HK1185088B - Humanized il-25 antibodies - Google Patents

Description

Humanized IL-25 antibodies

Background

Interleukin-25 (IL-25), also known as IL-17E, is a cytokine belonging to the IL-17 cytokine family and is secreted by type 2 helper T cells (Th2) and mast cells. IL-25 induces the production of other cytokines (including IL-4, IL-5, and IL-13) in multiple tissues and stimulates the expansion of eosinophils.

IL-25 is associated with chronic inflammation associated with the gastrointestinal tract, and the IL-25 gene has been identified as being located in a chromosomal region associated with an autoimmune disease of the intestine, such as Inflammatory Bowel Disease (IBD). Traditional therapies for treating IBD involve antibiotics or steroid-derived drugs; however, these drugs are currently not successfully used to induce or maintain clinical remission in patients.

IL-25 also showed upregulation in samples from asthmatics (asthma is a condition estimated to affect more than 3 billion people worldwide), suggesting that overexpression of this cytokine contributes to the pathogenesis of asthma and related disorders.

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

Disclosure of Invention

The present invention relates to target binding members, including antibodies and binding fragments thereof, directed against interleukin 25 (IL-25).

The present invention also relates to variants of the humanized (CDR-grafted) type RH2.5R71V of murine 2C3 antibody, also referred to herein using the terms "huDDG 91" and "M9". One such variant is referred to herein as the "M6 antibody" or "M6". M6 exhibited a variety of beneficial in vitro and in vivo properties, including, for example, enhanced binding affinity for IL-25 relative to the parent huDDG91 antibody, improved ability to inhibit the IL-25 receptor relative to the parent huDDG91 antibody in receptor and cell-based assays, and other properties, such as high expression levels, high solubility, no significant protein aggregation upon purification, and the absence of unwanted post-translational modifications, protein-protein interactions, and oxidation upon purification.

In one embodiment, the invention relates to a target binding member that binds IL-25, wherein the target binding member binds one or more amino acid sequences selected from the group consisting of: SEQ ID NO:17, amino acid residues 46-63 of SEQ ID NO:17 and amino acid residues 66-84 of SEQ ID NO:17 at amino acid residue 129-135. In particular embodiments, the target binding members of the invention bind to SEQ id no:17 and amino acid residues 56-63 of SEQ ID NO:17 at amino acid residues 66-74. In another embodiment, the target binding member comprises an antibody VL domain comprising CDR3 having amino acid sequence QQYLAFPYTF (SEQ ID NO: 8).

In another embodiment, the invention relates to a target binding member that binds IL-25, wherein the target binding member comprises:

a) an antibody VL domain comprising a heavy chain variable region having amino acid sequence SASQGISNYLN (SEQ id no:6) CDR1 having the amino acid sequence YTSSLHS (SEQ ID NO:7) and a CDR2 having amino acid sequence QQYLAFPYTF (SEQ ID NO:8) the CDR3 of (1); and

b) an antibody VH domain comprising a VH domain having the amino acid sequence GYTMN (SEQ ID NO:10) the CDR1 of (1), having the amino acid sequence LINPYNGGTSYNQNFKG (SEQ id no:11) and a CDR2 having an amino acid sequence EDYDGYLYFAMDY (SEQ id no:12) and CDR 3.

In particular embodiments, the target binding member comprises a polypeptide having the sequence of SEQ ID NO:5 and a VL domain having SEQ ID NO: 9. In other embodiments, the target binding member comprises a whole antibody.

In various embodiments, the invention also relates to isolated nucleic acids comprising nucleotide sequences encoding target binding members of the invention, expression vectors comprising such nucleic acids, and host cells carrying such expression vectors.

In another embodiment, the invention relates to a method of producing a target binding member comprising culturing a host cell of the invention under conditions suitable for production of the target binding member.

In other embodiments, the invention provides a composition comprising a target binding member of the invention and a pharmaceutically acceptable carrier.

In further embodiments, the invention encompasses methods of treating or preventing a disease or disorder, including (but not limited to) asthma and inflammatory bowel disease, in a subject in need thereof.

In other embodiments, the invention provides for the use of a target binding member of the invention, e.g., in the form of a pharmaceutical composition, for the treatment of a disease or disorder, including inflammatory disorders such as asthma (including allergic asthma) and inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis).

In another embodiment, the invention relates to a target binding member that competes for binding to IL-25 with a target binding member that binds to one or more amino acid sequences selected from the group consisting of SEQ ID NO:17, amino acid residues 46-63 of SEQ ID NO:17 and seq id NO:17 at amino acid residue 129-135. In particular embodiments, the target binding member has a binding affinity for human IL-25 of less than or equal to about 50 pM.

In another embodiment, the invention also relates to a target binding member of the invention comprising:

a) an antibody VL domain comprising a heavy chain variable region relative to SEQ ID NO:5 has an amino acid sequence of 1 to about 20 amino acid substitutions;

b) an antibody VH domain comprising a heavy chain variable region relative to SEQ ID NO:9 has an amino acid sequence of 1 to about 20 amino acid substitutions; or

c) Combinations thereof.

In another embodiment, the invention provides a method of producing a target binding member of the invention comprising:

(a) providing a starting repertoire of nucleic acids encoding VL domains, wherein the nucleic acids comprise a CDR3 encoding region to be replaced or lack a CDR3 encoding region;

(b) combining the starting library with a donor nucleic acid encoding a VL CDR3 having amino acid sequence QQYLAFPYTF (SEQ ID NO:8), wherein the donor nucleic acid is inserted into one or more nucleic acids in the library to form a product library of nucleic acids encoding a VL domain comprising a VL CDR3 having amino acid sequence QQYLAFPYTF (SEQ ID NO: 8);

(c) expressing the nucleic acids of the product pool to form a target binding member;

(d) selecting a target binding member that specifically binds to one or more sequences selected from the group consisting of SEQ ID NO:17, amino acid residues 56-63 of SEQ ID NO:17 and amino acid residues 66-74 of SEQ ID NO:17 at amino acid residue 129-135; and

(e) recovering the target binding member or the nucleic acid encoding the target binding member.

Drawings

This patent or patent application document contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the U.S. patent and trademark office upon request and payment of the necessary fee.

FIG. 1 shows the kappa light chain nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of the huDDG91/RH 2.5R71V antibody. The underlined amino acid sequences represent CDRs. Contains no leader sequence.

FIG. 2 shows the heavy chain nucleotide sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 4) of the huDDG91/RH 2.5R71V antibody. The underlined amino acid sequences represent CDRs. Contains no leader sequence.

FIG. 3 is a table showing the properties of 9 candidate monoclonal antibodies (including M6) identified by screening a 25 member combinatorial library based on the huDDG91/RH 2.5R71V antibody. Italics represent numbers obtained from different data sets. R71V G1 refers to huDDG91 antibody. Two rows of R71V G1 represent measurements from two different data sets. The residues with increased hydrophobicity are highlighted in yellow.

FIG. 4A shows the amino acid sequences of the light chain of the M6 antibody (SEQ ID NO: 5) and its CDRs (SEQ ID NOS: 6-8). The underlined amino acid sequences represent the positions of the CDRs. Contains no leader sequence. Amino acid residues in bold red indicate amino acid substitutions relative to the parent huDDG91/RH 2.5R71V sequence.

FIG. 4B shows the amino acid sequences of the heavy chain of the M6 antibody (SEQ ID NO: 9) and its CDRs (SEQ ID NOS: 10-12). The underlined amino acid sequences represent the positions of the CDRs. Contains no leader sequence.

FIG. 5 is a graph showing that M6 inhibits to a greater extent than huDDG91(M9) antibody in inhibiting IL-4/IL25 mediated IL-5 secretion by naive human CD4+ T cells stimulated with autologous dendritic cells in the presence of rhIL-4 and rhIL-25 for 4 days. Isotype IgG1 antibody (iso) was used as a control. n-4 donors.

FIGS. 6A and 6B are graphs showing that M6 antibody enhances the inhibition of IL-25 mediated GROa secretion in LS174T cells stimulated with recombinant human IL-25 for 24 hours, relative to huDDG91(M9) antibody. The graphs of fig. 6A and 6B represent teachings from two separate experiments.

FIG. 7A shows a sequence overlay of amino acid residues 1-78 of human IL-25(SEQ ID NO: 13), said human IL-25 being digested with pepsin and the reaction quenched with 2M urea, 1M TCEP (pH 3.0). The black line is the peptide observed.

FIG. 7B shows a sequence overlay of amino acid residues 79-146 (SEQ ID NO: 14) of human IL-25 digested with pepsin and quenched with 2M urea, 1M TCEP (pH 3.0). The black line is the peptide observed.

FIGS. 8A and 8B show the difference in the level of deuteration of different fragments of human IL-25 protein (SEQ ID NO: 15 and SEQ ID NO: 16) when the M6 antibody binds in H/D exchange experiments. Each square represents human IL-25 peptide and contains data for six time points: 150s and 500s at pH 6, and 150s, 500s, 1,500s, and 5,000s at pH 7. Dark blue indicates no protection upon binding of the M6 antibody. Other colors indicate that the deuterium concentration after solution forward/column reverse (on-solution/off-column) exchange is more than that after column forward/column reverse (on-column/off-column) exchange, as shown in the right inset. Deuterium attached to either of the first two amino acid residues of each ion is lost during analysis (digestion/separation/mass analysis in aqueous environment), which explains the small gaps in the H/D-Ex pattern.

FIGS. 9A-9F are graphs showing deuterium content in different fragments of human IL-25 after forward/reverse exchange at pH 6 and pH 7 using M6 antibody columns at 3 ℃. Blue indicates forward/reverse column exchange of the solution, and purple indicates forward/reverse column exchange. All exchange times were converted to equivalent values at pH 7, 23 ℃ (e.g. 150s at pH 6, 3 ℃ equals 1.85s at pH 7, 23 ℃). The human IL-25 residues represented in each fragment are shown at the top of each figure.

FIG. 10 shows the amino acid sequence of human IL-25(SEQ ID NO: 17).

Detailed Description

The invention is based, in part, on the identification of high affinity human IL-25 antibodies (referred to herein as "M6") (see example 1) and the identification of amino acid residues in human IL-25 to which M6 antibodies bind, as determined by hydrogen/deuterium (H/D) exchange mass spectrometry (see example 3). Thus, in one embodiment, the invention relates to a target binding member that binds IL-25, wherein the target binding member binds to one or more amino acid sequences selected from the group consisting of amino acid residues 46-63, 66-84 and 129-135 of human IL-25(SEQ ID NO: 17). Thus, a target binding member of the invention may bind to SEQ ID NO:17, amino acid residues 46-63 of SEQ ID NO:17, or amino acid residues 66-84 of SEQ id no:17, 129-135, or any combination thereof. In one embodiment, the target binding member of the invention binds to SEQ ID NO:17 and SEQ id no:17 from position 66 to position 74.

As used herein, a "target binding member" refers to any protein or peptide comprising a molecule having at least a portion of an immunoglobulin molecule comprising at least one Complementarity Determining Region (CDR) of a heavy or light chain or a ligand binding portion thereof that specifically binds a mammalian (e.g., human) IL-25 protein or a portion thereof. Such target binding members may also comprise at least a portion of an antibody heavy or light chain variable region, at least a portion of an antibody heavy or light chain constant region, at least a portion of an antibody framework region, or any combination thereof. Such target binding members modulate, reduce, antagonize, alleviate, ameliorate, block, inhibit, abrogate and/or interfere with the activity or binding of at least one IL-25, or the activity or binding of an IL-25 receptor, in vitro, in situ and/or in vivo. By way of non-limiting example, suitable target binding members of the invention can bind with high affinity to inhibitory and/or neutralizing epitopes of human IL-25 recognized by the M6 antibodies described herein.

Target binding members of the invention are not limited to binding only to SEQ ID NO:17 at amino acid residues 46-63, at amino acid residues 66-84 and/or at amino acid residues 129-135. Thus, in some embodiments, the target binding members of the invention may additionally bind to a polypeptide of SEQ ID NO:17 at amino acid residues 46-63, at amino acid residues 66-84 and/or at amino acid residues 129-135. In other examples, the target binding member of the invention may additionally bind to a polypeptide in human IL-25 that is flanked by SEQ ID NOs: 17 at amino acid residues 46-63, at amino acid residues 66-84 and/or at amino acid residues 129-135.

The invention also contemplates target binding members that bind to a polypeptide encompassed by SEQ ID NO:17, amino acid residues 46-63, amino acid residues 66-84 and/or amino acid residues 129-135, such as the amino acid sequence defined by SEQ ID NO:17 at least 5 amino acid residues from amino acid residues 46-63, amino acid residues 66-84 and/or amino acid residues 129-135. Exemplary portions of IL-25 to which a target binding member can bind include seq id NO:17 at amino acids 56-63 and/or at amino acids 66-74.

