate the overall structural and biological profiles. Of particular interest for substitution are hydrophobic residues selected from the group consisting of Val, Leu and Ile, or the group consisting of Met, Gly, Ser, Ala, Tyr and Trp. For example, substitutable residues may include Val, Leu, and Ile, or a group consisting of Met, Gly, Ser, Ala, Tyr, and Trp residues as set forth in SEQ ID NO: 2. Conservative cysteine residues at the positions in SEQ ID NO: 2 and SEQ ID NO: 11 are relatively non-replaceable.

The present invention also relates to antibodies that bind functional fragments of IL-31 polypeptides, and to nucleic acid molecules encoding such functional fragments. A “functional” IL-31 or fragment thereof, as defined herein, is characterized by its proliferation or differentiation activity, its ability to induce or inhibit specialized cellular functions, or its ability to specifically bind to an anti-IL-31 antibody or IL-31RA or with an antibody or IL-31RA / OSMRbeta heterodimers of these receptors (either soluble or immobilized). As described above, IL-31 is characterized by the structure of a four-helix globule containing helix A (amino acid residues 38-52), helix B (amino acid residues 83-98), helix C (amino acid residues 104-117), and helix D (amino acid residues 137-152), as presented in SEQ ID NO: 2. Thus, the present invention further relates to fusion proteins, including: (a) polypeptide molecules containing one or more helices, as described above; and (b) functional fragments containing one or more of these helices. The other polypeptide portion of the fusion protein can be represented by another cytokine in the form of a four-helix globule, such as IL-15, IL-2, IL-4 and GM-CSF, or an unnatural and / or unrelated secretory signal peptide that facilitates secretion of the fusion protein .

The present invention also relates to antibodies that bind to polypeptide fragments or peptides containing a portion of the IL-31 polypeptide with the epitope described herein. Such fragments or peptides may contain an “immunogenic epitope,” which is part of a protein that elicits an antibody response when a whole protein is used as an immunogen. Immunogenic epitope peptides can be identified using standard methods (see, for example, Geysen et al., Proc. Nat'l Acad. Sci. USA 81: 3998 (1983)). The binding of antibodies to these functional fragments leads to inhibition, blocking, neutralization and / or reduction of IL-31 signal transmission to its recognizing receptor.

In contrast, polypeptide fragments or peptides may comprise an “antigenic epitope”, which is a portion of a protein molecule to which an antibody can specifically bind. Certain epitopes consist of linear or continuous sections of amino acids, and the antigenicity of such an epitope is not disturbed by denaturing substances. It is known in the art that relatively short synthetic peptides that can mimic protein epitopes can be used to stimulate the production of antibodies against protein (see, for example, Sutcliffe et al., Science 219: 660 (1983)). Thus, the antigenic epitope-containing peptides and polypeptides of the invention are useful for the induction of antibodies (e.g., neutralizing antibodies) that bind to the polypeptides described herein. Hopp / Woods hydrophilicity profiles can be used to identify sites that have the greatest antigenic potential (Hopp et al., 1981, ibid., And Hopp, 1986, ibid.). For example, in human IL-31, hydrophilic sites include amino acid residues 54-59 in SEQ ID NO: 2, amino acid residues 129-134 in SEQ ID NO: 2, amino acid residues 53-58 in SEQ ID NO: 2, amino acid residues 35-40 in SEQ ID NO: 2 and amino acid residues 33-38 in SEQ ID NO: 2. For example, in mouse IL-31, hydrophilic sites include amino acid residues 34-39 in SEQ ID NO: 11, amino acid residues 46-51 in SEQ ID NO: 11, amino acid residues 131-136 in SEQ ID NO: 11, amino acid residues 158-163 in SEQ ID NO: 11 and amino acid residues 157-162 in SEQ ID NO: 11.

The epitope-containing antigenic peptides and polypeptides preferably contain at least four to ten amino acids, at least ten to fourteen amino acids, or from about fourteen to about thirty amino acids. SEQ ID NO: 2 or SEQ ID NO: 4. Such epitope-containing peptides and polypeptides can be prepared by fragmentation of an IL-31 polypeptide or chemical synthesis of peptides as described herein. Moreover, epitope selection can be carried out using phage display of random peptide libraries (see, for example, Lane and Stephen, Curr. Opin. Immunol. 5: 268 (1993); and Cortese et al, Curr. Opin. Biotechnol. 7: 616 (1996)). Standard methods for identifying epitopes and producing antibodies based on small peptides that contain an epitope are described, for example, Mole, "Epitope Mapping," in Methods in Molecular Biology, Vol. 10, Manson (ed.), Pages 105-116 (The Humana Press, Inc. 1992); Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies", in Monoclonal Antibodies: Production, Engineering, and Clinical Application, Ritter and Ladyman (eds.), Pages 60-84 (Cambridge University Press 1995), and Coligan et al. (eds.), Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley & Sons 1997).

Antibody activity, as described herein, can be determined by their ability to inhibit or reduce proliferation using a variety of assay methods that determine proliferation and / or binding to cells expressing IL-31RA receptor. Of particular interest are changes in IL-31-dependent cells. Suitable cell lines for generating IL-31-dependent cell lines include the IL-3-dependent BaF3 cell line (Palacios and Steinmetz, Cell 41: 727-734, 1985; Mathey-Prevot et al., Mol Cell. Biol. 6 : 4133-4135, 1986), FDC-P1 (Hapel et al., Blood 64: 786-790, 1984), and MO7e (Kiss et al., Leukemia 7: 235-240, 1993). Growth factor dependent cell lines can be obtained according to published methods (e.g., Greenberger et al., Leukemia Res. 8: 363-375, 1984; Dexter et al., Baum et al. Eds., Experimental Hematology Today, 8th Ann Mtg. Int. Soc. Exp. Hematol. 1979, 145-156, 1980).

The activity of anti-IL-31 antibodies described herein can be determined using a silicone-based biosensor microphysiometer that measures the rate of extracellular acidification or proton excretion associated with receptor binding and subsequent physiological cellular responses. An exemplary device is a Cytosensor ™ microphysiometer manufactured by Molecular Devices, Sunnyvale, CA. In this way, many cellular responses can be determined, such as cell proliferation, ion transport, energy production, inflammatory response, activation of the regulator and receptor, and the like. See, for example, McConnell, H.M. et al., Science 257: 1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol. 228: 84-108, 1997; Arimilli, S. et al., L Immunol. Meth. 212: 49-59, 1998; Van Liefde, I. et al., Eur. J. Pharmacol. 346: 87-95, 1998.

Antagonists are also suitable as research reagents for characterizing ligand-receptor interaction sites. Antagonists are useful for inhibiting the reproduction, proliferation, activation and / or differentiation of cells involved in the regulation of hematopoiesis. IL-31 activity inhibitors (IL-31 antagonists) include antibodies against IL-31 and soluble receptors for IL-31, as well as other peptide and non-peptide substances (including ribozymes).

Inhibition of IL-31 activity can be determined using a number of assay methods. In addition to the assay methods described herein, samples can be tested for inhibition of IL-31 activity using a variety of assays designed to determine receptor binding, stimulate / inhibit IL-31 dependent cell responses, or proliferate IL-31RA receptor-expressing cells.

IL-31 binding polypeptide can also be used to purify the ligand. The polypeptide is immobilized on a solid support, such as granules of agarose, cross-linked agarose, glass, cellulose resins, silica resins, polystyrene, cross-linked polyacrylamide or similar materials that are stable under the conditions of use. Methods for binding polypeptides to solid supports are known in the art and include amine chemical groups, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation. The resulting material is typically shaped into a column, and ligand-containing fluids are passed through the column one or more times to allow the ligand to bind to the receptor polypeptide. The ligand is then eluted using changes in salt concentration, chaotropic substances (guanidine-HCl), or pH to disrupt ligand-receptor binding.

Advantageously, an analysis system may be used in which a ligand binding receptor (or antibody, one member of a complement / anti-complement pair) or a binding fragment thereof, and a commercially available biosensor device (BIAcore, Pharmacia Biosensor, Piscataway, NJ) are used. Such a receptor, antibody, member of a complement / anti-complement pair or fragment is immobilized on the surface of the receptor chip. The use of this device is described in Karlsson, J. Immunol. Methods 145: 229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234: 554-63, 1993. A receptor, antibody, member or fragment is covalently attached, using the chemical group of an amine or sulfhydryl, to dextran fibers that attach to a gold film on a flow cell. The test sample is passed through the cell. If there is a ligand, epitope or the opposite member of the complement / anti-complement pair in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing changes in the refractive index of the medium, which is determined by the change in the surface plasmon resonance of the gold film. This system makes it possible to determine the rates of association and dissociation from which binding affinity can be calculated, and estimates of binding stoichiometry. Alternative ligand / receptor binding can be analyzed using SELDI ™ technology (Ciphergen, Inc., Palo Alto, CA).

Antibodies against IL-31 can be used to block the biological effects of the pro-inflammatory IL-31, and they are useful as anti-inflammatory drugs for a variety of diseases, as described in the present description. One skilled in the art will recognize that antigenic epitope-containing polypeptides contain a sequence of at least 6, preferably at least 9, and more preferably at least 15 to about 30 consecutive amino acid residues of the IL-31 polypeptide (e.g., SEQ ID NO: 2). Also included are polypeptides containing a larger fragment of an IL-31 polypeptide, i.e. from 30-100 residues up to a full-sized amino acid sequence. Antigens or immunogenic epitopes may also include attached labels, adjuvants, delivery agents, and carriers, as described herein. Suitable antigens include the IL-31 polypeptide encoded by SEQ ID NO: 2 from amino acid number 24 to amino acid number 164, or a continuous fragment thereof from 9 to 141 amino acids. Other suitable antigens include full-length and mature IL-31, A-D helices, and individual or compound helices A, B, C and D of the globule structure of the four IL-31 helices, as described herein. Peptides preferred for use as antigens are hydrophilic peptides, such as those predicted by one skilled in the art, based on the hydrophobicity schedule as described herein, for example, amino acid residues 114-119, 101-105, 126-131, 113 -118 and 158-162 in SEQ ID NO: 2; and amino acid residues 34-39, 46-51, 131-136, 158-163 and 157-162 in SEQ ID NO: 11. Moreover, IL-31 antigenic epitopes predicted by the Jameson-Wolf graph, for example, using the DNASTAR Protean program (DNASTAR, Inc., Madison, WI), are preferred antigens and can be easily determined by one skilled in the art.

Antibodies due to the immune response induced by inoculating an animal with these antigens can be isolated and purified as described herein. Methods for producing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications. CRC Press, Inc., Boca Raton, FL, 1982.

As will be apparent to one of ordinary skill in the art, polyclonal antibodies can be obtained by inoculation with a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice and rats, the IL-31 polypeptide, or a fragment thereof. The immunogenicity of the IL-31 polypeptide can be enhanced by the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete and incomplete adjuvant. Polypeptides suitable for immunization also include fusion polypeptides, such as a fusion polypeptide from IL-31 or part thereof with an immunoglobulin polypeptide or a maltose binding protein. The polypeptide immunogen may be a full-sized molecule or part thereof. If the polypeptide moiety is "hapten-like", then for immunization the moiety can advantageously be coupled to or bound to a macromolecular carrier (such as cochlear lymph lymphocyte hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid).

Antibodies are considered to be specifically binding if: 1) they have a threshold level of binding activity, and 2) they do not cross-react to a large extent with similar polypeptide molecules. The threshold level of binding is determined if the anti-IL-31 antibodies described herein bind to an IL-31 polypeptide, peptide or epitope with an affinity of at least 10 times that of the control (non-IL-31) polypeptide. Preferably, the antibodies have a binding affinity of (K _a ) of 10 ⁶ M ^-1 or more, preferably 10 ⁷ M ^-1 or more, more preferably 10 ⁸ M ^-1 or more, and most preferably 10 ⁹ M ^-1 or more. The binding affinity of an antibody can easily be determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949).

The absence of significant cross-reaction of anti-IL-31 antibodies with related molecules is indicated by detecting an IL-31 polypeptide but not related polypeptides using an antibody using standard Western blot analysis (Ausubel et al., Ibid.). Examples of known related polypeptides are those described in the prior art, such as orthologs and paralogs, and similar known members of the protein family. You can also screen using non-human IL-31 and mutant IL-31 polypeptides. Moreover, antibodies can be screened against known similar polypeptides to isolate a group that specifically binds to IL-31 polypeptides. For example, antibodies induced against IL-31 adsorb to related polypeptides attached to an insoluble matrix; antibodies specific for IL-31 pass through the matrix under appropriate buffer conditions. Screening makes it possible to isolate polyclonal and monoclonal antibodies that do not cross-react with known closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995). Screening and isolation of specific antibodies is well known in the art. See Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Principles and Practice, Goding, J.W. (eds.), Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984. Specific binding of anti-IL-31 antibodies can be detected by a number of methods in this area and the methods described below.

Monoclonal antibodies can be obtained, for example, by immunization of Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) with a purified mature recombinant human IL-31 polypeptide (amino acid residues from 27 (Leu) to 167 (Thr) in SEQ ID NO: 2) or a mouse ortholog derived from expression systems described herein. Rats receive an intraperitoneal (IP) injection of 100 μg of purified recombinant human IL-31 protein in complete Freund's adjuvant (Pierce, Rockford, IL) followed by secondary IP injections of 50 μg of purified recombinant protein in incomplete Freund's adjuvant every two weeks. From seven to ten days after the introduction of the third secondary injection, blood is collected from animals and serum is collected.

Characterization of samples of rat serum specific for human IL-31 is carried out by ELISA on a titer to specifically target antibodies to biotinylated human IL-31.

From 2 high titer rats, splenocytes and lymph node cells are collected and fused to SP2 / 0 myeloma cells (mice) using PEG 1500 in two separate fusion processes (4: 1 ratio for fusion of splenocytes and myeloma cells, "Antibodies A Laboratory Manual , E. Harlow and D. Lane, Cold Spring Harbor Press). After 10 days of cultivation after fusion, specific antibody-producing hybridoma pools are identified by ELISA. Hybridoma pools positive in both ELISA protocols are further analyzed for their ability to block or reduce the activity of purified recombinant IL-31 with respect to receptor binding ("neutralization assay").

Hybridoma pools for which positive results were obtained by ELISA alone or by ELISA and “neutralization assay” are cloned at least twice by limiting dilution.

Monoclonal antibodies purified from tissue culture medium are characterized for their suitability in an ELISA for the quantification of recombinant and natural human IL-31. Antibodies are selected and quantified.

Monoclonal antibodies purified from tissue culture medium are characterized by their ability to block or reduce the binding activity ("neutralization assay") of purified recombinant huIL-31 on BaF3 / MPL-IL-31 cells. In this way, a number of "neutralizing" monoclonal antibodies are identified. Hybridomas expressing the described neutralizing monoclonal antibodies against human IL-31 can then be deposited in the patent collection of the American Type Tissue Culture Collection (ATCC; Manassas VA) as initial deposited samples under the Budapest Treaty.

Monoclonal antibodies can be obtained by immunizing Lewis rats (Rockland Immunochemicals, Gilbertsville, PA) with cleaved and purified recombinant IL-31 fusion protein. Each rat is given an intraperitoneal (IP) injection of 100 μg of purified recombinant fusion protein in complete Freund's adjuvant (Pierce, Rockford, IL) followed by secondary IP injections of 50 μg of purified recombinant protein in incomplete Freund's adjuvant every two weeks for four weeks. After the first four weeks of immunization, auxiliary secondary intraperitoneal injections are carried out with 50 μg of cleaved purified recombinant protein attached to a carrier protein, which is cochlear lymphocytic hemocyanin (KLH, Pierce, Rockford, IL), in incomplete Freund's adjuvant every two weeks for four weeks. From seven to ten days after the fourth secondary injection, blood is collected from animals and serum is collected. Characterization of samples of rat serum specific for IL-31 is performed by ELISA using purified recombinant IL-31-Fc fusion protein as a specific target for an antibody and an unrelated fusion protein as a non-specific target for an antibody at a concentration of 500 ng / ml. Splenocytes were selected from one or more high titer rats and fused to SP2 / 0 myeloma cells (mice) in an optimized PEG mediated fusion protocol (Rockland Immunochemicals). After 12 days of cultivation after fusion, specific antibody-producing hybridoma pools are identified by ELISA using purified recombinant IL-31 fusion protein as a specific target for the antibody and an unrelated fusion protein as a non-specific target for the antibody, where each concentration is 500 ng / ml . Hybridoma pools that are positive only for a specific target for the antibody are further analyzed for their ability to block or reduce receptor binding activity ("neutralization assay") of recombinant huIL-31 on BaF3 / MPL-IL-31 cells and for binding ability in FACS analysis as a target for antibodies. Hybridoma pools for which positive results are obtained in an ELISA assay and positive results in either FACS or a “neutralization assay” are cloned at least twice by limiting dilution.

Five rat hybridomas with anti-mouse antibodies were similarly obtained and assigned the following clone designations: clone 271.9.4.2.6, clone 271.26.6.6.1, clone 271.33.1.2.2, clone 271.33.3.2.1 and clone 271.39.4.6.5 . See Example 1. Monoclonal antibodies produced by these clones were characterized by a number of methods, including sorting (ie, determining the ability of each antibody to inhibit binding by any other binding), relative affinity, and neutralization. It turned out that monoclonal antibodies were divided into two separate series, where clone 271.33.3.2.1 binds to an epitope different from the epitopes of the other four clones. The relative binding affinity for four of these monoclonal antibodies is described in Example 14.

The binding affinity of monoclonal antibodies can be achieved. A rat specific IgG-Fc gamma gamma antibody (Jackson) is immobilized on a Biacore CM5 chip. The assay is optimized to bind each mAb to the capture surface with anti-rat antibody, and then a series of IL-31 concentrations are passed through the mAb to determine association (Ka) and dissociation (Kd). After each analysis, the surface is regenerated to an antibody against rat antibodies by 2 injections of 20 mM HCl. Data is obtained for each sample, and evaluation software (BIAevaluation software version 3.2, Pharmacia BIAcore, Uppsala, Sweden) is used to evaluate the kinetics of binding of the anti-IL-31 antibody to the IL-31 protein.

mouse anti-IL-31 mouse mAbs can be prepared as follows. Six to twelve weeks old IL-31 knockout mice are immunized by intraperitoneal injection of 25-50 μg of human muFc soluble IL-31 protein mixed 1: 1 (v: v) with Ribi adjuvant (Sigma) according to a two-week schedule . Seven to ten days after the third immunization, blood samples are obtained by retroorbital blood sampling, serum is collected and evaluated for its ability to inhibit IL-31 binding in neutralization assays (for example, described in the present description) and stain transfected IL-31 cells 293 with respect to non-transfected cells in a FACS staining assay. Immunization of mice is continued and blood samples are taken and evaluated as described above until the neutralizing titers reach a plateau. Then, mice with the highest neutralizing titers are given an intravascular injection of 25-50 μg of soluble IL-31-Fc protein in PBS. Three days later, the spleen and lymph nodes from these mice are harvested and used to produce hybridomas, for example, using mouse myeloma cells (P3-X63-Ag8.653.3.12.11) or other suitable cell lines for this area, using standard methods known in the art (e.g., see Kearney, JF et al., J Immunol. 123: 1548-50, 1979; and Lane, RD J Immunol Methods 81: 223-8, 1985).

Primary screening for their ability to bind IL-31-muFc protein by ELISA using secondary stop reagents in the form of HRP conjugated goat anti-mouse light chain antibodies and mouse lambda for 8-8 days after fusion in hybridoma supernatants can be used to identify bound antibodies the mouse.

Biochemical confirmation that the target molecule, IL-31, recognized by the alleged anti-IL-31 mAbs, is indeed IL-31, is carried out by standard immunoprecipitation followed by SDS-PAGE analysis or Western blotting methods, both of which use soluble preparations membranes from IL-31 Baf3 cells transfected with respect to untransfected cells. mAbs are tested for their ability to specific immunoprecipitate or to Western blot with soluble IL-31-muFc protein.

Monoclonal antibodies purified from tissue culture medium were characterized by their ability to block or inhibit the ability of IL-31 to bind to its receptor in a neutralization assay. In this way twenty "neutralizing" monoclonal antibodies were identified. Ten of them were identified as “good neutralizers” after the first cloning cycle: clone 292.72.3, clone 292.118.6, clone 292.63.5, clone 292.64.6, clone 292.84.1, clone 292.109.4, clone 292.12.3, clone 292.51.5, clone 292.39.5 and clone 292.105.4. The other ten received the following clone symbols after the first cloning cycle: clone 294.35.2, clone 294.146.5, clone 292.152.4, clone 292.154.4, clone 294.154.5, clone 294.35.3, clone 291.78.4, clone 294.155.6 , clone 294.158.5, clone 294.163.2 and clone 294.144.3.

For these monoclonal antibodies, a second cloning cycle was carried out and again characterized by their ability to block or inhibit the ability of IL-31 to bind to its receptor in a neutralization assay. The clone designations after the second cloning cycle for “good neutralizers” were: clone 292.12.3.1, clone 292.63.5.3, clone 292.72.3.1, clone 292.84.1.6, clone 292.118.6.4, clone 292.64.6.5.5, clone 292.39.5.3 , clone 292.51.5.2, clone 292.109.4.4 and clone 292.105.4.1. Six of the other ten possess the following clone designations: clone 292.152.4.1, clone 294.158.5.2, clone 294.32.2.6.3, clone 294.144.3.5, clone 294.154.5.3 and clone 294.163.2.1.

Hybridomas expressing the neutralizing anti-human IL-31 monoclonal antibodies described above were deposited with the American Type Culture Collection (ATCC; Manassas VA) patent collection as initial deposited samples under the Budapest Treaty and were assigned the following ATCC registration numbers No: clone 292.12.3.1 (Patent Deposit Number in ATCC PTA-6815); clone 292.72.3.1 (Patent Deposit Number in ATCC PTA-6816); clone 292.63.5.3 (Patent Deposit Number in ATCC PTA-6829); clone 292.118.6.4 (Patent Deposit Number in ATCC PTA-6830); clone 294.163.2.1 (Patent Deposit Number in ATCC PTA-6831); clone 292.84.1.6 (Patent Deposit Number in ATCC PTA-6871); clone 294.35.2.6.3 (Patent Deposit Number in ATCC PTA-6872); clone 294.154.5.6 (Patent Deposit Number in ATCC PTA-6875); and clone 294.144.3.5 (Patent Deposit Number in ATCC PTA-6873).

A hybridoma expressing neutralizing anti-mouse IL-31 monoclonal antibodies described herein was deposited in the patent collection of the American Type Tissue Culture Collection (ATCC; Manassas VA) as an initial sample under the Budapest Treaty and was assigned ATCC registration number No: clone 271.26.6.6.1 (Patent Deposit Number in ATCC PTA-6874).

Monoclonal antibodies produced by these clone hybridomas can be cultured in a medium for growing from 90% Dulbecco medium modified by the Isca method with 2 mM L-glutamine, penicillin at a concentration of 100 μg / ml and streptomycin sulfate at a concentration of 100 μg / ml, and 10% clone I fetal serum (Hyclone Laboratories). Clone propagation can be carried out starting cultivation with 2 · 10 ⁵ cells / ml with maintenance between 1 · 10 ⁵ and 5 · 10 ⁵ cells / ml at 37 ° C and 5-6% CO. In subsequent transfers, cells can be adapted to serum-free conditions. Frozen cells are stored in 90% serum, 10% DMSO and stored in the gaseous phase in a device for freezing with liquid nitrogen.

Monoclonal antibodies in a tissue culture medium are characterized by their ability to block, inhibit, prevent, or reduce receptor binding when grown in the presence of purified recombinant human IL-31 proteins. For example, monoclonal antibodies produced by these clones have been characterized by a number of methods, including sorting (i.e. determining the ability of each antibody to inhibit binding by any other binding), relative affinity, and neutralization. It turned out that ten good neutralizing antibodies belong to one series, while other monoclonal antibodies are distributed into three separate series. In addition, eight of the good neutralizing antibodies are the IgG1 isotype and the other two are the IgG2a isotype.

Monoclonal antibodies from hybridoma supernatants were recovered using goat pAb against mouse Fc and the apparent binding affinity (EC50) of biotinylated IL-31 was determined. Under these assay conditions, good neutralizers have the smallest and comparable EC50 values of ~ 4 ng / ml Bt-IL31. The apparent affinity of weak neutralizers ranges from ~ 10 ng / ml to 236 ng / ml Bt-IL31.

Monoclonal antibodies obtained by the methods described herein can be tested for neutralization in a variety of ways. For example, a luciferase assay can be used as described in US Published Patent Application (See Publication Number 20030224487, Sprecher, Cindy et al., 2003). In addition, neutralization can be tested by measuring the decrease in the production of pro-inflammatory chemokines, such as TARC and MDC, by keratinocyte cultures in the presence of a ligand and a monoclonal antibody. Neutralization can also be determined by in vivo models, as described herein.

In one embodiment, the antibodies of the invention are antigen binding fragments of the human antibodies of the invention and include, but are not limited to, Fab, Fab 'and F (ab') 2, Fd, single chain Fv (scFv), single chain disulfide linked antibodies Fv bonds (sdFv), and fragments containing either the VL domain or the VH domain. Antigen binding fragments of antibodies, including single chain antibodies, may contain the variable region (s) separately or in combination with all or part of: the hinge region, domains C _H1 , C _H2 and C _H3 . This invention also relates to antigen-binding fragments containing also any combination of a variable region (s) with a hinge region, domains C _H1 , C _H2 and C _H3 .

In another embodiment, the antibodies of the invention may be monospecific, bispecific, trispecific, or more multispecific. Multispecific antibodies may be specific for different epitopes of a polypeptide according to the invention or may be specific both to a polypeptide according to the invention and to a heterologous epitope, such as a heterologous polypeptide or solid support material. See, for example, PCT Publication WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147: 60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148: 1547-1553 (1992).

The present invention also relates to genetically modified antibodies that are functionally equivalent to the antibodies described above. Modified antibodies that provide enhanced stability and / or therapeutic efficacy are preferred. Examples of modified antibodies include antibodies with conservative substitutions of amino acid residues, and one or more deletions or insertions of amino acids that do not result in a significant negative change in antigen binding property. Substitutions can vary from a change or modification of one or more amino acid residues to a complete redesign of the site, while maintaining the suitability for therapeutic purposes. Antibodies according to the invention can be modified post-translationally (e.g., acetylation and phosphorylation), or they can be modified synthetically (e.g., labeling).

Genetically modified antibodies also include chimeric antibodies derived from anti-IL-31 antibodies. Chimeric antibodies contain a variable region of a mouse or rat and a constant region of a person, so that the chimeric antibody has a longer half-life and is less immunogenic when administered to humans. A method for producing chimeric antibodies is known in the art. The variable regions of these antibodies can be linked to the constant region of human IgG to produce the desired chimeric antibody.

Genetically modified anti-IL-31 antibodies used according to the invention include a humanized variant of the antibodies described herein. Humanized antibodies contain the CDRs of a donor mouse immunoglobulin and framework regions of the human heavy and acceptor immunoglobulin light chain and light chain. A method of obtaining a humanized antibody is described in US patent No. 5301101; 5,585,089; 5693762 and 6180370 (all of which are incorporated into this description by reference in full). Then, the CDRs of these antibodies can be transplanted into any selected human framework region that is known in the art to obtain the desired humanized antibody.

Antibodies according to the invention can be described or characterized in terms of the epitope (s) or part (s) of the polypeptide according to the invention, which they recognize or specifically bind. The epitope (s) or polypeptide part (s) can be characterized as described herein, for example, at the N-terminal and C-terminal positions, in size in adjacent amino acid residues, or are presented in tables and sequences. Antibodies that specifically bind to any epitope or polypeptide of the invention may also be excluded. Thus, the present invention relates to antibodies that specifically bind the polypeptides according to the invention, and provides for their exclusion.

This invention also relates to antibodies that competitively inhibit the binding of a monoclonal antibody to a polypeptide according to the invention, preferably a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4. Competitive inhibition can be determined by any method known in the art, for example, using the competitive binding assays described herein. In preferred embodiments, the antibody competitively inhibits the binding of the monoclonal antibody of the invention by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% with the SEQ ID polypeptide NO: 2 or SEQ ID NO: 4.

This invention also relates to antibodies that competitively inhibit the binding of an antibody to an epitope according to the invention, as determined by any method known in the art, for determining competitive binding, for example, by immunoassay methods described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

Antibodies according to the invention include derivatives that are modified, namely, by covalently attaching any type of molecule to the antibody so that covalent attachment does not prevent the antibody from forming an anti-idiotypic response. By way of non-limiting example, derivative antibodies include antibodies modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization using known protective / blocking groups, proteolytic cleavage, binding to a cell ligand or other protein, and t .d. Any of a variety of chemical modifications can be carried out by known methods, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the derivative may contain one or more non-classical amino acids.

Antibodies according to the invention also include antibodies or fragments thereof which have a half-life (e.g., serum half-life) in a mammal, preferably in humans, more than 15 days, preferably more than 20 days, more than 25 days, more than 30 days, more than 35 days, more than 40 days, more than 45 days, more than 2 months, more than 3 months, more than 4 months or more than 5 months. The increased half-life of the antibodies of the invention or fragments thereof in a mammal, preferably in humans, leads to a higher serum titer of said antibodies or antibody fragments in a mammal and, thus, to a decrease in the frequency of administration of said antibodies or antibody fragments and / or to a decrease in the concentration of said antibodies or antibody fragments to be administered.

