EA042754B1 - NEUTRALIZING MONOCLONAL ANTIBODIES TO IL-25 AND THEIR USE - Google Patents

Description

Prerequisites for the creation of the invention

Interleukin-25 (IL-25), also known as IL-17E, belongs to the IL-17 family of cytokines and is secreted by T-helper type 2 (Th2) as well as mast cells. IL-25 induces the production of other cytokines, including IL-4, IL-5 and IL-13, in various tissues and stimulates an increase in the number of eosinophils.

The involvement of IL-25 in chronic inflammatory processes of the gastrointestinal tract and the presence of the IL-25 gene in the chromosomal region associated with the development of autoimmune bowel diseases, such as inflammatory bowel disease (IBD), have been noted. Conventional therapy for IBD is based on antibiotics or steroid drugs; however, these therapeutic approaches currently do not successfully achieve or maintain clinical remission in patients.

Increased expression of IL-25 has been noted in samples obtained from patients with asthma, a disease that is estimated to affect more than 300 million people worldwide; from which it can be concluded that the overexpression of this cytokine contributes to the pathophysiology of asthma and other related diseases.

Thus, there is a need to provide effective IL-25 antagonists that can be used to treat diseases and conditions characterized by overexpression of IL-25, including asthma and inflammatory bowel disease.

Statement of the Invention

In one aspect, the present document describes IL-25 related binding molecules that are useful in the treatment of diseases and disorders mediated by IL-25.

In one aspect, the present document describes isolated IL-25 binding molecules comprising a heavy chain variable domain containing one or more complementarity determining regions (CDRs) as defined in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9,

In addition, IL-25 binding molecules containing a heavy chain variable domain are described, wherein the heavy chain variable domain comprises SEQ ID NO: 13 and SEQ ID NO: 15

In one aspect, this document describes light chain variable domains containing one or more CDRs as defined in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

Also described herein are isolated IL-25 binding molecules comprising a light chain variable domain, wherein the light chain variable domain comprises SEQ ID NO: 14, SEQ ID NO 16;

In one aspect, the described IL-25 binding molecules may comprise a heavy chain variable domain and a light chain variable domain or complementarity determining regions of any of the preceding aspects.

Accordingly, IL-25 binding molecules containing one, two, or three heavy chain variable domain CDRs selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO : 7, SEQ ID NO: 8 and SEQ ID NO: 9, and one, two or three light chain variable domain CDRs selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

In addition, IL-25 binding molecules are described herein comprising a heavy chain variable domain, wherein the heavy chain variable domain comprises SEQ ID NO: 13 or SEQ ID NO: 15, and a light chain variable domain, wherein the light chain variable domain comprises SEQ ID NO: 14 or SEQ ID NO: 16.

Additionally, described herein are humanized IL-25 binding molecules comprising a heavy chain variable domain, wherein the heavy chain variable domain comprises SEQ ID NO: 17, and a light chain variable domain, wherein the light chain variable domain comprises SEQ ID NO: 18.

In one aspect, this document describes methods of treating, inhibiting or preventing airway inflammation due to rhinovirus infection, rheumatoid arthritis, osteoarthritis, bone erosion, intraperitoneal abscesses and adhesions, inflammatory bowel disease, allograft rejection, psoriasis, certain forms of cancer, angiogenesis, atherosclerosis, cystic fibrosis and multiple sclerosis, comprising administering a therapeutic amount of any of the IL-25 binding molecules from any of the preceding aspects.

Brief description of graphic materials

The accompanying graphics, which are included in and form part of this specification, illustrate certain embodiments and, together with the description, serve as examples of the compositions and methods described.

In FIG. 1 shows a summary of B cells from IL-25 immunized mice and their fluorescent sorting plot for B cell binding to IL-25.

In FIG. 2 shows denaturing gel electrophoresis for antibodies: lane 1: positive

- 1 042754 human IgG4 control, lane 2: kaleidoscopic prestained standard protein marker, lane 3: AVM109, lane 4: AVM125, lane 5: AVM109.2.

In FIG. 3 shows an analysis of the cellular potency of HT-29 monoclonal antibodies to IL-25.

In FIG. 4 shows an analysis of the HT-29 cell potency of the humanized monoclonal antibody AVM109.2 in IgG1, IgG2, and IgG4 isotypes.

In FIG. 5 shows surface plasmon resonance (SPR) data for anti-IL25 monoclonal antibodies and the estimated antibody affinity.

In FIG. 6 shows the effects of administration of AVM125 or a control antibody (IgG) on the total number of cells infiltrating the lungs, eosinophils, and macrophages in a rhinovirus infection model for allergic asthma in mice.

In FIG. 7 shows the amount of detectable rhinovirus RNA in the lungs of mice treated with control antibody (IgG) or AVM125.

In FIG. 8 shows the amount of detectable IL-5 RNA in the lungs of mice treated with control antibody (IgG) or AVM125.

In FIG. 9 shows the binding potency and affinity of humanized (CDR-grafted) versions of AVM125, referred to as AVM125.9 and AVM125.10, compared to chimeric AVM125 with fully murine variable regions.

Detailed description

Before disclosing and describing the present compounds, compositions, articles, devices and/or methods, it should be understood that, unless otherwise indicated, they are not limited to specific synthetic methods or specific methods of recombinant biotechnology, or, unless otherwise indicated, to specific reagents, since the above is, of course, subject to change. It should also be understood that the terms used herein are used only for the purpose of describing specific embodiments and are not restrictive.

A. Definitions.

In the present description and in the appended claims, the singular forms include the plural, unless otherwise clearly follows from the context. Thus, for example, the designation pharmaceutical carrier includes mixtures of two or more carriers, and the like.

As used herein, ranges may be expressed as from about one specific value and/or to about another specific value. When such a range is indicated, another embodiment includes a range from one particular value and/or to another particular value. Likewise, when values are indicated as approximate using the preceding word approximately, it is to be understood that a particular value generates another embodiment. It will be further understood that the endpoints of each of the ranges are significant both relative to the other endpoint and independently of the other endpoint. It should also be understood that a number of values are described herein, and that each value is described herein as well as approximately such a specific value in addition to such value itself. For example, if a value of 10 is described, then a value of approximately 10 is also described. It should also be understood that if a value is described that is less than or equal to a certain value, greater than or equal to a certain value, possible ranges between values are also described, which will be completely obvious to a person skilled in the art. For example, if a value of 10 is described, a value less than or equal to 10 and greater than or equal to 10 is also described. It should also be understood that throughout the text of this document, data is given in several different formats and that such data represents end points and starting points. and ranges for any combination of data points. For example, if specific data point 10 and specific data point 15 are described, it is clear that values greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, as well as values in the range of 10 to 15, are considered to be described. also understand that each unit between two specific units is described. For example, if 10 and 15 are described, 11, 12, 13, and 14 are also described.

In the present description and in the following claims, a number of terms will be used, which, in accordance with the definitions, have the following meanings:

Optional or optional means that the event or circumstance described below may or may not occur, and that the description includes cases where the specified event or circumstance occurs and cases where it does not.

This document provides references to various publications. The contents of these publications are incorporated herein by reference in their entirety for a more detailed description of the state of the art to which this disclosure relates. References cited herein are also individually and specifically incorporated in the text of this document by reference to the material contained therein, which is discussed in a proposal containing a particular link.

B. Compositions.

The components that will be used to prepare the described compositions are described, and

- 2 042754 also the compositions themselves, which will be used in the framework of the methods described herein. These and other materials are described herein, and it should be understood that when describing combinations, subpopulations, interactions, groups, etc. these materials, although specific reference to each individual and cumulative combination and permutation of such compounds cannot be explicitly described, each is specifically considered and described here. For example, when a particular anti-IL-25 antibody is described and discussed, and a number of modifications are discussed that may be applied to a number of molecules, including an anti-IL-25 antibody, all combinations and permutations of the anti-IL-25 antibody, and all possible modifications, are specifically meant, without exception. unless specifically stated otherwise. Thus, if a class of molecules A, B and C is described, as well as a class of molecules D, E and F, and an example of a combined molecule A-D is described, then even if each of them is not mentioned individually, it is understood that each of them individually and collectively means that combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are described. Any subpopulation or combination thereof is similarly described. Thus, for example, the subgroup A-E, B-F and C-E would be considered described. This concept applies to all aspects of this document, including, without limitation, the steps of the methods for obtaining and using the described compositions. Thus, if there are many additional steps that can be performed, it should be understood that each of these additional steps can be performed with any particular embodiment or combination of embodiments of the described methods.

As noted above, elevated levels of IL-25 have been associated with several conditions, diseases, or disorders, including airway inflammation, rheumatoid arthritis (RA), osteoarthritis, bone erosion, intraperitoneal abscesses and adhesions, inflammatory bowel disease (IBD), cystic fibrosis, rejection allograft, psoriasis, certain forms of cancer, angiogenesis, atherosclerosis and multiple sclerosis (PC).

Currently, the IL-17 family of cytokines includes IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (IL-25) and IL-17F. All IL-17 family cytokines contain four highly conserved cysteine residues that are involved in the formation of intrachain disulfide bonds and contain two or more cysteine residues that may be involved in the formation of intrachain disulfide bonds. The IL-17 family cytokines lack sequence similarity to any other known cytokines.

Type 2 cytokines play an important role in mediating protective immunity to parasitic helminth infection, regulating effector functions such as B cell growth and IgE secretion, inducing goblet cell hyperplasia and associated mucus production, eosinophilia, mastocytosis, and fibrosis. These cytokines play a critical role in regulating these effector functions, making them key therapeutic targets in asthma. Indeed, in mouse models overexpressing such cytokines, marked signs of asthma are observed. Thus, it was unexpectedly found that attempts to alleviate the symptoms of experimental asthma by blocking specific type 2 cytokines, other than inhibition of IL-13, were not found to be successful.

Inhibition of IL-13 suppresses both airway hyperresponsiveness (AHR) and airway inflammation, although the mechanisms of this effect are still unclear. However, given the complexity of the pathophysiology and the lack of understanding of the etiology of asthma, it is not certain that targeting individual pathways will ultimately be therapeutically successful.

Overexpression of IL-25/IL-17E has recently been shown to induce type 2 responses to in vivo treatment and enhance responsiveness to airway agonists. IL-25' ^/ ' mice failed to expel helminth parasites; which was a key indicator of ineffective type 2 response to treatment.

The authors of the present invention have received antibodies to IL-25 and identified an antibody molecule that binds to IL-25 with ultra-high affinity and specificity. Out of twenty-three screened blocking antibodies, two were identified, one of which had an extremely slow rate of dissociation.

binding molecules.

As used herein, the term binding molecule refers to an intact immunoglobulin, including monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized or human antibodies, as well as antibody fragments and functional variants, including an antigen-binding and/or variable domain containing an immunoglobulin fragment. , which conflicts with intact immunoglobulin due to specific binding to an immunoglobulin binding partner, such as IL-25.

In one aspect, the described IL-25 binding molecules may include an anti-IL-25 antibody (eg, an anti-IL-25 antibody). The term antibodies is used herein in a broad sense and includes polyclonal and monoclonal antibodies. As used herein, the term antibody includes, without limitation, a complete immunoglobulin (ie, intact antibody) of any class. In addition to intact immunoglobulin molecules, the term antibodies also

- 3 042754 includes fragments or polymers of such immunoglobulin molecules and human or humanized variants of immunoglobulin molecules or fragments thereof, provided that they are selected for their ability to interact with IL-25, so that the failure of IL-25 to interact with

IL-17RA and/or IL-17RB. In addition, antibodies are described that are associated with the described regions of IL-25 involved in the interaction between IL-25 and IL-17RA and/or IL-17RB.

Native antibodies are typically heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. The described IL-25 binding molecules, whether monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized or human antibodies, as well as fragments and functional variants of antibodies, may contain all or part of the light and heavy chains.