The region or epitope of IL-25 to which a target binding member of the invention binds can be determined using any of a variety of standard epitope mapping techniques well known in the art to which the invention pertains. Such techniques include, for example, site-directed mutagenesis in conjunction with binding assays, epitope mapping using peptide needles (e.g., Geysen et al, Peptides: Chem identify and Biological, Proceedings of the Twelfth American peptide zymosporium, p.519-523, Ed, G.R. Marshall, Ecom, Leiden, 1988(Geysen et al, "Peptides: chemistry and biology", Proceedings of the Twelfth American peptide workshop, pp.519-523, G.R. Marshall, Ecom, Leiden editors, 1988)), X-ray crystallography, and hydrogen/deuterium (H/D) exchange techniques (e.g., H/D exchange mass spectrometry).

As described herein, H/D exchange mass spectrometry is used to determine the epitope in human IL-25 that is recognized and bound by the M6 antibody (see example 3 and fig. 8A, 8B, and 9A-9F). Upon transfer from water to deuterium based solvent systems (heavy water), the protein will experience an increase in mass as its hydrogen atoms are progressively replaced by deuterons (heavier isotopes of hydrogen). The likelihood of a hydrogen/deuterium exchange event is largely determined by protein structure and solvent accessibility. H/D exchange mass spectrometry is used to measure exchange, and thus protein structure and solvent accessibility. When a small molecule or protein binding partner binds to a protein target, the target undergoes an experimentally observable change in exchange rate. The surface area that repels the solvent during complex formation is much slower to exchange. The excluded areas of solvent can be used to infer the location of the binding site. For example, in the case of antigen-antibody interactions, these changes highlight the position of the epitope.

Typically, a target binding member of the invention will comprise an antibody light chain variable region (VL) domain which pairs with an antibody heavy chain variable region (VH) domain to form an IL-25 binding domain. In making the invention described herein, it was found that changing the Complementarity Determining Regions (CDRs), particularly the CDR3 region, in the VL domain (SEQ ID NO: 1) of huDDG91 antibody to QQYLAFPYTF (SEQ ID NO:8) improved the binding affinity of the huDDG91 antibody. Thus, the invention described herein contemplates a polypeptide comprising SEQ id no:8 and target binding members comprising such VL domains. In some embodiments, the VL domain in the target binding member of the invention further comprises CDR1(SASQGISNYLN (SEQ ID NO: 6)) and CDR2(YTSSLHS (SEQ ID NO: 7)) regions from M6 and the huDDG91 antibody. Other VL CDR regions suitable for inclusion in a target binding member of the invention include, but are not limited to, any of the VL CDR1 regions shown in figure 3. In one embodiment, the target binding member of the invention comprises the VL domain of the M6 antibody SEQ ID NO: 5.

in some embodiments, the target binding members of the invention further comprise SEQ ID NOs: 10-12. Other VH CDR regions suitable for inclusion in target binding members of the invention include, but are not limited to, any of the VH CDR3 regions shown in fig. 3. In preferred embodiments, the target binding members of the invention include M6 and the VH domain of huDDG91 antibody SEQ ID NO: 9. the VH domain may be paired with multiple VL domains other than the VL domain of the M6 antibody (SEQ ID NO: 5). Preferably, the VH domain is paired with a VL domain (having the amino acid sequence of SEQ ID NO:8) comprising a CDR3 region.

The sequences of the CDRs of the M6 antibodies described herein can be modified by insertions, substitutions, and deletions and are included in the target binding members of the invention, so long as the target binding member (e.g., antibody) with the modified CDRs is capable of maintaining the ability to bind to human IL-25 and inhibit human IL-25. Maintenance of this activity can be determined by one of ordinary skill by performing the functional assays described herein. The CDRs in the target binding members of the invention correspond to SEQ ID NO: 6. the M6CDR represented by 7, 8, 10, 11 or 12 may have, for example, about 50% to about 100% homology, preferably about 80% to about 100% homology, more preferably about 90% to about 100% homology. In one embodiment, the CDRs in the target binding members of the invention are aligned with the corresponding CDRs in SEQ ID NOs: 6. the M6CDR represented by 7, 8, 10, 11 or 12 may have about 100% homology.

Target binding members according to the invention may bind IL-25 with an affinity substantially similar to, or greater than, that of the M6 antibody described herein. For example, a target binding member of the invention may have a binding affinity for IL-25 (e.g., human IL-25) of about 50pM (e.g., about 53pM) or less, e.g., about 45pM, about 40pM, about 35pM, about 30pM, about 25pM, or about 20 pM. The target binding member will typically have IL-25 specificity. Thus, the target binding member will not exhibit any significant binding to molecules other than its specific binding partner. For example, it has been found that the M6 antibody described herein does not cross-react with IL-17A, IL-17C, IL-17D or IL-17F. In some embodiments of the invention, it is a desirable feature of the target binding member to avoid such cross-reactivity with other cytokines involved in asthma and similar processes.

The affinity or avidity of the target binding member antigen may be determined experimentally using any suitable method. (see, e.g., Berzofsky et al, "Antibody-Antibody Interactions," InFundamental Immunology, Paul, W.E., Ed., Raven Press: New York, N.Y. (1984) (Berzofsky et al, "Antibody-Antigen Interactions," basic Immunology ", Paul, W.E., eds., Levin Press, N.Y., 1984); Kuby, Janis, Immunology, W.H.Freeman and Company: New York, N.Y. (1992) (Kuby, Janis, Immunology, W.H.Floriman, N.H.Freuman, N.Y., New York City, 1992), and the methods described herein). The measured affinity of a particular antibody-antigen interaction will be different if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of affinity and other antigen binding parameters (e.g., KD, Ka, KD) are preferably performed with standard solutions of antibodies and antigens, as well as standard buffers (e.g., the buffers described herein).

For example, specificity can be determined by a binding assay, such as an ELISA, Biacore assay, and/or Octet assay using a panel of antigens, and the like. Target binding members according to the invention may recognize IL-25 but not other members of the IL-17 family, in particular any of IL-17A, IL-17B, IL-17C, IL-17D and IL-17F; in one embodiment, all five of these molecules. Binding of a target binding member according to the invention to IL-25 can be abolished by competition with recombinant IL-25.

The binding affinity and neutralization potency of different target binding members can be compared under appropriate conditions.

The invention also relates to a target binding member that competes for binding to IL-25 with a target binding member of the invention that binds to one or more amino acid sequences selected from the group consisting of SEQ ID NO:17, amino acid residues 46-63 of SEQ ID NO:17 and amino acid residues 66-84 of SEQ ID NO:17 at amino acid residue 129-135. In particular embodiments, the target binding member has a binding affinity for human IL-25 of less than or equal to about 50 pM.

Competitive assays can be performed with target binding members (e.g., antibodies) of the invention to determine what proteins, antibodies, and other antagonists compete with the target binding members of the invention for binding to IL-25 and/or share epitopic regions. These assays are well known to those of ordinary skill in the art and are used to assess competition between antagonists or ligands for a limited number of binding sites on a protein, such as IL-25. The proteins and/or antibodies are immobilized or insolubilized before or after competition, and the sample bound to the IL-25 protein is separated from the unbound sample, for example by decantation (in the case of a pre-insolubilization of the protein/target binding member) or by centrifugation (in the case of a precipitation of the protein/antibody after the competitive reaction). Alternatively, competitive binding may be determined by whether binding or lack of binding of the target binding member to the protein alters function, e.g., whether the target binding member molecule inhibits or enhances, e.g., the enzymatic activity of the label. ELISA and other functional assays can be used, as is well known in the art.

Antibodies

Preferably, the target binding member of the invention is an antibody molecule. The term "antibody" is intended to encompass whole antibodies, digested fragments, specified portions, or variants thereof, including antibody mimetics or antibody portions that mimic the structure and/or function of an antibody or a specified fragment or portion thereof, including single chain antibodies and fragments thereof; each comprising at least one CDR. Functional fragments include antigen-binding fragments that bind mammalian IL-25. For example, antibody fragments capable of binding to IL-25 or a portion thereof are encompassed by the invention, including (but not limited to): fab fragments (e.g., obtained by papain digestion), Fab 'fragments (e.g., obtained by pepsin digestion and partial reduction), and F (ab')₂Fragments (e.g., obtained by pepsin digestion), facb fragments (e.g., obtained by plasmin digestion), pFc' fragments (e.g., obtained by pepsin or plasmin digestion), Fd fragments (e.g., obtained by pepsin digestion, partial reduction, and reaggregation), Fv or scFv fragments (e.g., obtained by molecular biology techniques) (see, e.g., Colligan, et al, eds., Current Protocols in Immunology, John Wiley)&Sons, inc., NY (19942001) (edited by Colligan et al, immunologic laboratory guidelines, john william father-son publishing company, new york, 1994, 2001); colliganet al.，Current Protocols in Protein Science，John Wiley &Sons, NY, n.y., (19972001) (Colligan et al, "guide to protein science laboratories," john william father publishing company, new york, 1997, 2001)).

Such fragments may be prepared by enzymatic cleavage, synthesis, or recombinant techniques known in the art and/or described herein. Various truncated forms of antibodies can also be produced using antibody genes in which one or more stop codons are introduced upstream of the natural termination site. For example, the code F (ab')₂The combined genes for the heavy chain portion were designed to include the CH encoding the heavy chain₁DNA sequence of a domain and/or hinge region. The various portions of the antibody can be chemically linked together by conventional techniques or can be prepared as a continuous protein using genetic engineering techniques.

The term "antibody" as used herein is also intended to encompass "chimeric" antibodies and "humanized" or "CDR-grafted" antibodies comprising any combination of the M6 CDRs described herein with one or more proteins or peptides derived from a non-murine source, preferably a human antibody. According to the present invention there is provided a chimeric or humanized antibody in which the CDRs are derived from the M6 antibody. Thus, in one embodiment, the human portion of the antibody may include a region that is substantially non-immunogenic in humans. The region of an antibody derived from a human antibody need not be 100% identical to the human antibody. In a preferred embodiment, as many human amino acid residues as possible are retained so that immunogenicity is negligible, but human residues may be modified as necessary to support the antigen binding site of CDR formation, while maximizing the degree of humanization of the antibody. These alterations or variations optionally and preferably maintain or reduce immunogenicity in humans or other species relative to the unmodified antibody. Humanized antibodies can be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and/or light chain) genes. In addition, when the antibody is a single chain antibody, it may comprise a linking peptide not present in a natural human antibody. For example, the Fv may comprise a linker peptide, e.g., 2 to about 8 glycine or other amino acid residues, that links the heavy chain variable region and the light chain variable region. Such linker peptides are considered to be of human origin.

Alternatively, the entire heavy and light chain variable regions of the M6 antibody in FIGS. 4A and 4B (SEQ ID NOS: 5 and 9) can be combined with human constant regions and framework regions to form target binding members of the invention.

The human gene encoding the constant (C) region of the target binding member of the invention may be derived from a human fetal liver bank by known methods. The human C region genes can be derived from any human cell, including those that express and produce human immunoglobulins. The human CH region may be derived from any known type or isotype of human H chain, including gamma, mu, alpha, and subtypes thereof, e.g., G1, G2, G3, and G4. Since the H chain isotype is responsible for various effector functions of an antibody, the choice of CH region will be guided by the desired effector function, e.g., activity of complement fixation or antibody-dependent cellular cytotoxicity (ADCC). In one embodiment, the CH region is derived from γ 1(IgG 1).

The human CL region may be derived from one of the human L chain isoforms κ or λ, preferably κ.

Genes encoding the C region of human immunoglobulins can be obtained from human cells by standard Cloning techniques (e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989) (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and Ausubel et al, eds., Current Protocols in Molecular Biology (1987, 1993) (Ausubel et al, eds., Molecular Biology Laboratory, 1987, 1993)). The human C region gene can be easily obtained from a known clone comprising genes representing two types of L chains, five types of H chains, and subclasses thereof. Chimeric antibody fragments, e.g. F (ab)₁)₂And Fab can be prepared by designing appropriately truncated chimeric H chain genes. For example, encoding F (ab)₁)₂The chimeric gene of the H chain portion of the fragment may comprise a gene encoding the H chainThe DNA sequence of the CH1 domain and hinge region, followed by a translation stop codon to produce a truncated molecule.

Generally, in one example, the chimeric antibodies, fragments and regions of the invention are produced by cloning DNA fragments encoding the H and L chain antigen binding regions of the M6 antibody and ligating these DNA fragments with DNA fragments encoding the CH and CL regions, respectively, to produce genes encoding chimeric immunoglobulins.

