HK1208182B - St2l antagonists and methods of use - Google Patents

Description

ST2L antagonists and methods of use

Cross Reference to Related Applications

The present application claims the benefit of U.S. application serial No. 13/798,204 filed on 3/13/2013, U.S. application serial No. 13/798,226 filed on 3/13/2013, U.S. provisional application No. 61/640,407 filed on 4/30/2012, and U.S. provisional application No. 61/640,238 filed on 4/30/2012, which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to ST2L antagonists, polynucleotides encoding the antagonists or fragments thereof, and methods of making and using the foregoing.

Background

ST2L (IL-1RL1 or IL-33 Ra) is a member of the Toll/IL-1 receptor family, expressed on the cell surface of a variety of immune cells, including T cells, NK/NKT cells, basophils, eosinophils, mast cells, and newly discovered non-B/non-T type 2 innate lymphocytes, nuocytes, and natural helper cells. ST2L expression can also be induced on Dendritic Cells (DCs), macrophages and neutrophils. ST2L is able to down-regulate the responsiveness of Toll-like receptors TLR2, TLR4 and TLR9, but is also able to induce type 2 cytokine release via activation by its ligand IL-33 and association with the accessory protein IL-1 RAcP. IL-33 has been described as an "alarm" because it is present in the nucleus of epithelial and endothelial cells in full-length form during homeostasis, but can be cleaved and released during cellular necrosis.

ST2L signaling requires the association of the accessory protein IL-1RAcP to a preformed ST2L/IL-33 complex. The IL-1 α/β signaling complex also shares the accessory protein IL-1 RAcP. Models of ST2L, the interaction of IL-33 and IL-1RAcP, and the interaction between IL-1R1 and IL-1RAcP have been proposed (Lingel et al, Cell 17: 1398-. Recently, ST2L/IL-33/IL-1RAcP was found to form a signaling complex with c-Kit (a receptor for Stem Cell Factor (SCF)) on mast cells. IL-33 induces cytokine production in primary mast cells in a SCF-dependent manner (Drube et al, Blood 115: 3899-906, 2010).

Activation of ST2L results in excessive type 2 cytokine responses (especially IL-5 and IL-13), mast cell and eosinophil activation, and airway hyperreactivity, and Th1 and Th17 responses have also been reported to be amplified by inducing IFN γ from NKT cells and IL-1 β and IL-6 from mast cells. Dysregulation of the ST2L/IL-33 pathway has been shown to be associated with a variety of immune-mediated diseases, including asthma, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, allergic rhinitis, nasal polyps, and systemic sclerosis (reviewed in Palmer and Gabay, Nat Rev Rheumatotol 7: 321-9, 2011 and Lloyd, CurrOpin Immunol 22: 800-6, 2010; Shimizu et al, Hum Molec Gen 14: 2919-27, 2005, Kamekura et al, Clin Exp Allergy 42: 218-28, 2012; Manetti et al, Ann Rheum Dis 69: 598-605, 2010).

Thus, there is a need for ST2L antagonists that are suitable for use in the treatment of ST2L mediated diseases and disorders.

Drawings

Figure 1 shows that binding of monoclonal antibody CNTO3914 by ST2L domain I inhibits airway hyperresponse in a model of pneumonia induced by intranasal administration of IL-33 when compared to isotype control CNTO 5516. Peaks in airway resistance were measured when Methacholine (MCH) was administered at elevated doses (mg/ml). CNTO3914/IL-33, relative to CNTO5516/IL-33,. p < 0.05; and CNTO3914/IL-33,. p < 0.001 relative to the IL-33 treated PBS group.

Figure 2 shows that binding of monoclonal antibody CNTO3914 by ST2L domain I inhibits bronchoalveolar lavage (BAL) cell recruitment in a model of pneumonia induced by intranasal administration of IL-33 when compared to isotype control CNTO 5516. P < 0.001.

FIG. 3 shows the dose-dependent inhibition of the release of mouse mast cell protease 1(MMCP-1) by ST2L domain I binding to monoclonal antibody CNTO3914 in cell-free BAL fluid in a model of pneumonia induced by intranasal administration of IL-33. P < 0.01, p < 0.001 relative to IL-33 treated CNTO5516 (isotype control).