Typically, in a complete antibody, each light chain is linked to the heavy chain by a single covalent disulfide bond, while the number of disulfide bonds between the heavy chains varies between immunoglobulin isotypes. Each heavy and light chain also has equidistant internal disulfide bridges. Each heavy chain has at one end a variable domain (V(H)) followed by several constant domains (C(H)). Each light chain has at one end a variable domain (V(L)), and at its other end a constant domain (C(L)); the light chain constant domain is opposite the first heavy chain constant domain, and the light chain variable domain is opposite the heavy chain variable domain. It is generally accepted that specific amino acid residues form the boundary between the variable domains of the light and heavy chains. The light chains of antibodies of any vertebrate species can be assigned, depending on the amino acid sequences of their constant domains, to one of two distinct types, called kappa (k) and lambda (l). Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be divided into different classes. There are five main classes of human immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further subdivided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Comparable classes for mice will be apparent to one skilled in the art. The heavy chain constant domains that correspond to the different classes of immunoglobulins are designated as alpha, delta, epsilon, gamma, and mu, respectively.

The term variable is used herein to describe certain heavy and light chain domains that differ in sequence among antibodies and are used to bind and specificize each particular antibody to its particular antigen. In this case, the variability is usually unevenly distributed over the variable domains of antibodies. Variable domains with higher levels of conservatism are called frameworks (FRs). The native heavy and light chain variable domains in each case contain four FR regions, mostly in the β-sheet configuration, connected by three complementarity determining regions (CDRs) that form loops connecting and in some cases forming part of the β-sheet structure. Variability is generally concentrated in the CDRs or hypervariable regions, which are found in both the light and heavy chain variable domains.

The CDRs in each strand are held together in close proximity by FR regions and together with the CDRs of the other strand contribute to the formation of an antibody antigen-binding site (see Rabat E.A. et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. ( 1987)). As used herein, the term complementarity-determining regions refers to sequences within the variable regions of binding molecules, such as immunoglobulins, that form an antigen-binding site that is complementary in shape and charge distribution to an epitope recognized on an antigen. CDR regions may be specific for linear epitopes, discontinuous epitopes, or conformational epitopes of proteins or protein fragments that are present on the protein either in its native conformation or, in some cases, on a denatured protein, for example, as a result of solubilization in SDS. Epitopes may also consist of post-translational modifications of proteins.

In addition, it is possible to replace one or more CDR residues or skip one or more CDRs. The scientific literature describes cases where one or two CDRs can be dispensed with in antibodies to ensure binding. In the work of Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only about a third of the CDR residues actually come into contact with the antigen. This work also found many antibodies in which one or two of the CDRs do not contain amino acids in contact with the antigen (see also Vajdos et al. 2002, J. Mol. Biol. 320:415-428).

Antigen non-contacting CDR residues can be determined based on previous studies (eg H60-H65 residues in CDRH2 are often not required) from Kabat CDR regions outside of Chothia's CDR by molecular modeling and/or empirically. If CDRs or their residues are omitted, they are usually replaced by amino acids occupying the corresponding

- 4 042754 position in another human antibody sequence, or a consensus of such sequences. Substitution positions within CDRs and amino acids can also be chosen empirically.

The constant domains are not directly involved in the binding of an antibody to an antigen, but perform various effector functions, such as influencing the involvement of the antibody in antibody-dependent cellular toxicity.

In one aspect, the present document describes isolated IL-25 binding molecules comprising a heavy chain variable domain containing one or more complementarity determining regions (CDRs) as defined in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, and/or SEQ ID NO: 9. For example, IL-25 binding molecules containing a heavy chain variable domain containing a number of CDRs as indicated in SEQ ID are described herein. NO: 1 and SEQ ID NO: 2; SEQ ID NO: 1 and SEQ ID NO: 3; SEQ ID NO: 2 and SEQ ID NO: 3; SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 7 and SEQ ID NO: 9; SEQ ID NO: 8 and SEQ ID NO: 9 and SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. In addition, IL-25 binding molecules containing a heavy chain variable domain are described, wherein the heavy chain variable domain chain contains SEQ ID NO: 13 or SEQ ID NO 15.

It should be understood and appreciated herein that the described heavy chain variable domain complementarity determining regions in the described IL-25 binding molecules may be contiguous or separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 , 36, 37, 38, 39 or 40 amino acids. Thus, IL-25 binding molecules containing at least two CDRs are described herein, wherein the first CDR is separated from the second CDR by 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. . For example, IL-25 binding molecules containing at least two CDRs are described herein, wherein the first CDR contains SEQ ID NO: 1 or SEQ ID NO: 7 and the second CDR contains SEQ ID NO: 2 or SEQ ID NO: 8 , and wherein the first CDR and the second CDR are separated by 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In addition, IL-25 binding molecules containing three CDRs are described herein, the first CDR comprising SEQ ID NO: 1 or SEQ ID NO: 7; the second CDR contains SEQ ID NO: 2 or SEQ ID NO: 8; and the third CDR contains SEQ ID NO: 3 or SEQ ID NO: 9; and wherein the second CDR and the third CDR are separated by 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

It should be understood and appreciated herein that IL-25 binding molecules may contain a light chain variable domain in place of, or in addition to, a heavy chain variable domain. Thus, in one aspect, the present document describes IL-25 binding molecules containing a light chain variable domain containing one or more CDRs as defined in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, and/or SEQ ID NO: 12. For example, IL-25 binding molecules containing a light chain variable domain containing a CDR as indicated in SEQ ID NO: 4 and SEQ ID are described herein. NO: 5; SEQ ID NO: 4 and SEQ ID NO: 6; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 10 and SEQ ID NO: 11; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID NO: 12 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12. In addition, isolated IL-25 binding molecules containing a light chain variable domain, wherein the light chain variable domain comprises SEQ ID NO: 14 or SEQ ID NO 16.

It should be understood and appreciated herein that the described light chain variable domain complementarity determining regions in the described IL-25 binding molecules may be contiguous or separated by 1, 2, 3, 4, 5, b, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 , 36, 37, 38, 39 or 40 amino acids. Thus, the present document describes IL-25 binding molecules containing a light chain variable domain, wherein such a light chain variable domain contains at least two CDRs, wherein the first CDR is separated from the second CDR 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. For example, IL-25 binding molecules containing at least two CDRs are described herein, wherein the first CDR contains SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16, or SEQ ID NO: 22, and the second CDR contains SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17, or SEQ ID NO: 23, and wherein the first CDR and second CDR are separated by 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. Also described herein are IL-25 binding molecules comprising a light chain variable domain, such a light chain variable domain containing three CDRs, the first CDR comprising SEQ ID NO: 4 or SEQ ID NO: 10; the second CDR contains SEQ ID NO: 5 or SEQ ID NO: 11; and the third CDR contains SEQ ID NO: 6 or SEQ ID NO: 12; and wherein the second CDR and the third CDR are separated by 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

In one aspect, it should be understood and meant herein that the disclosed IL-25 binding molecules may comprise both a heavy chain variable domain and a light chain variable domain. It should also be understood that said IL-25 binding molecules may comprise one, two or three heavy chain variable domain CDRs in combination with one, two or three light chain variable domain CDRs described herein. Accordingly, IL-25 binding molecules may contain one, two, or three heavy chain variable domain CDRs selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8 and/or SEQ ID NO: 9, and one, two or three light chain variable domain CDRs selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, and/or SEQ ID NO: 12. For example, IL-25 binding molecules containing a heavy chain variable domain are described herein, wherein the heavy chain variable domain comprises SEQ ID NO: 13 or SEQ ID NO 15, and a light chain variable domain, wherein the light chain variable domain comprises SEQ ID NO: 14 or SEQ ID NO: 16.

Accordingly, in one embodiment, an IL-25 binding molecule, designated AVM109, is provided that contains an antigen binding site comprising at least one immunoglobulin heavy chain (VH) variable domain that contains in its sequence the hypervariable regions CDR1, CDR2, and CDR3, wherein said CDR1 has the amino acid sequence of SEQ ID NO: 1 (SYWIE), said CDR2 has the amino acid sequence of SEQ ID NO: 2 (QILPGIGSTNYNEKFKG) and said CDR3 has the amino acid sequence of SEQ ID NO: 3 (GYGNYGDY); or their immediate CDR equivalents. In one aspect, an IL-25 binding molecule (such as, for example, AVM109) may also comprise at least one immunoglobulin light chain variable domain (V _L ) that contains in its sequence the CDR1', CDR2', and CDR3' hypervariable regions, wherein said CDR1' having the amino acid sequence of SEQ ID NO: 4 (RASESVDSYGNSFM), said CDR2' having the amino acid sequence of SEQ ID NO: 5 (RASNLES), and said CDR3' having the amino acid sequence of SEQ ID NO: 6 (QQSNEDPLT) or direct equivalents their CDR'.

In one aspect, an IL-25 binding molecule, designated AVM125, is also described, which contains an antigen-binding site containing at least one immunoglobulin heavy chain (VH) variable domain, which contains in its sequence the hypervariable regions CDR1, CDR2 and CDR3, and the specified CDR1 has the amino acid sequence of SEQ ID NO: 7 (TSGMGVG), said CDR2' has the amino acid sequence of SEQ ID NO: 8 (HIWWDDVKRYNPALKS) and said CDR3 has the amino acid sequence of SEQ ID NO: 9 (TLPHFFDY); or their immediate CDR equivalents. In one aspect, an IL-25 binding molecule (such as, for example, AVM125) may also comprise at least one immunoglobulin light chain variable domain (V _L ) that contains in its sequence the CDR1', CDR2', and CDR3' hypervariable regions, wherein said CDR1' has the amino acid sequence of SEQ ID NO: 10 (SASSSVSYMY), said CDR2' has the amino acid sequence of SEQ ID NO: 11 (RTSNLAS) and said CDR3' has the amino acid sequence of SEQ ID NO: 12 (KQYHSYPPTWT) or direct CDR' equivalents thereof .

As noted above, the IL-25 binding molecules described may also be antibody fragments. As used herein, the term antibody or fragments thereof encompasses chimeric antibodies and hybrid antibodies with bispecificity or multispecificity for an antigen or epitope, as well as fragments such as F(ab') ₂ , Fab', Fab, Fv, sFv, dAb, region fragments complementarity determining (CDR), single chain antibodies (scFv), bivalent single chain antibodies, diabodies, tribodies, tetrabodies, polypeptides that contain at least an immunoglobulin fragment that is configured to provide specific antigen binding to the polypeptide, etc., including hybrid pieces. Thus, antibody fragments are provided that retain the ability to bind their specific antigens. For example, antibody fragments that retain IL-25 binding activity are also included within the term antibody or fragment thereof. Such antibodies and fragments can be prepared using techniques known to those skilled in the art and can be screened for specificity and activity in accordance with the methods set forth in the examples, as well as in general methods for producing antibodies and screening antibodies for specificity and activity ( see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, (1988)).

The term antibody or fragments thereof also includes conjugates of antibody fragments and antigen-binding proteins (single-chain antibodies). Conjugated antibodies or fragments refer to antibodies or fragments that are operably linked or otherwise physically or functionally associated with an effector functional group or label, such as, but not limited to, a toxic compound, a radioactive compound, a fluorescent compound, a liposome, or an enzyme, as described, for example, in US patent No. 4704692, the contents of which are incorporated herein by reference.

Regardless of the structure, the antigen-binding fragments described herein can bind to the same antigen that is recognized by intact immunoglobulin. The antigen-binding fragment may comprise a peptide or polypeptide containing an amino acid sequence of at least 2 contiguous amino acid residues, at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues,

- 6 042754 at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 35 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of the binding molecule.

Such fragments, whether or not linked to other sequences, may also include insertions, deletions, substitutions, or other selective modifications of certain regions or specific amino acid residues, provided that the activity of the antibody or antibody fragment is not substantially altered or worse than unmodified antibody or antibody fragment. Such modifications may confer some additional properties such as the removal/addition of amino acids capable of forming a disulfide bond, increased biological longevity, altered secretory characteristics, and so on. In either case, the antibody or antibody fragment must have a bioactive property, such as specifically binding to its recognized antigen. Functional or active regions of an antibody or antibody fragment can be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods will be apparent to one of skill in the art and may include site-directed mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, M.J. Curr. Opin. Biotechnol. 3:348-354, 1992).