Antibodies or fragments thereof having an increased in vivo half-life can be obtained by methods known to those skilled in the art. For example, antibodies or fragments thereof with an increased in vivo half-life can be obtained by modifying (e.g., replacing, deleting or inserting) amino acid residues that are identified as being involved in the interaction between the Fc domain and the FcRn receptor (see, for example, international publications No. No. WO 97/34631 and WO 02/060919, which are incorporated herein by reference in full). Antibodies or fragments thereof with increased in vivo half-lives can be obtained by attaching a polymer molecule, such as high molecular weight polyethylene glycol (PEG), to the indicated antibodies or antibody fragments. PEG can be attached to antibodies or antibody fragments with or without a multifunctional linker, either by site-specific conjugation of PEG with the N- or C-terminus of said antibodies or antibody fragments, or via epsilon amino groups located on lysine residues. Derivatives with a linear or branched polymer are used that cause minimal loss of biological activity. In order to ensure proper conjugation of PEG molecules, the conjugation level is carefully monitored with antibodies by SDS-PAGE and mass spectrometry. Unreacted PEG can be separated from antibody-PEG conjugates, for example, by gel filtration or ion exchange chromatography.

It is understood that humanized antibodies constructed by this method may have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Conservative substitutions mean combinations such as gly, ala; val, ile, leu; asp glu; asn, gln; ser, thr; lys, arg; and phe, tyr.

Methods of humanizing non-human antibodies are well known in the art. Typically, humanized immunoglobulins, including humanized antibodies, are constructed by genetic engineering. Most of the humanized immunoglobulins that have been previously described (Jones et al., Suff. Cit.; Verhoeyen et al., Acc. Cit .; Riechmann et al., Acc. Cit.) Contain a framework region that is identical to the framework region of a particular chain human immunoglobulin, an acceptor, and three CDRs from a non-human immunoglobulin donor chain. Specifically, humanized antibodies are antibody molecules from antibodies of a non-human species that bind to a desired antigen having one or more complementarity determining regions (CDRs) of a non-human species and frame regions from a human immunoglobulin molecule.

The present invention relates to criteria by which a limited number of amino acids are selected in the frame region of a chain of a humanized immunoglobulin so that they are the same amino acids in the same positions as in the donor and not in the acceptor, in order to increase the affinity of the antibody containing the humanized chain immunoglobulin.

Antibodies according to the invention are obtained in part on the basis of a model which, in previous methods for producing antibodies (using, for example, mouse antibodies as a source of CDR), leads to two reasons for the loss of affinity:

(1) When combining a mouse CDR with a human framework region, the mouse amino acids in the framework region located close to the CDR are replaced with human amino acids. Without being bound by any theory, these altered amino acids may slightly deform the CDRs due to the fact that they create electrostatic or hydrophobic forces that are different from mouse donor antibodies, and deformed CDRs may not provide the same effective contact with the antigen as these CDRs provided in donor antibody.

(2) Amino acids in the original mouse antibody, which are located close to the CDR but are not part of them (i.e. are still part of the framework region), can provide contact with the antigen, which can contribute to affinity. These amino acids are lost during antibody humanization, since all amino acids of the skeleton region are human.

To avoid these problems and to obtain humanized antibodies that have a very high affinity for the desired antigen, one or more of the following principles according to the invention are applied for the construction of humanized immunoglobulins. In addition, the criteria can be used individually, or, if necessary, in combination, to achieve the desired affinity or other characteristics.

The principle is that a frame region from a particular human immunoglobulin, which is highly homologous to a donor immunoglobulin to be humanized, is used as an acceptor, or a consensus framework region of a plurality of human antibodies is used. For example, a comparison of the sequence of the variable region of the heavy (or light) chain of the mouse and the variable regions of the heavy (or light) chain of a person in a database (for example, the Identification Resource of Proteins of a National Biomedical Research Institution) shows that the degree of its homology with different parts of a person varies significantly. typically from about 40% to about 60-70%. Choosing one of the variable regions of the human heavy (respectively light) chains as the acceptor immunoglobulin, which is the most homologous to the variable region of the heavy (respectively light) chain of the donor immunoglobulin, less amino acids are replaced when the donor immunoglobulin is converted to humanized immunoglobulin. Thus, and also without any connection with any theory, it is believed that there is a small probability of amino acid substitution near the CDR, which changes their conformation. Moreover, the exact overall configuration of a humanized antibody containing a humanized immunoglobulin chain may be more closely related to the configuration of a donor antibody, which also reduces the likelihood of CDR deformation.

As a rule, to obtain the framework region of the heavy chain, and similarly for the light chain, one of the 3-5 most homologous sequences of the variable regions of the heavy chain in a representative collection of at least about 10 to 20 different human heavy chains is selected as an acceptor. Preferably, one of the 1-3 most homologous variable regions is used. The selected immunoglobulin acceptor chain most preferably has at least about 65% homology to the framework region with the donor immunoglobulin.

In many cases, it is considered preferable to use light and heavy chains from the same human antibody as the acceptor sequences to ensure that the humanized light and heavy chains are in acceptable contact with each other. In this case, donor light and heavy chains are compared only with chains of human antibodies with a known complete sequence, for example, antibodies Eu, Lay, Pom, Wol, Sie, Gal, Ou and WEA antibodies (Kabat et al., Art. ; sometimes the last few amino acids of the human chain are unknown, and they must be established by homology with other human antibodies). A human antibody is selected in which the sequences of the variable regions of the light and heavy chains, taken together, are, in general, the most homologous to the donor sequences of the variable region of the light and heavy chains. Sometimes, heavy chain sequences attach greater importance. Then, the selected human antibody provides acceptor sequences of both light and heavy chains. In practice, it is often found that the human antibody Eu plays this role.

In accordance with the so-called “best match” method, the variable domain sequence of a rodent antibody is screened against a whole library of known human variable domain sequences. The human sequence that is most similar to the rodent sequence is then taken as the human framework region (FR) for the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993); Chothia et al., J. Mol Biol., 196: 901 (1987)). Another method uses a specific framework region formed by the consensus sequence of all human antibodies of a particular subgroup of the light or heavy chain. The same framework region can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci, USA, 89: 4285 (1992); Presta et al., J. Immnol., 151: 2623 ( 1993)).

Regardless of how the acceptor immunoglobulin is chosen, higher activity can be achieved by selecting a small amount of amino acids in the frame region of the humanized immunoglobulin chain, which are the same amino acids at the same positions in the donor, and not in the acceptor. Often, framework region residues in human framework regions are replaced with the corresponding residue from the CDR of a donor antibody to alter, preferably improve, the binding of the antibody. These substitutions in the framework region are identified by methods well known in the art, for example, by modeling the interaction of CDR residues and the framework region to identify framework residues important for antigen binding and sequence comparisons to identify rare framework region residues at specific positions. (See, for example, Queen et al., US patent No. 5585089; Riechmann et al., Nature 332: 323 (1988), which are incorporated herein by reference in full). Antibodies can be humanized using various methods known in the art, including, for example, CDR transplantation (EP 239400; PCT publication WO 91/09967; US Patent Nos. 5,225,539; 5,530,101 and 5,550,089), hyperchimerization or surface modification (EP 592106; EP 519596; Padlan, Molecular Immunology 28 (4/5): 489-498 (1991); Studnicka et al., Protein Engineering 7 (6): 805-814 (1994); Roguska. Et al., PNAS 91: 969- 973 (1994)), and rearrangement of chains (US patent No. 5565332). Thus, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567), wherein essentially less than the entire human variable domain is replaced by the corresponding sequence of a non-human species.

The second principle is that the following categories determine which amino acids can be selected from the donor. Preferably, in many or all amino acid positions in one of these categories, a donor amino acid is actually selected.

Category 1: The amino acid position is in the CDR, as determined by Kabat et al., UK. Op.

Category 2: If the amino acid in the skeleton region of the human acceptor immunoglobulin is unusual (i.e., "rare", which, as used herein, indicates an amino acid found in this position in less than about 20%, however, as a rule, in less than approximately 10% of the sequences of the V region of the heavy (respectively light) chain in a representative database), and if the donor amino acid at this position is typical of human sequences (ie, “common”, which is used in the present herein indicates an amino acid occurring in more than about 25% but usually more than about 50% of sequences in a representative data base), then the donor amino acid may be selected rather than the acceptor. This criterion helps to ensure that the atypical amino acid in the human framework does not violate the structure of the antibody. Moreover, by replacing a rare amino acid with an amino acid from a donor antibody that is typical of human antibodies, a less immunogenic, humanized antibody can be obtained. All sequences of the variable regions of the human light and heavy chains are respectively grouped into “subgroups” of sequences that are particularly homologous to each other and have the same amino acids at certain critical positions (Kabat et al., J. Cit.). When determining that an amino acid in a human acceptor sequence is “rare” or “common” among human sequences, it is often preferable to consider only those human sequences that are in the same subgroup as the acceptor sequence.

Category 3: At positions immediately adjacent to one or more of the 3 CDRs in the primary chain sequence of the humanized immunoglobulin, the amino acid (s) of the donor, rather than the acceptor, can be selected. It is particularly likely that these amino acids interact with the amino acids in the CDR and, if selected from the acceptor, deform the donor CDRs and reduce affinity. Moreover, neighboring amino acids can interact directly with the antigen (Amit et al., Science, 233, 747-753 (1986), which is incorporated herein by reference) and it may be desirable to select these amino acids from the donor to maintain all contact with antigen, which provide the affinity of the original antibody.

Category 4: a 3-dimensional model, mainly of the original donor antibody, shows that certain amino acids outside the CDR are located close to the CDR and are highly likely to interact with the amino acids in the CDR through the formation of hydrogen bonds, van der Waals forces, hydrophobic interactions, etc. .d. At these amino acid positions, it is possible to select the amino acid of the donor immunoglobulin rather than the acceptor immunoglobulin. Amino acids in accordance with this criterion typically have a side chain atom within about 3 angstroms of some atom in the CDR, and they must contain an atom that can interact with CDR atoms in accordance with well-known chemical forces such as those listed above.

In the case of atoms that can form a hydrogen bond, a value of 3 angstroms is determined between their nuclei; however, for atoms that do not form a bond, a value of 3 angstroms is determined between their van der Waals surfaces. Thus, in the latter case, for atoms that should be considered capable of interacting, the nuclei should be within about 6 angstroms (3 + the sum of the van der Waals radii). In many cases, the nuclei are located at a distance of 4 or 5 to 6 angstroms. When determining the possibility of the interaction of an amino acid with a CDR, it is preferable not to consider the last 8 amino acids of the heavy chain CDR 2 as part of the CDR, because, from the point of view of structure, these 8 amino acids have the properties of part of the frame region.

Amino acids in the skeleton region that are capable of interacting with amino acids in the CDR and which, therefore, belong to category 4, can be detected in another way. The surface area of each amino acid of the framework region available for the solvent is calculated in two ways: (1) in the whole antibody and (2) in a hypothetical molecule consisting of an antibody with deleted CDRs. A significant difference between these values of approximately 10 square meters. angstroms or more, shows that the solvent’s access to the amino acid of the framework region is at least partially blocked by the CDR, and thus that the amino acid contacts the CDR. The surface area of the amino acid available to the solvent can be calculated based on a 3-dimensional model of the antibody using algorithms known in the art (e.g., Connolly, J. Appl. Cryst. 16, 548 (1983) and Lee and Richards, J. Mol. Biol 55, 379 (1971), both of which are incorporated herein by reference). Amino acids of the framework region can also sometimes interact indirectly with CDRs, by affecting the conformation of other amino acids of the framework region, which in turn contact the CDRs.

Amino acids are known at certain positions in the framework region to interact with CDRs in many antibodies (Chothia and Lesk, J. Mol. Biol. 196, 901 (1987), Chothia et al., Nature 342, 877 (1989), and Tramontano et al., J. Mol. Biol. 215, 175 (1990), all of which are incorporated herein by reference), especially at positions 2, 48, 64 and 71 of the light chain and 26-30, 71 and 94 of the heavy chains (numbering according to Kabat, suff. cit.), and thus, these amino acids are generally categorized as 4. Typically, the humanized immunoglobulins of the invention include donor amines slots (where different) from the category 4, in addition to these amino acids. Amino acids at positions 35 of the light chain and 93 and 103 of the heavy chain are also likely to interact with CDR. In all of these enumerated positions, it is preferable to choose a donor amino acid for a humanized immunoglobulin instead of an acceptor amino acid (if they differ). On the other hand, certain provisions that may be classified as category 4, such as the first 5 amino acids of the light chain, can sometimes be selected from an acceptor immunoglobulin without loss of affinity in a humanized immunoglobulin. Chothia and Lesk (op. Cit.) Determine CDR in a different way than Kabat et al. (art. cit.). In particular, CDR1 is defined as including residues 26-32. Thus, Riechmann et al. (UK. Op.) choose these amino acids from donor immunoglobulins.

It is also important that the antibodies are humanized while maintaining high affinity for the antigen and other positive biological properties. To achieve this, in accordance with a preferred method, humanized antibodies are obtained through a process of analysis of the source sequences and various conceptual humanized products using three-dimensional models of the source and humanized sequences. Computer programs are available that illustrate and display probable three-dimensional conformational structures of immunoglobulin sequences selected as candidates. The study of these images allows us to analyze the likely role of residues in the functioning of the candidate immunoglobulin sequence, i.e. analyze residues that affect the ability of the candidate immunoglobulin to bind its antigen. Thus, FR residues can be selected and combined from recipient and import sequences to achieve the desired antibody properties, such as increased affinity for the target antigen (s). Typically, CDR residues are directly and most significantly involved in influencing antigen binding. Computer programs for creating models of proteins such as antibodies are widely available and well known to those skilled in the art (see Levy et al., Biochemistry, 28, 7168-7175 (1989); Bruccoleri et al., Nature, 335, 564-568 (1988); Chothia et al., Science, 233, 755-758 (1986), all of which are incorporated herein by reference). They are not part of this invention. Indeed, due to the fact that all antibodies have similar structures, known antibody structures that are available from the Brookhaven Protein Database can be used, if necessary, as approximate models of other antibodies. Commercially available computer programs can be used to display images of these models on a computer monitor, to calculate the distance between atoms and to evaluate the likelihood of the interaction of various amino acids (see Ferrin et al., J. Mol. Graphics, 6, 13-27 (1988)).

In addition to the above categories, which determine when an amino acid in a humanized immunoglobulin can be selected from a donor, certain amino acids in a humanized immunoglobulin can be selected either from a donor or from an acceptor, if they relate to:

Categories 5: If the amino acid in this position of the donor immunoglobulin is “rare” for human sequences, and the amino acid in this position in the acceptor immunoglobulin is also “rare” for human sequences as defined above, then you can choose the amino acid in this position of the humanized immunoglobulin, which represents some amino acid "typical" for human sequences. The preferred choice is the amino acid that is most often found at this position in known human sequences belonging to the same subgroup as the acceptor sequence.

Humanized antibodies typically have at least three potential advantages over mouse antibodies or, in some cases, chimeric antibodies for use in the treatment of humans:

(1) Due to the fact that the effector part is human, it can interact better with other parts of the human immune system (for example, it is more efficient to destroy target cells by complement dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC)).

(2) The human immune system should not recognize the framework region or constant region of the humanized antibody as foreign, and thus the antibody response against such an injected antibody should be less than against a completely foreign mouse antibody or a partially foreign chimeric antibody.

(3) Injected mouse antibodies have been described to have a half-life in human bloodstream that is significantly shorter than the half-life of normal antibodies (D. Shaw et al., J. Immunol., 138, 4534-4538 (1987)). Injected humanized antibodies are believed to have a half-life more similar to naturally occurring human antibodies, allowing the administration of smaller and less frequent doses.

In one aspect, the present invention relates to the construction of humanized antibodies that are obtained by expression of recombinant DNA segments encoding heavy and light chain CDRs from donor immunoglobulin capable of binding to the desired antigen, such as IL-31, attached to DNA segments encoding acceptor framework regions person.

DNA segments, as a rule, additionally include DNA expression control sequences operably linked to humanized immunoglobulin coding sequences, including naturally associated or heterologous promoter regions. Expression control sequences are eukaryotic promoter systems in vectors capable of transformation or transfection into eukaryotic host cells, however, control sequences for prokaryotic hosts can also be used. After embedding the vector in an appropriate host, the host is kept under conditions suitable for the expression of nucleotide sequences at a high level, and, if desired, humanized light chains, heavy chains, light / heavy chain dimers, or whole antibodies binding fragments or can be isolated and purified. other forms of immunoglobulin (see S. Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NY, (1979), which is incorporated herein by reference).

Human constant region DNA sequences can be isolated in accordance with well-known methods from a variety of human cells, but preferably from immortalized B cells (see Kabat et al. And WP87 / 02671). The source of CDRs for producing immunoglobulins according to the invention are likewise monoclonal antibodies capable of binding to a particular antigen, such as IL-31, and obtained by well-known methods from any suitable source, which is a mammal, including mice, rats, rabbits, or from other vertebrates, capable of producing antibodies. Suitable cells that are the source of the DNA sequences of the constant region and the framework region, and host cells for expression and secretion of immunoglobulin, can be obtained from many sources, such as the American type culture collection (Catalog of Cell Lines and Hybridomas, sixth edition (1988) Rockville, Md. USA, which is incorporated herein by reference).

In addition to the humanized immunoglobulins specifically described herein, other "substantially homologous" naturally occurring modified immunoglobulins can be easily constructed and manufactured using various recombinant DNA methods well known to those skilled in the art. As the basis of the humanized immunoglobulins according to the invention, many different human framework regions can be used, alone or in combination. As a rule, gene modifications can be easily done through a variety of well-known methods, such as site-directed mutagenesis (see Gillman and Smith, Gene, 8, 81-97 (1979) and S. Roberts et al., Nature, 328, 731- 734 (1987), both of which are incorporated herein by reference).

Humanitariannet antibodies according to the invention include fragments, as well as whole antibodies. Typically, these fragments compete with the whole antibody from which they are derived for antigen binding. Fragments are typically associated with an affinity of at least 10 ⁷ M ^-1 , and more particularly 10 ⁸ or 10 ⁹ M ^-1 (i.e., in the same range as the whole antibody). Fragments of a humanized antibody include single heavy chains, light chains, Fab, Fab ', F (ab') _2, and Fv. Fragments are obtained by recombinant DNA methods, or by enzymatic or chemical cleavage of whole immunoglobulins.

For a more detailed description of the humanization of antibodies, see European Patents Nos. EP 239400, EP 592106 and EP 519596; international publications No. WO 91/09967 and WO 93/17105; U.S. Patent Nos. 5,225,539, 5530101, 5565332, 5585089, 5766886 and 6407213; and Padlan, 1991, Molecular Immunology 28 (4/5): 489,498; Studnicka et al., 1994, Protein Engineering 7 (6): 805,814; Roguska et al., 1994, PNAS 91: 969 973; Tan et al., 2002, J. Immunol. 169: 1119 25; Caldas et al., 2000, Protein Eng. 13: 353 60; Morea et al., 2000, Methods 20: 267 79; Baca et al., 1997, J. Biol. Chem. 272: 10678 84; Roguska et al., 1996, Protein Eng. 9: 895 904; Couto et al., 1995, Cancer Res. 55 (23 Supp): 5973s 5977s; Couto et al., 1995, Cancer Res. 55: 1717 22; Sandhu, 1994, Gene 150: 409 10; Pedersen et al., 1994, J. Mol. Biol. 235: 959 73; Jones et al., 1986, Nature 321: 522-525; Reichmann et al., 1988, Nature 332: 323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2: 593-596.

Various methods have been developed to obtain antibody fragments. Traditionally, these fragments are obtained by proteolytic cleavage of whole antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science, 229: 81 (1985)). However, at present, these fragments can be obtained using recombinant host cells. For example, antibody fragments can be isolated from phage libraries of antibodies described above. Alternatively, Fab'-SH fragments can be directly isolated from E. coli and chemically coupled to form F (ab ') fragments. Sub.2 (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another approach, F (ab '). Sub.2 fragments can be isolated directly from cultures of recombinant host cells. Other methods for producing antibody fragments are understood by a skilled person. In addition, examples of methods that can be used to produce single chain Fv and antibodies include methods described in US Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203: 46-88 (1991); Shu et al., PNAS 90: 7995-7999 (1993) and Skerra et al., Science 240: 1038-1040 (1988).

The present invention relates to antibodies fused by recombinant methods or chemically conjugated (including both covalent and non-covalent conjugation) with a polypeptide (or part thereof, preferably with at least 10, 20 or 50 amino acids of a polypeptide) according to the invention to produce fusion proteins. Thus, this invention also relates to immunoconjugates containing the antibody described herein conjugated to a cytotoxic substance such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin of bacteria, fungi, plants or animals, or fragments thereof), or a radioactive isotope (i.e., radio conjugate).

The present invention relates to antibodies fused by recombinant methods or chemically conjugated (including both covalent and non-covalent conjugation) with a polypeptide (or part thereof, preferably with at least 10, 20 or 50 amino acids of a polypeptide) according to the invention to produce fusion proteins. The merger does not have to be direct, but can be carried out using linker sequences. Antibodies may be specific for antigens other than polypeptides (or parts thereof, preferably at least 10, 20 or 50 amino acids of a polypeptide) according to the invention. For example, antibodies can be used to target the polypeptides of the invention to specific cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the invention with antibodies specific for specific cell surface receptors. Antibodies fused or conjugated to the polypeptides of the invention can also be used in in vitro immunological assays and purification methods using methods known in the art. See, for example, Harbor et al., Supra, and PCT publication WO 93/21232; EP 439095; Naramura et al., Immunol. Lett. 39: 91-99 (1994); U.S. Patent No. 5,474,981; Gillies et al., PNAS 89: 1428-1432 (1992); Fell et al., J. Immunol. 146: 2446-2452 (1991), which are incorporated by reference in their entirety.

In addition, the present invention relates to compositions containing the polypeptides of the invention (e.g., polypeptides containing an immunogenic or antigenic epitope) fused or conjugated to heterologous polypeptide sequences (e.g., antibody domains other than variable regions). For example, the polypeptides of the invention may be fused or conjugated to or part of an antibody Fc region. For example, the polypeptides of the invention (including fragments or variants thereof) can be fused to the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or parts thereof (C _H1 , C _H2 , C _H3, or any combination or part thereof), to produce chimeric polypeptides. A portion of an antibody fused to a polypeptide of the invention may comprise a constant region, a hinge region, a C _H1 domain, a C _H2 domain and a C _H3 domain, or any combination of whole domains or parts thereof. The polypeptides can also be fused or conjugated to the above parts of the antibody with the formation of multimers. For example, Fc regions fused to the polypeptides of the invention can form dimers by forming disulfide bonds between the Fc regions. More multimeric forms can be obtained by fusion of polypeptides with parts of IgA and IgM. Methods for fusing or conjugating polypeptides of the invention to antibody portions are known in the art. See, for example, US Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5349053; 5,447,851; 5,112,946; EP 307434; EP 367166; PCT Publication WO 96/04388; WO 91/06570; Ashkenazi et ah, Proc. Natl. Acad. Sci. USA 88: 10535-10539 (1991); Zheng et al., J. Immunol. 154: 5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89: 11337-11341 (1992) (these references are incorporated by reference in their entirety). As another non-limiting example, the polypeptides and / or antibodies of the invention (including fragments or variants thereof) may be fused to albumin (including, but not limited to, human recombinant serum albumin or fragments or variants thereof (see, for example, US Patent No. 5876969, issued March 2, 1999, EP 0413622 and US Patent No. 5766883, issued June 16, 1998, incorporated by reference in their entirety. Polypeptides and / or antibodies of the invention (including fragments or variant thereof) s) may be fused to either N-terminus or from the C-end of the heterologous protein (e.g., Fc-immunoglobulin polypeptide or human serum albumin polypeptide). Also in this invention include polynucleotides encoding the fusion proteins of the invention.

As described above, the polypeptides of the invention can be fused or conjugated to the above portions of an antibody to increase the half-life of the polypeptides in vivo or for use in immunological assays using methods known in the art. In addition, the polypeptides according to the invention can be fused or conjugated to the above parts of the antibodies to facilitate purification. One published example describes chimeric proteins consisting of the first two domains of a human CD4 polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (See, for example, EP 394827; Traunecker et al., Nature 331: 84-86 (1988)). Increased antigen delivery across the epithelial barrier to the immune system is indicated for antigens (e.g., insulin) conjugated to an FcRn binding partner, such as IgG or Fc fragments (see, for example, PCT Publications WO 96/22024 and WO 99/04813). Polypeptides of the invention fused or conjugated to an antibody having disulfide-linked dimeric structures (due to IgG) may also be more effective at binding and neutralizing other molecules than a monomeric secreted protein or protein fragment alone (Fountoulakis et al., J. Biochem. 270: 3958-3964 (1995)). In many cases, the Fc portion of a fusion protein is suitable for treatment and diagnosis, and thus can lead, for example, to improved pharmacokinetic properties (EP A 232262). Alternatively, it is desirable to remove the Fc portion after expression, detection, and purification of the fusion protein. For example, the Fc region may interfere with treatment and diagnosis if a fusion protein is used as an antigen for immunization. In drug development, for example, human proteins, such as hIL-5, were fused to Fc sites for high throughput screening assays to identify hIL-5 antagonists. (See D. Bennett et al., J. Molecular Recognition 8: 52-58 (1995); K. Johanson et al., J. Biol. Chem. 270: 9459-9471 (1995)). Such methods also include, but are not limited to, the use of bifunctional substances for conjugation (see, for example, US Pat. the contents of each of which is incorporated into this description by reference in full).

Moreover, it is possible to fuse the polypeptides of the invention (eg, antibodies or fragments thereof) with marker sequences, such as a peptide, to facilitate their purification. In a further embodiment, it is also possible to recombine nucleic acids encoding the polypeptides of the invention (including, but not limited to, nucleic acids encoding immunogenic and / or antigenic epitopes) with a gene of interest, such as an epitope marker (e.g., a hemagglutinin marker ( "HA") or flag marker), to facilitate the detection and purification of the expressed polypeptide. In preferred embodiments, the amino acid marker sequence is a hexahistidine peptide, such as a marker provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among other markers, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), for example, hexahistidine provides convenient purification of fusion protein. Other peptide markers suitable for purification include, but are not limited to, the HA marker, which corresponds to the epitope of the influenza virus hemagglutinin protein (Wilson et al., Cell 37: 767 (1984)) and the flag marker.

In addition, the present invention relates to antibodies or fragments thereof conjugated to a diagnostic or drug. Antibodies can be used for diagnostic purposes, for example, to monitor the development or progression of a tumor, as part of the clinical testing process, for example, to determine the effectiveness of the prescribed treatment regimen, diagnosis, detection and / or prevention. Detection can be simplified by attaching the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radioactive substances, positron-active metals used in various methods of positron emission tomography, and non-radioactive metal ions. See, for example, US Pat. No. 4,741,900, for metal ions that can be conjugated to antibodies for use in the diagnosis of the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase; examples of suitable complexes of prosthetic groups include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent substance includes luminol; examples of bioluminescent substances include luciferase, luciferin and equorin; and examples of suitable radioactive material include ¹²⁵ I, ¹³¹ I, ¹¹¹ In or ⁹⁹ Tc.

In addition, the antibody or fragment thereof can be conjugated to a therapeutic group, such as a cytotoxin, for example, a cytostatic or cytocidal substance, drug or radioactive metal ion. A cytotoxin or cytotoxic substance is a substance that causes cell death. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etopzid, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, digidroksiantratsindion, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, Tatracaine, lidocaine, propranolol and puromycin and their analogues or homologs. Medicines include, but are not limited to, antimetabolites (e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g. mechlorethamine, thiotepu, chlorambucil, melphalan, N-carmustine) lomustine (CCNU), cyclophosphamide, bisulfan, dibromannite, streptozotocin, mitomycin C and cis-dichlorodiamine-platinum (II) (DDP) cisplatin), anthracyclines (e.g. daunorubicin (formerly daunomycin) and doxorubicin (e.g., antibiotics) actinomycin), bleomycin, mitramycin and a tramitsin (AMC)), and antimitotic srestva (e.g., vincristine and vinblastine).

The conjugates according to the invention can be used to modify this biological response, a drug or a group of drugs should not be understood as limited to classical drugs. For example, a group of a drug may be a protein or polypeptide having the desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet growth factor, tissue plasminogen activator, thrombotic substance or anti-angiogenic substance, for example, angiostatin or endostatin; or biological response modifiers, such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte-macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

Antibodies can also be coupled to solid supports that are particularly suitable for immunological assays or purification of a target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Methods for conjugating such therapeutic groups to antibodies are well known, see, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62: 119-58 (1982).

Alternatively, the antibody can be conjugated to a second antibody to form an antibody heteroconjugate, as described by Segal in US Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

An antibody with or without a conjugated therapeutic group, administered alone or in combination with cytotoxic factor (s) and / or cytokine (s), can be used as a medicine.

In another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for use in pre-targeting a tumor, where the antibody-receptor conjugate is administered to a patient, and then an unbound conjugate is removed from the bloodstream using an excretory agent, followed by " ligand "(e.g., avidin), which is conjugated to a cytotoxic substance (e.g., a radionuclide).

To identify antibodies that bind to proteins or polypeptides of IL-31, you can use many methods known to specialists in this field. Illustrative assay methods are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: combined immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) , dot blot or western blot analysis, inhibitory or competitive analysis and sandwich analysis. In addition, antibodies can be screened for binding to the wild-type IL-31 protein or polypeptide relative to the mutant form.

Anti-IL-31 antibodies can be used to label cells that express IL-31; for isolation of IL-31 by affinity purification; for diagnostic tests to determine the levels of IL-31 polypeptides in the bloodstream; to detect or quantify soluble IL-31 as a marker for an associated pathology or disease; in analytical methods using FACS methods; for screening expression libraries; to obtain anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists for blocking IL-31 activity in vitro and in vivo. Suitable direct markers or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles, and the like; a characteristic feature of indirect markers may be the use of biotin-avidin pairs or other complement / anti-complement pairs as intermediates. The antibodies described herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates can be used for diagnostic or therapeutic methods of in vivo use. Furthermore, anti-IL-31 antibodies or fragments thereof can be used in vitro to detect denatured IL-31 or fragments thereof in assays, for example, Western blots or other assays known in the art.