Thus, in one embodiment, a fusion chimeric gene is produced comprising a first DNA segment encoding at least an antigen binding region of non-human origin, such as a functionally rearranged V-region, joined by a joining (J) segment to a second DNA segment encoding at least a portion of a human C-region.

The sequence of the variable region of the M6 antibody can be modified by insertions, substitutions and deletions as long as the target binding member maintains the ability to bind to human IL-25 and inhibit human IL-25.

For convenience, the numbering scheme of Kabat et al is adopted herein. Residues are indicated by lower case numbers or by the necessary hyphens to allow the sequences of the invention to be identical to the standard Kabat numbering sequence.

In the case of a CDR-grafted or humanized antibody (wherein the CDR regions of the M6 antibody are combined with human source regions), residues specific for the parent antibody (e.g., M6) may be retained in the FR regions according to the present invention. Residues that have been shown to have a critical role in humanization of other antibodies may also be retained. These guidelines can be followed to some extent, but it is necessary to support the antigen binding site of CDR formation while maximizing the degree of humanization of the antibody.

Methods for engineering or humanizing non-human or human antibodies can also be used and are well known in the art. Generally, a humanized or engineered antibody has one or more amino acid residues from a non-human source, such as (but not limited to) a mouse, rat, rabbit, non-human primate, or other mammal. These human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain, constant domain, or other domain of a known human sequence. Known human Ig Sequences are published, for example, in a number of public databases (e.g., the NCBI database of the National institutes of Health) or publications (e.g., Kabat et al, Sequences of Proteins of Immunological Interest, U.S. Dept. Health (1983) (Kabat et al, protein Sequences of Immunological Interest, U.S. department of Health, 1983)).

As is known in the art, such input sequences may be used to reduce immunogenicity, or to reduce, enhance or modify binding, affinity, association rate, dissociation rate, avidity, specificity, half-life or any other suitable characteristic. Generally, some or all of the non-human or human CDR sequences may be retained, while the non-human sequences of the variable and constant regions are replaced with human or other amino acids. The antibodies may also optionally be humanized, retaining a high affinity for the antigen and other advantageous biological properties. To achieve this goal, humanized antibodies can optionally be prepared by a process of analyzing the parental sequences and various contemplated humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are available that specify and display the likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this manner, FR residues can be selected from the consensus and input sequences and combined so that a desired antibody characteristic, such as increased affinity for the target antigen, can be achieved. Generally, CDR residues are directly and most significantly involved in affecting antigen binding. Humanization or engineering of antibodies of the invention can be performed using any known method, such as, but not limited to, Jones et al, Nature 321: 522(1986) (Jones et al, Nature, Vol. 321, p. 522, 1986); riechmann et al, Nature 332: 323(1988) (Riechmann et al, Nature, vol. 332, p. 323, 1988); verhoeyen et al, Science 239: 1534(1988) (Verhoeyen et al, science 239, page 1534, 1988); sims et al, j.immunol.151: 2296(1993) (Sims et al, J. Immunol, Vol. 151, p. 2296, 1993); chothia and Lesk, j.mol.biol.196: 901(1987) (Chothia and Lesk, journal of molecular biology, Vol. 196, p. 901, 1987); carter et al, proc.natl.acad.sci.u.s.a.89: 4285(1992) (Carter et al, Proc. Natl. Acad. Sci. USA, Vol. 89, p. 4285, 1992); presta et al, j.immunol.151: 2623(1993) (Presta et al, J. Immunol., Vol. 151, p. 2623, 1993); those methods described in U.S. Pat. Nos. 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539, 4,816,567, PCT/US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755, WO90/14443, WO90/14424, WO90/14430, EP 229246 (the entire disclosures of each patent, including the references cited therein, being incorporated herein by reference).

The human constant region of the target binding member of the invention may be of any type (IgG, IgA, IgM, IgE, IgD etc.) or isotype and may comprise a kappa or lambda light chain. In one embodiment, the human constant region comprises an IgG heavy chain or defined fragment, e.g., at least one isotype IgG1, IgG2, IgG3, or IgG 4. In another embodiment, the target binding member comprises an IgG1 heavy chain and an IgG1K light chain. An isolated target binding member of the invention may comprise an antibody amino acid sequence disclosed herein encoded by any suitable polynucleotide. Preferably, the target binding member binds to human IL-25, thus partially or substantially neutralizing at least one biological activity of the protein. The target binding member, or a specific portion or variant thereof, partially or preferably substantially neutralizes at least one biological activity of at least one IL-25 protein or fragment, thereby inhibiting activity mediated through IL-25 binding to an IL-25 receptor or through other IL-25 dependent or mediated mechanisms. As used herein, the term "neutralizing antibody" refers to an antibody that can inhibit IL-25 dependent activity by about 20-100%, preferably by at least about 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or more, depending on the assay. The ability of a target binding member to inhibit IL-25 dependent activity is preferably assessed by at least one suitable IL-25 protein or receptor assay described herein and/or known in the art.

At least one antibody of the invention binds to at least one epitope as specified herein to which the M6 antibody binds. The at least one epitope may comprise at least one antibody binding region comprising at least a portion of said protein, which epitope is preferably constituted by at least one extracellular, soluble, hydrophilic, external or cytoplasmic portion of said protein. Generally, a target binding member of the invention will comprise an antigen binding region comprising at least one complementarity determining region (CDR1, CDR2 and CDR3) of SEQ ID nos. 10, 11 and 12 or a variant of at least one heavy chain variable region and at least one human complementarity determining region (CDR1, CDR2 and CDR3) (SEQ ID nos. 6, 7 and 8) or a variant of at least one light chain variable region.

Target binding members that bind to human IL-25 and comprise defined heavy or light chain variable or CDR regions can be prepared using suitable methods, such as phage display (Katsube, Y., et al, Int J. mol. Med, 1 (5): 863868 (1998)) (Katsube, Y. et al, J. chem. Med., 1,5, p. 863-868, 1998)) or using transgenic animals, as known in the art and/or described herein. For example, an antibody, specified portion, or variant can be expressed in a suitable host cell using the encoding nucleic acid or portion thereof.

As noted above, the invention also relates to antibodies, antigen-binding fragments, immunoglobulin chains, and CDRs comprising amino acid sequences that are substantially identical to the M6 amino acid sequence described herein. Such anti-IL-25 antibodies may comprise one or more amino acid substitutions, deletions, or additions resulting from natural mutation or human manipulation, as described herein. Preferably, such antibodies or antigen binding fragments and antibodies comprising such chains or CDRs can bind to human IL-25 with high affinity (e.g., a KD of less than or equal to about 10-9M). Amino acid sequences that are substantially identical to the sequences described herein include sequences that include conservative amino acid substitutions as well as amino acid deletions and/or insertions. Conservative amino acid substitutions are those that replace a first amino acid with a second amino acid that has similar chemical and/or physical properties (e.g., charge, structure, polarity, hydrophobicity/hydrophilicity) as the first amino acid. Conservative substitutions include the replacement of one amino acid by another within the following group: lysine (K), arginine (R) and histidine (H); aspartic acid (D) and glutamic acid (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D, and E; alanine (a), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C), and glycine (G); F. w and Y; C. s and T.

The number of amino acid substitutions that can be made by the skilled person depends on many factors, including those described above. Generally, the number of amino acid substitutions, insertions, or deletions for any given anti-IL-25 antibody, fragment, or variant will not exceed 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6,5, 4,3, 2, or 1, e.g., 1-30, or any range or value therein, as specified herein.

Amino acids essential for function in the target binding members of the invention can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra, chapters 8 and 15; Cunningham and Wells, Science 244: 10811085 (1989)) (Cunningham and Wells, Science 244, vol.1081-1085, 1989)). The latter method introduces single alanine mutations at every residue of the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to, at least one IL-25 neutralizing activity. Sites of crucial antibody binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al, J.mol.biol.224: 899904 (1992) (Smith et al, J.Mol., J.M.biol., 224, p.899-904, 1992) and de Vos, et al, Science 255: 306312 (1992) (de Vos et al, Science, 255, p.306-312, 1992)).

Target binding members of the invention may include, but are not limited to, a member selected from the group consisting of SEQ ID NOs: 5-12 at least one portion, sequence or combination of 5 to all contiguous amino acids.

The target binding member may also optionally comprise SEQ ID NO:5 or 9, at least one of 70-100% contiguous amino acids.

In one embodiment, the amino acid sequence of the immunoglobulin chain or portion thereof (e.g., variable region, CDR) is identical to SEQ ID NO:5 or 9 has about 70-100% identity (e.g., 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, 100, or any range or value therein). For example, the amino acid sequence of the light chain variable region may be identical to SEQ ID NO:5, or the amino acid sequence of the heavy chain variable region may be compared to the amino acid sequence of SEQ ID NO:9 for comparison. Preferably, 70-100% amino acid identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or any range or value therein) is determined using a suitable computer algorithm known in the art.

In SEQ ID NO: exemplary heavy and light chain variable region sequences are provided in fig. 5 and 9. A target binding member of the invention, or a particular variant thereof, may comprise any number of contiguous amino acid residues from an antibody of the invention, wherein the number is selected from an integer from 10 to 100% of the number of contiguous residues in the target binding member. Optionally, the contiguous amino acid subsequence is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more amino acids in length, or any range or value therein. Furthermore, the number of subsequences may be any integer selected from 1 to 20, for example at least 2, 3, 4 or 5.

The skilled artisan will appreciate that the invention includes at least one biologically active antibody of the invention. The specific activity of a biologically active antibody is at least 20%, 30% or 40%, and preferably at least 50%, 60% or 70%, and most preferably at least 80%, 90% or 95% -1000% of the specific activity of the natural (non-synthetic), endogenous or related and known antibody. Methods for determining and quantifying measures of enzyme activity and substrate specificity are well known to those skilled in the art.

In another aspect, the invention relates to a target binding member as described herein modified by covalent attachment of an organic moiety. Such modifications can result in antibodies or antigen-binding fragments with improved pharmacokinetic properties (e.g., increased serum half-life in vivo). The organic moiety may be a linear or branched hydrophilic polymeric group, a fatty acid group, or a fatty acid ester group. In particular embodiments, the hydrophilic polymer group may have a molecular weight of about 800 to about 120,000 daltons, and may be a polyalkylene glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), a carbohydrate polymer, an amino acid polymer, or polyvinylpyrrolidone, and the fatty acid or fatty acid ester group may comprise about 8 to about 40 carbon atoms.

The modified target binding members of the invention may comprise one or more organic moieties covalently bonded, directly or indirectly, to the antibody. Each organic moiety bound to an antibody or antigen-binding fragment of the invention may independently be a hydrophilic polymeric group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" encompasses monocarboxylic acids and dicarboxylic acids. The term "hydrophilic polymeric group" as used herein refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Thus, antibodies modified by covalent attachment of polylysine are included in the present invention. Is suitable for repairingHydrophilic polymers that decorate the antibodies of the invention can be linear or branched and include, for example, polyalkanediols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc.), hydrophilic amino acid polymers (e.g., polylysine, polyarginine, polyaspartic acid, etc.), polyalkylene oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), and polyvinylpyrrolidone. Preferably, the hydrophilic polymer modifying the antibody of the invention has a molecular weight of about 800 to about 150,000 daltons as a separate molecular entity. For example, PEG may be used₅₀₀₀And PEG_20,000Wherein the subscript is the average molecular weight (daltons) of the polymer. The hydrophilic polymeric group may be substituted with 1 to about 6 alkyl, fatty acid, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acids or fatty acid ester groups can be prepared by employing suitable methods. For example, a polymer comprising amine groups may be coupled to carboxyl groups of a fatty acid or fatty acid ester, and activated carboxyl groups on the fatty acid or fatty acid ester (e.g., activated with N, N-carbonyldiimidazole) may be coupled to hydroxyl groups on the polymer.

Fatty acids and fatty acid esters suitable for modifying the antibodies of the invention may be saturated or may contain one or more units of unsaturation. Fatty acids suitable for modifying the antibodies of the invention include, for example, n-dodecanoic acid (C12, lauric acid), n-tetradecanoic acid (C14, myristic acid), n-octadecanoic acid (C18, stearic acid), n-eicosanoic acid (C20, arachidic acid), n-docosanoic acid (C22, behenic acid), n-triacontanoic acid (C30), n-tetracontanoic acid (C40), cis-9-octadecanoic acid (C18, oleic acid), all cis-5, 8, 11, 14-eicosatetraenoic acid (C20, arachidonic acid), suberic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include dicarboxylic acid monoesters containing a linear or branched lower alkyl group. The lower alkyl group may contain 1 to about 12, preferably 1 to about 6 carbon atoms.