As used herein, the term functional variant refers to a binding molecule that contains a nucleotide and/or amino acid sequence that has been changed by one or more nucleotides and/or amino acids from the nucleotide and/or amino acid sequences of the original binding molecule, and which is still performed. with the ability to compete for binding partner binding, for example, IL-25 (including IL-25), with the original binding molecule. In other words, modifications to the amino acid and/or nucleotide sequence of the original binding molecule do not significantly affect the binding characteristics - or do not change their binding molecule, which is encoded by the nucleotide sequence or contains the amino acid sequence, ie. the binding molecule is still configured to recognize and bind to its target. A functional variant may have conservative sequence modifications, including nucleotide and amino acid substitutions, additions and deletions. Such modifications may be introduced using standard techniques known to those skilled in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and may include natural as well as synthetic nucleotides and amino acids.

As described herein, binding molecules, antibodies, fragments, and variants are configured to specifically bind to an antigenic target, such as, for example, IL-25. As used herein, the term specific binding in relation to the interaction of a binding molecule, such as an antibody, and its binding partner, such as an antibody, means that the interaction is dependent on the presence of a specific structure, such as an antigenic determinant or epitope, on the binding partner. In other words, the antibody preferentially binds or recognizes a binding partner even if the binding partner is present in a mixture of other molecules. The binding may be mediated by covalent or non-covalent interactions, or a combination of both. In other words, the term specific binding means immunospecific binding to a particular antigen or fragment thereof, but not immunospecific binding to other antigens. A binding molecule that is immunospecifically bound to an antigen may be bound to other peptides or polypeptides of lower affinity, as determined by, for example, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), BIAcore, or other assay techniques known to those skilled in the art. in area. Binding molecules or fragments thereof that are immunospecifically bound to an antigen may cross-react with related antigens. Binding molecules or fragments thereof which are immunospecifically bound to an antigen preferably do not cross-react with other antigens.

In one aspect, the described antibodies or binding molecules described herein may be human antibodies or human binding molecules. The term human as applied to binding molecules as defined in this

- 7 042754 the present document refers to molecules that are either derived directly from a human or derived from a human sequence. If the binding molecule is derived from a human or derived from a human sequence and then modified, it should still be considered human, and this approach is used throughout the above description. In other words, the term human when applied to binding molecules is intended to include binding molecules with variable and constant regions derived from human germline immunoglobulin sequences based on variable or constant regions either not present in humans or human lymphocytes or present in a modified form. . Thus, human binding molecules may include amino acid residues not encoded by human germline immunoglobulin sequences, contain substitutions and/or deletions (e.g., mutations introduced, for example, by in vitro random or site-specific mutagenesis or by in vitro somatic mutation vivo). As used herein, the term based refers to a situation where a nucleic acid sequence can be exactly copied from a template or with minor mutations, such as using error-prone PCR methods or synthetically, exactly according to the template or with minor modifications. For the purposes of this document, semi-synthetic molecules based on human sequences are also considered to be human.

If necessary, antibodies are produced in organisms of other species and humanized for administration to humans. Humanized forms of non-human (eg, murine) antibodies may be chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab', F(ab') ₂ or other antibody antigen-binding subsequences) that contain the minimum sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from the complementarity determining region (CDR) of the recipient are replaced by residues from the complementarity determining region of a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired specificity, affinity and ability. In some cases, human immunoglobulin Fv backbone residues are replaced with corresponding non-human residues. Humanized antibodies may also contain residues that are not present in either the recipient antibody or the imported CDR or framework sequences. Typically, a humanized antibody will contain substantially all of at least one, and usually two, variable domains in which all or substantially all of the CDRs correspond to those in a non-human immunoglobulin, and all or substantially all of the framework the regions correspond to similar human immunoglobulin consensus sequences. A humanized antibody will also optimally contain at least a portion of an immunoglobulin (Fc) constant region, typically an immunoglobulin that is human (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988) and Presta, Curr Op Struct Biol 2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known to those skilled in the art. Typically, a humanized antibody contains one or more amino acid residues introduced into its sequence from a non-human source. Such non-human amino acid residues are often referred to as imported residues, which are usually derived from an imported variable region. Humanization can essentially be carried out in accordance with the method proposed by Winter et al. (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by using rodent CDRs or CDR sequences in place of the corresponding human antibody sequences. Accordingly, such humanized antibodies are chimeric antibodies (US Pat. No. 4,816,567) in which substantially less than an intact human variable domain is replaced by the corresponding sequence of a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced with residues from analogous sites in rodent antibodies.

The choice of both heavy and light chain human variable domains to be used in the preparation of humanized antibodies is very important to reduce antigenicity. According to the best fit method, the sequence of the variable domain of the rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence closest to that of the rodent is adopted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993) and Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a particular subset of light or heavy chains. The same scaffold can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992);

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Presta et al., J. Immunol., 151:2623 (1993)).

In some aspects, it may be important that the antibodies be humanized while retaining high antigen affinity and other beneficial biological properties. To achieve this goal, and in accordance with the preferred method, humanized antibodies are obtained by analyzing the original sequences and various conceptual humanized products using three-dimensional models of the original and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are well known to those skilled in the art. Computer programs exist that illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. The study of these images allows us to analyze the likely role of the 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 the consensus and imported sequence so as to obtain the desired antibody characteristic, such as increased affinity for the target antigen(s). As a rule, CDR residues are directly and essentially related to the effect on antigen binding (see WO 94/04679, published March 3, 1994).

In one specific aspect, a humanized version of AVM109, designated AVM109.2, contains some of the heavy and light chain CDRs of AVM109 and retains specific binding and neutralization of human, mouse, and monkey IL-25. The heavy and light chain variable regions are shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

Described are hybridoma cells that produce a monoclonal antibody. As used herein, the term monoclonal antibody refers to an antibody derived from a substantially homogeneous population of antibodies, ie. individual antibodies containing a population are identical, except for possible mutations of natural origin, which may be present in small quantities. As used herein, monoclonal antibodies generally include chimeric antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, with the remainder of the chain( i) identical or homologous to the corresponding sequences in antibodies derived from other species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that they exhibit the desired activity (see US patent No. 4816567 and Morrison et al., Proc. Natl Acad Sci USA 81:6851-6855 (1984)).

Monoclonal antibodies can be obtained by hybridoma methods such as those described in Kohler and Milstein, Nature, 256:495 (1975) or Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York (1988). In the hybridoma method, a mouse or other suitable host animal is typically immunized with an immunizing agent to generate lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. In an alternative embodiment, the lymphocytes may be immunized in vitro. The immunizing agent preferably contains IL-25. The generation of monoclonal antibodies has traditionally depended on the availability of purified protein or peptides for use as an immunogen. Recently, DNA-based immunization has shown promise as a way to induce strong immune responses and produce monoclonal antibodies. Within this approach, DNA immunizations can be used, wherein the DNA encoding the portion of IL-25 expressed as a fusion protein with human IgG1 is administered to the host animal according to methods known to those skilled in the art (e.g., Kilpatrick K. E., et al. Kilpatrick K. E. et al., High-affinity monoclonal antibodies to PED /PEA-15 generated using 5 μg of DNA Hybridoma 2000 Aug;19(4):297-302, which are incorporated herein in their entirety by reference to antibody production methods) and as described in the examples.

An alternative approach to immunizations using either purified protein or DNA is to use an antigen expressed in baculovirus. The advantages of such a system include ease of generation, high levels of expression, and post-translational modifications that are highly similar compared to those observed in mammalian systems. The use of such a system includes the expression of the domains of antibodies to IL-25 in the form of hybrid proteins. The antigen is generated by inserting a gene fragment into reading frame between the signal sequence and the mature protein domain of the nucleotide sequence of an anti-IL-25 antibody. This leads to the formation of foreign proteins on the surface of the virion. This method provides immunization with complete virus, eliminating the need to purify the target antigens.

Generally, if cells of human origin are needed, peripheral blood lymphocytes (PBLs) are used in methods for producing monoclonal antibodies, and if non-human mammalian sources are needed, spleen cells or lymph node cells are used. The lymphocytes are then fused with the immortalized cell line using

- 9 042754 a suitable fusion-inducing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), p. 59-103). Immortalized cell lines are typically transformed mammalian cells, including myeloma cells, from rodent, bovine, equine, or human origin. Typically, rat or mouse myeloma cell lines are used. The hybridoma cells can be cultured in a suitable nutrient medium, which preferably contains one or more substances that inhibit the growth or survival of non-fused, immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium will typically include hypoxanthine, aminopterine, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells. Preferred immortalized cell lines are those that provide efficient fusion, maintain a stable high level of antibody expression by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, CA, USA and the American Type Culture Collection, Rockville, MD, USA. The use of human myeloma and mouse-human heteromyeloma cell lines has also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63). The culture medium in which the hybridoma cells are cultured can subsequently be assessed for the presence of monoclonal antibodies directed against IL-25. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known to those skilled in the art and are described further in the examples below or in Harlow and Lane Antibodies, A Laboratory Manual Cold Spring Harbor Publications, New York (1988).

Once the desired hybridoma cells have been identified, clones can be subcloned by limited serial dilution techniques or by fluorescence activated cell sorting (FACS) cytometry procedures and grown using standard methods. Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. In an alternative embodiment, the hybridoma cells can be grown in vivo as an ascitic tumor in a mammal.

Monoclonal antibodies secreted by subclones can be isolated or purified from culture media or ascitic fluid by standard immunoglobulin purification methods such as, for example, protein A sepharose, protein G, hydroxyapatite, gel electrophoresis, dialysis, or affinity chromatography.

As used herein, the term isolated in relation to binding molecules refers to binding molecules that are substantially free of other proteins or polypeptides, especially free of other binding molecules having differing antigen specificity characteristics, and also substantially free of other cellular or tissue materials. and/or chemical precursors or other chemical compounds. For example, if the binding molecules are obtained using recombinant technologies, they preferably contain essentially no culture medium, and if the binding molecules are obtained by chemical synthesis, they preferably contain essentially no chemical precursors or other chemicals, i. they are separated from chemical precursors or other chemicals that were involved in protein synthesis. Substantially free preferably means that the binding molecule will typically contain about 50%, 60%, 70%, 80%, or 90% w/w of the sample, more typically about 95%, and preferably more than 99% pure.

Monoclonal antibodies can also be generated by recombinant DNA techniques such as those described in US Pat. No. 4,816,567. DNA encoding monoclonal antibodies can be readily isolated and sequenced by standard techniques (for example, oligonucleotide probes that are capable of specifically binding to genes coding heavy and light chains of mouse antibodies). Hybridoma cells act as the preferred source of such DNA. Once isolated, the DNA can be introduced into expression vectors which are then transfected into host cells such as monkey COS cells, Chinese Hamster Ovary (CHO) cells, plasmacytoma cells, or myeloma cells that would otherwise not produce immunoglobulin protein in order to achieve synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by replacing the constant domains of the human heavy and light chain coding sequence with mouse homologous sequences (US Pat. part. If necessary, such a non-immunoglobulin polypeptide is used to replace the constant domains of an antibody or the variable domains of one antigen-binding site of an antibody to create a chimeric bivalent antibody containing one antigen-binding site having specificity for IL-25 (including IL-25) and another antigen-binding site having specificity for another antigen.