FIG. 4 shows that IL-33-induced release of GM-CSF (FIG. 4A), IL-5 (FIG. 4B) and TNF α (FIG. 4C) from mouse bone marrow derived mast cells was inhibited in vitro by binding of the monoclonal antibody CNTO3914 to ST2L domain I. In parentheses, the CNTO3914 concentration used is shown as μ g/ml, and the IL-33 concentration is shown as ng/ml.

FIG. 5 shows that IL-33-induced prostaglandin D2 (PGD) of human umbilical cord blood-derived mast cells is inhibited by ST2L domain I binding to monoclonal antibody C2494(STLM62) at the indicated IL-33 and C2494 concentrations₂) And (4) releasing. MOX-PDG₂: methoxyamine-PGD₂。

FIG. 6 shows that GM-CSF (FIG. 6A), IL-8 (FIG. 6B), IL-5 (FIG. 6C), IL-13 (FIG. 6D) and IL-10 (FIG. 6E) release in human umbilical cord blood-derived mast cells (hBMC) was inhibited by ST2L domain I binding to monoclonal antibodies C2244 and C2494 at the indicated concentrations (μ g/ml) in the presence of 1ng/ml IL-33+100ng/ml SCF (stem cell factor) in StemPro-34 medium.

FIG. 7 shows the effect of GM-CSF (FIG. 7A), IL-8 (FIG. 7B), IL-5 (FIG. 7C), IL-13 (FIG. 7D) and IL-10 (FIG. 7E) release in human umbilical cord blood-derived mast cells by binding to monoclonal antibody C2519 or C2521 at the indicated concentrations (μ g/ml) of ST2L domain III in the presence of 1ng/ml IL-33+100ng/ml SCF in StemPro-34 medium.

FIG. 8 shows A) GM-CSF in human umbilical cord blood-derived mast cells (hCBMC) in the presence of 3ng/ml IL-33+100ng/ml SCF in RPMI/10% FCS medium, by binding of ST2L domain I to monoclonal antibody C2494 and ST2L domain III to monoclonal antibodies ST2M48(M48), ST2M49(M49), ST2M50(M50) and ST2M51 (M51); B) IL-8; C) IL-5; D) IL-13 and E) the effect of IL-10 release.

FIG. 9 shows the average percent (%) of GM-CSF, IL-5, IL-8, IL-10, and IL-13 release from human umbilical cord blood-derived mast cells induced by IL-33 and SCF using 50 μ g/ml or 2 μ g/ml of each antibody tested in anti-ST 2L antibody that binds either domain I (D1) or domain III (D3) of ST2L as indicated. Negative values indicate% activation.

FIG. 10 shows the heavy chain variable region (VH) and heavy chain CDR sequences of anti-ST 2L antibody, which was derived from a phage display library and subsequently affinity-maturation screened.

Figure 11 shows the light chain variable region (VL) and light chain CDR sequences of an anti-ST 2L antibody, which was derived from a phage display library and subsequently screened for affinity maturation.

Figure 12 shows the sequences of the VH and VL regions, and the heavy chain CDRs, of the anti-ST 2L antibody STLM208VH ST2H257HCDR3 variant.

FIG. 13 shows A) VH and B) VL sequences of anti-ST 2L antibodies, which were derived from a phage display library and subsequently screened by affinity maturation.

Figure 14 shows the division of C2494VH and VL antigen binding sites transferred to the human framework (the transferred portion is labeled HFA "human frame adaptation"). The Kabat CDRs are underlined and Chothia HV is indicated by dashed lines above the indicated transition HFA region. The numbering of the VH and VL residues is according to Chothia. Residues highlighted in grey in VH were not transferred in some HFA variants. C2494 VH: SEQ ID NO: 48; c2494 VH: SEQ ID NO: 52.

figure 15 shows CDR sequences of human framework-adapted (HFA) antibodies derived from C2494.

Figure 16A) serum levels of anti-ST 2L antibody CNTO 3914; B) inhibition of bronchoalveolar lavage (BAL) cell recruitment; C) inhibition of IL-6 secretion by IL-33 stimulated whole blood cells; D) in a 6 hour model of lung inflammation induced by intranasal administration of IL-33, MCP1 secretion from whole blood cells stimulated with IL-33 was inhibited by CNTO3914 24 hours after dosing. P < 0.05, p < 0.01, p < 0.001; NQ is below the detection limit; @ one data point is below the detection limit.