Suitable detectable molecules can be directly or indirectly attached to a polypeptide or antibody, and may include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles, and the like. Suitable cytotoxic molecules can be directly or indirectly attached to a polypeptide or antibody and can include bacterial or plant toxins (e.g. diphtheria toxin, saporin, Pseudomonas exotoxin, ricin, abrin, etc.), as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached, for example, by means of a chelating group). Polypeptides or antibodies can also be conjugated to cytotoxic drugs, such as adriamycin. For indirectly attaching a detectable or cytotoxic molecule, a detectable or cytotoxic molecule can be conjugated to a member of a complementary substance / anti-complementary substance pair, where another member is coupled to a polypeptide or part of an antibody. For these purposes, an illustrative complementary / anti-complementary substance pair is a biotin / streptavidin pair.

Binding polypeptides can also act as “antagonists” of IL-31 to block IL-31 binding and signal transmission in vitro and in vivo. These anti-IL-31 binding polypeptides may be useful for inhibiting IL-31 activity or protein binding.

Polypeptide-toxin fusion proteins and antibody-toxin fusion proteins can also be used to inhibit or destroy target cells or target tissues (for example, to target cancer cells or tissues). Alternatively, if the polypeptide has different functional domains (i.e., an activating domain or a receptor binding domain, plus a targeting domain), then a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, cytotoxic molecule, or complementary molecule to the type of cell or tissue of interest. In cases where a fusion protein with only one domain includes a complementary molecule, the anti-complementary molecule can be conjugated to a detectable or cytotoxic molecule. Such fusion proteins of a domain-complementary molecule, therefore, are a typical targeting carrier or a carrier for specific cell / tissue delivery of typical conjugates of an anti-complementary detectable / cytotoxic molecule.

In another embodiment, anti-IL-31-cytokine antibody fusion proteins can be used to kill in vivo target tissues (e.g., leukemia, lymphoma, lung cancer, colon cancer, melanoma, pancreatic cancer, ovarian cancer, skin cancer, blood and bone marrow, or other malignant tumors where IL-31 receptors are expressed) (See mainly Hornick et al., Blood 89: 4437-47, 1997). The described fusion proteins target the antibody to the desired region of action, thereby providing an increased local concentration of the antibody. Suitable IL-31 polypeptides or anti-IL-31 antibodies target an unwanted cell or tissue (i.e., a tumor or leukemia), and they increase the cytokine-mediated fusion of the target cell with effector cells. Suitable cytokines for these purposes include, for example, interleukin-2 and granulocyte macrophage colony stimulating factor (GM-CSF).

In another embodiment, if the IL-31 polypeptide, or anti-IL-31 antibody, targets vascular cells or tissues, such a polypeptide or antibody can be conjugated to a radionuclide and, in particular, a beta-emitting radionuclide to reduce restenosis.

Monoclonal antibodies of the invention can be determined by their ability to inhibit, block, or neutralize the IL-31 ligand, as determined by various in vivo models known in the art and described herein, including, but not limited to, the NC / Nga model, cutaneous Ova model, a model of chronic hypersensitivity and a chronic model with hapten.

Both T-cells directed to the skin and epidermal keratinocytes are involved in the pathology of skin diseases in humans. The expression of mRNA and IL-31 protein in humans is limited to the skin-directed population of CLA + T cells. Essentially, an IL-31 antagonist, including an antibody or receptor antagonist, will be useful in treating skin and epidermal diseases in which CLA + T cells are detected. Such diseases include, for example, atopic dermatitis, contact dermatitis, drug-induced allergic reactions, skin tropic viruses and the virus-associated itching, vitiligo, cutaneous T-cell lymphoma, focal alopecia, redheads, acne vulgaris, nodular pruritus and bullous pemphigoid. Chemokine markers such as TARC and MDC are suitable for determining the effect of a neutralizing monoclonal anti-IL-31 antibody. As presented in Example 15, the inhibitory effects of anti-IL-31 antibody treatment described herein can be determined by monitoring TARC and MDC levels.

Contact dermatitis

Allergic contact dermatitis is defined as a T-cell mediated immune response to an antigen that comes in contact with the skin. The CLA + T-cell population is believed to be involved in triggering dermatitis, since the allergen-dependent T-cell response is mainly limited to the CLA + cell population (See Santamaria-Babi, LF, et al., J Exp Med: 181, 1935, (1995) ) Recent data showed that only memory cells (CD45RО +) CD4 + CLA +, but not CD8 + T cells, in response to nickel, a common contact hypersensitivity allergen, proliferate and produce cytokines of both type 1 (IFN-) and type 2 (IL- 5). Moreover, the number of cells expressing CLA in combination with CD4, CD45RO (memory) or CD69 increases after specific stimulation with nickel, and they express chemokine receptors CXCR3, CCR4, CCR10, but not CCR6. See Moed H., et al., Br J Dermatol: 51, 32, (2004).

In animal models, it has been shown that allergic contact dermatitis is T-cell dependent and that T cells responsible for the allergic reaction migrate to the area where the allergen is applied. See mainly: Engeman T.M., et al., J Immunol: 164, 5207, (2000); Ferguson T.A. & Kupper T.S. J Immunol: 150, 1172, (1993); and Gorbachev A.V. & Fairchild R.L. Crit Rev Immunol: 21, 451 (2001). Since CLA + T cells produce IL-31, and stimulation of skin keratinocytes by IL-31 can induce pro-inflammatory chemokines such as TARC and MDC, IL-31 may be involved in the pathophysiology of contact dermatitis. Anti-IL-31 neutralizing antibodies were used in a mouse model of contact hypersensitivity. See example 8.

Thus, neutralizing IL-31 by the antibodies described herein can be used to improve the clinical outcome of contact hypersensitivity by inhibiting, reducing, preventing or blocking inflammation and / or combing associated with the disease.

Atopic dermatitis

Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease, and its frequency has increased dramatically over the past decades. Clinically, AD is characterized by severely itchy, often flaky plaques and papules that reflect a chronic relapsing course. The diagnosis of AD is mainly based on primary and secondary clinical signs. See Hanifin J.M., Arch Dermatol: 135, 1551 (1999). With histopathology, spongiosis, hyperkeratosis and focal parakeratosis in acute foci of damage are detected, while severe epidermal hyperplasia with hyperkeratosis and parakeratosis, acanthosis / hypergranulosis and perivascular dermal infiltration of lymphocytes and abnormalities in mast cells are a sign of mast cell damage.

T cells play a central role in triggering local immune responses in tissues, and data confirm that skin infiltrating T cells, in particular, can play a key role in triggering and maintaining immune responses with impaired regulation in the skin. Approximately 90% of the infiltrated T cells in inflammatory areas of the skin express cutaneous lymphocyte-associated Ag (CLA +), which binds E-selectin, an inducible endothelial adhesion molecule (described in Santamaria-Babi LF, et al., Eur J Dermatol: 14, 13, (2004)). A significant increase in circulating CLA + T cells has been documented among patients with AD compared with control individuals (See Teraki Y., et al., Br J Dermatol: 143, 373 (2000)), while other sources show that allergen extract preferably respond to CLA + memory T cells of patients with AD compared to the CLA- population (See Santamaria-Babi, LF, et al., J Exp Med: 181, 1935, (1995)). In humans, the pathogenesis of atopic skin disorders is associated with an increase in the number of CLA + T cells that express elevated levels of Th-2 type cytokines, such as IL-5 and IL-13, 9, 10. See Akdis M., et al., Eur J Immunol: 30, 3533 (2000); and Hamid Q., et al., J Allergy Clin Immunol: 98, 225 (1996).

NC / Nga mice spontaneously develop AD-like lesions that correspond to human AD in many aspects, including clinical course and signs, histopathology and immunopathology, when kept under conditions without a non-specific pathogen (non-SPF) at an age of approximately 6-8 weeks. In contrast, in NC / Nga mice that are kept under SPF conditions, skin lesions do not develop. However, the onset of spontaneous skin lesions and combing behavior can be synchronized in NC / Nga mice kept under SPF conditions by weekly intradermal injection of untreated dust mite antigen. See Matsuoka H., et al., Allergy: 58, 139 (2003). Thus, the development of AD in NC / Nga is an acceptable model for evaluating new drugs for the treatment of AD.

In addition to the NC / Nga spontaneous AD model, cutaneous sensitization of mice using OVA can also be used as a model for the induction of antigen-dependent thickening of the epidermis and dermis with mononuclear infiltrate in the skin of sensitized mice. This, as a rule, coincides with an increase in serum levels of general and specific IgE, however, normally in this model there is no skin barrier dysfunction or itching. See Spergel J. M., et al., J Clin Invest, 101: 1614, (1998). This protocol can be modified to induce dysregulation of the skin barrier and pruritus by sensitizing transgenic DO11.10 OVA TCR mice via OVA. An increased amount of antigen-specific T cells that can recognize a sensitizing antigen can increase the level of inflammation in the skin with the induction of visible scratching and lichenization / peeling of the skin.

Both the NC / Nga spontaneous AD model and the OVA cutaneous model in DO11.10 are used to study the expression of IL-31 and IL-31RA in AD, as well as the ability of the antibodies described herein to inhibit, reduce, or neutralize effects IL-31. See examples 9 and 10 of the present description. In one study using the in vivo NC / Nga model (Example 10), administration of rat antibodies against mouse IL-31 showed a reduction in scratching, but not a decrease in dermatitis. The antibodies described herein are useful for inhibiting dermatitis combing and pruritic diseases, including atopic dermatitis, nodular pruritus, and eczema. Inhibiting, reducing, or preventing combing alone can be effective in treating pruritus-related diseases, including but not limited to atopic dermatitis, nodular pruritus and eczema, since stopping the scratching will stop the progression of dermatitis, the development of which depends on scratching.

Additional models for determining the inhibitory effects of anti-IL-31 antibodies described herein are described in Umeuchi, H. et al., European Journal of Pharmacology, 518: 133-139, 2005; and in Yoo, J. et al., J. Experimental Medicine, 202: 541-549, 2005.

Thus, neutralizing IL-31 with the antibodies described herein can be used to improve the clinical outcome of dermatitis and pruritic diseases, including atopic dermatitis, nodular pruritus and eczema, by inhibiting, reducing, preventing or blocking inflammation and / or combing associated with the disease.

Drug-induced delayed skin allergic reactions

Drug-induced delayed skin allergic reactions are highly heterogeneous and can reflect many different pathophysiological processes. See Brockow K., et al., Allergy: 57, 45 (2002). It has been shown that the immunological mechanisms involved in these reactions are either mediated by antibodies or are cell-mediated. For immediate allergies to the drug, an IgE-mediated antibody response can be indicated by injection skin test and / or intradermal test after 20 minutes, while non-immediate drug reactions can occur more than one hour after the last taking the drug and is often mediated by T cells. Non-immediate T-cell-mediated delayed reactions can occur in patients with adverse drug reactions, such as penicillins. In patients with penicillin allergies, proliferative T-cell responses to penicillins are limited to a subpopulation of memory T-cells (CD45RO +) CLA +, while the subgroup CD45RO + CLA- shows the absence of a proliferative response. See Blanca M., Leyva L., et al., Blood Cells Mol Dis: 31, 15 (2003). Slow-type hypersensitivity reactions (HRT) can be artificially reproduced in mice, which allows the assessment of factors that may be involved in initiating and maintaining the HRT response. A neutralizing anti-IL-31 antibody may be effective in delayed-type hypersensitivity reactions. See example 51.

Toxic epidermal necrolysis (TEN) is a very rare but extremely severe drug reaction characterized by widespread apoptosis of the epidermis with extensive blisters. Studies have shown that lymphocytes that infiltrate blisters are CLA + T cells and they may exhibit cytotoxicity against epidermal keratinocytes. See Leyva L., et al.,. J Allergy Clin Immunol: 105, 157 (2000); and Nassif A., Bensussan A., et al., J Allergy Clin Immunol: 114, 1209 (2004). A system with transgenic mice, in which OVA is expressed under the control of the keratin-5 (K5) promoter in keratinocytes of the epidermis and mouse hair follicle, was created to obtain a TEN model in animals. OVA-specific CD8 + T cells, when adoptively transferred to K5-OVA mice, undergo activation and proliferation in the skin drainage lymph nodes and affect the skin of K5-OVA mice, resulting in the development of skin lesions that resemble TEN. See Azukizawa H., et al., Eur J Immunol: 33, 1879 (2003).

Thus, neutralizing IL-31 by the antibodies described herein can be used to improve the clinical outcome of TEN by inhibiting, reducing, preventing or blocking inflammation and / or combing due to disease.

Bullous pemphigoid

Bullous pemphigoid is a subepidermal disorder, which manifests itself in the form of subepidermal blisters with an infiltrate in the dermis of neutrophils and eosinophils. The diagnosis is characterized by the presence of antigen-specific antibodies against certain epidermal adhesion proteins and the dermal epidermal transition region. See Jordon R.E., et al., JAMA: 200, 751 (1967). In studies analyzing the role of T cells in the pathogenesis of bullous pemphigoid by analyzing PBL and T cells of skin blisters, a predominance of CLA + T cells expressing elevated levels of Th2 cytokines such as IL-4 and IL-13 was revealed. See Terald Y., et si., J Invest Dermatol: 111, 1097 (2001). In patients with bullous pemphigoid after treatment with systemic corticosteroids, the incidence of CLA + but not CLA- cells producing interleukin-13 is significantly reduced. A decrease in the number of CLA + cells after treatment with corticosteroids is associated with clinical improvement. See Teraki, ibid.

Thus, the neutralization of IL-31 by the antibodies described herein can be used to improve the clinical outcome of the bullous pemphigoid by inhibiting, reducing, preventing or blocking inflammation and / or scratching due to the disease.

Alopecia areata

Alopecia areata (AA) is understood to mean a tissue-limited autoimmune disease of hair follicles in which follicle activity is discontinued due to the continuous activity of lymphatic infiltrates. AA leads to areas of complete hair loss throughout the body, although in reality there is no loss of hair follicles, even in areas where hair is missing. Despite the absence of clinical signs of inflammation, skin biopsy specimens from areas of active disease show perifollicular lymphocytic inflammation, mainly from CD4 + cells, together with intrafollicular CD8 + infiltrate. See Kalish R.S. & Gilhar A. J Investig Dermatol Symp Proc: 8, 164 (2003).

Studies have shown that infiltrating the skin of the skull with CD4 + or CD8 + lymphocytes express CLA, and in the peripheral blood of individuals with AA, the percentage of CD4 + or CD8 + lymphocytes CLA + is significantly higher than in the peripheral blood of healthy control donors. Moreover, it was shown that patients with severe or progressive AA are significantly more CLA-positive compared with patients recovering from the disease, and a decrease in the percentage of CLA + cells corresponds to a good clinical course. See Yano S., et al., Acta Derm Venereal: 82, 82 (2002). Thus, these studies confirm that CLA + lymphocytes can play an important role in AA. Xenograft models have shown that activated T cells are likely to be involved in the pathogenesis of AA. In the damaged skin of the skull of patients with AA transplanted into nude mice, hair regrowth occurred, coinciding with the loss of infiltrating lymphocytes in the graft, and the transfer of activated T cells from the lesion sites to SCID mice can transfer hair loss to human skull explants in SCID mice. See Kalish R.S. & Gilhar A. J Investig Dermatol Symp Proc: 8, 164 (2003).

A variety of immunomodulatory drugs are part of the traditional treatment for this disorder, however, none of these drugs have a permanent effect. See Tang L., et al., J Invest Dermatol: 120, 400 (2003); Tang L., et al., (2004); and Tang L., et al., J Am Acad Dermatol: 49, 1013 (2003). However, their use in suitable animal models is a tool for analyzing the molecular mechanisms of therapeutic effects. See Shapiro J., et al., J Investig Dermatol Symp Proc: 4, 239 (1999); Tang L., et al., Old wine in new bottles: reviving old therapies for alopecia areata using rodent models (2003); and Verma D.D., et al., Eur J Dermatol: 14, 332 (2004).

Thus, the neutralization of IL-31 by the antibodies described in the present invention can be used to improve the clinical outcome of focal alopecia by inhibiting, reducing, preventing or blocking inflammation and / or combing caused by the disease.

Red Acne / Common Acne

Acne vulgaris, a violation of the apparatus of the hair follicles and sebaceous glands, are the most common skin problem of adolescents. It is believed that abnormalities of keratinization of the follicle cause acne. Red acne differs from ordinary acne in the presence of red papules, pustules and blisters and extensive telangiectosis, but the absence of comedones (white heads). Increased sebum secretion from the sebaceous glands is the main factor in the pathophysiology of common acne. Other sebaceous gland functions are also associated with the development of acne, including pro-inflammatory lipid lipids; various locally produced cytokines; near-iron peptides and neuropeptides, such as corticotropin releasing factor, which is produced by sebocytes; and substance P, which is expressed in nerve endings near acne-appearing healthy glands of patients. See Zouboulis CC. Clin Dermatol: 22, 360 (2004).

Although the pathophysiology of common blackheads and redheads remains unknown, clinical observations and histopathological studies suggest that inflammation of the hair follicle and sebaceous gland may be the central link in the pathogenesis of redheads and common blackheads. Previous studies analyzing subgroups of T cells that infiltrate lesions in redheads indicate that most T cells express CD4. See Rufli T. & Buchner S.A. Demiatologica: 169, 1 (1984).

CD4 + T cells produce IL-31, and a skin IHC analysis of IL-31 expression confirms that IL-31 is expressed in the sebaceous and sweat glands. Stimulation of epidermal keratinocytes by IL-31 induces chemokine expression, which is likely to lead to cell infiltration, confirming that IL-31 can lead to a pro-inflammatory response in the skin. See Dillon S.R., et al., Nat Immunol: 5, 752 (2004). Thus, IL-31 may be involved in the pathophysiology of red eels and common eels.

Thus, neutralizing IL-31 with the antibodies described herein can be used to improve the clinical outcome of common acne by inhibiting, reducing, preventing or blocking inflammation and / or combing caused by the disease.

Knotted Pruritus

Knotted pruritus is a rash of lichenized or excoriated nodules caused by uncupable itching, which is difficult to treat. Despite the fact that constant grinding leads to lichenization and combing to longitudinal abrasions, in individuals picking and squeezing their itchy irritated skin, there is a tendency to the appearance of significantly thickened papules, known as prurigo nodules. Although nodular pruritus is not characteristic of atopic dermatitis, many patients with these nodules also have an atopic reaction that manifests itself as allergic rhinitis, asthma, or food allergies. T cells represent the majority of the infiltrating cells in the lesions with pruritus, and these lesions often represent the most itchy lesions of the skin in patients with atopy.

Topical treatment of nodular pruritus with capsaicin, an anti-itch alkaloid that prevents the sensation of itching and pain by depleting neuropeptides, such as substance P, in the small sensitive nerves of the skin, is an effective and safe regimen that eliminates foci of skin damage. See Stander S., et al., J Am Acad Dermatol: 44, 471 (2001). Studies of pruritus-related response in NC / Nga mice using capsaicin administration have shown almost complete prevention of spontaneous development of lesions associated with dermatitis. Moreover, there was a significant suppression of the increase in serum IgE levels and the number of infiltrating eosinophils and mast cells in the damaged skin of mice that were injected with capsaicin was reduced. See Mihara K., et al., Br J Dermatol: 151, 335 (2004). Observations of this group have confirmed that combing can participate in the development of dermatitis by enhancing various immunological responses, implying, therefore, that preventing pruritus and / or pruritus-related scratching behavior may be an effective way to treat AD. See Mihara K., et al., Br J Dermatol: 151, 335 (2004). Thus, the anti-IL-31 antibodies described herein are useful in minimizing the effects of AD, nodular pruritus, and other pruritic diseases, since they have been shown to reduce scratching in NC / Nga mice.

Continuous delivery of IL-31 causes itching and alopecia in mice, followed by the development of foci of skin damage resembling dermatitis, confirming that IL-31 can cause itching. See Dillon S.R., et al., Nat Immunol: 5, 752 (2004). The involvement of IL-31 in the induction of pruritus-related responses was tested in two ways (i) by administering capsaicin to mice that were injected with IL-31, and (ii) by administering IL-31 to mice with Tac1 knockout, which significantly reduced nociceptive pain response due to the absence of expression of neuropeptides, in example 52. In addition, the possibility of neutralizing IL-31 in mice injected with IL-31 to prevent itching and alopecia was tested in example 12.

Thus, neutralizing IL-31 with the antibodies described herein can be used to improve the clinical outcome of nodular pruritus by inhibiting, reducing, preventing or blocking inflammation and / or combing caused by the disease.

Skin tropic viruses and virus-associated pruritus

Herpes simplex virus (HSV) specific CD8 + T cells in peripheral blood and HSV specific CD8 + T cells isolated from herpetic lesions express high levels of CLA, and expression in CD8 + T cells associated with herpes simplex virus expression CLA is missing. See Koelle D.M., et al., J Clin Invest: 110, 537 (2002). HSV-2 reactive CD4 + T lymphocytes also express CLA, however, at levels that are lower than previously observed levels for CD8 + T lymphocytes. See Gonzalez J.C., et al., J Infect Dis: 191, 243 (2005). Itching is also often associated with herpes virus infections (See Hung KY, et al., Blood Purif: 16, 147 (1998), although other viral diseases, such as HIV, are also associated with itchy foci of skin damage. Severe, non-stopping itching, often associated with erythematous-papular foci of skin damage and hypereosinophilia, is a condition observed in some HIV-infected patients without atopy 36. See Singh F. & Rudikoff D, Am J Clin Dermatol; 4, 177 (2003); and Milazzo R, Piconi S ., et al., Allergy: 54, 266 (1999).

The association of skin tropic viruses with pruritus and CLA + T cells suggests that IL-31 producing T cells may be involved in the pathophysiology of viral infections.

Thus, neutralizing IL-31 by the antibodies described herein can be used to improve the clinical outcome of pruritus associated with skin tropic viruses by inhibiting, reducing, preventing or blocking inflammation and / or combing caused by the disease.

Moreover, inflammation is the body's defensive response to fight against an invading agent. Inflammation is a cascading process in which many cellular and humoral mediators are involved. On the one hand, the suppression of the inflammatory response can lead to a weakening of the immune system in the host; however, if not controlled, inflammation can lead to serious complications, including chronic inflammatory diseases (for example, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, etc.), septic shock, and multiple organ failure. It is important that these various painful conditions have common mediators. The combination of diseases that are characterized by inflammation has a significant effect on the morbidity and mortality of a person. Thus, it is apparent that anti-inflammatory antibodies and binding polypeptides, such as anti-IL-31 antibodies and the binding polypeptides described in the present invention, may have significant therapeutic potential for a wide class of human and animal diseases, from asthma and allergies to autoimmune diseases and septic shock. Essentially, the use of anti-inflammatory antibodies against IL-31 and the binding polypeptides described herein can be used therapeutically as antagonists of IL-31 described herein, in particular in diseases such as arthritis, endotoxemia, inflammatory bowel disease, psoriasis, related diseases, etc.

1. Arthritis

Arthritis, including osteoarthritis, rheumatoid arthritis, arthritic joints as a result of damage, and the like, is a common inflammatory condition that can be positively affected by the therapeutic use of anti-inflammatory antibodies and binding polypeptides such as anti-IL-31 antibodies and binding polypeptides according to the invention. For example, rheumatoid arthritis (RA) is a systemic disease that affects the entire body and is one of the most common forms of arthritis. It is characterized by inflammation of the lining of the joint, which causes pain, stiffness, fever, redness and swelling. Inflammatory cells release enzymes that can break down bone and cartilage. As a result of rheumatoid arthritis, the joint covering the synovial membrane can damage and damage the bone and cartilage, which, among other physiological effects, leads to joint damage and severe pain. The affected joint may lose its shape and position, causing pain and loss of mobility.

Rheumatoid arthritis (RA) is an immune-mediated disease, in particular characterized by inflammation and subsequent tissue damage, resulting in severe disability and increased mortality. In the joints with rheumatoid arthritis, many cytokines are locally produced. Several studies have shown that IL-1 and TNF-alpha, two prototype pro-inflammatory cytokines, play an important role in the mechanisms involved in inflammation of the synovial membrane and in the progressive destruction of the joint. Indeed, the administration of TNF-alpha and IL-1 inhibitors in patients with RA leads to a significant improvement in the clinical and biological signs of inflammation and to a reduction in the radiological signs of bone ulceration and cartilage destruction. However, despite these encouraging results, a significant percentage of patients do not respond to these funds, confirming that other mediators are also involved in the pathophysiology of arthritis (Gabay, Expert. Opin. Biol. Ther. 2 (2): 135-149, 2002) . One of these mediators may be IL-31, and essentially a molecule that binds or inhibits IL-31, such as anti-IL-31 antibodies or binding partners, can act as a suitable therapeutic agent for reducing inflammation in rheumatoid arthritis and other arthritic diseases.

Several animal models of rheumatoid arthritis are known in the art. For example, in a model of collagen-induced arthritis (CIA), mice develop chronic inflammatory arthritis, which is closely related to human rheumatoid arthritis. Since CIA has common immunological and pathological features with RA, this has led to the fact that it is an ideal model for screening potential anti-inflammatory compounds in humans. The CIA model is a well-known mouse model that depends on both the immune response and the inflammatory response arising in this order. The immune response involves the interaction of B cells and CD4 + T cells in response to collagen, which is administered as an antigen, and leads to the production of antibodies against collagen. The inflammatory phase is the result of tissue responses through inflammatory mediators, resulting from the fact that some of these antibodies cross-react with their own mouse collagen and activate the complement cascade. The advantage of using the CIA model is that the main mechanisms of its pathogenesis are known. The corresponding epitopes for T-cells and B-cells on type II collagen have been identified and various immunological (e.g., delayed-type hypersensitivity and anti-collagen antibodies) and inflammatory (e.g. cytokines, chemokines and matrix-degrading enzymes) parameters associated with immuno-mediated arthritis have been identified. , and thus, they can be used to evaluate the effectiveness of the test compound in the CIA model (Wooley, Curr. Opin. Rheum. 3: 407-20, 1999; Williams et al, Immunol. 89: 9784-788, 1992; Myers et al., Life Sci. 61: 1861-78, 1997; and Wang et al., Immunol. 92: 8955-959, 1995).

The administration of soluble IL-31RA containing polypeptides (including the heterodimeric and multimeric receptors described herein), such as IL-31RA-Fc4, or other soluble and fusion proteins of IL-31RA to mice in this model of CIA was used to evaluate the use of IL-31RA to alleviate symptoms and change the course of the disease. Because the molecule that modulates the immune and inflammatory response, IL-31, can induce the production of SAA, which is involved in the pathogenesis of rheumatoid arthritis, IL-31 antagonists can reduce SAA activity in vitro and in vivo, and systemic or local administration of IL-31 antagonists. such as anti-IL-31 antibodies or binding partners, IL-31RA containing the polypeptides (including the heterodimeric and multimeric receptors described herein), such as IL-31RA-Fc4, or other soluble and fused IL-31RA proteins, can potentially suppress inflammatory em at RA. Other potential drugs include IL-31RA polypeptides, soluble heterodimeric and polymeric receptor polypeptides, or anti-IL-31 antibodies or binding partners of the invention, and the like.

2. Endotoxemia

Endotoxemia is a serious condition usually resulting from infectious agents such as bacteria and other agents of infectious diseases, sepsis, toxic shock syndrome, or in immunocompromised patients who have experienced opportunistic infections, and the like. Therapeutically useful anti-inflammatory antibodies and binding polypeptides, such as anti-IL-31 antibodies and binding polypeptides of the invention, may facilitate the prevention and treatment of endotoxemia in humans and animals. Other potential drugs, including IL-31RA polypeptides, soluble heterodimeric and multimeric receptor polypeptides, or anti-IL-31 antibodies or binding partners of the invention, and the like, may act as suitable drugs to reduce inflammation and pathological effects of endotoxemia.

Lipopolysaccharide-induced (LPS) endotoxemia involves many pro-inflammatory mediators that have pathological effects in infectious diseases, and LPS-induced endotoxemia in rodents is a widely used and acceptable model for studying the pharmacological effects of potential pro-inflammatory or immunomodulating agents. LPS produced in gram-negative bacteria is a major etiological factor in the pathogenesis of septic shock (Glausner et al., Lancet 338: 732, 1991). A shock-like condition can indeed be experimentally induced by a single injection of LPS in animals. Molecules produced by cells responding to LPS can affect pathogens directly or indirectly. Although these biological responses protect the host from invading pathogens, they can also be harmful. Thus, massive stimulation of innate immunity resulting from a severe infection with gram-negative bacteria leads to excessive production of cytokines and other molecules and to the development of a deadly syndrome, toxic shock syndrome, which is characterized by fever, hypotension, disseminated intravascular coagulation and multiple organ failure (Dumitru et al Cell 103: 1071-1083, 2000).

These toxic effects of LPS are mainly associated with the activation of macrophages, leading to the release of many inflammatory mediators. Among these mediators, TNF appears to play a key role, as shown by preventing LPS-related toxicity by the administration of neutralizing antibodies against TNF (Beutler et al., Science 229: 869, 1985). It was determined that the injection of 1 μg of E. coli LPS into a C57B1 / 6 mouse resulted in a significant increase in circulating IL-6, TNF-alpha, IL-1 and acute phase proteins (e.g., SAA) approximately 2 hours after injection. LPS toxicity appears to be mediated by these cytokines, as passive immunization against these mediators can lead to a reduction in mortality (Beutler et al., Science 229: 869, 1985). Potential immune system interventions for the prevention and / or treatment of septic shock include anti-TNF mAb, a receptor antagonist for IL-1, LIF, IL-10, and G-CSF. Because LPS induces the production of pro-inflammatory factors likely to lead to endotoxemia pathologies, neutralizing the activity of IL-31, SAA, or other pro-inflammatory factors by antagonizing the IL-31 polypeptide can be used to reduce symptoms of endotoxemia, such as those detected with endotoxin shock. Other potential drugs include IL-31RA polypeptides, soluble heterodimeric and multimeric receptor polypeptides, or anti-IL-31 antibodies or binding partners of the invention, and the like.