In vitro methods are also suitable for the production of monovalent antibodies. Cleavage of antibodies to obtain their fragments, in particular fragments of Fab, can be achieved by using standard techniques known to experts in the field. For example, digestion can be carried out using papain. Examples of digestion using papain are described in WO 94/29348, published December 22, 1994, US Pat. No. 4,342,566 and Harlow and Lane Antibodies, A Laboratory Manual Cold Spring Harbor Publications, New York, (1988). Cleavage of antibodies with papain typically results in two identical antigen-binding fragments, termed Fab fragments, each including one antigen-binding site and a residual Fc fragment. Treatment with pepsin results in the formation of a fragment, called the F(ab') ₂ fragment, which includes two antigen-binding sites and is still capable of antigen cross-linking.

Fab fragments resulting from antibody digestion also contain light chain constant domains and a first heavy chain constant domain. Fab' fragments differ from Fab fragments by the addition of several residues at the carboxyl terminus of the heavy chain domain, including one or more cysteines from the antibody hinge region. The F(ab) ₂ fragment is a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region. In this document, the designation Fab'-SH is used for Fab', in which the cysteine residue(s) of the constant domains contain a free thiol group. The antibody fragments were originally generated as a pair of Fab' fragments with hinged cysteines in between. Other forms of chemical bonding of antibody fragments are also known.

In an alternative embodiment, the disclosed antibodies can be generated using transgenic animals (eg, mice) that, when immunized, are able to produce the full complement of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been shown that a homozygous deletion in the antibody heavy chain junction region gene (J(H)) in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene template into such germline mutant mice upon antigen challenge will result in the production of human antibodies (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993 ); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993)). Human antibodies can also be produced using phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). In addition, the methods proposed by Cote et al. and Boerner et al., for the production of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1): 86-95 (1991)).

In addition, an isolated immunogenically specific paratope or antibody fragment is provided. A specific immunogenic epitope of an antibody can be isolated from a whole antibody by chemical or mechanical disruption of the molecule. The fragments thus obtained were tested to determine their immunogenicity and specificity by means of the methods presented herein. If necessary, immunoreactive paratopes can be synthesized directly. An immunoreactive fragment is defined as an amino acid sequence of at least about two to five consecutive amino acids derived from the amino acid sequence of an antibody.

One method of making proteins containing antibodies involves linking two or more peptides or polypeptides together using protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using the chemistry of Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonyl). (Applied Biosystems, Inc., Foster City, California). One skilled in the art will appreciate that a peptide or polypeptide corresponding to an antibody, for example, can be synthesized using standard chemical reactions. For example, a peptide or polypeptide can be synthesized without cleavage from its synthesis resin, wherein another antibody fragment can be synthesized and then cleaved from the resin, thereby providing access to a terminal group that is operably blocked on the other fragment. Using peptide condensation reactions, these two fragments can be covalently linked via a peptide bond at their carboxyl and amine end groups, respectively, to form an antibody or fragment thereof. (Grant G.A. (1992), Synthetic Peptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky M. and Trost B., Ed. (1993), Principles of Peptide Synthesis. Springer-Verlag Inc., NY In an alternative embodiment, the peptide or polypeptide is independently synthesized in vivo as described

- 11 042754 above. Once isolated, such independent peptides or polypeptides can be linked to form an antibody or fragment thereof by analogous peptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segments allows relatively short peptide fragments to be linked to produce large peptide fragments, polypeptides, or complete protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)). In an alternative embodiment, native chemical ligation of synthetic peptides can be used to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of two chemical reaction steps (Dawson et al., Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)). The first step is the chemoselective reaction of an unprotected synthetic alpha-thioester peptide with another unprotected segment of the peptide containing an amino-terminal Cys residue to produce a thioether-linked intermediate as the starting covalent product. Without changes in the reaction conditions, such an intermediate enters into a spontaneous, fast intramolecular reaction with the formation of a native peptide bond at the ligation site. The use of such a native chemical ligation method for the complete synthesis of a protein molecule has been demonstrated in the production of human interleukin-8 (IL-8) (Baggiolini M. et al. (1992), FEBS Lett. 307:97-101; Clark-Lewis I. et al., J. Biol. Chem., 269:16075 (1994); Clark-Lewis, I. et al., Biochemistry, 30:3128 (1991); Rajarathnam K. et al., Biochemistry, 33:6623-30 ( 1994)).

In an alternative embodiment, the unprotected peptide segments are chemically linked where the bond formed between the peptide segments by chemical ligation is not a natural (peptide) bond (Schnolzer, M. et al., Science, 256:221 (1992)). This technique has been used to synthesize protein domain analogues as well as large quantities of relatively pure proteins with full biological activity (deLisle Milton R.C. et al., Techniques in Protein Chemistry IV. Academic Press, New York, p. 257-267 (1992)).

In addition, antibody fragments having biological activity are described. The polypeptide fragments may be recombinant proteins obtained by cloning polypeptide-encoding nucleic acids in an expression system capable of producing polypeptide fragments thereof, such as an adenovirus or baculovirus expression system. For example, it is possible to determine the active domain of an antibody from a specific hybridoma, which can cause a biological effect associated with the interaction of the antibody with IL-25. For example, amino acids that do not appear to contribute to binding activity or specificity can be removed without loss of associated activity. For example, in various embodiments, amino or carboxyl terminal amino acids are sequentially removed from a native or modified non-immunoglobulin molecule or immunoglobulin molecule and the corresponding activity is assayed by one of the many available assay methods. In another example, an antibody fragment includes a modified antibody in which at least one amino acid has been replaced by a naturally occurring amino acid at a particular position, and a portion of either the amino terminal or carboxyl terminal amino acids or the interior of the antibody has been replaced by a polypeptide fragment or other functional group, such as biotin, which can simplify the purification of the modified antibody. For example, a modified antibody can be fused to a maltose-binding protein using either peptide chemistry or cloning of the corresponding nucleic acids encoding two polypeptide fragments into a single expression vector such that expression of the coding region results in a fusion polypeptide. The fusion polypeptide can be affinity purified by passing through an amylose affinity column and then the modified antibody receptor can be separated from the maltose binding region by cleavage of the fusion polypeptide using the specific protease factor Xa. (See, for example, New England Biolabs Product Catalog, 1996, p. 164.). Similar purification procedures also exist for the isolation of fusion proteins from eukaryotic cells.

Such fragments, whether or not linked to other sequences, include insertions, deletions, substitutions, or other selective modifications of certain regions or specific amino acid residues, provided that the activity of the fragment is not significantly altered or impaired from that of the unmodified antibody or antibody fragment. Such modifications may provide some additional properties, such as the removal or addition of amino acids capable of forming a disulfide bond to increase its biological durability, to change its secretory characteristics, etc. In either case, the fragment must have a biological property such as binding activity, regulation of binding in the binding domain, and so on. Functional or active regions of an antibody can be determined by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods will be apparent to one of skill in the art and may include site-directed mutagenesis of the nucleic acid encoding the antigen. (Zoller M.J. et al., Nucl. Acids Res. 10:6487-500 (1982).

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Various immunoassay formats can be used to select antibodies that selectively bind to a particular protein, variant, or fragment. For example, solid-phase ELISA variants are routinely used to select antibodies having selective immunoreactivity for a protein, protein variant, or fragment thereof. See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988), which describes immunoassay formats and conditions that can be used to determine selective binding. The binding affinity of a monoclonal antibody can, for example, be determined using the Scatchard assay described in Munson and Pollard, Anal. Biochem. 107:220 (1980).

In addition, an antibody reagent kit is provided, containing containers with a monoclonal antibody or fragments thereof, and one or more reagents for detecting binding of an anti-IL-25 antibody or fragment thereof to an IL-25 molecule. Reagents may include, for example, fluorescent labels, enzymatic labels, or other labels. The reagents may also include secondary or tertiary antibodies or reagents for enzymatic reactions, the enzymatic reactions resulting in a product that can be visualized.

As noted throughout the text of this document, in one aspect of this document, isolated IL-25 binding molecules are described that contain a heavy chain variable domain containing one or more CDRs as defined in SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: 3.

In addition, IL-25 binding molecules from any of the preceding aspects are described, wherein the CDR is SEQ ID NO: 1, SEQ ID NO: 2, and/or SEQ ID NO: 3.

In one aspect of this document, isolated IL-25 binding molecules from any preceding aspect are described, wherein the heavy chain variable domain comprises SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 1 and SEQ ID NO: 3; SEQ ID NO: 2 and SEQ ID NO: 3 and/or SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

In one aspect of this document, isolated IL-25 binding molecules from any preceding aspect are described wherein SEQ ID NO: 1 and SEQ ID NO: 2 are separated by 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

In one aspect of this document, isolated IL-25 binding molecules from any preceding aspect are described wherein SEQ ID NO: 2 and SEQ ID NO: 3 are separated by 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39 or 40 amino acids.

In one aspect, the present document describes isolated IL-25 binding molecules from any of the preceding aspects, wherein the heavy chain variable domain comprises SEQ ID NO: 13.

In one aspect of this document, isolated IL-25 binding molecules from any preceding aspect are described, further comprising a light chain variable domain containing one or more CDRs as defined in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 .

In one aspect of this document, isolated IL-25 binding molecules from any of the preceding aspects are described, wherein the light chain variable domain comprises SEQ ID NO: 4 and SEQ ID NO: 5; SEQ ID NO: 4 and SEQ ID NO: 6; SEQ ID NO: 5 and SEQ ID NO: 6 and/or SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

In one aspect of this document, isolated IL-25 binding molecules from any preceding aspect are described wherein SEQ ID NO: 4 and SEQ ID NO: 5 are separated by 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

In one aspect of this document, isolated IL-25 binding molecules from any preceding aspect are described wherein SEQ ID NO: 5 and SEQ ID NO: 6 are separated by 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39 or 40 amino acids.

In one aspect, the present document describes isolated IL-25 binding molecules from any of the preceding aspects, wherein the light chain variable domain comprises SEQ ID NO: 14.

In one aspect, the present document describes isolated IL-25 binding molecules from any preceding aspect comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises SEQ ID NO: 13 and the light chain variable domain comprises SEQ ID NO: 14.

In one aspect, the present document describes isolated IL-25 binding molecules containing a light chain variable domain containing one or more CDRs as defined in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

In one aspect of this document, isolated IL-25 binding molecules from any preceding aspect are described, wherein the light chain variable domain comprises SEQ ID NO: 4 and SEQ ID NO: 5; SEQ ID NO: 4 and SEQ ID NO: 6; SEQ ID NO: 5 and SEQ ID NO: 6 and/or SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

In one aspect of this document, isolated IL-25 binding molecules from any preceding aspect are described wherein SEQ ID NO: 4 and SEQ ID NO: 5 are separated by 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

In one aspect of this document, isolated IL-25 binding molecules from any preceding aspect are described wherein SEQ ID NO: 5 and SEQ ID NO: 6 are separated by 25, 26, 27, 28, 29, 30,

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31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids.

In one aspect, the present document describes isolated IL-25 binding molecules from any of the preceding aspects, wherein the light chain variable domain comprises SEQ ID NO: 14.

In one aspect, the present document describes isolated IL-25 binding molecules containing a heavy chain variable domain containing one or more CDRs as defined in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

In one aspect, the present document describes isolated IL-25 binding molecules from any of the preceding aspects, wherein the heavy chain variable domain comprises SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 7 and SEQ ID NO: 9; SEQ ID NO: 8 and SEQ ID NO: 9 and/or SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

In one aspect of this document, isolated IL-25 binding molecules from any preceding aspect are described wherein SEQ ID NO: 7 and SEQ ID NO: 8 are separated by 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

In one aspect of this document, isolated IL-25 binding molecules from any preceding aspect are described wherein SEQ ID NO: 8 and SEQ ID NO: 9 are separated by 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39 or 40 amino acids.

In one aspect, the present document describes isolated IL-25 binding molecules from any of the preceding aspects, wherein the heavy chain variable domain comprises SEQ ID NO: 15.

In one aspect of this document, isolated IL-25 binding molecules from any preceding aspect are described, further comprising a light chain variable domain containing one or more CDRs as defined in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 .