FIG. 17 Competition between various anti-ST 2L antibodies. A) The 30nM labeled C2244Fab competed with the indicated antibody for binding to ST2L-ECD coated on the microwell. C2244 competes with C2494, but not with C2539. B)10nM labeled C2494 competes with the indicated antibody for binding to ST2L-ECD coated on microwells. C2494 competes with STLM208 and STLM213, but not with C2539.

FIG. 18 shows a simplified H/D exchange diagram for human ST2-ECD (SEQ ID NO: 119) complexed with C2244 Fab. The areas protected by the antibodies are shown in different shades of gray. Fragments containing residues 18-31 (within the dashed square box) (corresponding to residues 35-48 of full length ST2L of SEQ ID NO: 1) were protected by Fab. The region containing residues 71-100 (within the solid box) (corresponding to residues 88-117 of SEQ ID NO: 1) is heavily glycosylated and is not covered by peptide.

Figure 19 shows the kinetics and affinity constants of ST2L domain I binding antibodies for the ST2L variants shown in the figure.

FIG. 20 shows A) GM-CSF of the anti-ST 2L antibody, STLM208, on primary human lung mast cells; B) IL-5; C) IL-8; D) inhibition of IL-13 secretion.

Disclosure of Invention

The present invention provides an isolated human or human adaptive antibody antagonist or fragment thereof that specifically binds to domain I (SEQ ID NO: 9) of human ST 2L.

The invention also provides a human adaptive antibody antagonist that specifically binds to human ST2L having the sequence: specific light and heavy chain variable region sequences, or specific heavy and light chain complementarity determining sequences.

The invention also provides human or human adaptive antibody antagonists that specifically bind human ST2L in defined epitope regions and/or have particular characteristics as described herein.

The invention also provides an isolated polynucleotide encoding a heavy chain variable region (VH) or a light chain variable region (VL) of the invention.

The invention also provides a vector comprising the isolated polynucleotide of the invention.

The invention also provides a host cell comprising a vector of the invention.

The invention also provides a method of producing an antibody of the invention, the method comprising culturing a host cell of the invention and recovering the antibody from the cell.

The invention also provides a pharmaceutical composition comprising an isolated antibody of the invention and a pharmaceutically acceptable vehicle.

The invention also provides a method of treating or preventing an ST 2L-mediated disorder, the method comprising administering to a patient in need thereof a therapeutically effective amount of an isolated antibody of the invention for a time sufficient to treat or prevent an ST 2L-mediated disorder.

The invention also provides a method of inhibiting a mast cell response in a patient, comprising administering to a patient in need thereof a therapeutically effective amount of an isolated antibody of the invention for a time sufficient to inhibit a mast cell response.

The invention also provides a method of inhibiting the interaction of IL-33 and ST2L in a subject, the method comprising administering to the subject an antibody that specifically binds domain I of ST2L in an amount sufficient to inhibit the interaction of IL-33 and ST 2L.

Detailed Description

All publications (including but not limited to patents and patent applications) cited in this specification are herein incorporated by reference as if fully set forth herein.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

As used herein, the term "antagonist" means a molecule that partially or completely inhibits the biological activity of ST2L by any mechanism. Exemplary antagonists are antibodies, fusion proteins, peptides, peptidomimetics, nucleic acids, oligonucleotides, and small molecules. Antagonists can be identified using the ST2L bioactivity assay described below. ST2L antagonists may inhibit the measured ST2L biological activity by 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

The term "ST 2L" or "huST 2L" or "human ST 2L" refers to a human ST2L polypeptide having the amino acid sequence set forth in genbank accession No. NP 057316. SEQ ID NO: 1 shows the amino acid sequence of full-length human ST 2L. As used herein, "ST 2L extracellular domain", "ST 2L-ECD" or "huST 2L-ECD" means having the amino acid sequence of SEQ ID NO: 1, amino acids 19-328. The huST2L-ECD has three Ig-like C2-type domains spanning SEQ ID NO: 1 (domain I, SEQ ID NO: 9), 123-202 (domain II, SEQ ID NO: 10) and 209-324 (domain III, SEQ ID NO: 11). "domain I" or "ST 2L domain I" or "huST 2L domain I" or "D1" refers to the first immunoglobulin-like domain on human ST2L having the sequence of seq id no:9, or a sequence shown in seq id no. "domain III" or "ST 2L domain III" refers to a third immunoglobulin-like domain on human ST2L having the amino acid sequence of SEQ ID NO: 11, or a sequence shown in fig. 11.