3. Inflammatory bowel disease. IBD

In the United States, approximately 500,000 people suffer from inflammatory bowel disease (IBD), which can affect either the colon and rectum (ulcerative colitis) or the small and large intestines (Crohn's disease). The pathogenesis of these diseases remains unclear, however, chronic inflammation of the affected tissues is involved in them. Potential drugs include IL-31RA polypeptides, soluble heterodimeric and multimeric receptor polypeptides, or anti-IL-31 antibodies or binding partners of the invention and the like, which can serve as a drug to reduce inflammation and pathological effects IBD and related diseases.

Ulcerative colitis (UC) is an inflammatory disease of the large intestine, commonly called the colon, characterized by inflammation and ulceration of the mucosa or the deepest intestine. This inflammation causes frequent colon emptying, leading to diarrhea. Symptoms include loose stools and associated abnormal cramps, fever, and weight loss. Although the exact cause of UC remains unknown, recent studies have suggested that the body’s natural defenses act against the body’s proteins, which the body perceives as foreign (“autoimmune response”). Perhaps due to the fact that they are similar to bacterial proteins of the intestine, these proteins can either initiate or stimulate the inflammatory process, which begins to destroy the lining of the intestine. As the lining is destroyed, ulcers form with the release of mucus, pus and blood. The disease usually begins in the rectum and can eventually spread throughout the large intestine. Repeated episodes of inflammation lead to a thickening of the intestinal wall and rectum with scar tissue. In the case of a serious illness, death of the colon tissue or sepsis can occur. Symptoms of ulcerative colitis vary in severity, and their onset can be gradual or sudden. Attacks can trigger many factors, including respiratory tract infections or stress.

Although there is currently no available treatment for UC, treatment methods focus on suppressing an abnormal inflammatory process in the lining of the intestine. Medicines are available for treating the disease, including corticosteroids, immunosuppressive drugs (e.g., azathioprine, mercaptopurine and methotrexate) and aminosalicylates. However, prolonged use of immunosuppressants, such as corticosteroids and azathioprine, can lead to severe side effects, including thinning of bones, cataracts, infection, and effects on the liver and bone marrow. In patients in whom modern medicines are unsuccessful, surgery is possible. Surgery involves removal of the entire colon and rectum.

There are several animal models that can almost mimic chronic ulcerative colitis. The most widely used model is the model of colitis induced by 2,4,6-trinitrobenzenesulfonic acid / ethanol (TNBS), which induce chronic inflammation and ulceration in the large intestine. When TNBS is introduced into the large intestine of sensitive mice via intrectal instillation, T-cell mediated immune responses are induced in the mucous membrane of the large intestine, leading, in this case, to massive inflammation of the mucous membrane, characterized by dense T-cell and macrophage infiltration through the entire wall of the large intestine . Moreover, this histopathological picture is accompanied by a clinical picture of progressive weight loss (emaciation), hemorrhagic diarrhea, rectal prolapse and thickening of the colon wall (Neurath et al. Intern. Rev. Immunol. 19: 51-62, 2000).

Another colitis model uses sodium dextran sulfate (DSS), which induces acute colitis, which is manifested by hemorrhagic diarrhea, weight loss, shortening of the large intestine, and ulceration of the mucous membrane with neutrophil infiltration. Colitis induced by DSS is histologically characterized by the infiltration of inflammatory cells into a lamina propria with hyperplasia of lymphoid tissue, focal destruction of crypts and ulceration of the epithelium. It is believed that these changes develop due to the toxic effect of DSS on the epithelium, and phagocytosis of the cells of their own plate and the production of TNF-alpha and IFN-gamma. Despite widespread use, several issues regarding the correspondence of DSS mechanisms to the disease in humans remain unresolved. DSS is considered as a T-cell independent model due to the fact that it is observed in T-cell-deficient animals such as SCID mice.

The introduction of antibodies against IL-31 or binding partners, soluble IL-31RA containing polypeptides (including heterodimeric and multimeric receptors), such as IL-31RA-Fc4, or other soluble and fused proteins of IL-31RA in these models with TNBS or DSS can use to evaluate the use of IL-31 antagonists to alleviate symptoms and alter the course of a gastrointestinal disease. IL-31 may be involved in the inflammatory response in colitis, and the neutralization of IL-31 activity by administration of IL-31 antagonists is a potential therapeutic approach for IBD. Other potential drugs include IL-31RA polypeptides, soluble heterodimeric and multimeric receptor polypeptides, or anti-IL-31 antibodies or binding partners of the invention, and the like.

4. Psoriasis

Psoriasis is a chronic skin condition that affects seven million Americans. Psoriasis occurs with abnormal growth of skin cells, which leads to inflamed, swollen and scaly areas of the skin, where old skin does not exfoliate quickly enough. Plaque-like psoriasis, the most common form, is characterized by inflamed areas of the skin (“lesion foci”) covered with silver-white scales. Psoriasis can be limited to a few plaques or involve a moderate to large area of the skin, most often appearing on the skin of the skull, knees, elbows and trunk. Although it is clearly visible, psoriasis is not a contagious disease. The pathogenesis of the disease includes chronic inflammation of the affected tissues. IL-31RA polypeptides, soluble heterodimeric and multimeric receptor polypeptides, or anti-IL-31 antibodies or binding partners of the invention and the like, can serve as a suitable drug to reduce inflammation and pathological effects in psoriasis and other inflammatory skin diseases allergies to the skin and mucous membranes, and related diseases.

Psoriasis is a T-cell mediated inflammatory disorder that can cause significant discomfort. It is a disease for which there is no cure and which affects people of all ages. Psoriasis affects approximately two percent of the population of Europe and North America. Despite the fact that patients with mild psoriasis can often control their disease through topical substances, more than one million patients worldwide need treatment with ultraviolet light and systemic immunosuppressants. Unfortunately, the inconvenience and risk of exposure to ultraviolet light and the toxicity of many drugs limit their long-term use. Moreover, patients usually have a relapse of psoriasis, and in some cases

IL-31 was isolated from tissue, which is known to have important immunological function and which contains cells that play a role in the immune system. IL-31 is expressed in selected CD3 + activated peripheral blood cells, and IL-31 expression has been shown to increase after T-cell activation. Moreover, the results of the experiments described in the examples section of the present description confirm that the polypeptides according to the invention can have an effect on the growth / reproduction of monocytes / macrophages, T cells, B cells, NK cells and / or on the level of differentiation of monocytes / macrophages , T cells, B cells, NK cells, or precursors of these cells. Factors that both stimulate proliferation of hematopoietic precursors and activate mature cells are mainly known, but additional growth factors may also be necessary for proliferation and activation. For example, it has been shown that IL-7 and Steele factor (c-kit ligand) are necessary for the formation of NK cell precursor colonies. IL-15 + IL-2 in combination with IL-7 and Steele factor were most effective (Mrozek et al., Blood 87: 2632-2640, 1996). However, unidentified cytokines may be necessary for the proliferation of certain subtypes of NK cells and / or NK cell precursors (Robertson et. Al., Blood 76: 2451-2438, 1990). Similarly, IL-31 can act alone, or in conjunction, or synergistically with other cytokines, enhancing the growth, proliferative expansion and modification of the differentiation of monocytes / macrophages, T cells, B cells or NK cells.

The present invention relates to a method for inhibiting the activation or differentiation of monocytes / macrophages. Monocytes are cells with incomplete differentiation that migrate to tissues, where they mature and turn into macrophages. Macrophages play a central role in the immune response, presenting antigen to lymphocytes, and play a supporting role as auxiliary cells for lymphocytes, secreting a number of cytokines. Macrophages can internalize extracellular molecules and, when activated, have an increased ability to destroy intracellular microorganisms and tumor cells. Activated macrophages are also involved in stimulating acute or local inflammation.

The tissue distribution of receptors for a given cytokine is a strict indicator of the potential areas of exposure of this cytokine. Expression of IL-31RA was observed in monocytes and B cells, with a significant increase in expression upon activation of CD3 +, CD4 + and CD8 + T cells. In addition, also two monocytic cell lines, THP-1 (Tsuchiya et al., Int. J. Cancer 26: 171-176, 1980) and U937 (Sundstrom et al., Int. J, were positive for IL-31RA expression. Cancer 17: 565-577, 1976).

It is described that the expression of OSMR is very wide (Mosley et al, JBC 271: 32635-32643, 1996). This distribution of IL-31RA and OSM receptors confirms the role of IL-31 in immune responses, especially in T-cell expansion upon activation, or its role in the monocytic / macrophage link of the immune system.

Thus, specific embodiments of the present invention are directed to the use of soluble IL-31RA / OSMR heterodimers as antagonists in inflammatory and immune diseases or conditions such as pancreatitis, type I diabetes (IDDM), pancreatic cancer, pancreatitis, Graves disease, inflammatory intestinal disease (IBD), Crohn’s disease, colon cancer and intestinal cancer, diverticulosis, autoimmune disease, sepsis, organ or bone marrow transplantation ha; inflammation due to trauma, surgery, or infection; amyloidosis; splenomegaly; graft versus host reaction; and when inhibiting inflammation, suppressing the immune system, reducing the proliferation of hematopoietic, immune, inflammatory or lymphoid cells, macrophages, T cells (including Th1 and Th2 cells, CD4 + and CD8 + cells), suppressing the immune response to a pathogen or antigen. Moreover, the presence of IL-31RA expression in activated cells of the immune system, such as activated CD4 + and CD19 + cells, indicates that the IL-31RA receptor may be involved in the body's immune defenses against foreign agents: such as microorganisms and cell debris, and may participate in immune responses in the process of inflammation and the formation of a malignant tumor. Essentially, antibodies and binding partners of the invention that are agonists or antagonists of IL-31RA receptor function, such as IL-31, can be used to modify the immune response and inflammation.

IL-31 may be useful in suppressing the immune system, such as the treatment of autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, diabetes mellitus, inflammatory bowel disease, Crohn's disease, etc. Suppression of the immune system can also be used to reduce rejection of transplanted or transplanted tissues or organs and to treat T-cell, B-cell or monocyte-specific leukemia or lymphomas and other malignant tumors by inhibiting the proliferation of damaged cell types. Moreover, IL-31 can be used to detect monocytes, macrophages and activated T cells and to facilitate the diagnosis of such an autoimmune disease, in particular in a disease state where an increase in the number or activation of monocytes occurs.

Polypeptides, Peptides, Anti-IL-31 Antibodies, etc. can also be used in diagnostic systems to determine circulating levels of IL-31. In a similar embodiment, antibodies or other substances that specifically bind to IL-31 polypeptides can be used to detect circulating IL-31 polypeptides. Elevated or decreased levels of ligand polypeptides may indicate pathological conditions, including a malignant tumor. IL-31 polypeptides may be involved in pathological processes and may be indirect markers of an associated disease.

Moreover, one skilled in the art will recognize that IL-31 antagonists are suitable. For example, in atherosclerotic plaques, one of the first anomalies is the localization of monocytes / macrophages among endothelial cells. These lesions can be prevented by the use of IL-31 antagonists. Anti-IL-31 antibodies (e.g., a neutralizing anti-IL-31 antibody), soluble IL-31RA receptors, heterodimers and multimers, and IL-31 binding partners can also be used as antagonists of IL-31. Moreover, monoblastic leukemia is associated with various clinical abnormalities that reflect the release of biological products of macrophages, their examples include high levels of lysozyme in serum and urine and high fever. Moreover, with such leukemia, an abnormal increase in monocytic cells occurs. These effects can probably be prevented by IL-31 antagonists, such as those described herein. Moreover, anti-IL-31 antibodies can be conjugated to molecules, such as toxic groups and cytokines, as described herein, to target the killing of monocytic leukemia cells.

Since the expression pattern of IL-31 is specific for T cells, macrophages and monocytes, and these diseases include abnormalities of monocytic cells, such as proliferation, functioning, localization and activation, the polynucleotides, polypeptides and antibodies of the invention can be used as diagnostic means for detecting such abnormalities of monocytic cells, and an indicator of the presence of diseases. Such methods include taking a patient’s biological sample, such as blood, saliva or biopsy, and comparing them with a normal control. To determine the relative levels or localization of IL-31 or cells expressing IL-31, i.e. monocytes, histological, cytological methods, flow cytometry methods, biochemical and other methods can be used in a patient’s sample compared to a normal control. A change in the level (increase or decrease) in the expression of IL-31, or a change in the number or localization of monocytes (for example, an increase in the number or infiltration of monocytic cells in tissues where they are normally not present) compared with the control, is an indicator of the presence of the disease. Such diagnostic methods may also include the use of radiometric, fluorescent and colorimetric labels attached to the polynucleotides, polypeptides or antibodies of the invention. Such methods are well known in the art and are described herein.

It has been shown that IL-31 is expressed by activated mononuclear cells, and it may be involved in the regulation of inflammation. Essentially, the polypeptides according to the invention can be evaluated and applied based on their ability to modify inflammation, or they can be used as a marker of inflammation. Methods for determining the pro-inflammatory and anti-inflammatory properties of IL-31 are known in the art and are described herein. Moreover, it may be involved in upregulating the production of acute phase reactants such as serum amyloid A (SAA), α1-antichymotrypsin and haptoglobin, and this expression of the IL-31RA receptor ligand may increase upon injection of lipopolysaccharide (LPS) in vivo, which involved in the inflammatory response (Dumoutier, L. et al., Proc. Nat'l. Acad. Sci. 97: 10144-10149, 2000). It is believed that the production of acute phase proteins such as SAA is a short-term survival mechanism where inflammation has a positive effect; however, maintaining the acute phase proteins for longer periods leads to chronic inflammation and can be harmful to human health. For a review, see Uhlar, CM and Whitehead, AS, Eur. J. Biochem. 265: 501-523, 1999, and Baumann H. and Gauldie, J. Immunology Today 15: 74-80, 1994. Moreover, the acute phase SAA protein is involved in the pathogenesis of several chronic inflammatory diseases, it is involved in atherosclerosis and rheumatoid arthritis and is a precursor of amyloid A protein deposited in amyloidosis (Uhlar, CM and Whitehead, supra). Thus, in the case where a ligand, such as IL-31, acts as a pro-inflammatory molecule and induces the production of SAA, antagonists may be suitable for the treatment of inflammatory disease and other diseases associated with the acute phase protein response induced by the ligand. The present invention relates to such antagonists. For example, a method of reducing inflammation involves administering to a mammal with inflammation an amount of an IL-31 composition, or an anti-IL-31 antibody (e.g., a neutralizing antibody), which is sufficient to reduce inflammation. Moreover, a method of suppressing an inflammatory response in a mammal with inflammation may include: (1) determining the level of serum protein of amyloid A; (2) administering a composition comprising an IL-31 polypeptide or anti-IL-31 antibody, as described herein, in an acceptable pharmaceutical carrier; (3) determining the level of whey protein of amyloid A after administration; (4) comparing the level of whey protein of amyloid A stage (1) with the level of whey protein of amyloid A stage (3), where the absence of an increase or decrease in the level of whey protein of amyloid A indicates the suppression of the inflammatory response.

Like IL-31, analysis of the tissue distribution of mRNA corresponding to the cDNA of its IL-31RA receptor showed that the mRNA level was highest in monocytes and prostate cells and increased in activated monocytes and activated CD4 +, activated CD8 + and activated CD3 + cells. Thus, the IL-31RA receptor is also involved in the induction of an inflammatory and immune response. Thus, specific embodiments of the present invention relate to the use of antibodies against IL-31 and IL-31, as well as soluble heterodimers of the IL-31RA receptor, as antagonists in inflammatory and immune diseases or conditions, such as pancreatitis, type I diabetes ( IDDM), pancreatic cancer, pancreatitis, Graves disease, inflammatory bowel disease (IBD), Crohn's disease, colon cancer and intestinal cancer, diverticulosis, autoimmune disease, sepsis, organ or bone marrow transplantation; inflammation due to trauma, surgery, or infection; amyloidosis; splenomegaly; graft versus host reaction; and when inhibiting inflammation, suppressing the immune system, reducing the proliferation of hematopoietic, immune, inflammatory or lymphoid cells, macrophages, T cells (including Th1 and Th2 cells, CD4 + and CD8 + cells), suppressing the immune response to a pathogen or antigen. Moreover, the presence of the IL-31RA receptor and the expression of IL-31 in activated cells of the immune system, such as activated CD3 +, monocytes, CD4 + and CD19 + cells, indicate that the IL-31RA receptor may be involved in the body's immune defenses against foreign agents: such as microorganisms and cell debris, and can participate in immune responses during inflammation and the formation of a malignant tumor. Essentially, IL-31 and anti-IL-31 antibodies of the invention, which are agonists or antagonists of IL-31RA receptor function, can be used to modify the immune response and inflammation.

IL-31 polypeptides that bind IL-31RA receptor polypeptides and antibodies thereto are suitable for:

1) Antagonism or blocking signal transmission through IL-31RA-containing receptors in the treatment of acute inflammation, inflammation resulting from trauma, tissue damage, surgery, sepsis or infection, and chronic inflammatory diseases such as asthma, inflammatory bowel disease (IBD), chronic colitis, splenomegaly, rheumatoid arthritis, recurrent acute inflammatory episodes (e.g. tuberculosis), and in the treatment of amyloidosis and atherosclerosis, Castleman’s disease, asthma and other diseases associated with ind tion of the acute phase response.

2) Antagonism or blocking signal transmission through IL-31RA receptors in the treatment of autoimmune diseases such as IDDM, multiple sclerosis (MS), systemic lupus erythematosus (SLE), myasthenia gravis, rheumatoid arthritis and IBD, to prevent or inhibit signal transmission in immune cells (e.g., lymphocytes, monocytes, white blood cells) via the IL-31RA receptor (Hughes C et al., J. Immunol 153: 3319-3325, 1994). Alternatively, antibodies, such as anti-IL-31 monoclonal antibodies (MAb), can also be used as an antagonist to reduce the number of unwanted immune cells for the treatment of autoimmune disease. Asthma, allergies, and other diseases can be treated with MAb against, for example, anti-IL-31 antibodies, soluble IL-3IRA receptors, or IL-31RA / CRF2-4 heterodimers, to inhibit the immune response or reduce the number of damaged cells. Blocking or inhibiting signal transduction via IL-31RA using the polypeptides and antibodies of the invention may also be useful in the case of diseases of the pancreas, kidneys, pituitary and neuronal cells. Positive effects can be exerted on IDDM, NIDDM, pancreatitis, and pancreatic carcinoma. IL-31RA can act as a target for treatment with a malignant tumor MAb, where antagonistic MAb inhibits cancer growth and directs immune-mediated cell killing (Holliger P, and Hoogenboom, H: Nature Biotech. 16: 1015-1016, 1998). Mabs against soluble monomers, homodimers, heterodimers, and multimers of the IL-31RA receptor may also be suitable for the treatment of nephropathies, such as glomerulosclerosis, membranous nephropathy, amyloidosis (which, in addition to other tissues, also affects the kidneys), renal arteriosclerosis, glomerulonephritis of various origins, kidney disease, as well as kidney dysfunction associated with SLE, IDDM, type II diabetes (NIDDM), kidney tumors, and other diseases.

3) Antagonism or triggering sigal transmission through IL-31RA receptors in the treatment of autoimmune diseases such as IDDM, MS, SLE, myasthenia gravis, rheumatoid arthritis and IBD. IL-31 can signal lymphocytes or other immune cells to differentiate, change proliferation, or change the production of cytokines or cell surface proteins that reduce autoimmunity. Specifically, modulating the response of T-helper cells to the altered pattern of cytokine secretion may deflect an autoimmune response in the direction of disease mitigation (Smith JA et al., J. Immunol. 160: 4841-4849, 1998). Similarly, IL-31 can be used to transmit signal, reduce the number and rejection of immune cells involved in asthma, allergies and atopic disease. Signaling through the IL-31RA receptor can also have a beneficial effect in diseases of the pancreas, kidney, pituitary, and neuronal cells. Positive effects can be exerted on IDDM, NIDDM, pancreatitis, and pancreatic carcinoma. IL-31RA can act as a target for treatment with pancreatic cancer MAb, where signal transmission through MAb inhibits cancer growth and targets immune-mediated cell killing (Tutt, AL et al., J Immunol. 161: 3175-3185, 1998). Similarly, T cell specific leukemias, lymphomas, plasma cell dysrasia (e.g., multiple myeloma) and carcinoma can be treated with monoclonal antibodies (e.g., neutralizing antibodies) against the soluble IL-31RA-containing receptors of the invention.

Anti-IL-31 antibodies, soluble monomeric, homodimeric, heterodimeric and multimeric IL-31RA receptor polypeptides described herein can be used to neutralize / block IL-31RA receptor ligand activity in the treatment of autoimmune disease, atopic disease, NIDDM, pancreatitis and dysfunction kidney, as described above.

Anti-IL-31 antibodies and soluble IL-31RA-containing receptors are useful as IL-31 antagonists. Such antagonistic effects can be achieved by directly neutralizing or binding their natural ligand. In addition to being used as antagonists, soluble receptors can bind IL-31 and act as a carrier or carrier proteins, in order to transport IL-31 to various tissues, organs and cells in the body. Essentially, soluble receptors can be fused or linked to molecules, polypeptides or chemical groups that direct the soluble receptor-ligand complex to a specific area, such as tissue, a specific immune cell, monocytes or tumor. For example, in case of acute infection or some malignant tumors, a positive effect may appear due to the induction of inflammation and the local response of acute phase proteins. Thus, the soluble receptors described herein or antibodies of the invention can be used to specifically target the pro-inflammatory ligand of IL-31. See Cosman, D. Cytokine 5: 95-106, 1993; and Fernandez-Botran, R. Exp. Qpin. Invest. Drugs 9: 497-513, 2000.

Typically, the dosage of the IL-31 polypeptide administered (or an analog or IL-31RA fusion protein) varies, depending on factors such as the patient’s age, body weight, height, gender, general medical condition, and previous medical history. Generally, it is desirable to provide the recipient with a dosage of an IL-31 polypeptide that is in the range of about 1 pg / kg to 10 mg / kg (amount of agent / patient body weight), although lower or higher doses can also be administered in certain cases. One of skill in the art can readily determine such dosages and their changes using methods known in the art.

The administration of an IL-31 polypeptide to a subject can be local, inhaled, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a local catheter, or by direct injection into the lesion site. When therapeutic proteins are administered by injection, administration can be carried out by continuous infusion or by single or multiple bolus injections.

Additional routes of administration include oral administration, mucosal administration, pulmonary administration, and transdermal administration. Oral delivery is suitable for polyester-based microspheres, zein-based microspheres, proteoidal microspheres, polycyanoacrylate-based microspheres and lipid-based systems (see, for example, DiBase and Morrel, "Oral Delivery of Microencapsulated Proteins," in Protein Delivery: Physical Systems , Sanders and Hendren (eds.), Pages 255-288 (Plenum Press 1997). The suitability of intranasal delivery is illustrated by such a method of administering insulin (see, for example, Hinchcliffe and Hlum, Adv. Drug Deliv. Rev. 35: 199 (1999)). Dry or liquid particles containing IL-31 can be prepared and inhaled using dry powder dispersants, liquid aerosol generators, or nebulizers (e.g. Pettit and Gombotz, TIBTECH 16: 343 (1998); Patton et al, Adv. Drug Deliv. Rev . 35: 235 (1999)). This approach is illustrated by the AERX diabetes control system, which is a pocket-sized electronic inhaler that delivers aerosolized insulin to the lungs. Studies have shown the delivery of 48,000 kDa proteins through the skin at therapeutic concentrations using low-frequency ultrasound treatment, which illustrates the suitability of transdermal administration (Mitragotri et al, Science 269: 850 (1995)). Transdermal delivery using electroporation is another method of administering a molecule having IL-31 binding activity (Potts et al, Pharm. Biotechnol. 10: 213 (1997)).

A pharmaceutical composition comprising a protein, polypeptide, or peptide having IL-31 binding activity can be formulated according to known methods to provide a pharmaceutically acceptable composition, wherein the therapeutic proteins are combined with a pharmaceutically acceptable carrier. The composition is said to be a “pharmaceutically acceptable carrier” if its administration can be tolerated by the recipient patient. One example of a pharmaceutically acceptable carrier is sterile phosphate buffered saline. Other suitable carriers are well known in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).

For therapeutic purposes, molecules having IL-31 binding activity and a pharmaceutically acceptable carrier are administered to the patient in a therapeutically effective amount. A combination of a protein, polypeptide or peptide having IL-31 binding activity and a pharmaceutically acceptable carrier is said to be administered in a “therapeutically effective amount” if the amount administered is physiologically significant. A substance is physiologically significant if its presence leads to a detectable change in the physiology of the recipient patient. For example, an agent used to treat inflammation is physiologically significant if its presence attenuates at least a portion of the inflammatory response.

A pharmaceutical composition comprising IL-31 (or an analog or IL-31 fusion protein) may be presented in liquid form, in the form of an aerosol, or in solid form. Examples of liquid forms are injectable solutions, aerosols, drops, topological solutions and oral suspensions. Illustrative solid forms include capsules, tablets, and controlled-release forms. An illustrative example of the latter form is mini-osmotic pumps and implants (Bremer et al, Pharm. Biotechnol. 10: 239 (1997); Ranade, "Implants in Drug Delivery", in Drug Delivery Systems, Ranade and Hollinger (eds.), Pages 95- 123 (CRC Press 1995); Bremer et al, "Protein Delivery with Infusion Pumps", in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), Pages 239-254 (Plenum Press 1997); Yewey et al, "Delivery of Proteins from a Controlled Release Injectable Implant ", in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)). Other solid forms include creams, pastes, other topological agents, and the like.

Antibodies against IL-31 described herein can also be formulated as immunoliposomes. Antibody-containing liposomes are prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); and US patents Nos. 4485045 and 4544545. Liposomes with increased circulation time are described in US patent No. 5013556.

Particularly suitable liposomes can be prepared by the reverse phase evaporation of a lipid composition containing phosphatidylcholine, cholesterol and a PEG derivative of phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters with a defined pore size to obtain liposomes with the desired diameter. Fab'fragments of an antibody of the invention can be conjugated to liposomes as described in Martin et al. J. Biol. Chem. 257: 286-288 (1982), by means of a disulfide exchange reaction. A chemotherapeutic agent (such as doxorubicin) is optionally contained in the liposome. See Gabizon et al. J. National Cancer Inst. 81 (19) 1484 (1989).

For storage, therapeutic antibody formulations are prepared by mixing an antibody of the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Phermaceuticals Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citrate, and other organic acid buffers; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt forming counterions, such as sodium; complexes with metals (for example, Zn-protein complexes); and / or non-ionic surfactants such as Tween ™, Pluronics ™ or polyethylene glycol (PEG).

The composition described in the present description may also contain more than one active compound, if necessary in the case of specific indications for treatment, preferably active compounds with additional activities that do not adversely affect each other. For example, it may also be desirable to provide antibodies that bind to IL-31 in a single formulation. Alternatively or additionally, the composition may comprise a chemotherapeutic agent or cytokine. Such molecules in combination are respectively presented in amounts that are effective for the intended purpose.

The active ingredients can also be enclosed in microcapsules obtained, for example, by coacervation or interfacial polymerization methods, for example, microxycapsules based on hydroxymethyl cellulose or gelatin and microcapsules based on polymethylmethacrylate, respectively, in colloidal systems for drug delivery (e.g., liposomes, microspheres microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such methods are described in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

Formulations intended for in vivo administration must be sterile. This is easily achieved by filtration through sterile filtration membranes.

You can get drugs with slow release. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing an antibody, wherein the matrices are in the form of shaped articles, such as films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly-2-hydroxyethyl methacrylate or polyvinyl alcohol), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene vinyl acetate degradable copolymers of lactic acid and glycolic acid, such as Lupron Depot ™ (injectable microspheres consisting of a copolymer of lactic acid and glycolic acid and leuprolide acetate), and poly-D - (-) - 3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid release molecules within 100 days, certain hydrogels release proteins for shorter periods of time. When encapsulated antibodies remain in the body for a long time, they can denature or aggregate as a result of exposure to moisture at 37 ° C, resulting in loss of biological activity and possible changes in immunogenicity. For stabilization, appropriate strategies can be developed, depending on the mechanism involved. For example, if it is revealed that the aggregation mechanism is an intermolecular S-S bond formation through thio-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilization from acidic solutions, controlling the degree of moisture, using appropriate additives and developing certain polymer matrix compositions.

One method for delivering therapeutic polypeptides to a subject intravenously, intraperitoneally, intrathecally, intramuscularly, subcutaneously, or by oral administration, inhalation, or intranasal administration is liposomes. Liposomes are microscopic vesicles that consist of one or more lipid bilayers surrounded by water chambers (see mainly Bakker-Woudenberg et al, Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl. 1): S61 (1993), Kim, Drugs 46: 618 (1993), and Ranade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” in Drug Delivery Systems, Ranade and Hollinger (eds.), Pp. 3-24 (CRC Press 1995 )). The composition of the liposomes is similar to the composition of the membranes, and as a result, the safe introduction of liposomes is possible, and they are biologically degradable. Depending on the method of preparation, liposomes can be single-layer or multilayer, and the diameter of the liposomes can vary from 0.02 μm to more than 10 μm. Many substances can be encapsulated in liposomes: hydrophobic substances are distributed in bilayers, and hydrophilic substances are distributed in the internal water space (s) (see, for example, Machy et al, Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and Ostro et al. American J. Hosp. Pharm. 46: 1516 (1989)). Moreover, the therapeutic availability of the encapsulated substance can be controlled by varying the size of liposomes, the number of bilayers, lipid composition, as well as the charge and surface properties of liposomes.