Modified target binding members may be prepared using suitable methods, for example by reaction with one or more modifying agents. The term "modifying agent" as used herein is meant to encompassSuitable organic groups for the activating group (e.g., hydrophilic polymers, fatty acids, fatty acid esters). An "activating group" is a chemical moiety or functional group that can react with a second chemical group under suitable conditions, thereby forming a covalent bond between the modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halogen (chloro, bromo, fluoro, iodo), N-hydroxysuccinimide ester (NHS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acryloyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. The aldehyde functional group can be coupled to an amine-or hydrazide-containing molecule, and the azide group can react with a trivalent phosphorus-containing group to form a phosphoramidate or a phosphoramidite linkage. Suitable methods for introducing activating groups into molecules are known in the art (see, e.g., Hermanson, G.T., bioconjugateTechniques, Academic Press: San Diego, Calif. (1996) (Hermanson, G.T., "Bio-Cross-linking technology", Academic Press, San Diego, Calif.)). The activating group may be bonded to an organic group (e.g., hydrophilic polymer, fatty acid ester) either directly or through a linker moiety, such as a divalent C wherein one or more carbon atoms may be replaced by a heteroatom (e.g., oxygen, nitrogen, or sulfur)₁-C₁₂A group. Suitable linker moieties include, for example, tetraethylene glycol, - - (CH)₂)₃--、--NH--(CH₂)₆--NH--、--(CH₂)₂- -NH- -and- -CH₂--O--CH₂--CH₂--O--CH₂--CH₂- -O- -CH- -NH- -. Modifiers comprising a linker moiety can be generated, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate. Removal of the Boc protecting group from the product by treatment with trifluoroacetic acid (TFA) can be carried out to expose a primary amine, which can be coupled to another carboxylic acid ester (as described), or can be reacted with maleic anhydride and the resulting product cyclized to give activation of the fatty acidA maleimide-based derivative. (see, e.g., WO 92/16221 to Thompson et al, the entire teachings of which are incorporated herein by reference.)

The modified target binding members of the invention may be produced by reacting a human antibody or antigen-binding fragment with a modifying agent. For example, the organic moiety can be bound to the antibody in a non-site specific manner by using an amine-reactive modifier (e.g., a NHS ester of PEG). Modified human antibodies or antigen-binding fragments can also be prepared by reducing disulfide bonds (e.g., intrachain disulfide bonds) of an antibody or antigen-binding fragment. The reduced antibody or antigen-binding fragment can then be reacted with a thiol-reactive modifier to produce the modified antibody of the invention. Modified human antibodies and antigen-binding fragments comprising an organic moiety bonded to a specific site of an antibody of the invention can be prepared using suitable methods, such as reverse proteolysis (Fisch et al, Bioconjugate Chem., 3: 147153 (1992) (Fisch et al, biochross chemistry, Vol.3, pp.147-153, 1992); Werlen et al, Bioconjugate-chem., 5: 411417 (1994); Werlen et al, biochross chemistry, Vol.5, pp.411-417, 1994); Kumaran et al, Protein Sci.6 (10): 22332241(1997) (Kuan mar et al, Protein science, Vol.6, pp.10, 2233-42), Itoh et al, Bioorg.cheg.m., 24(1), 5968 (Capture et al, Vol.6, pp.24, 1996, Capture et al, 1996, Capture et al, pp.24-24, 1996, Capture et al, Biocrosslinking chemistry, Vol.147-153, 1994), biotechnol, bioeng, 56 (4): 456463(1997) (Capillas et al, Biotechnology and bioengineering, Vol.56, No.4, p.456-463, 1997)), and Hermanson, G.T., Bioconjugate Techniques, Academic Press: san Diego, Calif. (1996) (Hermanson, G.T., "Bio-Cross-linking technology", academic Press, San Diego, Calif., 1996).

The target binding members used in the methods and compositions of the invention are characterized by high affinity binding to IL-25 and optionally have low toxicity. In particular, target binding members of the invention, wherein the individual components, e.g., variable, constant and framework regions, individually and/or collectively, optionally and preferably have low immunogenicity, are useful in the invention. The target binding members useful in the present invention are optionally characterized in that they can be used to treat patients for extended periods of time, can significantly reduce symptoms and have low and/or acceptable toxicity. Low or acceptable immunogenicity and/or high affinity, as well as other suitable properties, may help achieve a therapeutic result. "Low immunogenicity" is defined herein as producing a significant HAHA, HACA or HAMA response in less than about 75%, or preferably less than about 50%, of treated patients, and/or causing low titers (less than about 300, preferably less than about 100 as measured by a dual antigen enzyme immunoassay) in treated patients (Elliott et al, Lancet 344: 11251127 (1994) (Elliott et al, Lancet, Vol.344, p.1125-1127, 1994), which is incorporated herein by reference).

Bispecific, xeno-specific, xeno-conjugated or similar antibodies, which are monoclonal, humanized antibodies having binding specificity for at least two different antigens, may also be used. In this case, one binding specificity is directed to at least one IL-25 protein and the other binding specificity is directed to any other antigen. Methods of making bispecific antibodies are known in the art. Typically, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature 305: 537 (1983)) (Milstein and Cuello, Nature, Vol. 305, p. 537, 1983.) these hybridomas (quadromas) produce a mixture of possibly 10 different antibody molecules, only one of which has the correct bispecific structure, the purification of the correct molecule (usually by an affinity chromatography step) is rather cumbersome and the yield of the product is low, a similar method is described in, for example, WO 93/08829, U.S. Pat. Nos. 6,210,668, 6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453, 6,010,902, 5,989,530, 5,959,084, 5,959,083, 5,932,448, 48, 5,821,333, 5,807,706, 5,643,759, 5,601,819, 5,5966, 5,496,549, WO 4, 91/00360, 92/00373, WO 676, WO2,5968, 1986, 19823, WO 638, WO 634,676, WO 4,676, EP 03089, Traunecker et al, EMBO j.10: 3655(1991) (Traunecker et al, J. Eur. Mol. biol. org. J., Vol. 10, p. 3655, 1991), Suresh et al, Methods in enzymology 121: 210(1986) (Suresh et al, methods in enzymology, Vol.121, p.210, 1996), each of which is incorporated by reference herein in its entirety.

IL-25

Il-25, also known in the art as Il-17E, may be obtained from commercial sources (e.g., andes, MN, USA) or may be cloned or synthesized by reference to the sequences of Il-25 available in the art. The sequence of human IL-25(SEQ ID NO: 17) is shown in FIG. 3. For antibody production or use in immunoassays, any fragment or combination of fragments of the IL-25 protein (e.g., recombinant IL-25 protein), particularly those that are N-terminally truncated, can be used. For example, commercially available recombinant human IL-25(IL-17E) contains the mature protein sequence of Tyr 33-Gly 177 (accession No. Q9H293), and commercially available murine IL-25 contains the Val 17-Ala 169 residues of mouse IL-17E (accession No. NP 542767).

Nucleic acid molecules

Using the information provided herein, a nucleic acid molecule of the invention encoding at least one target binding member of the invention can be obtained using methods described herein or known in the art.

The nucleic acid molecules of the invention can be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA produced by cloning or synthesis, or any combination thereof. The DNA may be triplex, double stranded or single stranded or any combination thereof. Any portion of at least one strand of the DNA or RNA may be the coding strand, also referred to as the sense strand, or it may be the non-coding strand, also referred to as the antisense strand.

The isolated nucleic acid molecules of the invention may include nucleic acid molecules having an Open Reading Frame (ORF), optionally having one or more introns, such as, but not limited to, at least one designated portion of at least one CDR, such as CDR1, CDR2 and/or CDR3 of at least one heavy chain (e.g., SEQ ID NOS: 10-12) or light chain (e.g., SEQ ID NOS: 6-8); nucleic acid molecules having a coding sequence for an anti-IL-25 variable region (e.g., SEQ ID NO:5 or 9); and nucleic acid molecules having nucleotide sequences substantially different from those described above, but which, due to the degeneracy of the genetic code, still encode at least one anti-IL-25 antibody, as described herein and/or as known in the art. Of course, the genetic code is well known in the art. Thus, it will be apparent to those skilled in the art that such degenerate nucleic acid variants encoding a specific anti-IL-25 antibody of the present invention can be routinely produced. See, e.g., Ausubel et al (supra), and such nucleic acid variants are included in the present invention.

As noted herein, the nucleic acid molecules of the invention comprise nucleic acids encoding anti-IL-25 antibodies, which can include, but are not limited to, those nucleic acids that individually encode the amino acid sequences of antibody fragments; the coding sequence of the entire antibody or a portion thereof; the coding sequence for the antibody, fragment or portion and additional sequences, such as the coding sequence for at least one signal leader peptide or fusion peptide with or without additional coding sequences as described above; such as at least one intron; also included are additional non-coding sequences, including (but not limited to) non-coding 5 'and 3' sequences, such as transcribed, non-translated sequences that function in transcription, mRNA processing, including splicing and polyadenylation signals (e.g., ribosome binding and stabilization of mRNA); additional coding sequences that encode additional amino acids, such as those that provide additional functions. Thus, the sequences encoding the antibody may be fused to a marker sequence, e.g., a sequence encoding a peptide that may facilitate purification of the fused antibody comprising the antibody fragment or portion.

Polynucleotides which selectively hybridize to polynucleotides

The present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to the polynucleotides disclosed herein. Thus, the polynucleotides of this embodiment can be used to isolate, detect, and/or quantify nucleic acids comprising such polynucleotides. For example, the polynucleotides of the invention may be used to identify, isolate or amplify partial or full length clones in a deposited library. In some embodiments, the polynucleotide is an isolated genomic or cDNA sequence, or is complementary to a cDNA from a human or mammalian nucleic acid library.

Preferably, the cDNA library comprises at least 80% of the full-length sequence, preferably at least 85% or 90% of the full-length sequence, and more preferably at least 95% of the full-length sequence. cDNA libraries can be normalized to increase the appearance of rare sequences. Low or medium stringency hybridization conditions are generally, but not exclusively, used for sequences having reduced sequence identity relative to the complementary sequence. Medium and high stringency conditions can optionally be used for sequences with greater identity. Low stringency conditions allow for selective hybridization of sequences having about 70% sequence identity and can be used to identify orthologous or paralogous sequences.

Optionally, the polynucleotides of the invention will encode at least a portion of an antibody encoded by a polynucleotide described herein. The polynucleotides of the invention comprise nucleic acid sequences that can be used to selectively hybridize to polynucleotides encoding the antibodies of the invention. See, e.g., Ausubel (supra); colligan (supra), each of which is incorporated by reference herein in its entirety.

Construction of nucleic acids

The isolated nucleic acids of the present invention can be prepared using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, or a combination thereof, as is well known in the art.

The nucleic acid may conveniently comprise a sequence other than a polynucleotide of the invention. For example, a multiple cloning site comprising one or more endonuclease restriction sites can be inserted into a nucleic acid to aid in the isolation of the polynucleotide. In addition, translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the invention. For example, a hexahistidine tag sequence provides a convenient means for purifying the proteins of the invention. The nucleic acids of the invention are optionally, in addition to the coding sequences, vectors, adaptors or linkers for cloning and/or expression of the polynucleotides of the invention.

Additional sequences may be added to these cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve introduction of the polynucleotide into a cell. The use of cloning vectors, expression vectors, adapters and linkers is well known in the art. (see, e.g., Ausubel (supra); or Sambrook (supra))

Recombinant method for constructing nucleic acids

The isolated nucleic acid compositions of the present invention, e.g., RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using a variety of cloning methods known to those skilled in the art. In some embodiments, oligonucleotide probes that selectively hybridize under stringent conditions to a polynucleotide of the invention are used to identify a desired sequence in a cDNA or genomic DNA library. The isolation of RNA, and the construction of cDNA and genomic libraries, are well known to those of ordinary skill in the art. (see, e.g., Ausubel (supra); or Sambrook (supra))

Nucleic acid screening and isolation method

cDNA or genomic libraries can be screened with probes based on the sequences of the polynucleotides of the invention (e.g., those disclosed herein). Probes can be used to hybridize to genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. One skilled in the art will recognize that hybridization of various degrees of stringency can be used for the assay; and the hybridization or wash medium may be stringent. As the conditions for hybridization become more stringent, a greater degree of complementarity must exist between the probe and target where duplex formation occurs. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of partially denaturing solvents such as formamide. For example, the stringency of hybridization can be conveniently varied by varying the polarity of the reactant solution, for example, by manipulating the concentration of formamide in the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary depending on the stringency of the hybridization medium and/or wash medium. The degree of complementarity will optimally be 100% or 70 to 100% or any range or value therein. However, it is understood that minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and/or wash medium.