As used herein, the term "IL-33" includes full-length IL-33 (GenBank accession NP-254274 SEQ ID NO: 3) and variants and active forms thereof. IL-33 variants include proteins having the amino acid sequence set forth in genbank accession No. NP _001186569 and genbank accession No. NP _ 001186570. The active form of IL-33 includes "mature IL-33" having the amino acid sequence of SEQ ID NO: residue 112 and 270 of 3. Additional active forms include fragments of IL-33 having the amino acid sequence of SEQ ID NO: 3, residues 11-270, 115-270, 95-270, 99-270 or 109-270 (LeFrancais et al, ProcNatl Acad Sci (USA) 109: 1673-8, 2012), or any form or combination of forms isolated from cells endogenously expressing IL-33. An "IL-33 active form" is SEQ ID NO: 3, or a fragment or variant of IL-33.

As used herein, the term "antibody" is meant broadly and includes: immunoglobulin molecules, including polyclonal antibodies; monoclonal antibodies, including murine, human adapted, humanized and chimeric monoclonal antibodies; an antibody fragment; bispecific or multispecific antibodies formed from at least two intact antibodies or antibody fragments; dimeric, tetrameric or multimeric antibodies; and single chain antibodies.

Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as isotypes IgA₁、IgA₂、IgG₁、IgG₂、IgG₃And IgG₄. Antibody light chains of any vertebrate species can be assigned to one of two distinctly different types, namely κ (κ) and λ (λ), based on the amino acid sequences of their constant domains.

The term "antibody fragment" refers to a portion of an immunoglobulin molecule that retains heavy and/or light chain antigen binding sites, such as heavy chain complementarity determining regions (HCDR)1, 2, and 3, light chain complementarity determining regions (LCDR)1, 2, and 3, a heavy chain variable region (VH) or a light chain variable region (VL). Antibody fragments include the well-known Fab, F (ab') 2, Fd and Fv fragments as well as domain antibodies (dAbs) consisting of one VH domain. The VH and VL domains may be linked together via a synthetic linker to form various types of single chain antibody designs, where the VH/VL domains pair intramolecularly, or intermolecularly in the case where the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain fv (scfv) or diabody (diabody); for example, as described in International patent publication WO98/44001, International patent publication WO88/01649, International patent publication WO94/13804, International patent publication WO 92/01047.

The antibody variable region consists of a "framework" region into which three "antigen binding sites" are inserted. Different terms are used to define antigen binding sites: (i) three Complementarity Determining Regions (CDRs) in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3) are based on sequence variability (Wu and Kabat, J Exp Med 132: 211-50, 1970; Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health service, National Institutes of Health, Bethesda, Md., 1991). (ii) Three "hypervariable regions", "HVRs" or "HV" in VH (H1, H2, H3) and three in VL (L1, L2, L3), refer to regions of structural hypervariability in antibody variable domains, as defined by Chothia and Lesk (Chothia and Lesk, Mol Biol 196: 901-17, 1987). Other terms include "IMGT-CDR" (Lefranc et al, Dev company Immunol 27: 55-77, 2003) and "specificity determining residue usage" (SDRU) (Almagro, Mol Recognit 17: 132-43, 2004). The International ImmunoGeneTiCs (IMGT) database (http:// www _ imgt _ org) provides a standardized numbering and definition of antigen binding sites. The correspondence between CDR, HV and IMGT divisions is described in Lefranc et al, Dev company Immunol 27: 55-77, 2003.

As used herein, "Chothia residues" are antibody VL and VH residues, which are numbered according to Al-Lazikani (Al-Lazikani et Al, J Mol Biol 273: 927-48, 1997).

"framework" or "framework sequence" is the remaining sequence of the variable region except for those sequences defined as antigen binding sites. Since the antigen binding site can be defined by different terms as described above, the exact amino acid sequence of the framework depends on how the antigen binding site is defined.