Liposomes can be adsorbed onto cells of almost any type, and then slowly release the encapsulated substance. Alternatively, endocytosis of adsorbed liposomes by cells that are phagocytic can occur. After endocytosis, intralysosomal degradation of liposomal lipids and the release of encapsulated substances occurs (Scherphof et al, Ann. N.Y. Acad. Sci. 446: 368 (1985)). After intravenous administration, small liposomes (from 0.1 to 1.0 μm) are usually captured by cells of the reticuloendothelial system located mainly in the liver and spleen, while liposomes greater than 3.0 μm are deposited in the lung. The preferred capture of smaller liposomes by cells of the reticuloendothelial system was used to deliver chemotherapeutic agents to macrophages and to liver tumors.

The reticuloendothelial system can be bypassed in several ways, including the saturation of large doses of liposome particles or the selective inactivation of macrophages by pharmacological methods (Claassen et al, Biochim. Biophys. Acta 502: 428 (1984)). In addition, it was shown that the incorporation of phospholipid derivatives formed using glycolipids or polyethylene glycol into liposome membranes leads to a significant decrease in reticuloendothelial uptake (Allen et al, Biochim. Biophys. Acta 1068: 133 (1991); Allen et al, Biochim. Biophys Acta 1150: 9 (1993)).

Liposomes can also be prepared to target specific cells or organs by varying the phospholipid composition or incorporating receptors or ligands into liposomes. For example, liposomes made with a high non-ionic surfactant content were used to target the liver (Hayakawa et al., Japanese Patent 04-244018; Kato et al, Biol. Pharm. Bull. 16: 960 (1993)). These formulations were prepared by mixing soy phosphatidylcholine, α-tocopherol and ethoxylated hydrogenated castor oil (HCO-60) in methanol, concentrating the mixture in vacuo and then diluting the mixture with water. It was also shown that the composition of liposomes from dipalmitoylphosphatidylcholine (DPPC) with a mixture of sterilglucoside (SG) and cholesterol (Ch) obtained from soy is directed to the liver (Shimizu et al, Biol Pharm. Bull 20: 881 (1997)).

Alternatively, various ligands for targeting, such as antibodies, antibody fragments, carbohydrates, vitamins, and transport proteins, can be bound to the surface of the liposome. For example, to target receptors for asialoglycoprotein (galactose), which are expressed exclusively on the surface of liver cells, liposomes can be modified using branched-type galactosillipid derivatives (Kato and Sugiyama, Grit. Rev. Ther. Drug Carrier Syst. 14: 287 (1997) ; Murahashi et al, Biol. Pharro. Bull. 20: 259 (1997)). Similarly, it was shown by Wu et al, Hepatology 27: 772 (1998) that liposomes labeled with asialofetuin lead to a shortened plasma half-life of liposomes and significantly enhanced uptake of hepatic labeled liposomes with hepatocytes. On the other hand, the accumulation in the liver of liposomes containing branched-type galactosillipid derivatives can be inhibited by pre-injection of asialofetuin (Murahashi et al, Biol. Pharm. Bull. 20: 259 (1997)). Human serum albumin-based polyconitilated liposomes are another approach for targeting liposomes in liver cells (Kamps et al, Proc. Nat'l Acad. Sci. USA 94: 11681 (1997)). Moreover, Geho, et al., US Patent No. 4,630,044 describes a hepatocyte-directed liposomal vesicle delivery system that is specific for hepatobiliary receptors associated with specialized metabolic liver cells.

Polypeptides having IL-31 binding activity can be encapsulated in liposomes using standard protein microencapsulation methods (see, for example, Anderson et al, Infect. Immun. 31: 1099 (1981), Anderson et al, Cancer Res. 50: 1853 (1990), and Cohen et al, Biochim. Biophys. Acta 1063: 95 (1991), Alving et al "Preparation and Use of Liposomes in Immunological Studies", in Liposome Technology, 2nd edition, Vol. III, Gregoriadis (ed .), page 317 (CRC Press 1993), Wassef et al, Meth. Enzymol. 149: 124 (1987)). As indicated above, therapeutically acceptable liposomes may contain many components. For example, liposomes may contain lipid derivatives of polyethylene glycol (Allen et al, Biochim. Biophys. Acta 1150: 9 (1993)).

To maintain high systemic levels of therapeutic proteins, degradable polymer microspheres have been developed. The microspheres are made from degradable polymers such as a lactide-glycolide copolymer (PLG), polyanhydrides, polyorthoesters, biologically non-degradable ethyl vinyl acetate polymers in which the polymer contains proteins (Gombotz and Pettit, Bioconiugate Chem. 6: 332 (1995); Ranade; Roleade of Polymers in Drug Delivery ", in Drug Delivery Systems, Ranade and Hollinger (eds.), pages 51-93 (CRC Press 1995); Roskos and Maskiewicz," Degradable Controlled Release Systems Useful for Protein Delivery, "in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), Pages 45-92 (Plenum Press 1997); Bartus et al, Science 281: 1161 (1998); Putney and Burke, Nature Biotechnology 16: 153 (1998); Putney, Curr. Opin Chem. Biol. 2: 548 (1998)). Polyethylene glycol (PEG) coated nanospheres can also be carriers for intravenous administration of therapeutic proteins (see, for example, Gref et al, Pharm. Biotechnol. 10: 167 (1997)).

Specialists in this field can develop other dosage forms, as shown, for example, Ansel and Popovich, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th edition (Lea & Febiger 1990), Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th edition (Mack Publishing Company 1995), and Ranade and Hollinger, Drug Delivery Systems (CRC Press 1996).

By way of example, pharmaceutical compositions may be supplied as a kit containing a container that contains an IL-31 polypeptide or an IL-31 antagonist (for example, an antibody or antibody fragment that binds an IL-31 polypeptide). Therapeutic polypeptides can be provided in the form of an injectable solution in single or multiple doses, or as a sterile powder, which is diluted before injection. Alternatively, such a kit may include a dry powder dispersant, a liquid aerosol generator, or a nebulizer for administering a therapeutic polypeptide.

In one aspect, this invention relates to a monoclonal antibody comprising a monoclonal antibody that specifically binds a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, wherein the polypeptide is capable of binding to a monoclonal antibody produced by a hybridoma deposited in the American Type Culture Collection with the ATCC patent designation selected from: a) patent number of deposit in ATCC PTA-6815; b) Patent Deposit Number at ATCC PTA-6816; c) patent number of deposit in ATCC PTA-6829; d) Patent Deposit Number at ATCC PTA-6830; e) Patent Deposit Number at ATCC PTA-6831; f) Patent Deposit Number at ATCC PTA-6871; g) Patent Deposit Number at ATCC PTA-6872; h) Patent Deposit Number at ATCC PTA-6875; and i) Patent Deposit Number at ATCC PTA-6873. In this embodiment, the antibody neutralizes the interaction of IL-31 (SEQ ID NO: 2) with IL-31RA (SEQ ID NO: 5). In another embodiment, the antibody is (a) a monoclonal mouse antibody, (b) a humanized antibody formed from (a), or (c) a fragment of an antibody, or (d) a human monoclonal antibody. In another embodiment, the antibody further comprises a radionuclide, an enzyme, a substrate, a cofactor, a fluorescent marker, a chemiluminescent marker, a peptide marker, a magnetic particle or toxin. In another embodiment, the antibody further has pegylation. In another embodiment, the antibody is a neutralizing antibody. In another embodiment, administration of an antibody to a mammal inhibits, prevents, reduces, or blocks the pro-inflammatory activity of the polypeptide. In another embodiment, administration of an antibody to a mammal inhibits, prevents, reduces, or blocks scratching and dermatitis due to the pro-inflammatory activity of the polypeptide.

In another aspect, this invention relates to a monoclonal antibody that specifically binds a polypeptide containing the amino acid sequence of SEQ ID NO: 2, where the polypeptide is capable of binding to a monoclonal antibody produced by a hybridoma deposited in the American Type Culture Collection having an ATCC patent designation selected from: a) Patent Deposit Number in ATCC PTA-6815; b) Patent Deposit Number at ATCC PTA-6816; c) patent number of deposit in ATCC PTA-6871; d) the patent deposit number at ATCC PTA-6829; and e) the patent deposit number at ATCC PTA-6830.

In another aspect, this invention relates to a monoclonal antibody that specifically binds a polypeptide containing the amino acid sequence of SEQ ED NO: 2, where the polypeptide is capable of binding to a monoclonal antibody produced by a hybridoma deposited in the American Type Culture Collection having an ATCC patent designation selected from: a) Patent Deposit Number in ATCC PTA-6815; b) the patent deposit number in ATCC PTA-6816; and c) the patent deposit number in ATCC PTA-6871.

In another aspect, this invention relates to a monoclonal antibody that specifically binds a polypeptide containing the amino acid sequence of SEQ ID NO: 2, where the polypeptide is capable of binding to a monoclonal antibody produced by a hybridoma deposited in the American Type Culture Collection having an ATCC patent designation selected from: a) the patent deposit number of the ATCC PTA-6829; and b) the patent number of the deposit of the ATCC PTA-6830.

In another aspect, a monoclonal antibody is provided that specifically binds a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, where the polypeptide is capable of binding to a monoclonal antibody produced by a hybridoma deposited in the American Type Culture Collection having the ATCC patent designation selected from: a) a patent deposit numbers in ATCC PTA-6872; and b) the patent deposit number in ATCC PTA-6875.

In another aspect, a monoclonal antibody is provided that specifically binds a polypeptide containing the amino acid sequence of SEQ ID NO: 11, where the polypeptide is capable of binding to a monoclonal antibody produced by a hybridoma deposited in the American Type Culture Collection with the patent designation ATCC PTA-6874. In this embodiment, the antibody is a neutralizing antibody. In this embodiment, administration of an antibody to a mammal inhibits, prevents, reduces, or blocks the pro-inflammatory activity of the polypeptide. In this embodiment, administration of an antibody to a mammal inhibits, prevents, reduces, or blocks scratching and dermatitis due to the pro-inflammatory activity of the polypeptide.

In another aspect, this invention relates to a method for reducing inflammation in a mammal, comprising administering to the mammal an amount of an anti-IL-31 antibody described herein, by which inflammation is reduced.

In another aspect, this invention relates to a method for treating a mammal suffering from an inflammatory disease in which IL-31 is involved, comprising: administering an IL-31 antagonist to a mammal so that the inflammation is reduced, wherein the antagonist contains (i) an antibody, antibody fragment or binding a polypeptide that specifically binds a polypeptide or fragment of an IL-31RA polypeptide; or (ii) a polypeptide or fragment of an IL-31RA polypeptide; and where the inflammatory activity of IL-31 is reduced. In this embodiment, the disease is an inflammatory disease, including pruritus-related diseases. In this embodiment, the disease is atopic dermatitis. In this embodiment, the disease is nodular pruritus.

In another aspect, this invention relates to a method for reducing, inhibiting, or preventing the effect of pruritus in a mammal in which IL-31 is involved, the method comprising: administering an IL-31 antagonist to a mammal so that itching is reduced, wherein the antagonist contains (i) an antibody, fragment an antibody or a binding polypeptide that specifically binds a polypeptide or polypeptide fragment of a polypeptide containing the amino acid sequence of SEQ ID NO: 2, or a fragment thereof; and where there is a decrease in the itch-related activity of IL-31. In this embodiment, the disease of the mammal is atopic dermatitis. In this embodiment, the disease of the mammal is dermatitis. In this embodiment, the antibody is an antibody as described herein.

In another aspect, this invention relates to a method of reducing, inhibiting, or preventing the effect of pruritus in a mammal in which IL-31 is involved, comprising: administering an IL-31 antagonist to a mammal so that pruritus reduction in a mammal occurs, wherein the antagonist contains (i) an antibody, antibody fragment or binding polypeptide that specifically binds a polypeptide or polypeptide fragment of a polypeptide containing the amino acid sequence of SEQ ID NO: 2, or a fragment thereof; and where through this there is a decrease in the activity of IL-31 in relation to combing. In this embodiment, the disease of the mammal is atopic dermatitis. In this embodiment, the disease of the mammal is dermatitis. In this embodiment, the antibody is an antibody as described herein.

In another aspect, this invention relates to a monoclonal antibody that specifically binds a polypeptide containing the amino acid sequence of SEQ ID NO: 2, where the polypeptide is capable of binding to a monoclonal antibody produced by a hybridoma clone denoted by a number selected from: a) clone 292.12.3.1 (patent number deposit in ATCC PTA-6815); b) clone 292.63.5.3 (patent deposit number in ATCC PTA-6829); c) clone 292.72.3.1 (patent deposit number in ATCC PTA-6816); d) Clone 292.84.1.6 (Patent Deposit Number in ATCC PTA-6871) and e) Clone 292.118.6.4 (Patent Deposit Number in ATCC PTA-6830).

In another aspect, this invention relates to a monoclonal antibody that specifically binds a polypeptide containing the amino acid sequence of SEQ ID NO: 2, where the polypeptide is capable of binding to a monoclonal antibody produced by a hybridoma clone designated by a number selected from: a) clone 294.35.2.6.3 ( Patent Deposit Number in ATCC PTA-6872); b) clone 294.144.3.5 (patent deposit number in ATCC PTA-6873); c) clone 294.154.5.6 (patent number of deposit in ATCC PTA-6875) and d) clone 294.163.2.1 (patent number of deposit in ATCC PTA-6831).

In another aspect, this invention relates to a monoclonal antibody containing a monoclonal antibody capable of competing for the binding of a polypeptide containing the amino acid sequence of SEQ ID NO: 2, where the polypeptide is capable of binding a monoclonal antibody produced by a hybridoma deposited in the American type culture collection with the patent designation for the sample ATCC selected from: a) patent number of deposit in ATCC PTA-6815; b) Patent Deposit Number at ATCC PTA-6816; c) patent number of deposit in ATCC PTA-6829; d) Patent Deposit Number at ATCC PTA-6830; e) Patent Deposit Number at ATCC PTA-6831; f) Patent Deposit Number at ATCC PTA-6871; g) Patent Deposit Number at ATCC PTA-6872; h) Patent Deposit Number at ATCC PTA-6875; and i) Patent Deposit Number at ATCC PTA-6873.

In another aspect, a hybridoma is provided wherein the hybridoma is deposited in the American Type Culture Collection with an ATCC sample patent designation selected from: a) ATCC patent deposit number PTA-6815; b) Patent Deposit Number at ATCC PTA-6816; c) patent number of deposit in ATCC PTA-6829; d) Patent Deposit Number at ATCC PTA-6830; e) Patent Deposit Number at ATCC PTA-6831; f) Patent Deposit Number at ATCC PTA-6871; g) Patent Deposit Number at ATCC PTA-6872; h) Patent Deposit Number at ATCC PTA-6875; and i) Patent Deposit Number at ATCC PTA-6873.

In another aspect, the present invention relates to a method for producing an anti-IL-31 polypeptide antibody, comprising: inoculating an animal with a polypeptide selected from the group consisting of: (a) a polypeptide consisting of from 9 to 141 amino acids, where the polypeptide is identical to a continuous sequence amino acid residues in SEQ ID NO: 2, from amino acid number 24 (Ser) to amino acid number 164 (Thr); (b) a polypeptide as described above; (c) a polypeptide containing the amino acid sequence of SEQ ID NO: 2 from amino acids 38-52; (d) a polypeptide containing the amino acid sequence of SEQ ID NO: 2 from amino acids number 83-98; (e) a polypeptide containing the amino acid sequence of SEQ ID NO: 2 from amino acids number 104-117; (f) a polypeptide containing the amino acid sequence of SEQ ID NO: 2 from amino acids number 137-152; (g) a polypeptide containing the amino acid sequence of SEQ ID NO: 2 from amino acids 38-152; (h) a polypeptide containing the amino acid sequence of SEQ 3D NO: 2 from amino acids 24-164; (c) a polypeptide containing the amino acid sequence of SEQ ID NO: 11, from amino acids 38-52; (d) a polypeptide containing the amino acid sequence of SEQ ID NO: 11 from amino acids number 85-98; (e) a polypeptide containing the amino acid sequence of SEQ ID NO: 11 from amino acids number 104-118; (f) a polypeptide containing the amino acid sequence of SEQ ID NO: 11 from amino acids number 141-157; (g) a polypeptide containing the amino acid sequence of SEQ ID NO: 11 from amino acids 38-157; (h) a polypeptide containing the amino acid sequence of SEQ ID NO: 11 from amino acids number 24-163; (i) a polypeptide containing an antigenic epitope in accordance with the Hopp / Woods hydrophilicity profile of SEQ ID NO: 2 or SEQ ID NO: 11, where the profile is based on the advancement of a window of six residues, in-depth residues G, S and T and exposed residues H, Y and W do not count; and where the polypeptide elicits an immune response in an animal with antibody production; and isolation of the antibody from the animal.

In another aspect, the present invention relates to an antibody (eg, a neutralizing antibody) produced by a method as described above, wherein the antibody binds to a polypeptide of SEQ IDN NO: 2 or SEQ ID NO: 11. In one embodiment, the antibody described above specifically binds to the polypeptide shown in SEQ ID NO: 2 or SEQ ID NO: 11.

In another aspect, the present invention relates to a method for detecting the presence of IL-31 in a biological sample, comprising the steps of: (a) contacting the biological sample with an antibody or antibody fragment, as described above, wherein the contacting is carried out under conditions that allow the binding of the antibody or fragment antibodies with a biological sample, and (b) detecting either a bound antibody or a bound antibody fragment.

In another aspect, the present invention relates to a method for killing cancer cells, comprising obtaining ex vivo tissue or a biological sample containing cancer cells of a patient, or identifying cancer cells in vivo; the production of a polypeptide in a manner as described herein; formulating the polypeptide in a pharmaceutically acceptable carrier; and administering to the patient or exposure of the polypeptide to malignant cells; where the polypeptide causes cell death. In one embodiment, the method of killing cancer cells is as described above, wherein the polypeptide is further conjugated to a toxin. In one embodiment, the antibody is as described above, wherein the antibody is selected from the group consisting of: (a) a polyclonal antibody, (b) a mouse monoclonal antibody, (c) a humanized antibody formed from (b), (d) a fragment antibodies and (e) human monoclonal antibodies.

In another aspect, the present invention relates to an antibody or antibody fragment that specifically binds to a polypeptide comprising a sequence of amino acid residues selected from the group consisting of: (a) a polypeptide represented by residue 38 (Val) to 152 (Leu), as shown in SEQ ID NO: 2; (b) a polypeptide represented by residue 27 (Leu) to 164 (Thr), as shown in SEQ ID NO: 2; (c) a polypeptide represented by residue 24 (Thr) to 164 (Thr), as shown in SEQ ID NO: 2; and (d) a polypeptide represented by residue 1 (Met) to 164 (Thr), as shown in SEQ ID NO: 2. In another embodiment, the antibody is as described above, wherein the antibody further comprises a radionuclide, an enzyme, a substrate, a cofactor, a fluorescent marker, a chemiluminescent marker, a peptide label, a magnetic particle, a drug, or a toxin.

In another aspect, the present invention relates to a method of inhibiting IL-31-induced proliferation or differentiation of hematopoietic and hematopoietic progenitor cells, comprising culturing bone marrow or peripheral blood cells with a composition containing an amount of an antibody as described herein sufficient to reduce proliferation or differentiation of hematopoietic cells in bone marrow or peripheral blood cells compared with bone marrow cells or peripheral blood cultured in the absence of soluble receptor for the cytokine. In one embodiment, a method of inhibiting IL-31-induced proliferation or differentiation of hematopoietic cells and hematopoietic progenitor cells is as described above, wherein the hematopoietic cells and hematopoietic progenitor cells are lymphoid cells. In another embodiment, the method of inhibiting IL-31-induced proliferation or differentiation of hematopoietic cells and hematopoietic progenitor cells is as described above, wherein the lymphoid cells are macrophages or T cells.

In another aspect, the present invention relates to a method for reducing IL-31-induced inflammation, comprising administering to a mammal with inflammation an amount of an antibody composition, as described herein, sufficient to reduce inflammation.

In another aspect, the present invention relates to a method for suppressing an inflammatory response in a mammal with inflammation, comprising: (1) determining the level of the inflammatory molecule; (2) administering a composition comprising an antibody, as described herein, in an acceptable pharmaceutical carrier; (3) determining the level of the inflammatory molecule after administration; (4) comparing the level of the inflammatory molecule of stage (1) with the level of the inflammatory molecule of stage (3), where the absence of an increase or decrease in the level of the inflammatory molecule indicates a suppression of the inflammatory response. In one embodiment, the antibody is as described above, wherein the antibody further comprises a radionuclide, an enzyme, a substrate, a cofactor, a fluorescent marker, a chemiluminescent marker, a peptide label, a magnetic particle, a drug, or a toxin.

In another aspect, the present invention relates to a method of inhibiting IL-31-induced proliferation or differentiation of hematopoietic and hematopoietic progenitor cells, comprising culturing bone marrow or peripheral blood cells with a composition containing an amount of an antibody as described herein sufficient to reduce proliferation or differentiation of hematopoietic cells in bone marrow or peripheral blood cells compared with bone marrow cells or peripheral Coy blood cultured in the absence of soluble receptor for the cytokine. In one embodiment, a method of inhibiting IL-31-induced proliferation or differentiation of hematopoietic cells and hematopoietic progenitor cells is as described above, wherein the hematopoietic cells and hematopoietic progenitor cells are lymphoid cells. In another embodiment, the method of inhibiting IL-31-induced proliferation or differentiation of hematopoietic cells and hematopoietic progenitor cells is as described above, wherein the lymphoid cells are macrophages or T cells.

In another aspect, the present invention relates to a method for reducing IL-31-induced inflammation, comprising administering to a mammal with inflammation an amount of an antibody composition, as described herein, sufficient to reduce inflammation.

In another aspect, the present invention relates to a method for suppressing an inflammatory response in a mammal with inflammation, comprising: (1) determining the level of the inflammatory molecule; (2) administering a composition comprising an antibody, as described herein, in an acceptable pharmaceutical carrier; (3) determining the level of the inflammatory molecule after administration; (4) comparing the level of the inflammatory molecule of stage (1) with the level of the inflammatory molecule of stage (3), where the absence of an increase or decrease in the inflammatory molecule indicates a suppression of the inflammatory response.

In another aspect, the present invention relates to a method for treating a mammal suffering from an inflammatory disease in which IL-31 is involved, comprising: administering an IL-31 antagonist to the mammal so that there is a reduction in inflammation, wherein the antagonist is selected from the group consisting of an antibody or binding a polypeptide that specifically binds a polypeptide or fragment of an IL-31 polypeptide (SEQ ID NO: 2). In one embodiment, a method of treating a mammal suffering from an inflammatory disease is as described above, wherein the disease is a chronic inflammatory disease. In another embodiment, a method of treating a mammal suffering from an inflammatory disease is as described above, wherein the disease is a chronic inflammatory disease selected from the group consisting of: inflammatory bowel disease; ulcerative colitis; Crohn's disease; atopic dermatitis; eczema and psoriasis. In another embodiment, a method of treating a mammal suffering from an inflammatory disease is as described above, wherein the disease is an acute inflammatory disease. In another embodiment, a method of treating a mammal suffering from an inflammatory disease is as described above, wherein the disease is an acute inflammatory disease selected from the group consisting of: endotoxemia; septicemia; toxic shock syndrome and infectious disease. In another embodiment, a method of treating a mammal suffering from an inflammatory disease is as described above, wherein the antibody further comprises a radionuclide, an enzyme, a substrate, a cofactor, a fluorescent marker, a chemiluminescent marker, a peptide label, a magnetic particle, a drug, or a toxin.

In another aspect, the present invention relates to a method for detecting inflammation in a patient, comprising: obtaining tissue or a biological sample of a patient; incubating a tissue or biological sample with an antibody, as described herein, under conditions where the antibody binds to a complementary polypeptide in a tissue or biological sample; imaging an antibody bound to a tissue or biological sample; and comparing the levels of the antibody bound to the tissue or biological sample of the patient with a normal control tissue or biological sample, where an increase in the level of the antibody bound to the tissue or biological sample of the patient relative to the normal control tissue or biological sample indicates inflammation in the patient.

In another aspect, the present invention relates to a method for detecting inflammation in a patient, comprising: obtaining tissue or a biological sample of a patient; labeling a polynucleotide containing at least 14 consecutive nucleotides of SEQ ID NO: 1 or a complementary sequence of SEQ ID NO: 1; incubating a tissue or biological sample with a polynucleotide under conditions in which the polynucleotide hybridizes to a complementary polynucleotide sequence; visualization of the labeled polynucleotide in a tissue or biological sample; and comparing the level of hybridization of the labeled polynucleotide in the patient’s tissue or biological sample with normal healthy tissue or biological sample, where increased hybridization of the labeled polynucleotide with the patient’s tissue or biological image relative to normal healthy tissue or biological sample indicates inflammation in the patient.

This invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1

Obtaining rat MAb against mouse IL-31

A. Immunization and screening of rat serum

Four female 3-month old Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were immunized with mouse IL-31. Rats received primary immunization by intraperitoneal injection of ~ 290 μg of purified recombinant mouse IL-31 (produced by BHK cells) fused at the C-terminus with a peptide consisting of the EYMPME sequence (SEQ ID NO: 7) (hereinafter referred to as mIL31-CEE) in combination with Freund's complete adjuvant (Pierce, Rockford, IL) according to the manufacturer's instructions. After primary immunization, an additional 150 μg mIL-31-CEE was administered to each rat in incomplete Freund's adjuvant by the intraperitoneal method every two weeks for six weeks. Seven days after the third and fourth immunizations in rats, blood was taken through the retroorbital plexus and serum was isolated from the blood to analyze its ability to bind to mIL-31-CEE in solution and to inhibit (as determined by neutralization) the stimulating activity of mIL-31-CEE in relation to mouse IL-31RA transfected cell line.

The ability of rat antibodies against mouse IL-31 in antiserum to bind to mIL-31-CEE was evaluated using an antigen-trap ELISA. In this analysis, the wells of 96-well polystyrene ELISA plates were first coated with Fc-specific goat anti-rat IgG antibody (Jackson Immunoresearch) at a concentration of 500 ng / ml in an amount of 100 μl / well in 0.1 M Na ₂ CO ₃ pH 9.6 The plates were incubated overnight at 4 ° C, after which the unbound antibody was aspirated, and the plates were washed twice with PBS-Tween at 300 μl / well (0.137 M NaCl, 0.0027 M KCl, 0.0072 M Na ₂ HPO ₄ , 0.0015 M KH ₂ PO ₄ , 0.05% v / v polysorbate 20, pH 7.2). Wells were blocked by 200 μl / well with SuperBlock (Pierce, Rockford, IL) for 5 minutes at room temperature (RT), SuperBlock was shaken off the plate and blocking was repeated again, after which the plates were washed twice with PBS-Tween. Serial 10-fold dilutions of serum were obtained (in PBS-Tween plus 1% w / v bovine serum albumin (BSA) and 0.05% v / v Proclin 300, pH 7.2 = dilution buffer), starting from an initial dilution of 1: 1000 to 1: 1,000,000. Three samples of each dilution were then transferred to an assay plate, 100 μl / well, to bind rat IgG in serum to assay the plate through the Fc portion of the molecule. Normal rat serum served as a negative control. After 1 hour of incubation at RT, the wells were aspirated and the plates were washed twice as described above. Then, biotinylated mIL-31-CEE (biotin: protein 12: 1 molecular ratio) was added to the wells at a concentration of 500 ng / ml, 100 μl / well. After 1 hour of incubation at RT, the unbound biotinylated mIL-31-CEE was aspirated from the wells and the plates were washed twice. Then, horseradish peroxidase-labeled streptavidin (Pierce, Rockford, IL) was added to each well at a concentration of 500 ng / ml, 100 μl / well, and the plates were incubated at RT for 1 hour. After removing unbound HRP-SA, the plates were washed 5 times, 100 μl / well of tetramethylbenzidine (TMB) (BioFX Laboratories, Owings Mills, MD) was added to each well, and the plates were incubated for 5 minutes at RT. Color formation was stopped by adding 100 μl / well TMB stop reagent for 450 nm (BioFX Laboratories, Owings Mills, MD) and the absorbance for the wells was determined on a Molecular Devices Spectra MAX 340 at 450 nm.

The ability of rat antibodies against mouse IL-31 in antiserum to inhibit (as determined by neutralization) the mouse IL-31 stimulatory activity through a recognition receptor was evaluated using a cell neutralization assay using lafiferase Baf3 transfected with mIL-31RA / OSMRbeta reporter system (Baf3 / KZ134 / mcytor17 / mOSMRbeta), which can be activated by stimulating cells with mouse IL-31. For this analysis, an initial 1: 250 dilution of each antiserum in the assay medium was obtained (RPMI 1640, 10% ETF, 2 mM L-glutamine, 1 mM sodium pyruvate, 1 × penicillin-streptomycin-neomycin (Lavitrogen, Carlsbad, CA)), and then, for each of them, serial two-fold dilutions were carried out to a final dilution of 1: 32000. An equal volume of mIL-31-CEE diluted to 4 ng / ml in assay buffer was added to each dilution of antiserum to a final volume in wells of 100 μl. The antibody / cytokine mixtures were incubated at RT for 0.5 hours, after which the target cells were washed from their growth medium, brought to a density of 300,000 cells / ml and antibody / cytokine, 100 μl / well was added to the mixtures. An assay medium containing 2 ng / ml mIL-31 and not containing antiserum served as a negative control and showed the maximum stimulation that can be achieved in this assay. After incubation at 37 ° C, 5% CO ₂ for 16-24 hours, the plates were centrifuged at 1,500 rpm for 5 minutes, the medium was carefully shaken off and 25 μl of 1 × cell lysis buffer was added to each well (Promega, Madison , WI). The plates were gently shaken for 10 minutes at RT to ensure complete cell lysis, and then the lysates were transferred to solid white 96-well plates (Corning / Costar 3917, Acton, MA). 40 μl of luciferase assay substrate (Promega) was added to each well. Wells were then evaluated for luciferase activity (reflecting activation by the IL-31 STAT reporter construct) on a luminometer using a 5 second integration interval.