Methods for amplifying RNA or DNA are well known in the art and, based on the teachings and guidance presented herein, can be used in accordance with the present invention without undue experimentation. Known methods of DNA or RNA amplification include, but are not limited to, Polymerase Chain Reaction (PCR) and related amplification methods (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 to Mullis et al, U.S. Pat. Nos. 4,795,699 and 4,921,794 to Tabor et al, U.S. Pat. No.5,142,033 to Innis, U.S. Pat. No.5,122,464 to Wilson et al, U.S. Pat. No.5,091,310 to Innis, U.S. Pat. No.5,066,584 to Gylensten et al, U.S. Pat. No.4,889,818 to Gelfand et al, U.S. Pat. No.4,994,370 to Silver et al, U.S. Pat. No.4,766,067 to Biswas, U.S. Pat. No.4,656,134 to Ringold et al, and double-stranded RNA-mediated amplification using antisense RNA as a template for DNA synthesis (see, U.S. Pat. No.4,130, NASK 130, incorporated herein by reference). (see, e.g., Ausubel (supra); or Sambrook (supra))

For example, the sequences of polynucleotides of the invention and related genes can be amplified directly from genomic DNA or cDNA libraries using Polymerase Chain Reaction (PCR) techniques. For example, PCR and other in vitro amplification methods can also be used to clone nucleic acid sequences encoding proteins to be expressed, to prepare nucleic acids for use as probes to detect the presence of desired mRNA in a sample, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to guide the skilled artisan through in vitro amplification methods can be found in Berger (supra), Sambrook (supra), and Ausubel (supra), and U.S. Pat. No.4,683,202 to Mullis et al (1987); and Innis, et al, PCR Protocols A guides methods and Applications, eds, Academic Press Inc., San Diego, Calif. (1990) (edited by Innis et al, "PCR Protocols: guidelines for methods and Applications," Academic Press, San Diego, Calif., 1990). Commercial kits for genomic PCR amplification are known in the art. See, for example, Advantage-GC Genomic PCR Kit (Clontech). In addition, for example, the T4 gene 32 protein (Boehringer Mannheim) can be used to increase the yield of long PCR products.

Synthetic methods for constructing nucleic acids

Isolated nucleic acids of the invention can also be prepared by direct chemical synthesis by known methods (see, e.g., Ausubel et al, supra). Chemical synthesis generally produces single-stranded oligonucleotides that can be converted to double-stranded DNA by hybridization to a complementary sequence, or polymerization with a DNA polymerase using the single strand as a template. One skilled in the art will recognize that while chemical synthesis of DNA may be limited to sequences of about 100 or more bases, longer sequences may be obtained by ligating shorter sequences.

Recombinant expression cassette

The invention also provides recombinant expression cassettes comprising a nucleic acid of the invention. Nucleic acid sequences of the invention, such as cDNA or genomic sequences encoding an antibody of the invention, can be used to construct recombinant expression cassettes that can be introduced into at least one desired host cell. A recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to a transcription initiation regulatory sequence that will direct transcription of the polynucleotide in a predetermined host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the invention.

In some embodiments, an isolated nucleic acid that acts as a promoter, enhancer, or other element may be introduced at a suitable location (upstream, downstream, or in an intron) in a non-heterologous form of a polynucleotide of the invention in order to up-or down-regulate expression of the polynucleotide of the invention. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion, and/or substitution.

Vectors and host cells

The invention also relates to vectors comprising the isolated nucleic acid molecules of the invention, host cells genetically engineered with the recombinant vectors, and the production of at least one anti-IL-25 antibody by recombinant techniques well known in the art. See, e.g., Sambrook et al (supra); ausubel et al (supra), each incorporated by reference herein in its entirety.

The polynucleotide may optionally be linked to a vector containing a selectable marker for propagation in a host. Generally, the plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using a suitable packaging cell line and subsequently transduced into a host cell.

The DNA insert should be operably linked to a suitable promoter. The expression construct will also contain a transcription start site, a termination site, and a ribosome binding site for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct will preferably include a translation initiation codon at the beginning of the mRNA to be translated and a stop codon (e.g., UAA, UGA or UAG) at an appropriate position at the end of the mRNA, with UAA and UAG being preferred for mammalian or eukaryotic cell expression.

The expression vector will preferably, but optionally, include at least one selectable marker. Such labels include (for example, but are not limited to): for eukaryotic cell culture, Methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. nos. 4,399,216, 4,634,665, 4,656,134, 4,956,288, 5,149,636, 5,179,017), ampicillin, neomycin (G418), mycophenolic acid or glutamine synthetase (GS, U.S. Pat. No.5,122,464, 5,770,359, 5,827,739) resistance genes, and for escherichia coli (e.coli) and other bacterial or prokaryotic cultures, tetracycline or ampicillin resistance genes (the entire disclosures of which are incorporated herein by reference). Suitable media and conditions for the above-described host cells are known in the art. Suitable vectors will be apparent to the skilled person. Introduction of the vector construct into the host cell can be accomplished by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, or other known methods. Such methods have been described in the art, for example, Sambrook (supra), chapters 1, 4 and 16, 18; ausubel (supra), chapters 1, 9, 13, 15, 16.

At least one antibody of the invention may be expressed in a modified form (e.g., a fusion protein) and may include not only a secretion signal, but may also include additional heterologous functional regions. For example, regions of additional amino acids (particularly charged amino acids) may be added to the N-terminus of the antibody to improve stability and persistence in the host cell during purification or during subsequent handling and storage. Likewise, peptide moieties may be added to the antibodies of the invention to aid in purification. These regions may be removed prior to the final preparation of the antibody or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook (supra), chapters 17.29, 17.42 and 18.1, 18.74; ausubel (supra), chapters 16, 17 and 18.

One skilled in the art will recognize that many expression systems may be used to express nucleic acids encoding proteins of the present invention.

Alternatively, the nucleic acid of the invention may be expressed in a host cell by switching on (by manipulation) in the host cell comprising the endogenous DNA encoding the antibody of the invention. Such methods are well known in the art, for example, as described in U.S. Pat. nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, which are incorporated herein by reference in their entirety.

An example of a cell culture that can be used to produce an antibody, a particular portion or variant thereof, is a mammalian cell. The mammalian cell system will typically be in the form of a cell monolayer, but mammalian cell suspensions or bioreactors may also be used. A number of suitable host cell lines capable of expressing the entire glycosylated protein have been developed in the art, including COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL 1651), HEK293, BHK21 (e.g., ATCC CRL 10), CHO (e.g., ATCC CRL 1610), and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2/0-Ag14, 293 cells, HeLa cells, and the like, which are readily available from, for example, the American Type Culture Collection, assas, Va. Preferred host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC accession number CRL-1580) and SP2/0-Ag14 cells (ATCC accession number CRL-1851). In particularly preferred embodiments, the recombinant cell is a P3X63Ab8.653 or SP2/0-Ag14 cell.

The expression vector for these cells may include one or more of the following expression control sequences, such as, but not limited to: an origin of replication; promoters, such as the late or early SV40 promoter, the CMV promoter (U.S. Pat. No.5,168,062, 5,385,839), the HSV tk promoter, the pgk (phosphoglycerate kinase) promoter, the EF-1. alpha. promoter (U.S. Pat. No.5,266,491), at least one human immunoglobulin promoter; enhancers and/or processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., SV40 large T Ag poly A addition sites), and transcription terminator sequences. See, e.g., Ausubel et al (supra); sambrook et al (supra). Other cells useful for producing the nucleic acids or proteins of the invention are also known and/or may be obtained, for example, from the american type culture collection cell line and hybridoma catalogue (www.atcc.org) or other known or commercial sources.

When eukaryotic host cells are employed, polyadenylation or transcription termination sequences are typically incorporated into the vector. An example of a termination sequence is a polyadenylation sequence from the bovine growth hormone gene. Sequences for correct splicing of transcripts may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al, J.Virol.45: 773781 (1983)) (Sprague et al, J.Virol.45, pp.773-781, 1983)). In addition, gene sequences that control replication in the host cell can be integrated into the vector, as is known in the art.

Composition comprising a metal oxide and a metal oxide

The present invention also provides at least one target binding member composition comprising at least one, at least two, at least three, at least four, at least five, at least six, or more target binding members as described herein and/or known in the art, provided in a non-naturally occurring composition, mixture, or form. Such composition percentages are calculated as weight, volume, concentration, molarity, or molarity of a liquid or anhydrous solution, mixture, suspension, emulsion, or colloid, as known in the art or described herein.

The compositions of the present invention may further comprise any suitable and effective amount of at least one of a composition or pharmaceutical composition comprising at least one anti-IL-25 target binding member for administration to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy, optionally further comprising at least one agent selected from the group consisting of: at least one TNF antagonist (such as, but not limited to, a TNF antibody or fragment, a soluble TNF receptor or fragment, a fusion protein thereof, or a small molecule TNF antagonist), an antirheumatic agent (such as methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalazine), a muscle relaxant, an anesthetic (narcotic), a non-steroidal anti-inflammatory drug (NSAID), an analgesic, an anesthetic (anesthesic), a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial agent (such as an aminoglycoside, an antifungal agent, an antiparasitic agent, an antiviral agent, a carbapenem, a cephalosporin, a fluoroquinolone, a macrolide, a penicillin, a sulfonamide, a tetracycline, an additional antimicrobial agent), an antipsoriatic agent, a corticosteroid, an anabolic steroid, a diabetes-related agent, Minerals, nutrients, thyroid agents, vitamins, calcium-related hormones, antidiarrheals, antitussives, antiemetics, antiulcers, laxatives, anticoagulants, erythropoietins (e.g., erythropoietin α), filgrastims (e.g., G-CSF, oxyphenbutazone), sargramostim (GM-CSF, tumor), vaccinants, immunoglobulins, immunosuppressive agents (e.g., basiliximab, cyclosporine, daclizumab), growth hormones, hormone replacement drugs, estrogen receptor modulators, mydriatic agents, cycloplegics, alkylating agents, antimetabolites, mitotic inhibitors, radiopharmaceuticals, antidepressants, antimanics, antipsychotics, anxiolytics, hypnotics, sympathomimetic agents, stimulants, donepezil, tacrine, asthma medications, beta agonists, inhaled steroids, Leukotriene inhibitors, methylxanthines, cromolyn, epinephrine or the like, alpha-streptokinase (in bermuda), cytokines or cytokine antagonists. Non-limiting examples of such cytokines include, but are not limited to, any of IL-1 through IL-23. Suitable dosages are well known in the art. See, e.g., Wells et al, eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000) (edited by Wells et al, Handbook of drug therapy, 2nd Edition, alpton and Lange, stanford, connecticut); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia2000, Delluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000) (PDR Pharmacopoeia: Tarason Pocket Pharmacopoeia2000, hardcopy, Tarason Publishing Co., Loma, Calif.), each of which is incorporated by reference herein in its entirety.