In one aspect of this document, isolated IL-25 binding molecules from any of the preceding aspects are described, wherein the light chain variable domain comprises SEQ ID NO: 10 and SEQ ID NO: 11; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID NO: 12 and/or SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

In one aspect of this document, isolated IL-25 binding molecules from any preceding aspect are described wherein SEQ ID NO: 10 and SEQ ID NO: 11 are separated by 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

In one aspect of this document, isolated IL-25 binding molecules from any preceding aspect are described wherein SEQ ID NO: 11 and SEQ ID NO: 12 are separated by 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39 or 40 amino acids.

In one aspect, the present document describes isolated IL-25 binding molecules from any of the preceding aspects, wherein the light chain variable domain comprises SEQ ID NO: 16.

In one aspect, the present document describes isolated IL-25 binding molecules from any preceding aspect comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises SEQ ID NO: 15 and the light chain variable domain comprises SEQ ID NO: 16.

In one aspect, the present document describes isolated IL-25 binding molecules containing a light chain variable domain containing one or more CDRs as defined in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

In one aspect of this document, isolated IL-25 binding molecules from any preceding aspect are described, wherein the light chain variable domain comprises SEQ ID NO: 10 and SEQ ID NO: 11; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID NO: 12; and/or SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

In one aspect of this document, isolated IL-25 binding molecules from any preceding aspect are described wherein SEQ ID NO: 10 and SEQ ID NO: 11 are separated by 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

In one aspect of this document, isolated IL-25 binding molecules from any preceding aspect are described wherein SEQ ID NO: 11 and SEQ ID NO: 12 are separated by 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39 or 40 amino acids.

In one aspect, the present document describes isolated IL-25 binding molecules from any of the preceding aspects, wherein the light chain variable domain comprises SEQ ID NO: 16.

In one aspect, the present document describes isolated IL-25 binding molecules from any of the preceding aspects, wherein the heavy chain variable domain comprises SEQ ID NO: 17.

In one aspect, the present document describes isolated IL-25 binding molecules from any of the preceding aspects, wherein the light chain variable domain comprises SEQ ID NO: 18.

In one aspect, this document describes a method for treating, preventing and/or inhibiting airway inflammation due to rhinovirus infection, rheumatoid arthritis, osteoarthritis, bone erosion, intraperitoneal abscesses and adhesions, inflammatory bowel disease, allograft rejection, psoriasis, certain forms of cancer, angiogenesis, atherosclerosis. , cystic fibrosis, and multiple sclerosis, comprising administering a therapeutic amount of any of the IL-25 binding molecules from any of the preceding aspects.

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1. Homology/identity.

It should be understood that one way of determining any known variants and derivatives, or those that may appear, of the structure of the genes and proteins described herein, involves the definition of such variants and derivatives according to homology with specific known sequences. For example, SEQ ID NO: 13 defines the specific sequence of the IL-25 heavy chain variable domain. In particular, variants of these and other genes and proteins described herein are described, which may have at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 , 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology with the indicated sequence. Those skilled in the art will appreciate methods for determining the homology of two proteins or nucleic acids, such as genes. For example, homology can be determined after aligning two sequences such that the homology is at the highest level.

Another way to determine homology can be done using published algorithms. Optimal alignment of sequences for comparison can be performed using the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by means of the alignment homology algorithm from Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), using the similarity search method from Pearson and Lipman, Proc. Natl. Acad. sci. U.S.A. 85:2444 (1988), using computer implementations of such algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or based on browsing.

It should be understood that, as a rule, you can use any of the methods and that in some circumstances the results of such different methods may differ, but the person skilled in the art will understand that if the identity is established using at least one of these methods, it will be considered that the sequences have the claimed identity and will be described herein.

For example, as used herein, a sequence labeled as having a certain percentage of homology with another sequence refers to sequences that share the specified homology as calculated using any one or more of the calculation methods described above. For example, the first sequence has 80% homology according to this document with the second sequence if the first sequence is calculated to have 80% homology to the second sequence using the Zuker calculation method, even if the first sequence does not have 80% homology to the second sequence according to the results of calculations using any other calculation methods. In another example, the first sequence has 80% homology according to this document with the second sequence if the first sequence is calculated to have 80% homology with the second sequence using the Zuker calculation method and the Pearson and Lipman calculation method, even if the first the sequence does not have 80% homology with the second sequence as calculated using the calculation method from Smith and Waterman, the calculation method from Needleman and Wunsch, the calculation methods from Jaeger, or any of the other calculation methods. In another example, the first sequence has 80% homology herein with the second sequence if the first sequence is calculated to have 80% homology with the second sequence using each of the calculation methods (although in practice different calculation methods will often result in different percentages of calculated homology).

2. Peptides.

a) Protein variants.

As discussed herein, there are many variations of the IL-25 binding molecules, and IL-25 CDR binding, and heavy and light chain variable regions described herein, which are known and discussed herein. In addition to the known functional strain variants, there are derivatives of IL-25 binding molecules, and IL-25 binding CDRs, and heavy and light chain variable regions that also function in the described methods and compositions. Protein variants and derivatives are well known to those skilled in the art and may include modifications to the amino acid sequence. For example, amino acid sequence modifications typically fall into one or more of three classes: substitution, insertion, or deletion variants.

As used herein, the term insert refers to a change in amino acid or nucleotide sequence resulting in the addition of one or more amino acid or nucleotide residues, respectively, from the original molecule, often of natural origin. Insertions include fusions of amine and/or carboxyl termini, as well as insertions of single or multiple amino acid residues within a sequence. The inserts will typically be smaller inserts than those obtained from amine or carboxyl end fusions, for example on the order of one to four

- 15 042754 residues. Immunogenic derivatives of fusion proteins, such as those described in the examples, are made by fusion of a polypeptide large enough to render the target sequence immunogenic, by in vitro cross-linking, or by recombinant cell culture transformed with DNA encoding the fusion protein. Deletions are characterized by the removal of one or more amino acid residues from a protein sequence. Typically, no more than about 2 to 6 residues are removed from any one site in a protein molecule. Such variants are usually generated by site-directed mutagenesis of nucleotides in the DNA encoding the protein, thereby obtaining the DNA encoding the variant, followed by expression of the DNA in recombinant cell culture. Techniques for performing substitution mutations at predetermined sites in DNA having a known sequence are well known, such as M13 primer mutagenesis and PCR polymerase chain reaction mutagenesis. Amino acid substitutions are usually made up of single residues, but may appear in several different positions at the same time; insertions will typically be on the order of about 1 to 10 amino acid residues; and deletions will range from approximately 1 to 30 residues. Deletions or insertions are preferably carried out in adjacent pairs, ie. deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof can be combined to obtain the desired construct. Mutations should not move the sequence out of frame and preferably will not form regions of complementarity that could lead to the formation of an mRNA secondary structure. Substitutive variants are those in which at least one residue has been removed and another residue has been introduced in its place. Such replacements are usually carried out in accordance with the table. 2 and are called conservative substitutions.

Table 1

Abbreviations for amino acid names

Amino Acid Abbreviations

Alanine Ala A Alloisoleucine APe Arginine Arg R Asparagine Asn N Aspartic asp D acid Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine gly G Histidine His H Isoleucine lie I Leucine Leu L Lysine Lys TO Phenylalanine Phe F Proline Pro P Pyroglutamine pGlu acid Serene Ser S Threonine Thr T Tyrosine Tyr Y tryptophan trp w Valine Val V

table 2

Amino acid replacements

Examples of conservative substitutions of the original residues, the rest are known

specialists in areas. Ala Ser Arg Lys; Gln Asn gln; His asp Glu Cys Ser Gln asn, Lys Glu asp gly Pro His asn; Gln lie Leu; Val Leu Ile; Val Lys Arg; Gln Met Leu; lie Phe met; Leu; Tyr Ser Thr Thr Ser trp Tyr Tyr trp; Phe Val lie; Leu

- 16 042754

Significant changes in function or immunological identity are achieved by choosing substitutions that are less conservative than those shown in Table 1. 2, i.e. by selecting residues that differ even more in their effect on maintaining (a) the structure of the polypeptide backbone at the replacement site, such as in a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain . Conservative amino acid substitutions include those substitutions in which an amino acid residue is replaced by an amino acid residue with similar structural or chemical properties. Families of amino acid residues having similar side chains are known in the art. Such families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine , phenylalanine, tryptophan, histidine).

The substitutions that are generally expected to result in the greatest changes in the properties of proteins are those in which (a) a hydrophilic residue, such as seryl or threonyl, is replaced by or replaces a hydrophobic residue, such as leucyl, isoleucyl, phenylalanyl, valyl, or alanyl ; (b) the cysteine or proline is substituted for or replaces any other residue; (c) a residue having an electropositive side chain, such as lysyl, arginyl, or histidyl, is replaced by or substitutes for an electronegative residue, such as glutamyl or aspartyl, or (d) a residue having a bulky side chain, such as phenylalanine, is replaced by a residue having no side chain , for example glycine in this case, or replaces it; (e) an increase in the number of sulfonation and/or glycosylation sites.

The replacement of an amino acid residue with another with biologically and/or chemically similar properties is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be to replace one hydrophobic residue with another, or one polar residue with another. Such substitutions include combinations such as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variants of each explicitly described sequence are included in the mosaic polypeptides provided herein.

Substitution or deletion mutagenesis can be used to insert N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr) sites. In addition, deletions of cysteine or other labile residues may be desirable. Deletions or substitutions of potential sites of proteolysis, such as Arg, are carried out by removing one of the major residues or replacing one of them with glutaminyl or histidyl residues.

Some post-translational derivatives result from the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are often subject to post-translational deamidation to form the corresponding glutamyl and asparyl residues. In an alternative embodiment, these residues are deamidated in a slightly acidic environment. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of o-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 [1983]), acetylation of the N-terminal amine, and in some cases amidation of the C-terminal carboxyl.

It should be understood that one way to define variants and derivatives of the genes and proteins described herein is to define variants and derivatives based on homology/identity to specific known sequences, e.g. SEQ ID NOs: 13, 14, 15 and 16. In particular, variants are described. such and other proteins described herein having at least 70% or 75% or 80% or 85% or 90% or 95% homology with the specified sequence. Those skilled in the art will appreciate methods for determining the homology of two proteins. For example, homology can be determined after aligning two sequences such that the homology is at the highest level.

Another way to determine homology can be done using published algorithms. Optimal alignment of sequences for comparison can be performed using the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by means of the alignment homology algorithm from Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), using the similarity search method from Pearson and Lipman, Proc. Natl. Acad. sci. U.S.A. 85:2444 (1988), using computer implementations of such algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575, Science Dr., Madison, WI) or based on browsing .

The same types of homology can be established for nucleic acids, for example, using the algorithms described in Zuker, M. Science, 244:48-52, 1989, Jaeger et al., Proc. Natl. Acad. sci. USA, 86:7706-7710, 1989, Jaeger et al., Methods Enzymol. 183:281-306, 1989.

- 17 042754

It should be understood that descriptions of conservative mutations and homology can be combined in any combination, such as embodiments that have at least 70% homology with a particular sequence, and its variants are conservative mutations.

Since various proteins and protein sequences are discussed herein, it should be understood that nucleic acids that can encode such protein sequences are also described. This will include all degenerate sequences related to a particular protein sequence, i.e. all nucleic acids having a sequence encoding one specific protein sequence, as well as all nucleic acids, including degenerate nucleic acids, encoding the described variants and derivatives of protein sequences. Thus, while each specific nucleotide sequence is not necessarily described herein, it should be understood that all sequences are actually disclosed and described herein in light of the described protein sequence. In addition, for example, the described conservative derivative of several SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17 or 18, for example, the replacement of valine (V) with isoleucine (I). It should be understood that for such a mutation, all nucleotide sequences are also described that encode such a specific derivative of several SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17 or 18.