Both the ELISA assay of the “antigenic trap” type and the cell neutralization assay showed that all four rats developed a high antibody response to mIL-31, although one rat clearly developed a stronger response than the others. In general, the response measured by an "antigenic trap" ELISA was largely consistent with that found in a cell neutralization assay, confirming that IgG antibodies are primarily responsible for inhibition of mIL-31.

B. Merger

Four weeks after the last intraperitoneal immunization, rats with the most significant neutralization titer mIL-31 were last immunized with approximately 150 μg mIL-31-CEE in PBS by intravascular injection. After five days, the spleen and lymph nodes of these rats were collected, turned into a suspension of individual cells and fused to the Sp2 / 0 mouse myeloma cell line (Shulman, M. et al., Nature 276: 269-270, 1976) in the ratio of lymphoid cells: 4: 1 myeloma cells using PEG 1500 using standard methods known in the art (Harlow, E. and Lane, D. 1988. "Antibodies A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The fusion mixture was distributed into a series of 96-well flat bottom plates together with BALB / c thymocytes as a feeder layer (Oi, VT and Herzenberg, LA. 1980. "Selected Methods in Cellular Immunology", BB Mishell and SM Shiigi, eds. , pp. 351-372, Freeman, San Francisco). Water in the wells of the fusion plates was replenished once by replacing 70% of the medium and thymocytes after 4 days and again after two days by replacing only the medium. Wells were analyzed eight days after culturing the cells to be fused.

C. Screening after the merger

For initial screening, an “antigenic trap” type ELISA was used for mIL-31 as described above, except that hybridoma supernatants were tested without dilution and applied as replicas to assay plates from culture plates. Approximately 170 positive wells were identified. Supernatants from each of these wells, as well as several negative wells, were then evaluated for their ability to inhibit mIL-31 in the cell neutralization assay described previously. Each of them was tested at a final dilution of 1: 8 in the medium for analysis. In this dilution, the level of non-specific stimulatory components in hybridoma supernatants was sufficiently reduced to make a minimal contribution to the determined stimulation index. It turned out that most of the supernatants neutralize to some extent mIL-31, and approximately ten of them show essentially complete neutralization, while the rest show a range of inhibition up to a level where approximately the other ten appear to be very low, if possessed, neutralizing potential. Hybridoma cells in approximately 150 of the "antigenic trap" positive ELISA wells were successfully grown in culture in 24-well plates. When the density of the cultures of the 24-well plates was approximately 4-6 · 10 ⁵ cells / ml, the supernatant (approximately 1.5 ml) was collected and stored separately for each well and the cells from each well were frozen.

Each of the supernatants from 24-well plates was reassayed both in an "antigenic trap" type ELISA and in IL-31 cell neutralization assays used in post-fusion screening. In addition, these supernatants were also tested in an "antigenic trap" ELISA using the human IL-31 homolog also constructed with the EYMPME marker (SEQ ID NO: 7) fused to the C-terminus of the IL-31 molecule and produced in cells BHK. The results indicate that after expansion, most of the major supernatants in the wells retain their ability to recognize mouse IL-31 in solution. Among these positive antigen trap assay wells, mIL-31 inhibitory activity ranged from almost complete inhibition for about 20 supernatants to essentially no inhibition for 10-15 supernatants. In the antigenic trap assay, no cross-reactivity was detected against human IL-31-CEE, indicating that none of the anti-mIL-31 rat antibodies cross-reacted with the human IL31 homolog or did not recognize the CEE marker fused with the C-terminus of the mIL-31-CEE molecule.

D. Selection and cloning of hybridomas producing neutralizing MAb against mIL31

Cells from seven of the ten main wells with the highest neutralization of IL-31 (271.5.7, 271.9.4, 271.26.6, 271.27.4, 271.33.1, 271.33.3 and 271.39.4) were cloned to isolate the cloned hybridoma, producing neutralizing mAbs of interest. Cells were cloned in the presence of thymocytes in 96-well microtiter plates for cell culture using a standard low-density dilution approach (less than 1 cell per well) and monoclonality was evaluated prior to analysis by microscopic examination of the wells for single growth foci. After six days of cultivation, all wells on the plates were screened by an "antigenic trap" ELISA for IgG specific for mIL-31. Supernatants from 6-10 wells that were positive for specific mAbs were also obtained from wells with only one hybridoma growth colony, were collected from each cloned series and re-screened at various dilutions in an ELISA of the "antigenic trap" type, as well as in a cell neutralization assay, to identify the "best" producing mAb clone. The results of these tests showed that the corresponding producing mAb neutralizing hybrid clones were obtained in series 271.9.4, 271.26.6, 271.33.1, 271.33.3 and 271.39.4. The "best" clone in each of these series was re-cloned and the subclones were screened as described to obtain the end lines of hybrids 271.9.4.2.6, 271.26.6.6.1, 271.33.1.2.2, 271.33.3.2.1 and 271.39. 4.6.5. The rat IgG mAb isotype produced by each of these hybridomas was determined using ELISA using mouse biotinylated mAbs specific for rat IgG isotype. It was found that all five mAbs belong to the IgG1 subclass. Hybridoma 271.26.6.6.1 was deposited in the patent collection of the American Type Culture Collection (ATCC, Manassas, VA) as an initial sample under the Budapest Treaty and was assigned ATCC registration number No. PTA-6874.

Each of the fully cloned hybridomas producing mouse anti-IL-31 mAbs was adapted for growth in a hybridoma serum-free medium (Hybridoma-SFM, Invitrogen). The supernatant from high-density cultures grown in Hybridoma-SFM was centrifuged to remove cells and cell debris, filtered through a 0.2 μm filter, and the mAbs were purified by protein G chromatography using standard methods known in the art.

E. Assessment of the specific neutralizing activity of MAb in vitro

To assess the in vitro specific neutralizing activity of each of the five rat mAbs against mouse IL-31, each of the purified mAbs was tested in the cell neutralization assay described previously, except that mIL-31 was first added to the cells, and then this mixture was added to antibodies. In addition to neutralizing activity against mIL-31-CEE, mAbs were also evaluated for another form of mouse IL-31, designated c108smIL-31, in which the cysteine residue at position 108 was converted to a serine residue. The latter material was produced in E. coli without the C-EE peptide. Both forms of mIL-31 were used at a final concentration of 1 and 5 ng / ml. the mAbs were adjusted to a concentration of 20 μg / ml in the assay medium and two samples were tested as serial 10-fold dilutions in the range of the final concentration in the analyzed mixture from 10 μg / ml to 0.00001 μg / ml. IC ₅₀ values were determined using Softmax software (Molecular Devices). The results are presented in table 1 and show that mAb 271.26.6.6.1, 271.33.1.2.2 and 271.39.4.6.5 are the most effective neutralizing mAbs and have similar efficacy in this in vitro test. Interestingly, mAb 271.33.3.2.1 was significantly less effective against the c108smIL-31 mIL-31 variant than other mAbs, confirming the findings of studies on sorting by epitopes that this antibody in all probability has an epitope specificity different from the other four mAbs . The fact that this mAb was also significantly less effective with respect to the c108smIL-31 mIL-31 variant compared to the mIL-31-CEE variant confirms that the epitope recognized by 271.33.3.2.1 may partially include the glycosylation site on the molecule IL-31.

Table 1
Rat MAb activity against mouse IL-31 in cell neutralization in vitro Option Concentration, IC _fifty  (ng / ml) for mAb mIL-31 ng / ml 271.9.4.2.6 271.26.6.6.1 271.33.1.2.2 271.33.3.2.1 271.39.4.6.5 mIL31-CEE one eleven 3 2 13 2 5 thirty fourteen 13 36 12 c108smIL31 one 96 eleven 7 1248 9 5 190 43 33 2551 40

Example 2

Obtaining mouse mAb against human IL-31

A. Immunization and screening of mouse serum

Two groups of female BALB / c mice aged six to eight weeks, five animals each, were immunized with human IL-31. Mice in group 1 were immunized by intraperitoneal injection of purified recombinant human IL-31 fused at the C-terminus with a peptide consisting of the sequence EYMPME (SEQ ID NO: 7) (hereinafter referred to as IL-31-CEE) in combined with Ribi adjuvant according to the manufacturer's instructions. This protein was produced in BHK cells. Mice in group 2 were similarly immunized with a purified recombinant mutant form of human IL-31, in which the cysteine residue at position 108 was converted to a serine residue (hereinafter referred to as c108sIL-31). This material was produced in E. coli without the C-EE peptide. All mice were injected with 50 μg of protein every two weeks for 10 weeks. Seven to ten days after the third and fourth immunizations, blood was taken from mice via the retroorbital plexus and serum was separated from the blood to analyze its ability to inhibit the binding and subsequent stimulating activity of human IL-31 in relation to the cell line transfected with human IL-RA.

The ability of individual antisera to inhibit human IL-31 was evaluated using a luciferase neutralization assay using IL-31-CEE or c108sIL-31 and BaF3 / KZ134 / IL-31RA / OSMRbeta cells (see examples 3 and 4) with the following modifications. Separate antisera were titrated in two samples by serial 4-fold dilutions in a 96-well white flat-bottom polystyrene plate (Corning / Costar 3917), starting from the initial dilution of antisera 1: 250 in assay buffer (RPMI 1640, 10% ETS, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U / ml penicillin G sodium, 100 μg / ml streptomycin sulfate). In order to normalize to some extent the stimulating effect of mouse serum in this assay, all dilutions, except for the initial dilution of 1: 250, were performed in assay buffer with 0.2% normal BALB / c mouse serum. The volume of diluted antisera in each well was 100 μl. Cells were washed 1.5 times in assay buffer, adjusted to a concentration of 3 · 10 ⁶ / ml, combined with either IL-31-CEE (100 pg / ml) or c108sIL-31 (30 pg / ml), and then the mixture was added to tablets, 100 μl / well. The total volume for analysis was 200 μl / well and consisted of 30,000 cells, IL-31 at a final concentration of 50 pg / ml (IL-31-CEE) or 15 pg / ml (c108sIL-31) and antiserums at a final dilution of 1: 500 , 1: 2000, 1: 8000, 1: 32000, 1: 128000, 1: 512000, 1: 2048000 and 1: 8192000. The plates were incubated at 37 ° C, 5% CO ₂ for 16-24 hours, after which they were centrifuged at 1250 rpm for 5 minutes, the medium was gently shaken off and 25 μl of 1 × cell lysis buffer was added to each well. The plates were gently shaken for 10 minutes to ensure cell lysis, after which 40 μl of luciferase assay substrate was added to each well. Wells were then evaluated for luciferase activity (reflecting activation by the IL-31 STAT reporter construct) on a luminometer using a 4 second integration interval.

Mostly, it was found that in this assay, the antiserum of mice immunized with IL-31-CEE effectively inhibits the stimulatory activity of both IL-31-CEE and c108sIL-31, and vice versa. For this reason, further neutralization analyzes were performed only with IL-31-CEE.

The mice with the highest neutralization titers were used for a series of three fusions aimed at obtaining hybridomas producing monoclonal antibodies that can with high efficiency neutralize the interaction of IL-31 with its recognizing receptor.

B. Merger

Merger 291

In the first fusion, lymph node cells of one mouse immunized with IL-31-CEE and one mouse immunized with c108sIL-31 were used. Each of these animals was immunized for the sixth time with 15 μg of the corresponding immunogen diluted in PBS and administered by intravascular injection three weeks after their last immunization. After three days, the spleen and lymph nodes from these mice were collected, combined, turned into a suspension of individual cells (4.69 × 10 ⁸ cells in total), and then merged with a clone of the mouse myeloma cell line P3-X63-Ag8.653 ( Kearney, JF et al., J Immunol. 123: 1548-50, 1979) (denoted as P3-X63-Ag8.653.3.12.11) in the ratio of lymphoid cells: myeloma cells 2.3: 1 by 3 ml of PEG 1500 over 2 minutes 55 seconds using standard methods known in the art (Lane, RD J Immunol Methods 81: 223-8, 1985). The fusion mixture was distributed into a series of 96-well flat-bottom plates, fed once by replacing 70% of the medium after 4 days, and analyzed after 6 days.

Merger 292

For the second fusion, lymphoid cells of one mouse immunized with IL-31-CEE were used. In addition to the five intraperitoneal immunizations mentioned above, this mouse was given an intravascular injection of 15 μg of IL-31-CEE in PBS three weeks after intraperitoneal injection and a similar injection three weeks after the first intravascular immunization. After three days, the spleen and lymph nodes were treated (a total of 2.27 · 10 ⁸ cells) and fused to P3-X63-Ag8.653.3.12.11 in the ratio of lymphoid cells: myeloma cells 2: 1 with 1.8 ml PEG 1500 in for 2 minutes 55 seconds, as described above. The fusion mixture was distributed into a series of 96-well flat-bottom plates, fed once by replacing 70% of the medium after 4 days, and analyzed after 5 days.

Merger 294

For the latter fusion, lymphoid cells from one mouse immunized with c108sIL-31 only were used. In addition to the five intraperitoneal immunizations described previously, this mouse was given an intravascular injection of 15 μg c108sIL-31 in PBS six weeks after the last intraperitoneal injection, and then again two weeks after the first intravascular injection. After three days, the spleen and lymph nodes were treated (total 1,586 × 10 ⁸ cells) and fused to P3-X63-Ag8.653.3.12.11 in the ratio of lymphoid cells: myeloma cells 2: 1 with 1.5 ml of PEG 1500 for 2 minutes 55 seconds as described above. The fusion mixture was distributed into a series of 96-well plates with a flat bottom, recharged twice by replacing 70% of the medium after 4 and 6 days, and analyzed 3 days after the recharge.

C. Screening after the merger

After all three fusions, screening was performed using the IL-31 cell neutralization assay described above, with two modifications. First, instead of diluted antiserum in assay plates, 100 μl of supernatant was sown as replicas from each well of the fusion plates on the assay plates. Secondly, the final concentration of IL-31-CEE in the assay mixture was 150 pg / ml.

In addition to the neutralization assay, several plates were screened after each fusion, randomly selected, for IgG antibodies that could bind to IL-31-CEE, which had previously been adsorbed onto polystyrene ELISA plates. The purpose of this analysis was to identify the main wells containing a hybridoma producing an anti-IL31 antibody that either poorly neutralizes IL-31 or does not neutralize this cytokine at all. In this analysis, the wells of 96-well polystyrene ELISA plates were first coated with IL31-CEE at a concentration of 200 ng / ml in an amount of 50 μl / well in 0.1 M Na ₂ CO ₃ , pH 9.6. The plates were incubated overnight at 4 ° C, after which the unbound antigen was aspirated and the plates were washed twice with PBS-Tween (0.137 M NaCl, 0.0027 M KCl, 0.0072 M Na ₂ HPO ₄ , 0.0015 M KH ₂ PO ₄ , 0.05% v / v polysorbate 20, pH 7.2) in an amount of 300 μl / well. Wells were blocked by PBS-Tween + 1% BSA in an amount of 200 μl / well for 1 hour at room temperature (RT). The blocking solution was then shaken off the plates and 50 μl of conditioned medium from each well was seeded as replicas in the wells of the assay plate. The plates were incubated for 1 hour at RT, after which the unbound antibody was aspirated and the plates were washed twice with PBS-Tween in an amount of 300 μl / well. Fc-specific antiserum with HRP conjugated goat anti-mouse IgG antibodies (Jackson Immunoresearch) was diluted 1: 5000 in PBS-Tween + 1% BSA and added to the wells of the assay plates, 100 μl / well. After incubation for 1 hour at RT, an unbound second stage antibody was aspirated from the wells and the plates were washed 5 times. Then, tetramethylbenzidine (TMB) (BioFX Laboratories, Owings Mills, MD) was added to each well in an amount of 100 μl / well and the plates were incubated for 5 minutes at RT. Color formation was stopped by adding TMB stop reagent for 450 nm (BioFX Laboratories, Owings Mills, MD) at 100 μl / well and absorbance values for the wells were determined on a Molecular Devices Spectra MAX 340 at 450 nm.

The results of neutralization assays showed that 52 and 139 wells after fusion 291 and 292, respectively, contained an antibody that inhibits at least 40% of the stimulating activity of IL-31. A more accurate determination of positivity was carried out after fusion 294, where it was found that 131 wells contained an antibody that inhibited at least 70% of the activity of IL-31. The results of ELISA assays for the detection of mAbs that bind to IL-31-CEE showed that in most cases, if it was found that the main well contains an IgG antibody that binds to IL-31-CEE, then it also contains an antibody that inhibits IL-31-CEE in a cell neutralization assay. However, some wells were positive in the ELISA assay, but showed relatively weak inhibitory ability in the cell neutralization assay and were retained for later analysis, as described below.

Hybridoma cells grown in positive main wells were cultured in 24-well plates. When the density of cultures from 24-well plates was approximately 4-6 · 10 ⁵ cells / ml, the supernatant (approximately 1.5 ml) was collected and stored separately for each well and the cells from each well were frozen.

D. Selection and cloning of the main wells for isolation of hybridomas producing effective MAb that neutralize IL-31

Each of the new 24-well supernatants was reassayed for IL-31 neutralization ability using the IL-31 cell neutralization assay used in post-fusion screening. Each supernatant was analyzed undiluted and two samples. The results indicate that after cultivation, most of the supernatants from the main wells retained inhibitory activity equal to or better than the activity detected by screening the original main wells. In order to better determine which of the new supernatants from the main wells has the highest inhibitory ability, those supernatants for which approximately 90% or the best inhibition of IL-31-CEE were shown in this assay were reanalyzed by testing dilutions of supernatants in a cell assay neutralization. Supernatants were titrated by serial 3-fold dilutions in fusion medium in assay plates to obtain 100 μl in each well of the following dilutions: without dilution, 1: 3, 1: 9, 1:27, 1:81 and 1: 243, and analysis was performed as described above for screening after fusion. Although the supernatants with the highest inhibitory ability were clearly identified in this analysis, it was not possible to determine which of them had the highest specific activity, since the concentration of anti-IL31 antibody in the supernatants was unknown.

To solve the problem of the concentration of a specific antibody, all supernatants were tested by titration using either serial 2-fold or 4-fold dilutions using IL-31-CEE in the direct ELISA assay described above. After plotting the data in Microsoft EXCEL, the dilution of the supernatant was determined, resulting in half the maximum optical density (OD) observed in the analysis, which, in turn, is an approximate indicator of the relative concentration of anti-IL-31 antibody in the supernatant.

Combining the neutralization assay data with the ELISA assay, the relative specific activity of each supernatant was determined and twenty major wells with the highest specific neutralization activity of IL-31 were identified. Interestingly, all the most effective neutralizing IL-31 main wells were obtained after fusion 292. In the neutralization data for these main wells, it was noted that, despite the fact that the maximum inhibition of IL-31 was achieved for each supernatant without dilution and dilution 1 : 3, the absolute degree of neutralization was slightly higher for some supernatants relative to others, which was reflected in slightly lower relative values of luciferase units. This was interpreted as slightly differing degrees of "complete" neutralization of IL-31. Combining this observation with relative specific neutralization activity for each main well, the list of the most effective neutralizing main wells was reduced to the estimated top ten. They included 292.12, 292.39, 292.51, 292.63, 292.64, 292.72, 292.84, 292.105, 292.109 and 292.118.

Cells from each of these top ten neutralizing IL-31 main wells were cloned to isolate a cloned hybridoma producing neutralizing mAbs of interest. Cells were cloned into 96-well cell culture microtiter plates using a standard low-density dilution approach (less than 1 cell per well) and monoclonality was evaluated prior to analysis by microscopic examination of the wells for single growth foci. The cloning medium consisted of a fusion medium in which there was no HAT component (IMDM, 10% FC1 serum, 2 mM L-glutamine, 1 × penicillin / streptomycin, 10% hybridoma cloning factor (Roche Applied Science)). Six to eight days after plating, supernatants were screened in all wells by ELISA with IL-31-CEE bound on a plate. Cells from 4-6 wells in each group in which the supernatant was highly positive for specific mAb and where only one colony of the hybridoma grew, were grown in cultures in 24-well plates and the obtained supernatants were retested by ELISA with IL- bound on the plate 31-CEE and through cell analysis of the neutralization of IL-31. Based on these results, the alleged most effective neutralizing clone in each group was re-cloned and screened by ELISA as described above. If 95% or more of the positive growth wells were also positive for the specific mAb, further subcloning attempts were considered optional and the hybridomas were recognized as clonal. Among the obtained clones, for only one main well, 292.64, a second subcloning cycle was carried out necessary to achieve the required percentage positivity for specific mAbs. Cells from 5-6 wells in each final group of subclones in which the supernatant was highly positive for a specific mAb and where only one hybridoma colony grew, were grown in 24-well plate cultures. Each clone of the hybridoma was then adapted for growth in a medium lacking a hybridoma cloning factor (IMDM, 10% FC1 serum, 2 mM L-glutamine, 1 × penicillin / streptomycin) by distributing cells in the latter medium with an acceptable cell density. After adaptation, subclone supernatants of each group were collected and screened by ELISA with IL-31-CEE bound on the plate. Based on the titer taking into account the cell density during the collection of supernatants, the “best” final clone was selected, which led to the selection of the following group of final clones: 292.12.3.1; 292.39.5.3; 292.51.5.2; 292.63.5.3; 292.64.6.5.5; 292.72.3.1; 292.84.1.6; 292.105.4.1; 292.109.4.4 and 292.118.6.4.

The mouse IgG isotype mAb produced by each of these hybridomas was determined using the IsoStrip mouse monoclonal antibody test (Roche Applied Science). It was found that all mAbs belong to the IgG1 subclass, with the exception of 292.72.3.1 and 292.84.1.6, which, as shown, belong to the IgG2a subclass.

E. In vitro assessment of the specific neutralizing activity of effective MAb neutralizing IL-31

To assess the specific activity of effective mouse mAbs against human IL-31 in relation to in vitro neutralization, the depleted supernatant from each clone of the first cycle was re-tested in the cell neutralization assay described previously. First, the amount of IgG in each supernatant was determined by HPLC analysis in a protein G column using a mAb with a known concentration as a standard. Each supernatant was then diluted to an IgG concentration of 1 μg / ml in the same medium as that used to obtain the depleted supernatant. Then, the diluted supernatants were titrated by 10-fold serial dilution in medium to obtain a concentration range from 1 μg / ml to 0.0001 μg / ml and three samples were tested in the previously described cell neutralization assay with a final concentration of 300 pg IL-31-CEE / ml (18.27 pM). The results of the analysis are presented in table 2, where the mAbs are presented in order from the most effective to the least effective. MAb 292.84.1 and 292.12.3 were the most effective neutralizers, followed by mAb 292.72.3 and 292.63.5, which turned out to be approximately two times less effective than the first two. For the remaining mAbs, it was shown that they are approximately 6–9 times less effective than the two best mAbs.

table 2
In vitro evaluation of effective neutralizing MAb against IL31
by activity in relation to specific neutralization MAb IC _fifty  (ng / ml) IC _fifty  (pM) * 292.84.1 1,077 ± 0,021 7.18 ± 0.14 292.12.3 1.197 ± 0.044 7.98 ± 0.29 292.72.3 1,997 ± 0,027 13.31 ± 0.18 292.63.5 2.819 ± 0.034 18.79 ± 0.23 292.51.5 6.478 ± 0.130 43.19 ± 0.87 292.118.6 6.550 ± 0.154 43.67 ± 1.03 292.109.4 8.286 ± 0.151 55.24 ± 1.01 292.39.5 8.633 ± 0.481 57.55 ± 3.20 292.105.4 9.144 ± 0.281 60.96 ± 1.87 292.64.6 10.05 ± 0.295 67.00 ± 1.97 * using the molecular weight of the antibody = 150,000 daltons

Based on the above results, hybridomas 292.84.1.6, 292.12.3.1, 292.72.3.1, 292.63.5.3 and 292.118.6.4 were selected as reflecting different levels of specific neutralizing ability for deposit in the patent collection of the American Type Culture Collection (ATCC, Manassas , VA) as initial samples under the Budapest Treaty, and they were assigned the following ATCC registration numbers No.№: clone 292.12.3.1 (Patent Deposit Number in ATCC PTA-6815); clone 292.72.3.1 (Patent Deposit Number in ATCC PTA-6816); clone 292.63.5.3. (Patent Deposit Number in ATCC PTA-6829); clone 292.118.6.4 (Patent deposit number in ATCC PTA-6830) and clone 292.84.1.6 (Patent deposit number in ATCC PTA-6871).

F. Selection and cloning of the main wells for isolation of hybridomas producing MAb with weak neutralization of IL-31

In preparing mAbs against human IL-31, it was of interest to isolate mAb pairs that can be used in a sandwich assay to quantify the amount of IL-31 in various liquids. Such mAb pairs typically have specificity for different epitopes on the target molecule and preferably do not cross-compete for binding to this molecule. To isolate candidate pairs of such mAbs, a pairing strategy was chosen from mAbs with excellent neutralization of IL-31 and a mAb that has little, if any, ability to neutralize, suggesting that two such different functional antibodies are most likely not binding non-overlapping ( spatially separated) epitopes.

As indicated previously for screening after fusion, after each fusion, several plates were screened by ELISA with IL-31-CEE bound on the plate to identify positive anti-IL-31 antibody supernatants. Comparison of the ELISA results with the results of the cell neutralization analysis showed the main wells that contained an antibody that binds well to IL-31-CEE but does not have high neutralization of this molecule in the cell neutralization analysis. As was done with the most effective neutralizing main wells, hybridoma cells grown in these main wells were grown in culture in 24-well plates. When the density of the cultures in the 24-well plates was approximately 4-6 · 10 ⁵ cells / ml, the supernatant (approximately 1.5 ml) was collected and stored separately for each well and the cells from each well were frozen.

These new supernatants were tested using a cell neutralization assay (serial 3-fold dilutions, starting from the supernatant without dilution and continuing to a final dilution of 1: 243), as well as using an "antigenic trap" type ELISA assay similar to that used for development of a rat mAb against mouse IL-31 with the following differences: 1) Fc-specific sheep antibody against mouse IgG (Jackson Immunoresearch) (1 μg / ml) as an antibody to coat the tablet, 2) the plates were blocked once with PBS-Tween + 1% BSA for 1 hour, 3) Each supernatant was added to the wells one at a time (serial 3-fold dilutions, starting from the supernatant without dilution and continuing to a final dilution of 1: 2187), and 4) addition of biotinylated IL-31-CEE (molar ratio of biotin: protein 3: 1) 200 ng / ml. It was believed that this assay is more suitable than binding of an antibody to a plate-bound IL-31-CEE, since it evaluated the ability of an antibody to bind to IL-31-CEE in solution, which is a property necessary for a good antibody for ELISA type a sandwich. In fact, it was found that a number of supernatants of the main wells containing an antibody that binds well to the IL-31-CEE plate bound poorly recognized this molecule in solution.

To select the wells from which the corresponding antibody-secreting hybridomas were cloned, 10 wells were identified (291.78, 292.152, 292.154, 294.35, 294.144, 294.146, 294.154, 294.155, 294.158 and 294.163), whose supernatants weakly neutralized IL-31-CEE in the neutralization assay (less than 50%, but preferably only 1-20%), and also relatively well bound to IL-31-CEE in solution (as determined by OD higher than 2.0 when the supernatant concentration was not diluted). Cloning, screening of clones and selection of the best clone of the first cycle was performed as described above for wells with effective neutralization.

To reduce the number of hybridomas selected for further subcloning, two new analyzes were performed with the depleted supernatant of each of the selected clones of the first cycle. The first evaluation method was an attempt to separate the mAbs into groups based on epitope specificity (“sorting”) using a Biacore 3000 surface plasmon resonance device. One of the supernatants with a good neutralizing mAb (292.64.6) and 10 weaker supernatants of the first cycle clones were analyzed relative to each other, which led to the identification of four alleged "series". Series 1 consisted only of a good neutralizing antibody. Series 2 consisted of 292.152.4, 292.154.4, 294.35.2, 294.146.5 and 294.154.5. Series 3 included 291.78.4, 294.155.6, 294.158.5 and 294.163.2. Series 4 contained only 294.144.3. The second analysis included repeating the ELISA as an “antigenic trap”, only this time, due to the higher concentration of specific antibodies in the supernatants compared to the original supernatants of the main cultures in 24-well plates, we obtained a more complete titration curve and were able to determine EC ₅₀ for binding antibodies to IL-31-CEE. The results are presented in table 3 (together with the results of sorting and% inhibition of activity against IL-31-CEE in the cell neutralization assay), and they indicate a wide range of EC ₅₀ values (and, as a consequence, the mAb affinity for IL-31-CEE ) for various mAb.

Table 3
The total data for the supernatant of the culture of the clone of the first cycle of the less effective neutralizing IL-31 hybrid MAb Epitope Series (Biacore) EC _fifty  (ng / ml) for the “antigenic trap" IL-31-CEE % neutralization of IL31 activity in cell analysis (undiluted supernatant) 291.78.4 3 10 94 292.152.4 2 182 25 292.154.4 2 57.7 76 294.35.2 2 22.2 82 294.144.3 four 13,4 75 294.146.5 2 10,2 94 294.154.5 2 15.8 88 294.155.6 3 172 53 294.158.5 3 119 31 294.163.2 3 24.2 47 292.64.6 one 3.4 one hundred

Given the selection of hybridomas producing mAbs, which 1) were in series by epitopes that differ from the mAb series with good neutralization (292.64.6) and 2) had the highest affinity, as shown by the low EC ₅₀ in the antigenic trap analysis , only clones of the first cycle 294.35.2, 294.144.3, 294.154.5 and 294.163.2 were assigned the status of the final clone, as described above for more effective neutralizing mAbs. This further work on subcloning led to the selection of the following group of end clones: 294.35.2.6.3; 294.144.3.5; 294.154.5.6 and 294.163.2.1.