"human antibody" or "fully human antibody" refers to an antibody comprising variable and constant region sequences derived from human immunoglobulin sequences. The human antibodies of the invention may include substitutions, and thus may not be exact copies of expressed human immunoglobulin or germline gene sequences. However, the definition of "human antibody" does not include antibodies in which the antigen-binding site is derived from a non-human species.

A "human-adapted" antibody or a "human framework-adapted (HFA)" antibody refers to an antibody adapted according to the method described in U.S. patent publication No. us2009/0118127, and also refers to an antibody that grafts an antigen-binding site sequence derived from a non-human species to a human framework.

"humanized antibody" refers to an antibody in which the antigen binding site is derived from a non-human species and the variable region framework is derived from human immunoglobulin sequences. Humanized antibodies may include substitutions in the framework regions, and thus the framework may not be an exact copy of the expressed human immunoglobulin or germline gene sequence.

The term "substantially identical" as used herein means that the amino acid sequences of the variable regions of the two antibodies being compared are identical or have "insubstantial differences". Insubstantial differences are substitutions of 1, 2, 3,4, 5, 6, 7, 8, 9,10, or 11 amino acids in the antibody or antibody variable region sequence that do not adversely affect the properties of the antibody. Amino acid sequences substantially identical to the variable region sequences disclosed herein are within the scope of the present application. In some embodiments, the sequence identity may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Percent identity can be determined, for example, by pairwise alignment using the default settings of the AlignX module of Vector NTI v.9.0.0(Invitrogen, Carslbad, CA). The protein sequences of the invention can be used as query sequences to perform public or patent database searches, for example, to identify related sequences. An exemplary program for performing such a search is the XBLAST or BLASTP program using default settingsSequence (http _// www _ ncbi _ nlm/nih _ gov), or GenomeQuest^TM(GenomeQuest, Westborough, MA) kit.

As used herein, the term "epitope" refers to a portion of an antigen to which an antibody specifically binds. Epitopes are usually composed of a chemically active (such as polar, non-polar or hydrophobic) surface moiety, such as an amino acid or a polysaccharide side chain, and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitopes can be composed of contiguous and/or noncontiguous amino acids that form conformational space units. For discontinuous epitopes, the amino acids of different parts of the linear sequence of the antigen are in close proximity in three dimensions by folding of the protein molecule. An exemplary epitope is the amino acid sequence of SEQ ID NO: domain I of huST2L shown at 9.

As used herein, the term "paratope" means a portion of an antibody to which an antigen specifically binds. Paratopes may be linear in character or may be discontinuous, formed by the spatial relationship between non-contiguous amino acids of an antibody, rather than a linear series of amino acids. "paratope" and "paratope" of the heavy chain or "paratope amino acid residue of the light chain" and "paratope amino acid residue of the heavy chain" refer to the antibody light chain and heavy chain residues, respectively, that are in contact with the antigen.

As used herein, the term "specifically binds" or "specifically binds" refers to an antibody that binds to a predetermined antigen with a higher affinity than to other antigens or proteins, typically, an antibody is 1 × 10^-7Dissociation constant (K) of M or less_D) Bound to a predetermined antigen, e.g. 1 × 10^-8M or less, 1 × 10^-9M or less, 1 × 10^-10M or less, 1 × 10^-11M or less, or 1 × 10^-12M or less, usually K_DK for its binding to a non-specific antigen (e.g. BSA, casein or any other specific polypeptide)_DAt least ten times. The dissociation constant can be measured using standard procedures. However, an antibody that specifically binds to a predetermined antigen may be cross-reactive to other related antigens, e.g. to the same from other species (homologous)Predetermined antigens, such as humans or monkeys, e.g. cynomolgus monkeys (cynomolgus).

As used herein, "bispecific" refers to an antibody that binds to two different antigens or two different epitopes within an antigen.

As used herein, "monospecific" refers to an antibody that binds to an antigen or an epitope.

The term "in combination" as used herein means that the agents described may be co-administered to an animal in admixture, simultaneously as a single agent or sequentially in any order as a single agent.

As used herein, "inflammatory condition" refers to an acute or chronic local or systemic response to a noxious stimulus, such as a pathogen, damaged cells, physical injury, or stimulus, which response is mediated in part by the activity of cytokines, chemokines, or inflammatory cells (e.g., neutrophils, monocytes, lymphocytes, macrophages), and which inflammatory condition is characterized in most cases by pain, redness, swelling, and impairment of tissue function.