It should be understood that there are many amino acid and peptide analogues that can be included in the described compositions. For example, there are many D-amino acids or amino acids that have a different functional substituent than the amino acids shown in Table. 1 and 2. Opposite stereoisomers of naturally occurring peptides as well as stereoisomers of peptide analogues are described. Such amino acids can be easily incorporated into polypeptide chains by inserting data on the selected amino acid into tRNA molecules and generating genetic constructs that use, for example, amber codons to introduce the amino acid analog into the peptide chain in a site-specific manner.

Molecules can be produced that resemble peptides but are not bound by a natural peptide bond. For example, linkages for amino acids or amino acid analogs may include -CH ₂ NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis- and trans-), -COCH2-, -CH(OH) CH ₂ - and -CHH2SO- (these and other links can be found in Spatola, AF in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983) Spatola, AF, Vega Data (March 1983), vol. 1, Issue 3, Peptide Backbone Modification (general revies), Morley, Trends Pharm. Sci. (1980), pp. 463-468, Hudson D. et al. , Int. J. Pept. Res., 14:177-185 (1979) (-CHNH-, -CH2CH2-); Spatola et al., Life Sci. 38:12431249 (1986) (-CHH2-S); Hann J. Chem. Soc. Perkin Trans. I, 307-314 (1982) (-CH-CH-, cis and trans-; Almquist et al., J. Med. Chem. 23:1392-1398 (1980) ( -COCH2-); Jennings-White et al Tetrahedron Lett 23:2533 (1982) (-COCH2); Szelke et al., European appln., EP 45665 CA (1992); 97:39405 (1982) (-CH (OH)CH ₂ -); Holladay et al., Tetrahedron Lett., 24:4401-4404 (1983) (-C(OH)CH ₂ -); and Hruby Life Sci., 31:189-199 (1982 ) (-CH ₂ -S-), each of which is incorporated herein by reference. A particularly preferred non-peptide bond is -CH ₂ NH-. It should be understood that peptide analogs may have more than one atom between bond atoms, eg β-alanine, γ-aminobutyric acid, and the like.

Amino acid and peptide analogs often have more pronounced or desirable properties, such as higher process economics, greater chemical stability, improved pharmacological properties (half-life, uptake, potency, potency, etc.), altered specificity (e.g., broad spectrum of biological activity), reduced antigenicity, etc.

D-amino acids can be used to build more stable peptides because peptidases and the like do not recognize D-amino acids. Systematic substitution of one or more amino acids in the consensus sequence with a D-amino acid of the same type (eg, D-lysine instead of L-lysine) can be used to produce more stable peptides. Cysteine residues can be used to cyclize or link together two or more peptides. This approach can be convenient for anchoring peptides in certain conformations.

In one aspect, the described IL-25 binding molecules may further comprise a label. The labels used herein may include a fluorescent dye, one of a binding pair such as biotin/streptavidin, a metal (e.g., gold), a radioactive substituent, or an epitope label that is configured to specifically interact with a detectable molecule. , for example, with the formation of a colored substrate or fluorescence. Compounds suitable for introducing a detectable label into proteins include fluorescent dyes (also known herein as fluorochromes and fluorophores) and enzymes that react with colorimetric substrates (eg, horseradish peroxidase). The use of fluorescent dyes is generally preferred for the practice of the present invention since they can be detected in very small amounts.

Fluorophores are compounds or molecules that luminesce. Typically, fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength. Representative examples of fluorophores include, without limitation, 1,5-IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxynaphthofluorescein; 5-carboxytetramethylrhodamine (5-TAMRA); 5-hydroxytryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-carboxyrodamine 6G; 6-CR 6G; 6-JOE; 7-amino-4-methylcoumarin; 7-aminoactinomycin D (7-AAD); 7-hydroxy-4-1-methylcoumarin; 9-amino-6chloro-2-methoxyacridine (ASMA); ABQ; fuchsin sour; acridine orange; acridine red; acridine yellow; acriflavin; acriflavin for SITSA Felgen stain; aequorin (photoprotein); AFP - autofluorescent protein - (Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; alizarin complexone; alizarin red; allophycocyanin (APC); AMC, AMCA-S; aminomethyl coumarin (AMSA); AMSA-X; aminoactinomycin D; aminocoumarin; aniline blue; anthocylstearate; ARS-Su7; APTRA-VTS; APTS; astrozone brilliant red 4G; astrozone orange R; astrozone red 6V; astrozone yellow 7 GLL; atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; auramine; aurophosphine G; aurophosphine; VAO 9 (bis-aminophenyloxadiazole); BCECF (high pH); BCECF (low pH); berberine sulfate; beta-lactamase; BFP GFP with blue shift (Y66H); blue fluorescent protein; BFP/GFP FRET; biman; bis-benzemide; bis-benzimide (Hoechst); bis-BTC; blancophore FFG; blancophore SV; VOVO™-1; VOVO™-3; Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FLATP; Bodipy Fl-ceramide; Bodipy R6G SE; BodipyTMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TRATP; Bodipy TR-X SE; BO-PRO™-1; BO-PRO™-3; Brilliant Sulfoflavin FF; BTC BTC-5N; calcein; calcein blue; Calcium Crimson; Calcium Green; Calcium Green-1 Ca ²⁺ - dye; Calcium Green-2 Ca ²⁺ ; Calcium Green-5N Ca ²⁺ ; Calcium Green-C18 Ca ²⁺ ; Calcium Orange; calcofluor white; carboxy-X-rhodamine (5-ROX); cascading blue™; cascading yellow; catecholamine; CCF2 (GeneBlazer); CFDA; CFP (blue fluorescent protein); CFP/YFP FRET; chlorophyll; chromomycin A; chromomycin A; CL-NERF; CMFDA; celenterazine; celenterazine cp; coelenterazine f; celenterazine fcp; celenterazine h; celenterazine hcp; celenterazine ip; coelenterazine n; celenterazine O; coumarin phalloidin; C-phycocyanin; CPM I methylcoumarin; STS; STS formazan; Cy2™; Su3.18; Su3.5™; Cy3™; Su5.18; Su5.5™; Su5™; Su7™; blue GFP; cyclo-AMP fluorosensor (FiCRhR); dubsil; dansil; dansyl amine; dansil cadaverine; dansyl chloride; dansil DHPE; dansyl fluoride; DAPI; dapoxyl; dapoxyl 2; dapoxyl 3'-DCFDA; DCFH (dichlorodihydrofluorescein diacetate); DDAO; DHR (dihydrorhodamine-123); Di-4-ANEPPS; Di-8-ANEPPS (disproportionate); DiA (4-Di 16-ASP); dichlorodihydrofluorescein diacetate (DCFH); DiD lipophilic label; DiD (DilC18(5)); DIDS; dihydrorhodamine 123 (DHR); Dil(DilC18(3)); I dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC18(7)); DM-NERF (high pH); DNP; dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF97; eosin; erythrosin; erythrosin ITC; ethidium bromide; ethidium homodimer-1 (EthD-1); eucrisin; Eukolight; europium (111) chloride; EYFP; fast blue; FDA; Felgen (pararosanilin); FIF (formaldehyde-induced fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; fluorescein (FITC); fluorescein diacetate; Fluoro Emerald; Fluoro-Gold (hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF; genacryl brilliant red B; Genacryl Brilliant Yellow 10GF; genacryl pink 3G; genacryl yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); redshift GFP (rsGFP); GFP native with excitation outside UV (wtGFP); GFP native, UV-excited (wtGFP); GFPuv; glyoxalic acid; granular blue; hematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; hydroxycoumarin; hydroxystilbamidine (FluoroGold); hydroxytryptamine; Indo-1, high concentration of calcium; Indo-1, low calcium; indodicarbocyanine (DiD); indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JOPRO-1; LaserPro; laurodan; LDS 751 (DNA); LDS 751 (RNA); leucophore PAF; Leucophor SF; Leucophor W.S.; lyssamine rhodamine; lyssamine rhodamine B; calcein/ethidium homodimer; LOLO-1; LO-PRO-1; lucifer yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red (phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; Magnesium Green; Magnesium Orange; malachite green; Marina Blue; I Maxilon Brilliant Flavin 10 GFF; maxilon brilliant flavin 8 GFF; merocyanine; methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; mithramycin; monobrombimane; monobrombimane (mBBr-GSH); monochlorbiman; MPS (methyl green pyronine stilbene); NBD; NBD amine; Nile red; nitrobenzoxydiazole; norepinephrine; nuclear fast red; i nuclear yellow; nilosan brilliant flavin E8G; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; pacific blue; pararosaniline (Felgen);

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PBFI; PE Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-Texas Red (red 613); phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; phosphine 3R; photoresist; phycoerythrin B [PE]; phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; pontochrome black and blue; RORO-1; RORO-3; PO-PRO-1; PO-I PRO-3; primulin; procyon yellow; propidium iodide (PI); RuMRO; pyrene; pyronine; pyronin B; pyrosal brilliant flavin 7GF; QSY7; chiacrine mustard; resorufin; RH 414; Rhod-2; rhodamine; rhodamine 110; Rhodamine 123; rhodamine 5 GLD; rhodamine 6G; rhodamine B; rhodamine B 200; rhodamine B extra; rhodamine BB; Rhodamine BG; rhodamine green; rhodamine phallicidin; rhodamine: phalloidin; rhodamine red; Rhodamine WT; bengal pink; R-phycocyanin; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; sapphire GFP; SBFI; serotonin; sevron brilliant red 2B; sevron brilliant red 4G; sevron I brilliant red B; sevron orange; sevron yellow L; sgBFP™ (Super Intense BFP); sgGFP™ (Super Intensive GFP); SITS (primulin; stilbene isothiosulfonic acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; Spectrum Aqua; Spectrum Green; SpectrumOrange; Spectrum Red; SPQ (6-methoxy-N-(3-sulfopropyl)quinoline); stilbene; sulforodamine B and C; sulforodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21;

SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45;

SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83;

SYTO 84; SYTO 85; SYTOX blue; SYTOX green; SYTOX orange; tetracycline; tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ conjugate; thiadicarbocyanine (DiSC3); thiazine red R; thiazole orange; thioflavin 5; thioflavin S; thioflavin TON; thiolite; thiozolic orange; Tinopol CBS (calcofluor white); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; Tricolor (PE-Cy5); TRITC tetramethylrhodamine isothiocyanate; true blue; Tru Red; Ultralite; uranine B; uvitex SFC; wt GFP; WW 781; X-rhodamine; XRITC; xylene orange; Y66F; Y66H; Y66W; yellow GFP; YFP; YO-PRO-1; YO PRO 3; YOYO-1; YOYO-3; Sybr Green; thiazole orange (mutually chelating dyes); semiconductor nanoparticles such as quantum dots; or a framework fluorophore (which can be activated with light or other source of electromagnetic energy), or a combination thereof.

A modifying group that can be incorporated into or directly attached to any of the compounds described herein via halogenation. Examples of radionuclides that are used in the present embodiment include, without limitation, tritium, iodine-125, iodine-131, iodine-123, iodine-124, astatine-210, carbon-11, carbon-14, nitrogen-13 , fluoro-18. In another aspect, the radionuclide may be linked to a linking group or linked with a chelating group, which is then linked to the link directly or via a linker. Examples of radionuclides that are used in this aspect include, without limitation, Tc-99m, Re-186, Ga-68, Re-188, Y-90, Sm-153, Bi-212, Cu-67, Cu- 64 and Cu-62. Radiolabeling procedures similar to those described are used in daily practice in the radiopharmaceutical industry.

Radiolabeled compounds are used as imaging agents to diagnose a neurological disease (eg, neurodegenerative disease) or psychiatric disorder, or to monitor the progression or treatment of such a disease in a mammal (eg, a human). The radiolabeled compounds described herein can be readily used in combination with imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT).