Such anti-cancer or anti-infective agents may also include a toxin molecule associated, bound, co-formulated, or co-administered with at least one antibody of the invention. The toxin may optionally act to selectively kill pathological cells or tissues. The pathological cells may be cancer cells or other cells. Such toxins may be, but are not limited to, purified or recombinant toxins or toxin fragments comprising at least one functional cytotoxic domain of a toxin, such as at least one selected from ricin, diphtheria toxin, venom toxin, or bacterial toxin. The term toxin also includes endotoxins and exotoxins produced by any naturally occurring, mutant or recombinant bacterium or virus that can cause any pathological condition in humans and other mammals, including toxin shock, which can lead to death. Such toxins may include, but are not limited to, enterotoxigenic e.coli heat-labile enterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin (Shigella), Aeromonas enterotoxin (Aeromonas) enterotoxin, toxic shock syndrome toxin-1 (TSST-1), Staphylococcal enterotoxin (staphyloccal) a (sea), b (seb), or c (sec), Streptococcal enterotoxin (streptococcus), and the like. Such bacteria include, but are not limited to, strains of the following bacteria: enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (e.g., serotype 0157: H7 strain), Staphylococcus (e.g., Staphylococcus aureus (Staphylocccus aureus), Staphylococcus suppurativa (Staphylocccus pyenens)), Shigella (e.g., Shigella dysenteriae (Shigella dysenteriae), Shigella flexneri (Shigella flexneri), Shigella pallidus (Shigella boydii) and Shigella sonnei (Shigella sonnei), Salmonella (e.g., Salmonella typhi (Salmonella typhi), Salmonella cholera (Salmonella cholera-suris), Salmonella enteritidis (Salmonella enteritidis)), Clostridium (e.g., Clostridium perfringens (Clostridium), Clostridium difficile (Clostridium difficile), Clostridium botulinum (Clostridium), Clostridium fetida (e.g., Clostridium perfoliatum (Clostridium), Campylobacter (Campylobacter) strain (e.g., Campylobacter coli (Campylobacter) strain (Campylobacter) such as the strain, and the like, Aeromonas (e.g., Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae, Aeromonas shigelloides, Yeissomas shigelloides, Yersinia enterocolitica, Vibrio (Vibrio cholerae), Vibrio (Vibrio parahaemolyticus), Klebsiella, Pseudomonas aeruginosa, and Streptococcus). See, e.g., Stein, ed., INTERNAL MEDICINE, 3rd ed., pp 113, Little, Brown and co., Boston, (1990) (Stein editions, science, 3rd edition, pages 1-13, litter and braun publishers, Boston, 1990)); evans et al, eds., Bacterial Infections of Humans: epidemic and Control, 2d. Ed., pp 239254, Plenum Medical Book Co., New York (1991) (edited by Evans et al, human bacterial infection: Epidemiology and Control, 2nd edition, p.239-; mandell et al, Principles and Practice of Infectious Diseases, 3d.Ed., Churchill Livingstone, N.Y. (1990) (Mandell et al, Principles and Practice of Infectious Diseases, 3rd edition, Cugilles Liwenston Press, New York, 1990); berkow et al, eds., The Merck Manual, 16th edition, Merck and Co., Rahway, N.J., 1992 (edited by Berkow et al, "Merck medical Manual", 16th edition, Merck corporation, Lawei, New Jersey, 1992); wood et al, FEMS Microbiology Immunology, 76: 121134 (1991) (Wood et al, Association of European microbiology, microbial immunology, Vol.76, pp.121-134, 1991); marrack et al, Science, 248: 705711 (1990) (Marrack et al, science, Vol.248, pp.705-711, 1990), the entire contents of these references are incorporated herein by reference.

The compositions of the present invention may also comprise at least one of any suitable adjuvants, such as, but not limited to: diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants and the like. Pharmaceutically acceptable adjuvants are preferred. Non-limiting examples and methods of preparing such sterile solutions are well known in the art, such as, but not limited to, Gennaro, Ed., Remington's pharmaceutical sciences, 18th Edition, Mack Publishing Co., Easton, Pa.)1990(Gennaro, ed., Remington pharmaceutical sciences, 18th Edition, Mike Publishing company, Iston, Pa.). Pharmaceutically acceptable carriers suitable for the mode of administration, solubility and/or stability of the anti-IL-25 target binding member, fragment or variant composition may be selected in a conventional manner, as is well known in the art or as described herein.

Pharmaceutical excipients and additives useful in the compositions of the present invention include (but are not limited to): proteins, peptides, amino acids, lipids and carbohydrates (e.g. sugars including mono-, di-, tri-, tetra-and oligosaccharides; derivatised sugars such as sugar alcohols, aldonic acids, esterified sugars etc.; and polysaccharides or sugar polymers), pharmaceutical excipients and additives may be present alone or in combination, having 1-99.99% by weight or volume, alone or in combination. Exemplary protein excipients include serum albumin, such as Human Serum Albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/antibody components that may also play a role in buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

Carbohydrate excipients suitable for use in the present invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), inositol, and the like. Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose and raffinose.

The composition may also include a buffering agent or a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; tris hydrochloride or phosphate buffer. Preferred buffers for use in the compositions of the present invention are organic acid salts, such as citrate.

Additionally, the compositions of the present invention may include polymeric excipients/additives such as polyvinylpyrrolidone, polysucrose (polymeric sugar), dextrates (e.g. cyclodextrins, such as 2-hydroxypropyl- β -cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g. polysorbates, such as "TWEEN 20" and "TWEEN 80"), lipids (e.g. phospholipids, fatty acids), steroids (e.g. cholesterol) and chelating agents (e.g. EDTA).

These and further known pharmaceutical excipients and/or additives suitable for use in the composition according to the invention are known in the art, for example in "Remington: the Science & Practice of Pharmacy, 19th ed., Williams & Williams, (1995) ("Remington: pharmaceutical sciences and practices", 19th edition, Williams and Williams publishers, 1995), and "Physician's DeskReference", 52nd ed., Medical Economics, Montvale, N.J. (1998) ("physicians' case head reference", 52nd edition, Medical Economizer, N.J. (1998), The disclosure of which is incorporated herein by reference in its entirety. Preferred carrier or excipient materials are carbohydrates (e.g. sugars and sugar alcohols) and buffers (e.g. citrate) or polymeric agents.

Therapeutic applications

In one aspect, the invention provides a method of preventing or reducing airway hyperresponsiveness in a subject (e.g., a human) in need of treatment, the method comprising administering to the subject a target binding member, particularly an antibody molecule that binds IL-25. In another aspect, the invention provides a method of preventing, alleviating or treating asthma in a subject in need of such treatment, the method comprising administering to the subject a target binding member, particularly an antibody molecule that binds IL-25. Asthma includes allergic asthma.

The above methods may be practiced using target binding members (including compositions thereof) according to the invention for binding to and preferably antagonizing the action of IL-25, with therapeutic potential in various diseases and disorders in which IL-25 plays a role. In addition to asthma, such diseases include other conditions associated with inflammation, such as Inflammatory Bowel Disease (IBD), e.g., crohn's disease and ulcerative colitis. The method may also be carried out using other target binding members (including compositions thereof) that bind IL-25, which target binding members are obtainable according to the methods described in the accompanying examples below.

Target binding members according to the invention (including compositions thereof) may be used in therapeutic (including prophylactic treatment) or diagnostic methods in human or animal subjects. Such therapeutic or diagnostic methods, which may include prophylactic treatment, may comprise administering to the subject an effective amount of a target binding member of the invention. Exemplary diseases and disorders are discussed further below.

Also provided is the use of a target binding member of the invention (including compositions thereof) in the manufacture of a medicament for administration to a human or animal subject.

Clinical indications in which anti-IL-25 target binding members may be used to provide a therapeutic benefit include any condition in which IL-25 has a pathological consequence. Thus, the target binding members of the invention may generally be used in the treatment of any condition associated with an unwanted Th2 response or type 2 response. For example, the target binding members of the invention may be used in the treatment of allergy and asthma, particularly asthma.

anti-IL-25 treatment can be given by injection (such as intravenous injection) or by local delivery methods. anti-IL-25 can be delivered by gene-mediated techniques. Alternative formulation strategies may provide formulations suitable for the oral or suppository routes. The route of administration may be determined by the physicochemical characteristics of the treatment, by special consideration of the disease, to optimize efficacy or to minimize side effects.

According to the present invention, the provided compositions may be administered to an individual. Administration is preferably carried out in a "therapeutically effective amount", which is sufficient to show a beneficial effect to the patient. Such a benefit may be at least an improvement in at least one symptom. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decision of dosage etc. is the responsibility of general practitioners and other physicians. Appropriate antibody dosages are well known in the art; see Ledermann j.a.et al (1991) int.j.cancer 47: 659-; bagshawe k.d.et al (1991) antibodies, Immunoconjugates and Radiopharmaceuticals 4: 915-922(Bagshawe K.D. et al, 1991, antibodies, immunoconjugates and radiopharmaceuticals, Vol.4, page 915-922).

The precise dosage will depend on a variety of factors, including whether the antibody is for diagnosis or treatment, the size and location of the area to be treated, the exact nature of the antibody (e.g., whole antibody, fragment, or diabody), and the nature of any detectable label or other molecule attached to the antibody. A typical antibody dose will be in the range of 0.5mg-1.0g, and the antibody may optionally be administered intravenously by bolus injection or infusion for several hours to achieve the desired dose. Other modes of administration include intravenous infusion over several hours to achieve a similar total cumulative dose. This is the dose for a single treatment of an adult patient, which can be scaled up for children and infants, and for other antibody formats also scaled up for molecular weight. Treatment may be repeated at daily, twice weekly, or monthly intervals, at the discretion of the physician.

Other modes of administration utilize a pre-coating of the indwelling device, or otherwise incorporate the indwelling device, for which the optimal amount of antibody will be determined by appropriate experimental methods.

In some preferred embodiments of the invention, the antibody molecule is a monomeric fragment, such as f (ab) or scFv. Such antibody fragments may have the advantage of a relatively short half-life and less risk of platelet activation, which may be triggered by receptor aggregation. The aggregation that causes platelet activation can be, for example, aggregation of IL-25 molecules or aggregation of IL-25 with Fc γ RII molecules.

If whole antibodies are used, it is preferred that the form is one that does not activate and/or destroy platelets. The IgG4 isotype, or alternatively, the "designer" isotype derived from the IgG1 backbone (novel Fc gene construct WO99/58572, Clark, Armour, Williamson) is the preferred choice. Smaller antibody fragments, such as F (ab') 2, may be used. Furthermore, whole antibodies or fragments (such as F (ab') 2 or diabodies) with bi-epitope specificity (such as for the epitope recognized by scFv 2C 3) can be used. While this example may promote receptor aggregation, a high association rate with a single receptor may eliminate this problem.

The target binding member of the invention will typically be administered in the form of a pharmaceutical composition which may comprise at least one component in addition to the target binding member.

The target binding members of the invention may be administered alone or in combination with other therapeutic modalities, simultaneously or sequentially, depending on the condition to be treated. Other treatment modalities may include administration of appropriate doses of analgesics such as non-steroidal anti-inflammatory drugs (e.g., aspirin, acetaminophen, ibuprofen, or ketoprofen) or opioids such as morphine; administering an antiemetic; or administering at least one other compound active in asthma, typically a bronchodilator that produces airway relaxation or enhances mucus clearance, e.g. a beta-agonist (such as salbutamol, salmeterol), disodium cromoglycate, a steroid or a PDEIV inhibitor.

Measurement method

The invention provides methods comprising eliciting or allowing binding of a target binding member provided herein to IL-25. As noted, such binding can occur in vivo, such as after administration of the target binding member or nucleic acid encoding the target binding member, or can occur in vitro, such as in ELISA, Biacore assays, Octet assays, western blots, immunocytochemistry, immunoprecipitation, or affinity chromatography.

The amount of target binding member bound to IL-25 can be determined. Quantification may be correlated with the amount of antigen in the test sample, which may be of diagnostic importance.

The activity of the antibody on the sample can be determined by any suitable method. Radioimmunoassay (RIA) is a viable method. The radiolabeled antigen is mixed with unlabeled antigen (test sample) and allowed to bind to the antibody. The bound antigen is physically separated from the unbound antigen and the amount of radioactive antigen bound to the antibody is determined. The more antigen in the test sample, the less radioactive antigen is bound to the antibody. Competitive binding assays can also be performed with nonradioactive antigens using antigens or analogs linked to reporter molecules. The reporter molecule may be a fluorescent dye, phosphor or laser dye with spectrally separated absorption or emission characteristics. Suitable fluorescent dyes include fluorescein, rhodamine, phycoerythrin, and texas red. Suitable chromogenic dyes include diaminobenzidine.

Other reporter molecules include macromolecular colloidal particles or particulate matter such as colored, magnetic, or paramagnetic latex beads, as well as biologically or chemically active agents that can directly or indirectly produce a detectable signal that can be visually observed, electronically detected, or otherwise recorded. These molecules may be enzymes which catalyze reactions such as colour development or change or cause changes in electrical properties. They may be of the molecular excitation type, such that electronic transitions between energy states result in characteristic spectral absorption or emission. They may include chemical entities used in conjunction with biosensors. A biotin/avidin or biotin/streptavidin and alkaline phosphatase detection system may be employed.

The signal generated by a single antibody-reporter conjugate can be used to derive quantifiable absolute or relative data for binding of the relevant antibody in the sample (both the conventional sample and the test sample).

The present invention also provides a method of measuring antigen levels in a competition assay using a target binding member as described above, that is, a method of measuring antigen levels in a sample by employing a target binding member provided by the present invention in a competition assay. In this case it may not be necessary to physically separate the bound antigen from the unbound antigen. One possible approach is to link the reporter molecule to the target binding member such that a physical or optical change occurs upon binding. The reporter molecule may directly or indirectly produce a detectable, and preferably measurable, signal. The linkage of the reporter molecule may be direct or indirect, covalent (e.g., via a peptide bond), or non-covalent. Linkage via a peptide bond may be the result of recombinant expression of the fusion gene encoding the antibody and reporter molecule.

The invention also provides for direct measurement of the level of antigen by employing a target binding member according to the invention, for example in a biosensor system.