The mouse IgG isotype mAb produced by each of these hybridomas was determined using the IsoStrip Monoclonal Mouse Antibody Test (Roche Applied Science). It was found that all four mAbs belong to the IgG1 subclass. Samples of each of the four hybridomas were deposited in the patent collection of the American Type Culture Collection (ATCC, Manassas, VA) as initial samples under the Budapest Treaty, and they were assigned the following ATCC registration numbers No.: clone 294.163.2.1 (Patent Deposit Number in ATCC PTA-6831); clone 294.35.2.6.3 (Patent Deposit Number in ATCC PTA-6872); clone 294.154.5.6 (Patent deposit number in ATCC PTA-6875) and clone 294.144.3.5 (Patent deposit number in ATCC PTA-6873).

G. Cross-reaction of mouse mAb against human IL-31 with mouse IL-31

All first-cycle clone supernatants for ten effective neutralizing mAbs, as well as ten less effective neutralizing mAbs, were tested for their ability to recognize mouse IL-31 by ELISA with mIL-31-CEE bound on the plate. No cross-reactivity was detected with any mAb, indicating that the mAbs appear to be specific for human IL-31. Moreover, these results showed that none of the antibodies recognized the CEE peptide marker common to the C-terminus of the recombinant IL-31 molecules of both humans and mice used in these studies, and this determination was not performed for the supernatants of the original wells.

Example 3

IL-31RA luciferase assay / OSMRbeta receptor

Plasmid KZ134 was designed with complementary oligonucleotides that contain the transcription factor-binding STAT elements of 4 genes, which include the modified inducible Sis c-fos element (m67SIE or hSIE) (Sadowski, H. et al., Science 261: 1739-1744, 1993), p21 SIE1 from the p21 WAF1 gene (Chin, Y. et al., Science 272: 719-722, 1996), a corresponding mammary element of the β-casein gene (Schmitt-Ney, M. et al., Mol. Cell Biol. 11: 3745-3755, 1991) and an inducible STAT element of the Fcg RI gene (Seidel, H. et al., Proc. Natl. Acad. Sci. 92: 3041-3045, 1995). These oligonucleotides contain Asp718-XhoI compatible ends and are ligated using standard methods into a firefly luciferase recipient vector with the c-fos promoter (Poulsen, LK et al., J. Biol. Chem. 273: 6229-6232, 199S ), cleaved by these enzymes and containing a selective marker of neomycin. Plasmid KZ134 was used for stable transfection of BaF3 cells using standard methods of transfection and selection to obtain the BaF3 / KZ134 cell line.

A stable BaF3 / KZ134 indicator cell line expressing full-length IL-31RA or IL-31RA / OSMRbeta receptor was obtained. Clones were diluted, seeded and selected using standard methods. Clone screening was performed by luciferase assay (see B, below) using conditioned medium with human IL-31 or purified IL-31 protein as an inducer. Clones were selected with the highest luciferase response (via STAT luciferase) and with the lowest background. Stably transfected cell lines were selected. The cell lines were called BaF3 / KZ134 / IL-31RA or BaF3 / KZ134 / IL-31RA / OSMRbeta depending on the receptors transfected into the cell line.

Similarly, BHK cell lines were also prepared using the method described herein and were used in the luciferase assays described herein. Cell lines were called BHK / KZ134 / IL-31RA or BHK / KZ134 / IL-31RA / OSMRbeta depending on receptors transfected into the cell line.

The BaF3 / KZ134 / IL-31RA and BaF3 / KZ134 / IL-31RA / OSMRbeta cells were centrifuged and washed with mIL-3-free medium. Cells were centrifuged and washed 3 times to ensure the removal of mIL-3. Then the cells were counted on a hemocytometer. Cells were plated in a 96-well plate in an amount of 30,000 cells per well in a volume of 100 μl per well using mIL-3-free medium. The same process was used for non-transfected BaF3 / KZ134 cells for use as a control in a subsequent analysis. BHK / KZ134 / IL-31RA or BHK / KZ134 / IL-31RA / OSMRbeta cells were seeded in a 96-well plate in an amount of 15,000 cells per well in 100 μl of medium. BHK / KZ134 parent cells were used as a control.

STAT activation of BaF3 / KZ134 / IL-31RA, BaF3 / KZ134 / IL-31RA / OSMRbeta, BHK / KZ134 / IL-31RA, or BHK / KZ134 / IL-31RA / OSMRbeta STAT cells was evaluated using conditioned medium or purified protein. One hundred microliters of diluted conditioned medium or protein are added to the BaF3 / KZ134 / IL-31RA, BaF3 / KZ134 / IL-31RA / OSMRbeta, BHK / KZ134 / IL-31RA or BHK / KZ134 / IL-31RA / OSMRbeta cells. Analysis using a conditioned medium is simultaneously performed on untransfected BaF3 / KZ134 or BHK / KZ134 cells as a control. The total analyzed volume is 200 μl. The assay plates are incubated at 37 ° C, 5% CO ₂ for 24 hours, and then BaF3 cells are pelleted by centrifugation at 2000 rpm for 10 minutes, and the medium is aspirated and 25 μl of lysis buffer (Promega) is added. For BHK cell lines, a centrifugation step is not necessary because they are adherent cell lines. After 10 minutes at room temperature, the activation of the STAT reporter construct was measured in the plates by reading on a luminometer (Labsystems Luminoskan, model RS), in which 40 μl of luciferase assay substrate (Promega) was added with integration for five seconds.

Example 4

Analysis of luciferase on transformed human epithelial cell lines through transient infection with the STAT / SRE adenovirus reporter gene

Inhibition, reduction and / or neutralization of IL-31 activity can be determined by luciferase assay. For example, transformed human cell lines can be seeded in 96-well flat bottom plates in an amount of 10,000 cells / well in a normal growth medium, as described for each cell type. The next day, cells are infected with the adenovirus reporter construct, KZ136, with a multiplicity of infection of 5000. In addition to the serum response element, the KZ136 reporter also contains STAT elements. The total volume is 100 μl / well using DMEM supplemented with 2 mM L-glutamine (GibcoBRL), 1 mM sodium pyruvate (GibcoBRL) and 1 × insulin transferrin-selenium additive (GibcoBRL) (hereinafter referred to as serum-free) Wednesday). Cells are cultured overnight.

On the next day, the medium is removed and 100 μl of induction medium is replaced. The induction medium is human IL-31 diluted in serum-free medium at a concentration of 100 ng / ml, 50 ng / ml, 25 ng / ml, 12.5 ng / ml, 6.25 ng / ml, 3.125 ng / ml and 1.56 ng / ml. A positive control in the form of 20% ETS is used to verify the analysis and to confirm that the adenovirus infection is successful. Cells are induced for 5 hours, after which the medium is aspirated. The cells are then washed in PBS in an amount of 50 μl / well, and then lysed in 1 × cell lysis buffer (Promega) in an amount of 30 μl / well. After incubation for 10 minutes at room temperature, a lysate of 25 μl / well was transferred to opaque white 96-well plates. Then read the plates on the luminometer using 5-second integration with injection of luciferase substrate (Promega) in an amount of 40 μl / well.

Example 5

Inhibition of cytokine production by anti-IL-31 antibodies

IL-31 has been shown to stimulate IL-6 production in DU145 (disease cell lines and normal cell lines); and also stimulates A549 cell lines (for disease and normal) with stimulation of IL-8 production, and reduces IL-8 production in U2OS cell lines (for disease and normal). See US Published Patent Application (see Publication Number 20030224487, Sprecher, Cindy et al, 2003). Essentially, the activity of monoclonal antibodies against IL-31, described in the present description, can be measured by inhibition, decrease and / or neutralization of the activity of IL-31 in these epithelial cell lines with the disease.

A. Inhibition of the production of cytokines of epithelial cell lines with a disease cultured with IL-31 person

Cells are seeded in a 6-well plate (Costar) with a density of 4.5 · 10 ⁵ cells per well and cultured in an appropriate medium for growth. Cells are cultured with test reagents; 100 ng / ml IL-31 (with and without antibody), 10 ng / ml interferon-gamma (IFN-gamma) (R&D Systems, Minneapolis, MN), 10 ng / ml tumor necrosis factor alpha (TNF-alpha) (R&D Systems, Minneapolis, MN), 10 ng / ml IL-1beta (R&D Systems, Minneapolis, MN) or 100 μg / ml lipopolysaccharide (LPS) (Sigma). Supernatants were harvested after 24 and 48 hours and scored for cytokines: GM-CSF (granulocyte macrophage colony stimulating factor), IL-1b, IL-6, IL-8, MCP-1 (Chemoattractant Macrophage-1 Protein) and TNFa. Multiplex antibody kits with granules from BioSource International (Camarillo, CA) are used to measure cytokines in samples. Reading for analysis is performed on a Luminex-100 device (Luminex, Austin, TX) and the data is analyzed using MasterPlex software (MiraiBio, Alameda, CA). Cytokine production (pg / ml) was determined for each cell line in the samples after 24 hours of cultivation.

B. Inhibition of cytokine production by normal human epithelial cell lines cultured with human IL-31

Cells with a density of 1 · 10 ⁵ cells per well are seeded in a 24-well plate and cultured with test reagents; 1000 ng / ml, 100 ng / ml and 10 ng / ml IL-31 (with and without antibody), 10 ng / ml TNFa, 10 ng / ml OSM, 10 ng / ml IFNa, 10 ng / ml TGFb or 10 ng / ml lymphotactin. Supernatants were harvested after 24 and 48 hours and scored for cytokines: IL-6, IL-8, MCP-1, MIP-1a, RANTES and eotaxin. Cytokines are analyzed as described previously. Cytokine production (pg / ml) was determined for each cell line in the samples after 48 hours of cultivation.

C. Inhibition of cytokine production by human cell lines with a disease co-cultured with human IL-31 and IFN-gamma

Cells are seeded in a 24-well plate with a density of 2 · 10 ⁵ cells per well and co-cultured with 10 ng / ml IFN-gamma +/- IL-31 at a concentration of 100 ng / ml, 10 ng / ml or 1 ng / ml. Supernatants were harvested after 24 and 48 hours and f was evaluated (with and without antibody), or IL-8 and MCP-1. Cytokine production (pg / ml) was determined for each line in the samples after 24 hours of cultivation.

Example 6

Inhibition of the effects of IL-31 on the adhesion of U937 monocytes to the monolayer of transformed bone marrow endothelial cells (TRBMEC)

It has been shown that IL-31 may affect the initial attachment of U937 cells to endothelial monolayers. In particular, IL-31 has a synergistic effect with TNF-alpha and further enhances the adhesion of U937. See US Published Patent Application (See Publication Number 20030224487, Sprecher, Cindy et al., 2003). Thus, inhibition of IL-31 with anti-IL-31 antibodies described herein can be determined using the following assay.

Transformed bone marrow endothelial cells (TRBMEC) are seeded in 96-well tissue clusters (Falcon) with a density of 25,000 / well in M131 medium (Cascade Biologies) supplemented with microvascular growth supplement (MVGS) (Cascade Biologies). When the monolayer merges (after 24 hours), the cells switch to M199 (Gibco-Life Technologies), supplemented with 1% fetal calf serum (Hyclone). Recombinant human IL-31 (test reagent) is added in various concentrations (from 0.4 to 10 ng / ml) with or without antibody to test the effect of the protein on the interaction of immune cells-endothelial cells, leading to adhesion. 0.3 ng / ml tumor necrosis factor (TNF-alpha R&D Systems), a well-known pro-inflammatory cytokine, in addition to IL-31, was added to some wells to test the effect of the protein on endothelial cells under conditions of inflammation. TNF-alpha at a concentration of 0.3 ng / ml was separately used as a positive control and the concentration used reflects approximately 70% of the maximum effect of TNF-alpha in this system, i.e. it does not induce maximum attachment of U937 cells (human monocyte-like cell line) to the endothelium. For this reason, under these conditions, both positive and negative regulation of the effects of TNF-alpha can be detected. Basal adhesion levels with and without TNF-alpha are used as a baseline to evaluate the effects of test reagents.

After incubation of endothelial cells with test reagents overnight, U937 cells stained with 5 μM calcein-AM fluorescent marker (Molecular Probes) are suspended in RPMI 1640 (without phenol red) supplemented with 1% ETS and plated in an amount of 100,000 cells / well on the washed TRBMEC monolayer. After 30 minutes, fluorescence levels were measured at excitation / emission wavelengths of 485/538 nm (Molecular Devices, CytoFluor application microplate reader), before and after washing the wells three times with warm RPMI 1640 (without phenol red) to remove non-adherent U937. Fluorescence before washing (total) and after washing (specific for attachment) is used to determine percent attachment (value for attachments / value for general · 100 =% attachment).

Example 7

Biological Analysis Protocol IL-31

BAF3 cells transfected with hzCYTOR17 (IL-31RA), hOSMRB and KZ134 are grown to 5 · 10 ⁵ and 1 · 10 ⁶ cells / ml. Cells were washed with assay medium (RPMI 1640, 10% ETS, L-glutamine, sodium pyruvate, and Pen / Strep (all from Gibco)) and resuspended in an amount of 3 × 10 ⁵ cells / ml in assay medium. In a 96-well opaque plate, hIL-31 standards were titrated in two samples from 600 pg / ml to 9.38 pg / ml in assay medium by serial dilutions of 1: 2, 100 μl / well. Standards for quality control of two samples at a concentration of 350 pg / ml and 35 pg / ml in 100 μl are added to the plate. Test samples are repeatedly diluted 1: 2 or 1: 4 and two samples are added to the sample wells. 100 μl of washed BAF3 cells are added to each well to a final concentration of 3 · 10 ⁴ cells / well. The plate is incubated for 16-24 hours at + 37 ° C in an incubator with 5% CO ₂ . The plate is centrifuged at 1200 rpm for 5 minutes, the medium is shaken off and lysis buffer in the amount of 25 μl / well (Promega) is added to each well. After 10 minutes, the tablet is read on a luminometer (Berthold). The luminometer adds a mixture of substrate for luciferase in an amount of 40 μl / well (Promega) and integrates the luminescence within 4 seconds. The luminescence indices are transferred to a spreadsheet, where they are analyzed and converted to IL-31 picograms at 10 ⁶ cells per ml volume.

Example 8

The involvement of IL-31 in triggering and maintaining contact hypersensitivity in vivo

Method I

The shaved mid-back of BALB / c mice is stained with 25 μl of 0.5% DNFB (2,4-dinitrofluorobenzene, Sigma, St. Louis MO) dissolved in acetone: olive oil (4: 1) using a pipettor. In the control group with the carrier, only 25 μl of a mixture of acetone: olive oil is administered. After 5 days, the mice are anesthetized with isoflurane in the inhalation chamber and the auricles of both experimental and control animals are measured using an engineering micrometer (Mitutoyo) to obtain the initial values. The mice are then immunized by applying 10 μl of 0.25% DNFB in acetone: olive oil (4: 1) to both sides of each ear of all mice. Contact hypersensitivity is measured after 24 hours and 48 hours as the difference between the right ear (immunized) and the left ear (non-immunized). All measurements are carried out using an engineering micrometer. Background values are determined from the difference in ear enlargement between the immunized and non-immunized ears of unexposed mice.

Before killing, whole blood and serum are sampled for FACS and / or ELISA and the ears are taken for histology.

Method II (Induction of Th2 Responses)

The shaved mid-back of BALB / c mice is stained with 100 μl of 0.5% FITC (fluorescein-isothiocyanate) in 1: 1 acetone / dibutyl phthalate solution (available on MSDS) using a pipettor on days 1, 2 and 8. On day 13, mice are anesthetized with isofluorane in an inhalation chamber and both ears of experimental animals are measured using an engineering micrometer (Mitutoyo) to obtain the initial values. Mice are immunized by applying 25 μl of 0.5% FITC (in a 1: 1 acetone / dibutyl phthalate mixture) to the dorsal surface of each ear. Contact hypersensitivity is determined after 24 hours and 48 hours, as the difference between the right ear (immunized) and the left ear (non-immunized). All measurements are carried out using an engineering micrometer. Background values are determined by the difference in ear increase between the immunized and non-immunized ears of unexposed mice. Before killing, whole blood and serum are sampled for FACS and / or ELISA and the ears are taken for histology.

Method III (induction of Th1 responses)

The shaved mid-back of BALB / c mice is stained with 25 μl of 2% oxazalone (in a 4: 1 acetone / olive oil mixture) using a pipettor. On day 7, mice are anesthetized with isofluorane in an inhalation chamber and the auricles of both experimental and control animals are measured using an engineering micrometer (Mitutoyo) to obtain the initial values. Mice are immunized by applying 8 μl of oxazalone to the dorsal surface of each ear. Contact hypersensitivity is measured after 24 hours and 48 hours as the difference between the right ear (immunized) and the left ear (non-immunized). All measurements are carried out using an engineering micrometer. Background values are determined from the difference in ear enlargement between the immunized and non-immunized ears of unexposed mice. Before sacrifice, whole blood and serum are sampled for FACS and / or ELISA and the ears taken for histology.

The involvement of IL-31 in triggering and maintaining contact hypersensitivity is tested using the antibodies described herein against IL-31, both during the sensitization and immunization experiment phases.

Example 9

The involvement of IL-31 in atopic dermatitis in vivo

Methods I (Sensitization of NC / Nga Mice)

Male NC / Nga mice were purchased from CRL Japan. On delivery, the mice were 4 weeks old and were placed in SPF quarantine for 4 weeks for acclimatization. At the time of antigen sensitization, mice were approximately 10–11 weeks old. Mice were anesthetized with isofluorane and their backs were shaved with electric shearing machines. Approximately 10 μg of the extract of Dermatophagoides pteronyssinus (Dp) (Indoor Biotechnologies, special order) was injected subcutaneously in the back of the neck 3 times a week for 5 to 6 weeks until the mice developed skin lesions. Control animals received intradermal injections of 10 μl of PBS 3 times a week. Dp extract was prepared according to the method of Matsuoka and colleagues. Matsuoka H., et al., Allergy: 58, 139 (2003). Briefly, 595 mg of lyophilized extract of depleted Dp culture was dissolved in 12 ml of sterile PBS (Gibco). Dp was mixed in a 50 ml Falcon tube on a shaker for 30 minutes. The extract was centrifuged for 10 minutes at 2000 rpm and the supernatant was collected and aliquoted into 1 ml cryovials and stored at -20 ° C.

Methods II (sensitization of mice DO11.10)

Transgenic mice DO11.10 were removed from their own colony, and by the beginning of sensitization with antigen, their age was between 9.5 and 14 weeks. 24 hours before cutaneous sensitization, mice were anesthetized with isofluorane and the entire body (back and stomach) of the mice was shaved using an electric shearing machine. Then, a strip of skin was exposed to the mice by means of an Elastin keeper tape (Johnson and Johnson) on the back. 1 cm ² sterile gauze flaps were moistened with either 500 μg ovalbumin (Calbiochem 32467) or sterile PBS (Gibco) and attached to the back of the mice on the left with DuoDerm Extra Thin Dressing (ConvaTec 187932). The flap and dressing were then covered with an Elastin keeper tape cuff so that the mice could not remove or damage the flaps. The flaps were kept for 7 days and removed. Mice were retested two weeks before the start of the next cutaneous sensitization cycle. A total of three sensitizations were performed to mice in one week.

results

An immunohistochemical analysis of IL-31RA expression in damaged and intact skin from NC / Nga dust mite particles and OVA sensitized animals DO11.10 showed that keratinocytes of the epidermis are expressed in IL-31RA mice, however, significant differences in expression levels between animal sensitized antigens cannot be detected. compared to animals sensitized by PBS.

Example 10

Inhibition of pruritus by administration of rat antibodies against mouse IL-31

In another study in NC / Nga mice, mild to moderate NC / Nga mice that already showed dermatitis were given access to male NC / Nga mice (SLC, Tokyo) at 4 weeks of age. After seven weeks, the mice were divided into three groups and the following injections were performed. Every fifth day for seven weeks, mice in one group were injected with rat antibodies against mouse IL-31 in an amount of 10 mg / kg; mouse serum albumin was injected in one group; and in the third group, no administration was performed. Mice were evaluated clinically twice a week for the severity of skin lesions. Specifically, skin lesions were evaluated (0 = no lesion; 1 = back lesion; 2 = back and muzzle; 3 = back, muzzle and ears) and severity of lesions (0 = normal skin; l = scaly and dry; 2 = nodular lesions; 3 = hemorrhagic lesions). In addition, combing was evaluated. Combing in mice with the fingers of their hind legs was automatically determined and objectively evaluated using Micro Act (Neuroscience, Tokyo, Japan). A small teflon-coated magnet was implanted subcutaneously in the dorsal side of both hind legs of the mice under anesthesia using a ketalar / xylazine mixture. Mice with magnets were placed in a surveillance chamber surrounded by round coils. The electric current induced by the movement of the magnets was amplified and recorded. In the analysis program, the following conditions were used for combing registration: Threshold (B) 0.1; interval between events (s) 0.2; maximum frequency (Hz) 20.0; minimum frequency (Hz) 2.0 and minimum duration (s) 1.5. It should be noted: in the model in NC / Nga mice, in mice with dermatitis-specific pruritus sensation, prolonged (> 1.5 s), but not short-term combing (0.3-1.5 s) is associated. In general, the main measured indicators were combing, dermatitis and body weight.

results

Given the entire period, treatment with rat antibody against mouse IL-31 did not change the main indicators. However, an additional analysis revealed a significant reduction in scratching by administering an anti-IL-31 antibody between 22-43 days. During this period of time, as for the entire period, the introduction of antibodies against IL-31 did not affect the development of lesions, similar to lesions in atopic dermatitis, and did not normalize weight gain in sick animals, possibly due to the delayed onset of clinical manifestations or neutralizing autoantibodies at the end of treatment. In this experiment, combing, but not dermatitis, was already elevated among most animals at the point in time when the administration of the antibody began.

Example 11

The involvement of IL-31 in HRT in vivo

Ways

To obtain a HRT response, mice were sensitized to the antigen on day 0 by subcutaneous immunization at the base of the tail with 100 μg of ovalbumin (OVA) in Freund's complete adjuvant (CFA, total volume 50-100 μl). After one week, mice were anesthetized with isofluorane in the inhalation chamber and both ears of the experimental and control animals were measured using an engineering micrometer (Mitutoyo) to obtain the initial measurements. Mice were immunized intradermally with 10 μg OVA in PBS in a total volume of 10 μl into the left auricle, directly under the skin, without entering any veins. As a control, mice were also injected with 10 μl of PBS in the right auricle. In some cases, in a separate control group, in which an intradermal injection of OVA was performed in the ear, local corticosteroids could also be administered as a positive control of reaction inhibition. 24 and 48 hours after immunization, mice were anesthetized and ear thickness was measured. The results were expressed as: specific ear increase = (measurement corresponding to 24 hours, measurement corresponding to 0 hours) for an experimental ear - (measurement corresponding to 24 hours, measurement corresponding to 0 hour) for an ear of negative control. Induction, a symptom of HRT, is detected 18 hours after injection of a sensitizing antigen and is maximum after 24-48 hours. The latent period with the appearance of palpable induction is the reason for the name of this answer to be "slow type".

results

IL-31 transgenic mice were tested for HRT, however, due to an increase in the ear thickness of non-immunized IL-31 transgenic animals, it was not possible to detect statistically significant differences in HRT between IL-31 Tg animals and wild-type controls. Animals with IL-31 receptor knockout were also tested in the HRT response, and it was not possible to detect significant differences in HRT response between animals with receptor knockout and wild-type animals.

Example 12

The involvement of IL-31 in the induction of an itchy response

Methods I (Administration of capsaicin to mice after administration of IL-31)

BALB / c animals at ten weeks of age (CRL) were anesthetized and a long-acting analgesic was injected, bupranorphine hydrochloride, subcutaneously in an amount of 0.1 mg / kg, and then 0.25 ml of capsaicin solution was injected at a concentration of 4 mg / ml in 10 % ethanol + 10% Tween-80 in saline subcutaneously in the back of the neck. Anesthesia continued in animals for at least 30 minutes after neurotoxin administration. Forty-eight hours later, 14-day osmotic pumps were implanted subcutaneously for continuous delivery of IL-31 in an amount of 20 μg / day for 14 days. Mice were monitored every day for 6 days for alopecia and itching using the following criteria: 0 = no combing, the animal looks normal, 1 = thinning of hair in small areas, brushing is noted, 2 = slight loss of hair (small areas), combing, 3 = moderate loss of hair, combing; and 4 = severe loss of hair, excessive combing.

The results showed that while mice that were not injected with capsaicin showed an average scratching / wool loss of 2.625 after three days of IL-31 delivery, mice that were injected with capsaicin showed a significantly lower value of 1. Thus, in mice that were injected with capsaicin before delivery of IL-31, both a decrease in the occurrence of combing and hair loss and a lower value of the intensity of combing and hair loss after six days of the experiment were shown. These data suggest that IL-31 induces some neuronal component, which leads to alopecia and pruritus induced by IL-31. Thus, neutralizing IL-31 can reduce the incidence and intensity of pruritus, and thus dermatitis, in patients suffering from skin disorders in which itching is involved.

Methods II

Tac1 null-homozygous mice do not express substance P or neurokinin A. These mice have significantly reduced nociceptive responses to pain with mitigation of intense stimuli and are thus a useful tool to study the contribution of tachykinin peptides to pain / itching and conditions of inflammatory disease. Twelve-week-old Tac1 knockout mice were implanted with 14-day osmotic pumps delivering 1 μg / day IL-31 protein and observed every day for alopecia and itching using the following criteria: 0 = no scratching, the animal looks normal, 1 = thinning of hair in small areas, combing is noted, 2 = slight loss of hair (small areas), combing, 3 = moderate loss of hair, combing and 4 = severe loss of hair, excessive combing.

The results of this study indicate that Tac1-deficient mice were less susceptible to IL-31-induced combing / hair loss compared to wild-type control mice. While 100% (10/10) wild-type mice developed signs of combing and hair loss on day 6 of IL-31 administration, only 33.3% (2/6) of Tac1-deficient mice showed signs of combing and loss wool at the same time. These data show that IL-31 induces a neuronal component that results in a combing / hair loss phenotype in mice that were injected with IL-31, and neutralizing IL-31 can reduce the frequency and intensity of combing associated with dermatitis.

Methods III (Introduction of a neutralizing antibody against IL-31)

Normal female BALB / c (CRL) mice aged about 8 to 12 weeks were subcutaneously implanted with 14-day osmotic pumps (Alzet, # 2002) delivering mIL-31 in an amount of 1 μg / day. Groups of mice received intraperitoneal (i.p.) injections of rat anti-mouse IL-31 monoclonal antibody in an amount of 10 mg / kg (200 μg / mouse) twice a week, starting 1 week before IL-31 delivery. In the control groups of mice, intraperitoneal injections of the vehicle (PBS / 0.1% BSA) were carried out with identical dosing regimens. Mice were evaluated for alopecia and itching every day using the following criteria: 0 = no combing, the animal looks normal, 1 = thinning of hair in small areas, brushing is noted, 2 = slight loss of hair (small areas), combing, 3 = moderate loss of hair , combing and 4 = severe hair loss, excessive combing.

In all experiments, in mice that were injected with rat mAb anti-mIL-31, the onset of symptoms was slowed down by about 5-7 days, and they had lower overall rates of alopecia and pruritus. In all groups of mice that were injected with mAb (regardless of the frequency of dosing or dose concentration), alopecia and pruritus developed similar to control mice by 13 days of the study. These data confirm that neutralization of IL-31 can slow the onset of the response in the form of combing / hair loss induced by IL-31.

Example 13

Characterization of monoclonal mouse antibodies against human IL-31

A panel of 21 clonal hybridomas producing monoclonal antibodies (mAb) specific for (recombinant) human interleukin 31 (IL31) was obtained. Ten hybridomas producing high neutralization mAbs were isolated. They were divided into two subseries, based on studies of competitive binding. Eleven hybridomas producing weakly neutralizing mAbs were isolated. They were divided into 4 additional series based on competitive binding studies. One mAb worked well in western blotting, and it also worked well in immunohistochemistry. Three mAbs suitable for use as competitive antagonists have been identified. Monoclonal antibodies suitable for use in sandwich immunological assays have also been identified.

A. Epitope Sort by Biacore

Materials: the immobilized antibody was an Fc-γ-specific goat antibody against mouse IgG (Jackson # 115-005-071); the blocking antibody was an Fc fragment of polyclonal IgG (Jackson Labs.); antigen: rIL31 produced in BHK with the CEE affinity marker; Biacore 1000; NHS-activated Biacore CM5 chip (Biacore, PN BR-1000-14).

Method: Studies were performed using a Biacore 1000 ™ system. For methods of program analysis used BIAlogue v. 1,2. The goat antibody against mouse Fc was covalently immobilized on a Biacore CM5 chip through lysine residues to form an active binding surface for research. The supernatants of the conditioned media of the clones of each of the 21 anti-huIL31 mouse mAbs were first injected onto the goat anti-mouse Fc antibody surface so that the primary mAbs to be tested could bind to the anti-mouse Fc antibody surface in a suitable orientation. Then, the remaining binding sites were blocked by injection of insignificant Fc fragments of polyclonal IgG. The antigen was then injected (rIL31) and immobilized on the surface with the primary antibody. Then, another injection of one of the supernatants of conditioned media of clones of each of the 21 anti-huIL31 mouse mAbs was performed to test the binding of the secondary antibody. If the primary and secondary mAbs competed for the same antigen binding site, the second mAb did not bind. If two mAbs did not compete for the same binding site on the antigen, then binding of the second mAb occurred.