As used herein, the term "ST 2L-mediated inflammatory disorder" refers to an inflammatory disorder that is caused, at least in part, by inappropriate activation of the ST2L signaling pathway. Exemplary ST 2L-mediated inflammatory conditions are asthma and allergy.

As used herein, the term "ST 2L-mediated condition" encompasses all diseases and medical conditions in which ST2L plays a direct or indirect role in the disease and medical condition, including initiation, development, progression, persistence, or pathology of the disease or condition.

As used herein, the term "ST 2L biological activity" refers to any activity that occurs as a result of binding of ST2L ligand IL-33 to ST 2L. Exemplary ST2L biological activity results in NF-. kappa.B activation in response to IL-33. NF-. kappa.B activation can be determined using a reporter gene assay when ST2L is induced with IL-33 (Fursov et al, Hybridoma 30: 153-62, 2011). Other exemplary ST2L biological activities result in the proliferation of Th2 cells, or the secretion of pro-inflammatory cytokines and chemokines, such as IL-5, GM-CSF, IL-8, IL-10, or IL-13. The release of cytokines and chemokines from cells, tissues or in the circulation can be measured using well known immunoassays, such as ELISA immunoassays.

The term "vector" refers to a polynucleotide capable of replication within a biological system or of movement between such systems. Vector polynucleotides typically contain elements such as origins of replication, polyadenylation signals, or selectable markers that function to facilitate replication or maintenance of these polynucleotides in a biological system. Examples of such biological systems may include cells, viruses, animals, plants, and biological systems reconstituted with biological components capable of replicating vectors. The polynucleotide comprising the vector may be a DNA or RNA molecule or a hybrid of such molecules.

The term "expression vector" refers to a vector that can be used to direct the translation of a polypeptide encoded by a polynucleotide sequence present in an expression vector in a biological or reconstituted biological system.

The term "polynucleotide" refers to a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemical means. Double-stranded DNA and single-stranded DNA, and double-stranded RNA and single-stranded RNA are typical examples of polynucleotides.

The term "polypeptide" or "protein" refers to a molecule comprising at least two amino acid residues joined by a peptide bond to form a polypeptide. Small polypeptides of less than 50 amino acids may be referred to as "peptides".

The following conventional one-letter and three-letter amino acid codes are used herein:

composition of matter

The present invention provides antibodies that specifically bind to ST2L and inhibit the biological activity of ST2L, and uses of such antibodies. The inventors have surprisingly found that an antibody that binds to domain I of human ST2L (SEQ ID NO: 9) blocks IL-33/ST2L interaction and inhibits a range of ST2L biological activities, including IL-33-induced mast cell responses, whereas an antibody that binds to domain III of human ST2L (SEQ ID NO: 11) does not block IL-33/ST2L interaction, although it is inhibitory to a range of ST2L biological activities. However, domain III binding antibodies have reduced or no inhibitory effect on IL-33-induced mast cell responses, or in some cases stimulate IL-33-induced mast cell responses.

In some embodiments described herein, an antibody that blocks IL-33/ST2L interaction and inhibits a range of ST2L biological activities including IL-33-induced mast cell responses, binds to an epitope within domain I of human ST2L (RCPRQGKPSYTVDW; SEQ ID NO: 210) and optionally ST2L amino acid residues T93 and F94 (residue numbering according to SEQ ID NO: 1).

The term "mast cell response" or "mast cell activity" refers to IL-33-induced cytokines such as GM-CSF, IL-8, IL-5, IL-13, and IL-10, and allergic mediators such as prostaglandin D₂Release from mast cells.

The present invention provides novel antigen binding sites that bind to domain I of human ST2L as described herein. The structure used to carry the antigen binding site is typically an antibody VH or VL.