Labeling can be done directly or indirectly. With direct labeling, the antibody to be detected (an antibody to the molecule of interest) or the molecule to be detected (a molecule that can be bound by an antibody to the molecule of interest) includes a label. The detection of a label indicates the presence of a detectable antibody or a detectable molecule, which in turn indicates the presence of the molecule of interest or an antibody to the molecule of interest, respectively. With indirect labeling, an additional molecule or functional group interacts with the immunocomplex or is formed at its site. For example, a signal-generating molecule or functional group, such as an enzyme, may be linked to or associated with a detectable antibody or detectable molecule. The signal generating molecule can then generate a detectable signal at the site of the immunocomplex. For example, the enzyme, when a suitable substrate is added, can form a visible or detectable product at the site of the immunocomplex. This form of indirect labeling is used in solid phase ELISA.

In another example of indirect labeling, an additional molecule (which may be called a binding agent) interacts with the immunocomplex, which can bind to the molecule in question or to an antibody (primary antibody) to the molecule in question, such as a secondary antibody to the primary antibody. The additional molecule may contain a label, or a signal-generating molecule, or a functional group. The additional molecule may be an antibody, which may thus be referred to as a secondary antibody. When linking secondary

A so-called sandwich of the first (or primary) antibody and the molecule in question can be formed with a primary antibody. Immune complexes can interact with a labeled, secondary antibody under conditions that effectively promote and for a certain time are sufficient for the formation of secondary immune complexes. Washing of the secondary immune complexes can then typically be performed to remove any non-specifically bound labeled secondary antibodies, allowing detection of the remaining label in the secondary immune complexes. The additional molecule may also be or include one of a pair of molecules or functional groups that can bind to each other, such as a biotin/avidin pair. In such an embodiment, the antibody to be detected or the molecule to be detected must include the other partner of the pair.

Other options for indirect labeling include detection of primary immune complexes in a two step approach. For example, a molecule (which may be referred to as a first binding agent), such as an antibody with binding affinity for the molecule in question or the corresponding antibody, can be used to form secondary immune complexes as described above. After washing, the secondary immune complexes can come into contact with another molecule (which can be called a second binding agent) with a binding affinity for the first binding agent again under conditions that effectively promote and for a certain time are sufficient for the formation of immune complexes (thus, forming tertiary immune complexes). The second binding agent may be coupled to a detectable label or signal-generating molecule or functional group, allowing detection of the thus formed tertiary immune complexes. Such a system can provide signal amplification.

3. Pharmaceutical carriers/delivery of pharmaceutical products.

As described above, the compositions may also be administered in vivo in a pharmaceutically acceptable carrier (also referred to herein as a pharmaceutically acceptable excipient). By pharmaceutically acceptable is meant a material that is not biologically or otherwise inert, i.e., such material can be administered to a subject with a nucleic acid or vector without any undesirable biological effects or inherently destructive interaction with any of the other components pharmaceutical composition in which it is contained. As is well known to those skilled in the art, such a carrier will naturally be chosen to minimize any degradation of the active ingredient and minimize any adverse side effects in the subject. Thus, in one aspect, the present document describes a pharmaceutical composition containing any of the IL-25 binding molecules described herein.

The compositions may be administered orally, parenterally (eg, intravenously), by intramuscular injection, by intraperitoneal injection, intradermally, extracorporeally, by topical application, and the like, including topical intranasal administration or administration by inhaler. As used herein, the term topical intranasal administration means delivery of compositions to the nose and nasal passages through one or both nostrils and may include delivery using a spray or droplet mechanism, or by aerosolization of a nucleic acid or a vector. Administration of the compositions via an inhaler may be via the nose or mouth via delivery using a nebulization mechanism or a droplet delivery mechanism. Delivery can also be made directly to any area of the respiratory system (eg the lungs) via intubation. The exact number of compositions required will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the allergic disorder requiring treatment, the particular nucleic acid or vector used, the route of administration, and the like. Therefore, it is not possible to specify the exact amount for each composition. However, the specific amount may be determined by one of ordinary skill in the art based on the ideas presented herein.

Parenteral administration of the composition, if used, is typically by injection. Injectable compositions may be prepared in standard forms, either as liquid solutions or suspensions, or as solid forms suitable for dissolution or suspension in a liquid prior to injection, or as emulsions. Recently, a revised approach for parenteral administration includes the use of a slow or sustained release system in such a way as to maintain a constant dosing. See, for example, US Pat. No. 3,610,795, which is incorporated herein by reference.

The materials may be in solution, suspension (eg, incorporated into microparticles, liposomes, or cells). They can be targeted to a specific cell type through the use of antibodies, receptors, or receptor ligands. The references below provide examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60: 275-281, (1989); Bagshawe, et

- 21 042754 al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol. 42:2062-2065 (1991)). Carrier media such as invisible and other antibody-conjugated liposomes (including lipid-mediated targeting for colon carcinoma), receptor-mediated targeting of DNA via specific cellular ligands, lymphocyte-regulated targeting to tumor, and highly specific targeting of therapeutic retroviruses to mouse glioma cells in vivo. The references below provide examples of the use of this technology to target delivery of specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220 (1989) and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187 ( 1992)). As a rule, receptors are involved in endocytosis processes, both constitutive and ligand-induced. Such receptors accumulate in clathrin-coated pits, enter the cell in clathrin-coated vesicles, pass through an acidified endosome where receptors are sorted, and then either regenerate to the cell surface, enter storage within the cell, or are degraded in lysosomes. Internalization processes perform a variety of functions such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic introduction of viruses and toxins, dissociation and degradation of the ligand, and regulation at the receptor level. Many receptors are characterized by more than one intracellular process depending on cell type, receptor concentration, ligand type, ligand valence, and ligand concentration. A review is available on the molecular and cellular mechanisms of receptor-mediated endocytosis (Brown and Greene, DNA and Cell Biology, 10:6, 399-409 (1991)).

a) Pharmaceutically acceptable carriers.

Compositions, including antibodies, can be used for therapy in combination with a pharmaceutically acceptable carrier.

Acceptable carriers and formulations thereof are described in Remington: The Science and Practice of Pharmacy ( ^19th ed.) ed. AR Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, a suitable amount of a pharmaceutically acceptable salt is included to make a formulation isotonic. Examples of pharmaceutically acceptable carriers include, without limitation, saline, Ringer's solution, and dextrose solution. The pH is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Additional carriers include sustained release formulations such as semi-permeable matrices of solid hydrophobic polymers containing the antibody, the matrices being in the form of molded articles such as films, liposomes, or microcapsules. It will be apparent to those skilled in the art that certain carriers may be more preferred depending on, for example, the route of administration and the concentration of the composition being administered.

Pharmaceutical carriers are known to those skilled in the art. Most often, they are standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and physiological pH buffers. The compositions may be administered intramuscularly or subcutaneously. Other compounds will be administered in accordance with standard procedures used by those skilled in the art.

In addition to the chosen molecule, pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surfactants, and the like. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anesthetics, and the like.

The pharmaceutical composition may be administered in a number of ways depending on whether topical or systemic treatment is required and the area being treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal), oral, inhalation or parenteral, for example in the form of intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavitaryly, or intradermally.

Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions.

Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including physiological and buffer media. Carrier media for parenteral administration include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or fixed oils. Vehicles for intravenous administration include fluid and nutrient loss compensators, electrolyte compensators (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobial, antioxidant, chelating agents and inert gases, and the like.

Topical formulations may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powdered or oily bases, thickeners, and the like may be necessary or desirable.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous vehicles, capsules, sachets or tablets. The presence of thickeners, flavors, diluents, emulsifiers, dispersing agents, or binders may be desirable.

Some compositions can potentially be administered in the form of a pharmaceutically acceptable acid or base addition salt formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, hydrothianoic acid, sulfuric acid and phosphoric acid, as well as organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid and fumaric acid, or by reacting with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, as well as organic bases such as mono-, di-, trialkyl- and arylamines and substituted ethanolamines.

4. Therapeutic uses and methods.

Effective dosages and schedules for administering the compositions can be determined empirically and such determinations can be made by those skilled in the art. Dosage ranges for administering the compositions are large enough to achieve the desired effect, which provides an effect on the symptoms of the disorder. The dose should not be so high as to cause adverse side effects such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary depending on the age, condition, sex and degree of disease of the patient, route of administration, or use of other drugs in the treatment regimen, and will be determined by one of ordinary skill in the art. With any contraindications, the dose can be adjusted individually by the attending physician. The dose may vary and may be administered in one or more doses per day for one or more days. Recommendations can be found in the literature for appropriate dosages for certain classes of pharmaceutical products. For example, guidance on selecting appropriate dosages for antibodies can be found in the literature on therapeutic applications of antibodies, for example, Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985), ch. 22 and p. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977), p. 365-389. The standard daily dose of antibodies in monotherapy may vary from about 1 to 100 μg/kg of body weight or more per day, depending on the factors listed above.

In one aspect, it should be understood and understood herein that the disclosed IL-25 binding molecules can be used to treat, prevent, or inhibit an inflammatory condition or disease, such as, for example, rhinovirus infection, airway inflammation, rheumatoid arthritis (RA) , osteoarthritis, bone erosion, intraperitoneal abscesses and adhesions, inflammatory bowel disease (IBD), allograft rejection, psoriasis, certain forms of cancer, angiogenesis, atherosclerosis, and multiple sclerosis (MS). Thus, in one aspect, the present document describes methods of treating, preventing or inhibiting rhinovirus infection, airway inflammation, rheumatoid arthritis (RA), osteoarthritis, bone erosion, intraperitoneal abscesses and adhesions, inflammatory bowel disease (IBD), allograft rejection, psoriasis, certain forms of cancer, angiogenesis, atherosclerosis and multiple sclerosis (PC), including the introduction of any of the binding IL-25 molecules described herein. For example, the present document describes methods of treating, preventing or inhibiting rhinovirus infection, airway inflammation, asthma, cystic fibrosis, rheumatoid arthritis (RA), osteoarthritis, bone erosion, intraperitoneal abscesses and adhesions, inflammatory bowel disease (IBD), allograft rejection, psoriasis, certain forms of cancer, angiogenesis, atherosclerosis, and multiple sclerosis (PC), comprising administering to the subject one, two, three or more IL-25 binding molecules containing a heavy chain variable domain containing one or more complementarity determining regions (CDRs), as defined in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. For example, methods of treating, inhibiting or prevention of rhinovirus infection, airway inflammation, asthma, cystic fibrosis, rheumatoid arthritis (RA), osteoarthritis, bone erosion, intraperitoneal abscesses and adhesions, inflammatory bowel disease (IBD), allograft rejection, psoriasis, certain forms of cancer, angiogenesis, atherosclerosis, and multiple multiple sclerosis (PC), comprising administering to the subject one or more IL-25 binding molecules containing a heavy chain variable domain containing a CDR as defined in SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 1 and SEQ ID NO: 3; SEQ ID NO: 2 and SEQ ID NO: 3; SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 7 and SEQ ID NO: 9; SEQ ID NO: 8 and SEQ ID NO: 9 and/or SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9; In one aspect, the disclosed methods of treating, preventing, or inhibiting may include administering to a subject IL-25 binding molecules comprising a heavy chain variable domain, the heavy chain variable domain comprising SEQ ID NO: 13 or SEQ ID NO: 27.

It should also be understood and appreciated herein that the disclosed methods for treating, preventing or inhibiting rhinovirus infection, airway inflammation, asthma, cystic fibrosis, rheumatoid arthritis (RA), osteoarthritis, bone erosion, intraperitoneal abscesses and adhesions, inflammatory bowel disease (IBD), allograft rejection, psoriasis, certain forms of cancer, angiogenesis, atherosclerosis, and multiple sclerosis (MS) may include administering to a subject suffering from said condition or disease, IL-25 binding molecules containing a light chain variable domain instead of a heavy chain variable domain, or in addition to it. In one aspect, the present document describes methods of treating, preventing or inhibiting rhinovirus infection, airway inflammation, asthma, cystic fibrosis, rheumatoid arthritis (RA), osteoarthritis, bone erosion, intraperitoneal abscesses and adhesions, inflammatory bowel disease (IBD), allograft rejection, psoriasis, certain forms of cancer, angiogenesis, atherosclerosis, and multiple sclerosis (PC) in a patient, comprising administering to the subject one, two, three or more IL-25 binding molecules containing a light chain variable domain containing one or more CDRs as defined in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, and/or SEQ ID NO: 12. For example, methods of treating, preventing, or inhibiting, comprising administering to the subject one or more IL-25 binding molecules comprising a light chain variable domain comprising a number of CDRs as defined in SEQ ID NO: 4 and SEQ ID NO: 5; SEQ ID NO: 4 and SEQ ID NO: 6; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 10 and SEQ ID NO: 11; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID NO: 12 and/or SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12. In addition, methods are described herein in which IL-25 binding molecules contain a variable a light chain domain, wherein the light chain variable domain comprises SEQ ID NO: 14 or SEQ ID NO: 16.