Those skilled in the art will be able to select the appropriate mode or combination based on their preferences and general knowledge.

Examples of the invention

Exemplary embodiments of the invention are described as follows:

example 1: construction of high affinity humanized IL-25 antibody M6

huDDG91 is a humanized (CDR grafted) version of the murine 2C3 monoclonal anti-IL 25 antibody, selected for use as the parent molecule for constructing variants with improved binding affinity for IL-25. The kappa light chain sequence (excluding the leader sequence) of huDDG91 is shown in FIG. 1 (SEQ ID NO: 2), where the amino acid sequences of the CDR loops (as defined by Kabat) are underlined. The sequence of the heavy chain of huDDG91 (SEQ ID NO: 4) is shown in FIG. 2, where the amino acid sequences of the CDRs (as defined by Kabat) are underlined. huDDG91 is also known as RH2.5_ R71V. The construction and characterization of the murine 2C3 monoclonal anti-IL 25 antibody is described in the following patents: international patent application No.: PCT/GB2008/001365, the entire contents of which are incorporated herein by reference.

To obtain variants of huDDG91, phage display libraries based on huDDG91 sequences were constructed using standard methods. The Fab library was panned against biotinylated IL-25. Fab was selected that showed comparable or higher binding (as determined by ELISA) to huDDG91(IgG 4).

Fab was converted to mAb in the following manner. Combinatorial libraries containing 5 different light chains and 5 different heavy chains (including huDDG91 light and heavy chains) were designed. The light and heavy chains used in the library differ from each other by the light chain CDR1 and CDR3 amino acid sequences and the heavy chain CDR3 sequences. A total of 25 mabs (IgG1) were constructed using these light and heavy chains, which were expressed on a small scale in HEK293 cells, and these mabs were purified. Each mAb was tested for IL-25 binding affinity, cytostatic, and receptor inhibitory properties as follows:

● the binding affinity of IL-25 was determined using a standard IL-25ELISA and Biacore binding assay. Selection of K for human IL-25_DK of less than 50pM and for human IL-17A, C, D and F_DCandidates greater than 100 nM;

● cytostatic was assessed using standard TK-10 human renal cancer cell assay (IL-25 response; IL-25 and IL-8 readings; differences between soluble IL-25R and parental mAb could be distinguished) and CD4+ T cell assay. For the TK-10 assay, mAbs exhibiting IC50 lower than the parental huDDG91 antibody and > 90% inhibition of IL-25 release at 10nM mAb concentration were selected. For the CD4+ T cell assay, mabs were selected: its inhibitory activity on IL-5 secretion is greater than or equal to that exhibited by the parent huDDG 91;

● improved microsphere-based electrochemiluminescence immunoassay (ECLIA) methods were used to assess receptor inhibition. Candidates were selected that reduced IL-25 binding to the receptor by more than three-fold the IC50 value compared to the parent huDDG91 antibody at 10nM mAb concentration.

Based on these criteria, 9 candidate mabs were selected (fig. 3). When expressed in HEK293 and purified using standard protocols, all candidates showed acceptable expression levels and exhibited the desired SE-HPLC and SDS-PAGE profiles. These 9 candidates were then transiently expressed in Chinese Hamster Ovary (CHO) cells on a 10L scale, purified using Mab Select SuRe (GE Healthcare Life Sciences), buffer-exchanged to Phosphate Buffered Saline (PBS) and concentrated. In each of these 9 candidate mAbs, there was a significant improvement in IL-25 binding affinity compared to huDDG 91. The expression level, class (SDS-PAGE), solubility, aggregation (SE-HPLC), post-translational modifications, protein-protein interactions and oxidation of each mAb were also tested using conventional techniques and assays. When expressed in Chinese hamster ovary cells and purified and processed under standard conditions, various candidate mAbs precipitate during concentration and exhibit low yields and/or undesirable SDS-PAGE and SE-HPLC profiles.

One mAb (designated M6) was selected for further study based on excellent expression levels, high solubility, no significant protein aggregation upon purification, and the absence of undesirable post-translational modifications, protein-protein interactions, and oxidation upon purification.

Example 2: characterization of the M6 antibody

Materials and methods

LS174T human colonic epithelial cell assay

Human colonic epithelial cell line LS174T (CL-188) was obtained from ATCC (Manassas, Va.) and 5% CO at 37 deg.C₂Maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen, Carlsbad, Calif.) supplemented with 10% FBS, penicillin and streptomycin, cells were seeded at a density of 1 × 105 cells/ml in a total volume of 100 μ l onto 96-well tissue culture plates and allowed to adhere overnight IL-25 antibodies (M6 and M9) were pre-complexed with recombinant human IL-25 protein (Centocor), where a fixed amount of IL-25 protein (10ng/ml final concentration) was mixed with varying amounts of anti-IL-25 antibody titrated into a three-fold or half-log dilution series and incubated at 37 ℃ for 1 hour, the cell culture medium was gently aspirated and replaced with IL-25 protein/IL-25 antibody complex, the cells were then incubated overnight for 18-22 hours, the plates were then centrifuged at 1200rpm for 2 minutes, to eliminate residual contamination of cell debris, the supernatant was collected and plated with an ELISA plate (Andy organism of Minneapolis, Minn.)&D Systems, Minneapolis, MN)) analysis of GROa。

Results

Sequence analysis of M6 showed that a total of 3 amino acid substitutions occurred relative to the huDDG91 sequence, all of which were present in the CDR3 of the light chain (see fig. 3). The amino acid sequences of the light chain (SEQ ID NO: 5) and heavy chain (SEQ ID NO: 9) of M6 and the CDRs thereof are shown in FIG. 4.

Binding affinity of M6 to human IL-25 was determined by Biacore to be 3 to 5 times higher than that of huDDG91 to human IL25 (FIG. 3). In addition, M6 has selectivity for IL-25, because it does not bind to human IL-17A, C, D or F. Species cross-reactivity of M6 was assessed using cynomolgus monkey I2-25cynoIL-25 and rodent (mouse) IL-25. Species cross-reactivity with cynoIL-25 is indicated by within 5-fold human IL-25 affinity, receptor ligand inhibition, and TK-10 cell-related assays. These criteria were used to determine that M6 binds to both cyno-and mouse IL-25.

Using the cell-related assay method described in example 1, M6 was shown to have enhanced receptor inhibition relative to huDDG91, while its IC50 showed a 3-fold greater reduction compared to huDDG91 (fig. 3). Furthermore, using the cell-related assay method described in example 1, M6 was shown to be enhanced in cytostatic properties of human IL25 compared to huDDG91 (fig. 3 and 5). This result was confirmed by LS174T human colonic epithelial cell assay performed according to the method described above (fig. 6A and 6B).

Example 3: H/D exchange Pattern of human IL-25 epitope recognized by M6

Disclosure of Invention

ExSar (Monmouth Junction, New Jersey) by ExSar corporation was used^TMHydrogen/deuterium exchange mass spectrometry was used to map the putative epitope of human IL-25 recognized by the M6 antibody.

Materials and methods

Optimization of digestion/isolation of IL-25

(1) Thawing of IL-25 on ice

(2) Aliquots were aliquoted at 18X 100. mu.L and one tube was left, and the remainder was frozen.

(3) mu.L of 0.28mg/mL (16.6. mu.M) of IL-25 was mixed with 30. mu.L of aqueous PBS (pH 7.0) → [ IL-25] ═ 0.07mg/mL (4.2. mu.M)

(4) Mixing 40 μ L of (3) with different quenching solutions

(5) Inject 55 μ L into ExSAR System

(6) The sample was passed through an immobilized pepsin column with buffer A (0.05% aqueous TFA) at a flow rate of 200. mu.L/min

(7) The peptide fragments were loaded onto a reverse phase trap column and desalted using buffer A at a flow rate of 200. mu.L/min for 3 minutes

(8) The pepsin peptides were separated by C18 chromatography column for 23 min using a linear gradient of 13% → 40% buffer B (95% acetonitrile, 5% H2O, 0.0025% TFA)

(9) Detection of peptides using mass spectrometry

M6 immobilization

Experimental procedure for M6 immobilization

<Binding of antibodies on resin>

(1) Thaw 1.5mL of 0.96mg/mL M6 on ice

(2) Using 400. mu.L (re-freezing unused material)

(3) 4mg of NaCNBH₃(87.5. mu. mol) into a 1.5mL screw-capped bottle → 40mg NaCNBH per 1g POROS AL₃

(4) Add 400. mu.L of antibody solution to the vial

(5) Ensuring NaCNBH₃Adding POROS AL resin after dissolving

(6) 100mg of POROS AL was added to the bottle

(7) Incubating the coupling reaction solution at room temperature for 3 hours while shaking

(8) Adding 2.8M Na with total volume of 400 μ L in 5 × 80 μ L portions₂SO₄Hourly part → [ antibodies ]]＝0.48mg/mL，[NaCNBH₃]＝5.0mg/mL[Na₂SO₄]＝1.4M。

(9) The mixture was shaken at room temperature overnight

(10) The mixture was washed through the filter funnel using a large amount of PBS buffer (pH 7.0)

<End capping>

(11) Preparation of end-capping solution: 250 μ L of ethanolamine (FW 61.08, density 1.012g/mL, 8.3mmol, about 1M) was mixed into 3.5mL of PBS (pH 7.0) and the pH of this solution was adjusted to 7.2 using glacial acetic acid (about 200 μ L). The final volume of the solution was close to 4 ml.

(12) Dissolve 4mg NaCNBH3 in 500. mu.L capping solution → [ NaCNBH ]₃]Not greater than 8mg/mL, [ ethanolamine ]]About 1M.

(13) Resuspending the washed dry resin in NaCNBH₃In solution

(14) Shaking at room temperature for 2 hours

(15) The mixture was filtered through a filter funnel and rinsed with copious amounts of PBS (pH 7.0).

(16) The resin cake was resuspended in 0.75mL of PBS buffer (pH 7.0).

(17) The coupling material was stored in a refrigerator at 4 ℃.

Experimental procedure for binding Capacity testing of M6 chromatography columns

<Preparation of buffer>

(1) A50 mM aqueous solution of citrate, pH 6.0, was prepared

(2) A50 mM citrate, 2mM Foscholine-12 aqueous solution was prepared at pH 6.0

(3) Preparation of an aqueous PBS solution at pH 7.0

(4) Any of (1), (2) or (3) is used as "buffer H"

<Binding Capacity test>

(5) mAb column (104. mu.L) was packed with 600. mu.L of POROS resin-coupled M6 using 500. mu.L/min flow rate of buffer A (0.05% aqueous TFA) and 2.1 mm. times.30 mm stainless steel jacket

(6) The antibody column was placed in a reservoir bath of a refrigerator set at 3 ℃ with tubing set for input and output of reagents from the column and capture of reagents in and out of the column. A2 micron filter head was aligned with the input line to filter the reagents and sample.

(7) The antibody column was equilibrated with 2X 250. mu.L of "buffer H". The pH of the solution at the end of the line was tested using pH paper to ensure that the pH was neutral.

(8) mu.L of 0.28mg/mL (16.6. mu.M) of IL-25 was mixed with 30. mu.L of "buffer H" → [ IL-25] → 4.1. mu.M (corresponding to 166pmol)

(9) The mixture was injected into the equilibrated antibody column.

(10) 200 μ L of "buffer H" (placed in a 500 μ L Hamilton syringe) was transferred into the column using a syringe pump at 3 ℃ and fractions of 5X 40 μ L were collected.

(11) 200 μ L of 0.8% formic acid was transferred to the column using a syringe pump at 3 ℃ and fractions of 5X 40 μ L were collected.

(12) Control injections were prepared in glass lined tubes: mu.L of 0.28mg/mL (16.6. mu.M) IL-25 was mixed with 30. mu.L of "buffer H

(13) All the liners containing the fraction or control samples were centrifuged to allow all the liquid on the liner wall to settle. The sample bottle was capped and labeled, and the liner was placed inside the bottle.

(14) The neutral and acidic fractions and the control sample were kept in the stacked cold plate 4, placed in the order of control sample, neutral washes 1, 2, 3, 4,5 and acidic washes 1, 2, 3, 4,5 in odd numbered positions from 3 to 23.

(15)11 capped empty bottles were placed in the stacked cold plate 4, in order at even positions from 4 to 24.

(16) The fractions were mixed using 20. mu.L of 2M urea, 1M TCEP (pH 3.0).

(17) The 55 μ L of quench solution was injected into the ExSAR system without the need for a pepsin chromatographic column.