The antibodies of the invention as described herein can be isolated human or human adaptive antibody antagonists or fragments thereof that specifically bind to domain I of human ST2L (SEQ ID NO: 9). An exemplary antibody that binds to domain I of human ST2L (SEQ ID NO: 9) is antibody STLM15(C2244), which comprises the amino acid sequences set forth in SEQ ID NOs: 23. HCDR1, HCDR2 and HCDR3 sequences of 27 and 31 and SEQ ID NOs: 35. LCDR1, LCDR2 and LCDR3 sequences of 39 and 43; or antibody C2494(STLM62) comprising the amino acid sequences set forth in SEQ ID NOs: 24. 28 and 32 and HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 36. LCDR1, LCDR2 and LCDR3 sequences of 40 and 44 (table 3). Further exemplary antibodies that bind to domain I of human ST2L are the antibodies shown in table 16 and fig. 13, e.g., antibodies STLM103, STLM107, STLM108, STLM123, STLM124, STLM208, STLM209, STLM210, STLM211, STLM212, and STLM 213. Exemplary human antibody antagonists are shown in fig. 12 and 13. Exemplary human-adapted antagonists are shown in table 14.

In some embodiments described herein, an isolated human or human adaptive antibody antagonist that specifically binds to domain I of human ST2L (SEQ ID NO: 9) or a fragment thereof blocks the IL-33/ST2L interaction.

Antibodies can be tested for their ability to block the IL-33/ST2L interaction by standard ELISA. For example, plates were coated with the extracellular domain of human ST2L (huST2L-ECD) and incubated with antibodies, after which binding of biotinylated IL-33 on the plates was measured. An antibody that "blocks the IL-33/ST2L interaction" or "inhibits the IL-33/ST2L interaction" is an antibody that reduces the signal from biotinylated IL-33 bound to a plate by at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% when compared to IL-33 binding in the absence of antibody at an antibody concentration of 50 μ g/ml in an ELISA assay using huST2L-ECD coated plates.

Inhibition of mast cell response by antibodies can be tested by assessing the inhibitory activity of the antibodies against, for example, GM-CSF, IL-5, IL-10, or IL-13 release from human cord blood-derived mast cells or primary human lung mast cells using standard methods and the methods exemplified below. An antibody that "inhibits mast cell response" or "inhibits mast cell activity" as described herein is one that reduces IL-33-induced secretion of GM-CSF, IL-5, IL-13, or IL-10 by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, when compared to mast cells not treated with the antibody, at a concentration of 10 μ g/ml,98%, 99% or 100% antibody. In general, mast cells can be derived from human umbilical cord blood or lung parenchyma and small airway CD34 by well-known methods and as exemplified below⁺A progenitor cell. Mast cell culture conditions can affect the measure of% inhibition of antibodies, so culture and test conditions can be kept standard using, for example, stempo-34 medium during differentiation at 6-10 weeks of age. Mast cells were stimulated daily with 10ng/ml IL-4, 10ng/ml IL-6 and 100ng/ml SCF 4 days prior to cytokine release assays. For cytokine release assays, mast cells can be resuspended in fresh StemPro-34 medium, or RPMI containing 10% antibiotic-free FCS and 100ng/ml SCF. Seeding densities suitable for assay were 65,000 to 75,000 cells per 0.16 ml/well. Exemplary antibodies of the invention that inhibit mast cell responses as described herein are antibodies STLM15, STLM62, and STLM 208. The antibody CNTO3914 binds to mouse ST2L domain I without cross-reactivity to human ST2L and inhibits mast cell response in mouse cells.

One skilled in the art will appreciate that mast cell response also includes the release of: IL-1 and IL-32, as well as chemokines (such as CCL1, CCL4, CCL5, CCL18, and CCL23), and allergic mediators (such as cysteinyl leukotrienes, histamine), and various mast cell proteases (including tryptase, chymase, carboxypeptidase, and cathepsin G). Antibodies of the invention as described herein can be tested for their ability to inhibit these additional mast cell responses using standard methods. Antibodies of the invention that bind domain I of ST2L and block IL-33/ST2L interactions as described herein, when tested at a minimum concentration of 10 μ g/ml under these conditions, are expected to inhibit these additional mast cell responses by at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more.

The antibodies of the invention as described herein bind to human ST2L with a constant of between about 5 × 10^-12M to about 7 × 10^-10Dissociation constant (K) between M_D) Between about 2 × 10⁶M^-1s^-1To about 1 × 10⁸M^-1s^-1The binding rate constant (K) to human ST2L in between_on) Or between about 1 × 10^-6s^-1To about 1 × 10^-2s^-1Dissociation rate constant (K) for human ST2L between_off) For example, an antibody of the invention as described herein is administered at less than about 7 × 10^-10M, less than about 1 × 10^-10M, less than about 5 × 10^-11M, less than about 1 × 10^-11M or less than about 5 × 10^-12K of M_DIn combination with human ST 2L.