In one aspect, it is to be understood and understood herein that the described IL-25 binding molecules for use in the described methods of treatment, prevention and/or inhibition may comprise both a heavy chain variable domain and a light chain variable domain. It should also be understood that said IL-25 binding molecules used in the described methods of treatment, prevention and/or suppression may include one, two or three heavy chain variable domain CDRs in combination with one, two or three of a number of light chain variable domain CDRs. circuits described in this document. Accordingly, an IL25 binding molecule for use in the disclosed methods for treating, preventing or inhibiting rhinovirus infection, airway inflammation, asthma, cystic fibrosis, rheumatoid arthritis (RA), osteoarthritis, bone erosion, intraperitoneal abscesses and adhesions, inflammatory bowel disease (IBD), allograft rejection, psoriasis, certain forms of cancer, angiogenesis, atherosclerosis, and multiple sclerosis (PC) may contain one, two, or three of a number of heavy chain variable domain CDRs selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8 and/or SEQ ID NO: 9, and one, two or three light chain variable domain CDRs selected from the group consisting of SEQ ID NO: 4 , SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11 and/or SEQ ID NO: 12. For example, methods for treating rhinovirus infection, airway inflammation, asthma, cystic fibrosis, rheumatoid arthritis (RA), osteoarthritis, bone erosion, intraperitoneal abscesses and adhesions, inflammatory bowel disease (IBD), allograft rejection, psoriasis, certain forms of cancer, angiogenesis, atherosclerosis, and multiple sclerosis (MS), including administration of binding ILs to the subject -25 molecules containing the variable domain of the heavy chain; wherein the heavy chain variable domain comprises SEQ ID NO: 13, SEQ ID NO: 15 and/or SEQ ID NO: 17 and a light chain variable domain; wherein the light chain variable domain comprises SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18.

As used herein, the term treating, treating, or administering treatment refers to a method of alleviating one or more effects of a disease or condition (such as, for example, an inflammatory condition or cancer) in a subject. Thus, in the described method, treatment may refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of a detected infection or symptom of an infection. For example, a method of providing treatment for an inflammatory condition or cancer is considered a treatment if a 10% improvement in one or more symptoms of the condition or cancer is observed in a subject compared to a control. Thus, the reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction from 10% to 100% from baseline or control levels. It should be understood that such treatment does not necessarily mean the cure or complete elimination of the condition or disease or the symptoms of the condition or disease. It should be understood and meant herein that the treatments discussed herein may be prophylactic or therapeutic. Accordingly, in one aspect, methods are provided for treating or alleviating the severity of an inflammatory disease or condition in

- 24 042754 subject, including the introduction of the subject binding IL-25 molecules. In addition, methods for preventing or slowing the development of an inflammatory disease or condition in a subject are described, comprising administering an IL-25 binding molecule to the patient.

As used herein, the terms prevent, prevent, and prevent against infection refer to an action, e.g., administration, of a therapeutic agent (e.g., a composition described herein) that occurs before or about the same time that a subject begins to experience one or more symptoms of the infection, and which inhibits or delays the development or aggravation of, or delays the recurrence of, one or more symptoms of the infection. As used herein, references to reduction, mitigation or inhibition include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more from the control level. For example, the methods described are considered to be prevention if there is an approximately 10% delay in the development, exacerbation, or recurrence of an inflammatory condition or disease, or symptoms of an inflammatory condition or disease, in a subject compared to control subjects who did not receive an IL-25 binding molecule to mitigate the inflammatory condition. or disease.

It should be understood and understood herein that the methods of treatment, prevention, and inhibition described herein are intended to include both therapeutic and prophylactic applications of the IL-25 binding molecules described herein.

C. Examples.

The following examples are intended to provide fully disclosed information and descriptions to those skilled in the art as to how the compounds, compositions, articles, devices and/or methods claimed in the claims were made and evaluated, and these examples are intended purely for illustration purposes. of the present invention and do not limit the present description. Efforts have been made to ensure the accuracy of the numbers (eg quantities, temperatures, etc.), but some errors and deviations in experiments should be taken into account. Unless otherwise indicated, parts are parts by weight, temperature is expressed in °C or equal to ambient temperature, and pressure is equal to or approximates atmospheric.

Example 1

Transgenic mice overexpressing mouse Ig-Alpha, mouse Ig-Beta and human interleukin 6 were intraperitoneally injected with recombinant human IL-25 (R&D Systems) at two week intervals. After the formation of a noticeable immune response, which was registered by the results of serum ELISA, lymph nodes, spleen cells and red bone marrow cells were collected, B cells with surface expression of IgM isotype antibodies were extracted using magnetic beads, and the remaining cells were sorted by their ability to bind IL- 25 using the MoFlo Fluorescent Activated Cell Sorter (FIG. 1).

IL-25 positive binding cells were sorted into 96-well plates, single cell RT-PCR was performed to amplify the variable regions, and the variable regions were cloned into expression vectors containing a human IgG4 heavy chain constant region or a human IgK light chain constant region. The resulting heavy and light chain clone pairs were transfected into HEK293 cells, and the resulting antibody protein was purified on protein A resin (FIG. 2) and analyzed for its ability to neutralize recombinant human IL-25 activity in HT-29 cells (FIG. 3). Humanized versions of AVM109 (designated AVM109.2) were also tested in the cell potency assay for HT-29 (FIG. 4).

The affinity of antibodies to IL-25 was measured by surface plasmon resonance (SPR) using Biacore T-100 (GE Healthcare). (Fig. 5, 9) Antibodies were immobilized on carboxymethylated dextran on flow cells 2-4 on a CM5 chip (flow cell 1 was used as a standard). rhIL25 or rmIL15 in HBS-P buffer were run on the chip at increasing concentrations (2.5, 5.0, 10, 20 and 40 nM) with an injection time of 120 s for each concentration. After the 5th injection, HBS-P buffer was run through each flow cell and IL-25 was allowed to dissociate for 20 minutes. The surface was regenerated by exposure for 30 s to 10 mM glycine HCl pH 1.5. A single cycle kinetic analysis was performed for each antibody minus the standard cuvette.

AVM125 was purified and administered to mice in a model of rhinovirus infection in allergic asthma. Mice were sensitized with low-LPS chicken egg ovalbumin (OVA 50 μg in 2 mg aluminum adjuvant). Mice were then challenged with 50 μg of ovalbumin (OVA) intranasally (i/n) for three consecutive days. Immediately after the last OVA challenge, mice were injected intraperitoneally (ip) with AVM125 or an isotype control (IgG). 4 hours after the monoclonal antibody dose, mice were infected i/n with 2.5x106 TCID ₅₀ RV1B. Inflammatory responses were assessed on the third day after injection. Cell recruitment in bronchoalveolar lavage fluid was then determined and lung mRNA expression for IL-5 was assessed by quantitative PCR with SYBR Green chemistry expressed in Log2 coordinates (fold change) relative to saline/phosphate buffered saline (PBS) control. 3 days after

- 25 042754 infections.

Total RNA was later extracted from preserved mouse lung apical lobes with an RNA kit (Qiagen) followed by cDNA synthesis (OMNISCRIPT® RT kit, Qiagen). RV-1B RNA genomic primers and probe sequences: sense 5'-GTGAAGAGCCSCRTGTGCT-3' (SEQ ID NO: 19) 50 nm, antisense 5'-GCTSCAGGGTTAAGGTTAGCC-3' (SEQ ID NO: 20) 300 nm and probe-5' -FAM-TGAGTCCTCCGGCCCCTGAATG-TAMRA-3' (SEQ ID NO: 21) 100 nm. An ABI 7500 Taqman (ABI) was used to analyze PCR reactions. RNA was quantified using a standard curve that was generated from plasmid DNA amplification and expressed as copies per µl of DNA reaction.

D. Sequences.

SEQ ID NO: 1

SYWIE

SEQ ID NO: 2

QILPGIGSTNYNEKFKG

SEQ ID NO: 3

GYGNYGDY

SEQ ID NO: 4

RASESVDSYGNSFM

SEQ ID NO: 5

RASNLES

SEQ ID NO: 6

QQSNEDPLT

SEQ ID NO: 7

TSGMGVG

SEQ ID NO: 8

HIWWDDVKRYNPALKS

SEQ ID NO: 9

TLPHFFDY

SEQ ID NO: 10

SASSSVSYMY

SEQ ID NO: 11

RTSNLAS

SEQ ID NO: 12

KQYHSYPPTWT

SEQ ID NO: 13

EVKVVESGADLMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGQILPGIGSTN

YNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARGYGNYGDYWGQGTTVTVSS

SEQ ID NO: 14

DIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLE

SGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPLTFGAGTKLELKR

SEQ ID NO: 15

QVTLKVSGPGILQPSQTLSLTCSFSGFSLNTSGMGVGWIRQPSGKGLEWLAHIWWDDVK

RYNPALKSRLTISKDTSGSQVFLKIASVDTADTATYYCARTLPHFFDYWGQGTTLTVSS

SEQ ID NO: 16

DIQMTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKSGSSPKPWIYRTSNLASGVPA

RFSGSGSGTSYSLTISSMEAEDAATYYCKQYHSYPPTWTFGGGTKLEIKR

SEQ ID NO: 17

QVQLVQSGAEVKKPGASVKVSCKASGYTFSSYWIEWVRQAPGQGLEWIGQILPGIGSTN

YNEKFKGRVTITADTSTSTVYMELSSLRSEDTAVYYCARGYGNYGDYWGQGTTVTVSS

SEQ ID NO: 18

DIVLTQSPASLAVSPGQRATITCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLE

SGVPARFSGSGSGTDFTLTINPVEAQDTANYYCQQSNEDPLTFGAGTKLELKR

SEQ ID NO: 19

GTGAAGAGCCSCRTGTGCT

SEQ ID NO: 20

- 26 042754

GCTSCAGGGTTAAGGTTAGCC

SEQ ID NO: 21

TGAGTCCTCCGGCCCCTGAATG

Claims (7)

1. An isolated IL-25 binding molecule comprising a heavy chain variable domain containing three CDRs as defined in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, and a light chain variable domain containing three CDRs, as defined in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12. 2. An isolated IL-25 binding molecule according to claim 1, wherein the heavy chain variable domain comprises SEQ ID NO: 15. 3. An isolated IL-25 binding molecule according to claim 1, wherein the light chain variable domain comprises SEQ ID NO: 16. 4. An isolated IL-25 binding molecule according to claim 1, comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises SEQ ID NO: 15 and the light chain variable domain comprises SEQ ID NO: 16. 5. A method of treating rhinovirus infection, airway inflammation, rheumatoid arthritis, osteoarthritis, bone erosion, intraperitoneal abscesses and adhesions, inflammatory bowel disease, allograft rejection, psoriasis, certain forms of cancer, angiogenesis, atherosclerosis, cystic fibrosis, and multiple sclerosis, comprising administering a therapeutic the amount of any of the IL-25 binding molecules of claim 1. 6. An isolated IL-25 binding molecule according to claim 1, wherein the heavy chain variable domain contains an amino acid sequence having at least 90% identity to SEQ ID NO: 15. 7. An isolated IL-25 binding molecule according to claim 1, wherein the light chain variable domain contains an amino acid sequence having at least 95% identity to SEQ ID NO: 16.