(18) The sample was loaded onto the trap column using buffer a (0.05% TFA) at a flow rate of 200 μ L/min, desalted for 3 min, and then eluted with a linear gradient of 13% to 40% buffer B (95% acetonitrile, 5% H2O, 0.0025% TFA) for 23 min.

(19) Using mass spectrometry at MS 1: the eluate was analyzed in bar graph mode.

Experimental procedure for solution Forward/column reverse exchange

<Preparation of buffer>

(1) A50 mM citrate, 2mM Foscholine-12 aqueous solution was prepared at pH 6.0

(2) An aqueous solution of PBS, 2mM Foscholine-12, pH 7.0 was prepared

(3) An aqueous solution of PBS was prepared at pH 7.0

(4) Use of (1) to (3) as "exchange liquid H"

(5) Preparation of "exchange liquid HH": one part of aqueous PBS solution with pH 7.0 was mixed with 3 parts of "exchange solution H

(6) A50 mM citrate, 2mM Foscholine-12, heavy aqueous solution was prepared at pH 6.0

(7) PBS, 2mM Foscholine-12 in heavy water, pH 7.0 was prepared

(8) A heavy aqueous solution of PBS was prepared at pH 7.0

(9) Use of (6) to (8) as "exchange liquid D"

(10) Preparation of "exchange liquid HD": 1 part of an aqueous PBS solution having a pH of 7.0 was mixed with 3 parts of "exchange solution D

<Forward exchange of solution>

(1) The mAb column (104. mu.L bed volume) was placed in a 3 ℃ cooling box and equilibrated

(2) mAb column was washed with 2X 250. mu.L of 0.8% formic acid

(3) The mAb column was washed with 2X 250. mu.L of "exchange HD" to equilibrate the column

(4) mu.L of 0.28mg/mL (16.6. mu.M) of IL-25 was mixed with 30. mu.L of "exchange solution D" at 3 → [ IL-25] ═ 0.07mg/mL (4.2. mu.M), [ D2O ] ═ 75% (start timer, record forward exchange time)

(5) Incubating the mixture at 3 ℃ for 150, 500, 1,500, or 5,000 seconds

(6) The mixture (40. mu.L) was injected into a mAb column

(7) Wash mAb column at 3 ℃ using 100. mu.L of "exchange HD

<Reverse exchange of columns>

(8) Using 200. mu.L of cooled "exchange liquid HH" (stop forward exchange time and start recording reverse exchange time once water contacts the column)

(9) Incubation at 23 ℃ for 75, 250, 750 or 2,500 seconds

<Elution is carried out>

(10) mu.L of cooled 0.8% formic acid was injected onto the mAb column (once the acid entered the column, stop reverse exchange time)

(11) An additional 40. mu.L of cooled 0.8% formic acid was injected to elute the antigen from the mAb column

(12) The 40 μ L fraction was collected using a glass liner

<Analysis of>

(13) mu.L of cooled 2M urea, 1M TCEP (pH 3.0) was added to the 40. mu.L fraction

(14) A55 μ L quench-exchanged sample was injected into an ExSAR system equipped with a pepsin column and a C18 column (pepsin column 104 μ L bed volume; flow rate through pepsin column 200 μ L/min; buffer B flowed through C18 column at a gradient of 13% -40% for 23 min). Digestion time was set at 3 minutes.

(15) Using mass spectrometry at MS 1: analysis of eluate in Profile mode

Experimental procedure for column Forward/column reverse exchange

<Column forward exchange>

(1) The mAb column (104. mu.L bed volume) was placed in a 23 ℃ cooling box and equilibrated

(2) mAb column was washed with 2X 250. mu.L of 0.8% formic acid

(3) Equilibration of the column by washing the mAb column with "exchange HH

(4) mu.L of 0.28mg/mL (16.6. mu.M) of IL-25 was mixed with 30. mu.L of "exchange solution H" at 3 → [ IL-25 → []＝0.07mg/mL(4.2μM)，[D₂O]＝0％

(5) Injecting the mixture into a mAb chromatographic column

(6) Washing was carried out with 100. mu.L of "exchange liquid HH

(7) 200 μ L of "exchange solution HD" was passed through the mAb column (forward exchange time was recorded at the beginning) to initiate the forward exchange reaction

(8) Incubation of mAb column at 3 ℃ for 150, 500, 1,500, or 5,000 seconds

< reverse exchange column > same as 5.2

< elution > same as procedure IV, steps 10-14 above.

< assay > same as procedure IV, step 15 above.

Experimental procedure for complete deuteration experiments

<Preparation of fully deuterated samples>

(1) mu.L of 0.28mg/mL (16.6. mu.M) IL-25 was mixed with 30. mu.L of "exchange solution D

(2) Heating the mixture at 60 deg.C for 3 hr

(3) Cooling it to room temperature

(4) 40 μ L of the mixture was loaded onto a mAb chromatography column

(5) 100 μ L of "exchange solution HD" was injected into the mAb column

< elution > same as procedure IV, steps 10-14 above.

< assay > same as procedure IV, step 15 above.

Any deuterium attached to the first two amino acid residues of each ion during analysis (digestion/separation/mass analysis in an aqueous environment) is absent. This explains the small gaps in the H/D-Ex pattern in FIGS. 8A and 8B. Non-deuterated experiments, forward-exchange experiments and fully-deuterated experiments were performed for each protein. The non-deuteration experiments were used to identify the precise m/z of the ions and each ion that contained no deuterium. A complete deuteration experiment was used to identify deuterium depletion of each ion during analysis (digestion/separation/mass analysis in an aqueous environment). In these types of experiments, the number of deuterons before LCMS analysis and after forward exchange reactions or forward-reverse exchange reactions can be back-calculated. For the forward-reverse crossover experiments, the reverse crossover time was half the forward crossover time. This is because the intrinsic H → D exchange rate is half of the intrinsic D → H exchange rate at the same pH reading.

Results

IL-25 digestion

IL-25 was digested by pepsin under different conditions. When 2 parts of the diluted IL-25 solution were quenched with 1 part of 2M urea, 1MTCEP (pH 3.0) and digested using a pepsin column at a rate of 200. mu.L/min, the digestion of IL-25 by pepsin was optimized. The most ideal separation conditions are: a linear gradient of 13% to 40% buffer B in buffer a (95% acetonitrile, 5% H2O, and 0.0025% TFA) was passed through the C18 column for 23 minutes. The sequence coverage of IL-25 after pepsin digestion was 100% (═ 146/146; fig. 7A and 7B).

Immobilization of M6 and binding Capacity testing of M6 chromatography columns

The IL-25 sample that was not digested with pepsin showed a chromatographic peak at 8.5 minutes. The sample appeared clean. M6 was successfully coupled to POROS AL resin by schiff base chemistry. ExSAR tested the binding capacity of M6 chromatography columns under three different conditions, as shown in table 1. The full peaks were then tested at m/z 1875(+18) and 1985(+ 17). In all tested binding conditions, no IL-25 could be eluted using a neutral eluent, indicating that all loaded IL-25(166pmol) bound to the M6 column under neutral conditions. IL-25 can bind non-specifically to M6 chromatography columns. In the absence of detergent, acid wash can only recover 17-18% of the loaded IL-25. When IL-25 is repeatedly loaded, the back pressure of the M6 chromatographic column will gradually increase. The recovery was improved by addition of Foscholine-12.

TABLE 1 conditions used to test the binding capacity of the M6 antibody column

The column between "temperature" and "neutral" is entitled "pepsin".

Epitope identification

<Forward exchange assay for IL-25 in solution>

The forward exchange experiments for IL-25 in solution were performed at 23 ℃ and pH 7.

IL-25 is shown to be a relatively dynamic protein.

<IL-25 Forward/reverse exchange experiments with and without M6 chromatography columns>

The fragments containing amino acid residues 56-63 and 66-74 were observed to be the most protective (FIGS. 8A and 9C; Table 2). Similar fragments containing amino acid residues 46-63 and 66-84 showed consistent weak protection (FIGS. 8A, 9C and 9D; Table 2). It was observed that the fragment containing amino acid residues 129-135 was boundary-protected (FIGS. 8B, 9E and 9F; Table 2).

TABLE 2 different human IL-25 after forward/reverse exchange experiments at pH 7 and pH 8 at 23 ℃ Differences in the level of fragment tritiation。

The teachings of all patents, published patent applications, and references cited herein are incorporated by reference in their entirety.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims (26)

1. A target binding member that binds IL-25, wherein the target binding member comprises:

a) an antibody VL domain comprising CDR1 having amino acid sequence SASQGISNYLN (SEQ ID NO:6), CDR2 having amino acid sequence YTSSLHS (SEQ ID NO:7) and CDR3 having amino acid sequence QQYLAFPYTF (SEQ ID NO: 8); and

b) an antibody VH domain comprising CDR1 having the amino acid sequence GYTMN (SEQ ID NO:10), CDR2 having the amino acid sequence LINPYNGGTSYNQNFKG (SEQ ID NO:11) and CDR3 having the amino acid sequence EDYDGYLYFAMDY (SEQ ID NO: 12).

2. The target binding member of claim 1, wherein the target binding member comprises an antibody constant region.

3. The target binding member of claim 2, wherein the antibody constant region is an IgG1 constant region or an IgG4 constant region.

4. The target binding member of claim 3, wherein the target binding member comprises a whole antibody.

5. The target binding member of claim 1, wherein the target binding member comprises an antibody fragment selected from the group consisting of a Fab antibody fragment, a F (ab')₂Antibody fragments and scFv antibody fragments.

6. The target binding member of claim 1, wherein the VL domain comprises SEQ ID NO 5 and the VH domain comprises SEQ ID NO 9.

7. An isolated nucleic acid comprising a nucleotide sequence encoding the target binding member of claim 1.

8. An expression vector comprising the nucleic acid of claim 7, wherein the nucleic acid is operably linked to a promoter.

9. A host cell carrying the expression vector of claim 8.

10. A method of producing a target binding member, the method comprising culturing the host cell of claim 9 under conditions suitable for production of the target binding member.

11. The method of claim 10, further comprising isolating the target binding member.

12. The method of claim 10, further comprising formulating the target binding member into a composition comprising at least one additional component.

13. A composition comprising the target binding member of claim 1 and a pharmaceutically acceptable carrier.

14. The composition of claim 13, wherein the composition comprises a lyophilized powder.

15. Use of an effective amount of the target binding member of claim 1 in the manufacture of a medicament for treating or preventing asthma in a subject in need thereof.

16. Use of an effective amount of the target binding member of claim 1 in the manufacture of a medicament for treating or preventing inflammatory bowel disease in a subject in need thereof.

17. Use of an effective amount of the target binding member of claim 1 in the manufacture of a medicament for treating or preventing ulcerative colitis in a subject in need thereof.

18. Use of an effective amount of a target binding member according to claim 1 in the manufacture of a medicament for the treatment or prevention of crohn's disease.

19. The target binding member of claim 1 for use in the treatment and prevention of asthma.

20. The target binding member according to claim 1 for use in the treatment and prevention of inflammatory bowel disease.

21. The target binding member of claim 20, wherein the inflammatory bowel disease is ulcerative colitis or crohn's disease.

22. A method of generating a target binding member for IL-25, wherein the target binding member binds to one or more sequences selected from the group consisting of amino acid residues 56-63 of SEQ ID NO:17, amino acid residues 66-74 of SEQ ID NO:17 and amino acid residues 129-135 of SEQ ID NO:17, the method comprising:

(a) providing a starting repertoire of nucleic acids encoding VL domains, wherein the nucleic acids comprise a CDR3 encoding region to be replaced or lack a CDR3 encoding region;

(b) combining the starting library with a donor nucleic acid encoding a VL CDR3 having amino acid sequence QQYLAFPYTF (SEQ ID NO:8), wherein the donor nucleic acid is inserted into one or more nucleic acids in the library to form a product library of nucleic acids encoding a VL domain comprising a VL CDR3 having amino acid sequence QQYLAFPYTF (SEQ ID NO: 8);

(c) expressing the nucleic acids of the product repertoire to form a target binding member;

(d) selecting a target binding member that specifically binds to one or more sequences selected from the group consisting of amino acid residues 56-63 of SEQ ID NO. 17, amino acid residues 66-74 of SEQ ID NO. 17, and amino acid residues 129-135 of SEQ ID NO. 17; and

(e) recovering the target binding member or the nucleic acid encoding the target binding member.

23. The method of claim 22, wherein the nucleic acids of the product repertoire are co-expressed with nucleic acids encoding a VH domain.

24. The method of claim 23, wherein the VH domain comprises SEQ ID NO 9.

25. The method of claim 22, 23 or 24, wherein the target binding member comprises a whole antibody.

26. The target binding member of claim 1, wherein the target binding member comprises a humanized antibody.