Each supernatant was tested as a primary mAb in combination with an entire set of mAb. Control cycles were performed to show the absence of a secondary mAb response in the absence of a primary mAb or antigen. Each mAb tested against itself was used as a negative control to establish the background signal level. Data was combined using BioEvaluation 3.2 RCI software and then uploaded to Excel ™ for data processing. Between each test cycle, a pair of monoclonal antibodies, the goat anti-mouse Fc antibody surface was regenerated by washing 2 x 30 s with 50 mM HCl.

mAbs with high neutralization (10) and mAbs with weak neutralization (11) were studied in two separate panels, except that when studying mAbs with weak neutralization, mAbs from hybridoma 292.64.6 were included as mAbs, which are mAbs with high neutralization. In addition, after studying the relative affinity indices and neutralization data, taking into account the epitope series, for the first two sorting panels, a third sorting panel was carried out for 8 “selected” mAbs, including neutralizing mAbs with both high and low neutralization.

Results: Mating of three panels of monoclonal antibodies was tested with mAb analysis with both high neutralization and low neutralization. In the first panel, all mAbs with high neutralization were tested against each other, and in the second panel, all mAbs with weak neutralization (and a representative mAb with high neutralization from hybridoma 292.64.6) were tested against each other. A third panel was performed to directly evaluate the pairing of the mAb series with both high neutralization and low neutralization, selected for further evaluation.

In addition to varying the absolute response (RU) defined for different mAb pairs, several mAb pairs worked differently when the orientation of the pairs changed (i.e., one mAb from a pair was used as a primary rather than a secondary mAb). This behavior has been observed frequently and is mainly characteristic of mAbs, presumably having overlapping epitopes.

Upon completion of the analysis of the first two panels, only four different series were obtained. For the first panel, all highly neutralized antibodies were grouped together to make up one series (series 1: 292.12.3, 292.39.5, 292.51.5, 292.63.5, 292.64.6, 292.72.3, 292.84.1 , 292.105.4, 292.109.4, 292.118.6). In the second panel, weakly neutralized antibodies were grouped into 3 additional main series (series 2: 294.35.2, 294.146.5, 292.152.4, 292.152.4, 294.154.5; series 3: 294.35.3, 291.78.4, 294.158 .5, 294.155.6, 294.163.2; series 4: 294.144.3), which differed from the only representative of series # 1 (292.64.6). In addition, the many mAb pairs evaluated in panel 2 showed a clear asymmetry of the response depending on the orientation in which the mAb was tested, indicating some overlap in binding sites. For two mAbs from series 3, 294.35.3 and 291.78.4, signs of competition with mAbs from series 2 were shown when tested as a secondary mAb. Also in panel 2, for an antibody with a high neutralization of 292.64.6 (series 1), competition with mAb in series 3 is shown when tested as a primary antibody.

After completing the analysis of the initial two sorting panels, the mAb group with the highest neutralization and the mAb with the highest affinity with the weakest neutralization were selected for further evaluation, and they were tested against each other in panel 3. The results for the third panel were completely consistent with the previous analyzes, showing that antibodies with high neutralization were sorted together and antibodies with weak neutralization were divided into certain multiple series. In addition, the third panel showed a partial overlap of the binding of two mAbs from series 2 (294.35.2 and 292.154.4) with a subgroup of mAbs assigned to series 1 (292.12.3, 292.72.3, 292.84). This led to the isolation of subseries 1A and 1B in series 1. In panel 3, the mAbs from the hybridomas 292.163.2 (series 3) and 292.144.3 (series 4) were sorted separately.

B. Sorting by epitope in a microtiter plate (competitive ELISA untagged)

Materials: The immobilized antibody was a goat anti-mouse IgG antibody (Fc-γ specific) Jackson # 115-005-071; the blocking antibody was mouse IgG1, (ZymoGenetics); supernatants of conditioned hybridomas were used for this study; the antigen was rIL31 produced in BHK with the CEE affinity marker biotinylated using a sulfo-NHS-biotin kit (Pierce, Rockford, IL); 96-well Nunc Maxisorp plates (Nalge Nunc, Rochester, NY); ELISA B (PBS, 0.1% Tween 20, 1% BSA); streptavidin-HRP (Pierce, Rockford, IL); TMB substrate (BioFX Laboratories, Owings Mills, MD)

Method: this method is similar to the unlabeled competitive ELISA (LFC-ELISA) described by Nagata et al (2004). In this method, biotinylated rIL31 is used for sorting by epitope. The titration microplates were coated with Fc-γ-specific goat anti-mouse IgG antibody (Jackson ImmunoResearch # 115-005-071) at a concentration of 1 μg / ml in an amount of 100 μl / well diluted in ELISA B (PBS, 0.1% Tween 20 , 1% BSA). After binding of this antibody to coat for 3 hours at ambient temperature, each conditioned mAb-containing medium was diluted in ELISA B to give an approximate mAb concentration of 0.5 μg / ml and was allowed to bind to goat antibody coated mouse anti-IgG plates overnight at 4 ° C (mAb # 1). Similarly, a second set of conditioned media (mAb # 2) was diluted in polystyrene test tubes to a mAb concentration of approximately 0.5 μg / ml in ELISA B, mixed with rIL31 biotinylated antigen at a concentration of 50 ng / ml and incubated overnight at 4 ° C. After incubation of mAb # 1 with a coating antibody, the plates were blocked with an unrelated antibody to saturate unoccupied binding sites on the plate. A mixture of mAb # 2-biotin-rIL31 was added to the plate and binding was achieved. As a control in the analysis (noncompetitive), biotinylated rIL31 was added at a concentration of 50 ng / ml directly (without preliminary incubation with mAb # 2) in wells containing immobilized mAb # 1. After incubation with the biotinylated IL31-mAb # 2 complex, streptavidin-HRP (Pierce, Rockford, IL) was added to the plate at a concentration of 0.5 μg / ml. The plates were developed using a TMB substrate (BioFX Laboratories, Owings Mills, MD), and the absorbance in individual wells at 450 nm was measured using a plate reader (Molecular Devices SpectraMax340, Sunnyvale, CA). If mAb # 1 recognized an epitope other than the mAb # 2 epitope, then the biotin-rIL31-mAb # 2 complex bound to the plate, which led to reading with high absorption. If mAb # 1 recognized the same epitope as mAb # 2, that biotin-rIL31-MAb # 2 complex did not bind to the plate, resulting in a low absorbance reading.

Results: a full panel of 21 mAb was tested against themselves simultaneously in a series of six 96-well titration microplates. The absorbance at 450 nm (A450 nm) was recorded for each combination and orientation and combined. In all cases, for combinations of mAbs with the same mAbs, low absorbance values were obtained. In addition to the control in the form of the same mAb, a positive control was tested, which was rIL31-biotin (without competitive mAb # 2) at a concentration of 50 ng / ml using each primary mAb on each plate. Two positive control samples were obtained for each primary mAb, and duplicate samples were similar. The absorption values obtained for wells with a positive control (without mAb # 2) for 9 of 11 mAb with weak neutralization (from series 2, 3 or 4) were less than the absorption values measured for control samples for the remaining mAbs. This was probably due to a lower affinity (higher EC ₅₀ ) for these mAbs. To simplify the interpretation of the results, the absorption values were normalized in such a way that the maximum measured absorption for any combination of mAb in one row (immobilized mAb (mAb # 1) remained constant) was assigned a value of 100%. Normalized values belonged to two different groups: one large group of values turned out to be lower than 32%, which corresponds to competition, and the second large group of values turned out to be higher than 32%, which corresponds to the absence of competition. A 32% threshold value was used to determine each combination and orientation of the mAb in one or two different categories (non-competitive and competitive). Using grouped data, sorting was automatically performed based on the hierarchical grouping algorithm. As it was revealed during sorting experiments using Biacore, some pairs of mAbs were grouped differently depending on which mAb was used as the primary mAb. A high level of rigor, requiring competition in both directions, was used to determine mAbs in the main series. Then, less stringent criteria requiring competition in only one direction were used to simplify the determination of mAb in the subseries, based on small differences in characteristics. Normalized data were grouped according to the definition in 5 main series, based on criteria of high stringency. In addition, the combinations and orientations of mAbs were color coded (white for competitive and dark for non-competitive) to simplify the visualization of interactions that define mAbs in the subseries.

Sorting results by competitive ELISA are consistent with batch definitions based on research results using Biacore. Using strict sorting conditions, the following series were defined:

Series # 1 (containing all mAbs with high neutralization): 292.72.3, 292.64.6, 292.63.5, 292.51.5, 292.39.5, 292.12.3. 292.118.6, 292.109.4, 292.105.4, 292.84.1

Series # 2: 294.35.2, 294.154.5, 294.146.5, 292.154.4, 292.152.4

Series # 3: 294.35.3, 294.163.2, 294.158.5, 294.155.6

Series # 4: 294.144.3

Series # 5: 291.78.4

Considering series # 1, for mAbs from 3 hybrid series # 1 (292.72.3, 292.12.3, 292.84.1) competition with 3 mAbs from series # 2 was shown when used as a secondary mAb (mAb # 2). Based on these characteristics, series 1 was divided into two sub-series; # 1A and # 1B, where the # 1B series contained mAbs that compete with the Series 2 mAbs for binding. Series # 1A contains 292.64.6, 292.63.5, 292.51.5, 292.39.5, 292.118.6, 292.109.4, 292.105.4. Series # 1B contains 292.72.3, 292.12.3, and 292.84.1.

Looking at series # 2, three different interactions were observed that were used to separate series # 2 into 3 sub-series. Series # 2A contains mAbs from hybridoma 294.146.5, which competes with all mAbs from both series # 1 and # 2. Series # 2B contains mAbs from the hybrid 294.35.2 and 294.154.5, which compete with the (secondary) mAbs from the series # 1. Series # 2C contains mAbs from hybrid 292.154.4 and 292.152.4, which compete with mAbs from series # 4 when used as a secondary mAb.

Considering series # 3, # 4 and # 5: mAb series # 3 (from hybrid 294.35.3, 294.163.2.1 and 294.155.6) compete with mAb from both series # 4 and # 5 when they are used as secondary mAb. The mAbs in series # 4 (from hybridoma 294.144.3.5) and # 5 mainly differ in their interaction with mAbs in subseries # 2C (and in the specific binding properties of mAbs from hybridoma 294.144.3 on western blots).

When a full panel of 21 mAb was tested simultaneously using LFC-ELISA, series 1 and subseries # 1A and # 1B were clearly identified. Partial overlap of series # 2 and # 3 was also revealed when the full mAb panel was tested at the same time, and series # 2 was divided into 3 sub-series. One small deviation from the determination of the series based on Biacore data was the determination of mAb from hybrid 291.78.4 and 294.144.3 in two different series, when strict criteria were used with respect to LFC-ELISA data. Based on studies of Biacore, mAb from hybridoma 291.78.4 was identified in series # 3.

C. Measurement of affinity on tablets

Materials: The immobilized polyclonal antibody was a goat anti-mouse IgG antibody (Fc-γ specific) (Jackson ImmunoResearch West Grove, Pennsylvania # 115-005-071); the blocking polyclonal antibody was a mouse antibody against human IgG (Jackson ImmunoResearch, # 209-005-082); supernatants of conditioned hybridomas were used for this study; the antigen was rIL31 produced in BHK with the CEE affinity marker biotinylated using the Sulfo-NHS-Biotin kit (Pierce, Rockford, JJL); 96-well Nunc Maxisorp plates (Nalge Nunc, Rochester, NY); ELISA B (PBS, 0.1% Tween 20, 1% BSA); streptavidin-HRP (Pierce, Rockford, IL); TMB substrate (BioFX Laboratories, Owings Mills, MD).

Method: This method is similar to the method described by van Heyningen (1986). The microtiter plates were coated with Fc-γ-specific goat anti-mouse IgG antibody (Jackson ImmunoResearch # 115-005-071) at a concentration of 1 μg / ml in an amount of 100 μl / well diluted in ELISA B (PBS, 0.1% Tween 20, 1% BSA). After binding of this antibody to coat for 3 hours at ambient temperature, each purified monoclonal antibody supernatant was diluted in ELISA B to give an approximate mAb concentration of 1 μg / ml and binding to the plate was ensured for 1 hour at ambient temperature. Serial dilutions from 500 ng / ml to 0 ng / ml of rIL31 biotinylated antigen in ELISA B were obtained and added to the wells. After incubation with biotinylated antigen, streptavidin-HRP (Pierce, Rockford, IL) was added to the plate at a concentration of 0.5 μg / ml. Plates were developed using a TMB substrate (BioFX Laboratories, Owings Mills, MD) and absorbance was measured for individual wells at 450 nm using a plate reader (Molecular Devices Spectra Max340, Sunnyvale, CA). Values for duplicates were averaged and the data was analyzed by four-parameter fitting. The “C” value for the 4-parameter fit is known as the apparent EC ₅₀ (ng / ml), and it represents the concentration of biotin-rIL31, which causes half of the maximum response in the analysis.

Results: A four-parameter fit was obtained from the experimental curves for all 21 mAb. The concentration of biotin-rIL31, causing half the maximum response (EC ₅₀ ) in the analysis, ranged from 3.3 to 236 ng / ml, where all 10 mAb with high neutralization showed low and comparable EC ₅₀ values (3.3-4.4 ng / ml).

D. Western blotting

Materials: the antigen was rIL31 produced in BHK with the CEE affinity marker; 4-12% bis-Tris NuPAGE gels (Invitrogen, Carlsbad, CA); non-reducing sample buffer (Invitrogen, Carlsbad, CA); Molecular Weight Standards were SeeBlue (Invitrogen); 1 × buffer for MES analysis (Invitrogen); Buffer A for Western Blotting (50 mM Tris pH 7.4, 5 mM EDTA, 150 mM NaCl, 0.05% Igepal, 0.25% Gelatin); 0.2 μm nitrocellulose membranes (Invitrogen); sheep antibodies against mouse HRG IgG (Amersham, Piscataway, NJ); Purified rabbit affinity pAb specific for rIL31 (ZymoGenetics); donkey anti-rabbit IgP antibody-HRP (Amersham); Lumi-Light Plus chemiluminescent reagent (Roche, Mannheim, Germany); Lumi-Imager (Mannheim-Boehringer).

Method: In this study, the ability of a mAb to detect denatured / reduced rIL31 in a Western blot was evaluated. RIL31 antigen was mixed with either reducing or non-reducing sample buffer, heated at 70 ° C for 10 min, and then applied to 4-12% NuPAGE bis-Tris gels in an amount of 100 ng / lane. The molecular weight standards were SeeBlue (Invitrogen), and electrophoresis was performed in 1 × MES assay buffer (Invitrogen). Protein strips in gels were transferred onto 0.2-μm nitrocellulose membranes (Invitrogen) and blocked overnight in 2.5% skimmed milk powder in Western Blot buffer A (50 mM Tris pH 7.4, 5 mM EDTA, 150 mM NaCl, 0.05% Igepal, 0.25% gelatin). Each monoclonal antibody was applied to nitrocellulose membranes in an approximate mAb concentration of 0.1 μg / ml. Then, a secondary horseradish peroxidase conjugated antibody (sheep anti-mouse IgG antibody-HRP; Amersham, Piscataway, NJ) was applied to the blots. As a positive control, a rabbit polyclonal antibody specific for IL-31 (ZymoGenetics) at a concentration of 0.1 μg / ml and a secondary antibody conjugated to horseradish peroxidase (donkey anti-rabbit IgP antibody-HRP; Amersham) were applied to a separate western blot ) Western blot bands were detected by Lumi-Light Plus reagent (Roche, Mannheim, Germany) and chemiluminescence was recorded by Lumi-Imager (Mannheim-Boehringer).

Results: Western blot analysis of mAb against IL31 showed that only three of the 21 hybridomas had mAbs that detected the denatured rIL31 antigen. The strongest signal was obtained from a mAb produced by hybridoma 294.144.3. This mAb also detected reduced / denatured rIL31 more efficiently than unreduced / denatured rIL31. The signal intensity observed for this mAb was similar to the intensity obtained for control in the form of a polyclonal antibody. It should be noted that the mAb from hybridoma 294.144.3 refers to a series that is different from the series of other evaluated mAbs. Monoclonal antibodies from two other hybridomas (292.84.1 and 292.64.6) showed unreduced / denatured rIL31 very weakly, however, restored / denatured rIL31 was not detected. This weak signal was not reproduced on the scanned blots. Both of these mAbs are series # 1 neutralizing antibodies.

Example 14

Relative binding affinity of rat monoclonal antibodies against mouse antibodies for ligand IL-31

The relative binding affinity of the four rat MAb against the ligand Ms-IL-31 was determined as follows. Clone 271.26.6.6.1, clone 271.33.3.2.1, clone 271.33.1.2.2 and clone 271.39.4.6.5 were evaluated. Fc-γ-specific goat anti-rat IgG antibody (Jackson) was immobilized on a Biacore CM5 chip. After preliminary testing, the analysis was further optimized by linking each MAb to a surface to trap anti-rat antibodies, and then a series of IL-31 ligand concentrations were passed through the MAb to determine association and dissociation. After each analysis, the surface was regenerated to an immobilized anti-rat antibody by 2 injections of 30 mM HCl. Data were obtained for each MAb and software was used to evaluate relative kinetics.

The relative kinetics data obtained by evaluating the MAb antigen binding curves are presented in Table 4.

Table 4 Clone 271.26.6.6.1 271.33.3.2.1 271.33.1.2.2 271.39.4.6.5 ka (M-1s-1) 5.61E + 05 1.43E + 05 8.19E + 05 8.94E + 05 kd (s-1) 3,6E-04 5.46E-04 4.21E-04 6.21E-04 KD (M) 6.42E-10 3.81E-9 4.73E-10 6.94E-10 Chi2 0,101 0.341 0.0917 1.92

Example 15

Decrease in TARC and MDC in response to anti-IL-31 antibody in mouse AD model

Method I

Six week old male NC / Nga mice (CRL Japan) were intradermally sensitized with 50 μg of house dust mite extract (D. pteronyssinus, Indoor Biotechnologies) three times a week in the back and AD-like lesions were evaluated. After 5 weeks of sensitization, the mice were sacrificed and the right ears were excised and placed in a separate well of a 48-well culture dish (Corning) supplemented with RPMI + 2% ETS (GIBCO Invitrogen). The plates were placed in incubators with 5% CO ₂ and controlled humidity. After 24 hours, supernatants were collected and frozen at -20 ° C until further analysis.

Method II

Twelve week old female NC / Nga mice (CRL Japan) were intradermally sensitized with 10 μg SEB (Toxin Technology) in the ear and back three times a week. Mice were evaluated for AD-like lesions. After 5 weeks of sensitization, the mice were sacrificed and a 6 mm puncture biopsy was taken from the injected ear of each mouse and placed in a separate well of a 48-well culture dish supplemented with RPMI + 2% ETS. The plates were placed in incubators with 5% CO ₂ and controlled humidity. After 24 hours, supernatants were collected and frozen at -20 ° C until further analysis.

In the groups of mice in both studies, either a rat monoclonal antibody against mouse IL-31 at a concentration of 10 mg / kg or a vehicle was administered intraperitoneally twice a week, starting from 1 to 2 weeks of sensitization.

The concentrations of TARC and MDC in supernatant samples after 24 hours were measured using a conventional ELISA (R&D Systems).

TARC and MDC concentrations were lower in ear supernatants from mice injected with anti-IL-31 antibody compared to control mice in both studies, however, these results were not statistically significant when analyzed by ANOVA, possibly due to the small sample size. When combining and analyzing all the data from both experiments, a statistically significant difference between the groups of introduction was revealed.

Example 16

Administration of a neutralizing IL-31 antibody

Normal female BALB / c (CRL) mice aged about 8 to 12 weeks were subcutaneously implanted with 14-day osmotic pumps (Alzet, # 2002) delivering mIL-31 in an amount of 1 μg / day. In groups of mice, intraperitoneal (i.p.) injections of rat monoclonal antibody against mouse IL-31 in an amount of 10 mg / kg (200 μg / mouse) were performed twice a week, starting 1 week before IL-31 delivery. In the control groups of mice, intraperitoneal injections of the vehicle (PBS / 0.1% BSA) were carried out with identical dosing regimens. Mice were evaluated for alopecia and itching every day using the following criteria: 0 = no combing, the animal looks normal, 1 = thinning of hair in small areas, brushing is noted, 2 = slight loss of hair (small areas), combing, 3 = moderate loss of hair , combing and 4 = severe hair loss, excessive combing.

In all experiments, mice injected with anti-mIL-31 rat mAb had a delayed onset of symptoms by about 5 to 7 days and lower overall rates for alopecia and pruritus. In all groups of mice that were injected with mAb (regardless of the frequency of dosing and dose concentration), alopecia and pruritus similar to control mice developed by 13 days of the study. These data confirm that neutralization of IL-31 can slow the appearance of the combing / loss of hair response induced by IL-31.

Based on the foregoing, it should be borne in mind that, despite the fact that for the purposes of illustration, the present description describes specific embodiments of this invention, various modifications can be made without departing from the essence and scope of this invention. Thus, this invention is not limited to anything other than the appended claims.

Claims (39)

1. Monoclonal antibody against IL-31 or its functional fragment, where the monoclonal antibody is produced by a hybridoma deposited in the American type culture collection, having a patent number of deposit in ATCC selected from
a) patent number of deposit in ATCC PTA-6815;
b) the patent number of the deposit in ATCC PTA-6816;
c) patent number of deposit in ATCC PTA-6829;
d) patent number of the deposit in ATCC PTA-6830;
e) patent number of deposit in ATCC PTA-6831;
f) the patent number of the deposit in ATCC RTA-6871;
g) the patent number of the deposit in ATCC PTA-6872;
h) patent number of deposit in ATCC PTA-6875; and
i) the patent number of the deposit in ATCC PTA-6873, and specifically binds a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. 2. A monoclonal antibody against IL-31 or a functional fragment thereof, wherein the monoclonal antibody is produced by a hybridoma deposited in the American Type Culture Collection having a ATCC patent deposit number selected from
a) patent number of deposit in ATCC PTA-6815;
b) the patent number of the deposit in ATCC PTA-6816;
c) the patent number of the deposit in ATCC PTA-6871;
d) patent number of deposit in ATCC PTA-6829; and
e) the patent number of the deposit in ATCC PTA-6830,
and specifically binds a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. 3. A monoclonal antibody against IL-31 or a functional fragment thereof, wherein the monoclonal antibody is produced by a hybridoma deposited in the American Type Culture Collection having a ATCC patent deposit number selected from
a) patent number of deposit in ATCC PTA-6815;
b) the patent number of the deposit in ATCC PTA-6816; and
c) the patent number of the deposit in ATCC RTA-6871,
and specifically binds a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. 4. A monoclonal antibody against IL-31 or a functional fragment thereof, wherein the monoclonal antibody is produced by a hybridoma deposited in the American Type Culture Collection having a ATCC patent deposit number selected from
a) patent number of the deposit in ATCC PTA-6829; and
b) the patent number of the deposit in ATCC PTA-6830,
and specifically binds a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. 5. A monoclonal antibody against IL-31 or a functional fragment thereof, wherein the monoclonal antibody is produced by a hybridoma deposited in the American Type Culture Collection having a ATCC patent deposit number selected from
a) patent number of the deposit in ATCC PTA-6872; and
b) the patent number of the deposit in ATCC PTA-6875, and
specifically binds a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. 6. The antibody or antibody fragment according to claims 1 or 2-5, wherein the antibody or antibody fragment neutralizes the interaction of IL-31 (SEQ ID NO: 2) with IL-31RA (SEQ ID NO: 5). 7. An antibody or antibody fragment according to claims 1 or 2-5, wherein the antibody or antibody fragment is (a) a monoclonal mouse antibody, (b) a humanized antibody derived from (a), or (c) an antibody fragment, or ( d) human monoclonal antibody. 8. The antibody or antibody fragment according to claims 1-5, wherein the antibody or antibody fragment contains a radionuclide, an enzyme, a substrate, a cofactor, a fluorescent marker, a chemiluminescent marker, a peptide marker, a magnetic particle or toxin. 9. The antibody or antibody fragment according to claims 1-5, wherein the antibody or antibody fragment contains pegylation. 10. The antibody or antibody fragment according to claims 1-5, wherein the antibody or antibody fragment is neutralizing. 11. The antibody or antibody fragment according to claims 1-5, where the introduction of an antibody or antibody fragment to a mammal inhibits, reduces or blocks the pro-inflammatory activity of the polypeptide. 12. The antibody or antibody fragment according to claims 1-5, wherein administration of the antibody or antibody fragment to a mammal inhibits, reduces, or blocks scratching and dermatitis due to the pro-inflammatory activity of the polypeptide. 13. The monoclonal antibody of claim 1, wherein the monoclonal antibody or antibody fragment is produced by a hybridoma deposited in the American Type Culture Collection having a patent deposit number in ATCC 6815. 14. The monoclonal antibody according to claim 1, wherein the monoclonal antibody or antibody fragment is produced by a hybridoma deposited in the American Type Culture Collection having a patent deposit number in ATCC 6816. 15. The monoclonal antibody according to claim 1, where the monoclonal antibody or antibody fragment is produced by a hybridoma deposited in the American type culture collection, having a patent deposit number in ATCC 6829. 16. The monoclonal antibody according to claim 1, where the monoclonal antibody or antibody fragment is produced by a hybridoma deposited in the American type culture collection, having a patent deposit number in ATCC 6830. 17. The monoclonal antibody according to claim 1, where the monoclonal antibody or antibody fragment is produced by a hybridoma deposited in the American type culture collection, having a patent deposit number in ATCC 6831. 18. The monoclonal antibody according to claim 1, where the monoclonal antibody or antibody fragment is produced by a hybridoma deposited in the American type culture collection, having a patent deposit number in ATCC 6871. 19. The monoclonal antibody of claim 1, wherein the monoclonal antibody or antibody fragment is produced by a hybridoma deposited in the American Type Culture Collection having a patent deposit number in ATCC 6872. 20. The monoclonal antibody according to claim 1, where the monoclonal antibody or antibody fragment is produced by a hybridoma deposited in the American Type Culture Collection, having a patent deposit number in ATCC 6875. 21. The monoclonal antibody according to claim 1, where the monoclonal antibody or antibody fragment is produced by a hybridoma deposited in the American type culture collection, having a patent deposit number in ATCC 6873. 22. The antibody or antibody fragment according to claims 1 or 2-5 for inhibiting, reducing or blocking alopecia areata or dermatitis. 23. The antibody or antibody fragment according to claims 1 or 2-5, wherein administration of the antibody to a mammal inhibits, reduces or blocks the production of pro-inflammatory chemokines. 24. The antibody or antibody fragment of claim 23, wherein the pro-inflammatory chemokines are TARC or MDC. 25. The antibody or antibody fragment according to claims 1 or 2-5, wherein administration of the antibody to a mammal inhibits, decreases, or blocks the desire in the mammal to itch. 26. The antibody or antibody fragment according to claims 1 or 2-5, where the antibody or antibody fragment is a Fab molecule or a molecule (Fab ′) ₂ . 27. The antibody or antibody fragment according to claims 1 or 2-5, wherein the antibody or antibody fragment is a single chain molecule. 28. A method of reducing inflammation in a mammal, comprising administering to the mammal an amount of an IL-31 antibody according to claim 1, whereby the inflammation is reduced. 29. The method of claim 28, wherein the inflammation is a symptom of vitiligo, cutaneous T-cell lymphoma, alopecia areata, redheads, acne vulgaris, prurigo nodosa or bullous pemphigoid, and where the inflammation is reduced. 30. A method of treating a mammal suffering from an inflammatory disease in which IL-31, including
administering an IL-31 antagonist to a mammal in order to reduce inflammation, the antagonist comprising the antibody or antibody fragment of claim 1; and
this leads to a decrease in the inflammatory activity of IL-31. 31. A method of reducing, inhibiting, or minimizing pruritus in a mammal in which IL-31 is involved, comprising
administering an IL-31 antagonist to a mammal so that itching is reduced, the antagonist comprising the antibody or antibody fragment of claim 1;
and thus there is a decrease in the itching activity of IL-31. 32. A method of reducing, inhibiting, or minimizing pruritus in a mammal in which IL-31 is involved, comprising administering an IL-31 antagonist to a mammal, the antagonist comprising the antibody or antibody fragment of claim 1, whereby administering an IL-31 antagonist reduces the animal has a desire to itch. 33. The method according to PP.28-32, where the disease is an inflammatory disease, including diseases associated with pruritus. 34. The method according to PP.28-32, where the disease is atopic dermatitis. 35. The method according to PP.28-32, where the disease is a knotty pruritus. 36. The method according to PP.28-32, where the antibody is an antibody according to PP.1-5. 37. A monoclonal antibody or a functional fragment thereof, wherein the monoclonal antibody is produced by a hybridoma clone indicated by a number selected from
a) clone 292.12.3.1 (patent number of the deposit in ATCC PTA-6815);
b) clone 292.63.5.3 (patent number of the deposit in ATCC PTA-6829);
c) clone 292.72.3.1 (patent number of the deposit in ATCC PTA-6816);
d) clone 292.84.1.6 (patent number of the deposit in ATCC PTA-6871); and
e) clone 292.118.6.4 (patent number of the deposit in ATCC PTA-6830),
and specifically binds a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. 38. A monoclonal antibody or a functional fragment thereof, wherein the monoclonal antibody is produced by a hybridoma clone indicated by a number selected from
a) clone 294.35.2.6.3 (patent number of the deposit in ATCC PTA-6872);
b) clone 294.144.3.5 (patent number of the deposit in ATCC PTA-6873);
c) clone 294.154.5.6 (patent number of the deposit in ATCC PTA-6875); and
d) clone 294.163.2.1 (patent number of the deposit in ATCC PTA-6831),
and specifically binds a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. 39. A hybridoma producing an anti-IL-31 antibody deposited in the American Type Culture Collection, having a patent deposit number in ATCC PTA-6815.

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