The antibodies of the invention as described herein cross-react with cynomolgus monkey (cyno) ST2L (SEQ ID NO: 2) and bind to cynomolgus monkey (cyno) ST2L with a constant between about 3 × 10^-12M to about 2 × 10^-9Dissociation constant (K) between M_D) Between about 4 × 10⁶M^-1s^-1To about 1 × 10⁸M^-1s^-1Binding rate constant (K) to cyno ST2L_on) Or between about 7 × 10^-5s^-1To about 1 × 10^-1s^-1Dissociation rate constant (K) for cyno ST2L in between_off) For example, an antibody of the invention as described herein is administered at less than about 2 × 10^-9M, less than about 1 × 10^-9M, less than about 1 × 10^-10M, less than about 1 × 10^-11M or less than about 3 × 10^{- 12}K of M_DIn combination with cyno ST 2L.

The affinity of the antibody for ST2L can be determined experimentally using any suitable method. Such methods may utilize ProteOn XPR36, Biacore 3000 or KinExA instruments, ELISA or competitive binding assays known to those skilled in the art. The measured affinity of a particular antibody/ST 2L interaction may be different if measured under different conditions (e.g., osmolarity, pH). Thus, affinity and other binding parameters (e.g., K)_D、K_on、K_off) The measurement of (a) is preferably performed with standardized conditions and standardized buffers such as the buffers described herein. Those skilled in the art will appreciate that the internal error (measured as standard deviation, SD) of affinity measurements using, for example, Biacore 3000 or ProteOn, is typically 5-3 of the measurement within typical detection limitsWithin 3% the term "about" thus reflects the standard deviation typical in an assay, e.g., 1 × 10^-9K of M_DHas a typical SD of at most. + -. 0.33 × 10^-9M。

Antibodies that bind human ST2L with the desired affinity and optionally cross-react with cyno ST2L may be selected from a library of variants or fragments by panning with human and/or cyno ST2L, and optionally by further antibody affinity maturation. Antibodies may be identified based on their inhibition of ST2L biological activity using any suitable method. Such methods can be performed using reporter gene assays or assays that measure cytokine production, using well known methods and as described herein.

One embodiment of the invention is an isolated antibody antagonist that specifically binds human ST2L, comprising:

SEQ ID NO：160(X₁X₂X₃MX₄) Heavy chain complementarity determining region (HCDR)1(HCDR 1); wherein

X₁S, F, D, I, G or V;

X₂is Y or D;

X₃a, D or S; and is

X₄S, F or I;

SEQ ID NO：161(X₅IX₆GX₇GGX₈TX₉YADSVKG) (HCDR 2); wherein

X₅A, S, T, Y or D;

X₆s, R, E, K, G or A;

X₇s, E or N;

X₈s, R, E, G, T, D or A; and is

X₉Y, D, N, A or S; and

SEQ ID NO：162(X₁₀X₁₁WSTEGSFFVLDY) (HCDR 3); wherein

X₁₀D, A, R, N, Q, P, E, I, H, S, T or Y; and is

X₁₁P, A, H, Y, E, Q, L, S, N, T, V, or I.

Another embodiment of the invention is an isolated antibody antagonist that specifically binds human ST2L, comprising:

SEQ ID NO：163(RASQSVDDX₁₂LA) light chain complementarity determining region (LCDR)1(LCDR 1); wherein

X₁₂Is A or D;

SEQ ID NO: LCDR 2(LCDR2) of 90 (DASNRAT); and

SEQ ID NO：164(QQX₁₃X₁₄X₁₅X₁₆X₁₇X₁₈t) LCDR 3(LCDR 3); wherein

X₁₃Is F or Y;

X₁₄y, I or N;

X₁₅n, G, D or T;

X₁₆is W or A;

X₁₇is P or absent; and is

X₁₈Is L or I.

Comprises SEQ ID NO: 160. the antibodies of the invention of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences 161, 162, 163, 90 and 164, respectively, can be prepared by well-known mutagenesis methods using, for example, SEQ ID NOs: 78. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of 81, 84, 87, 90 and 92 were prepared as templates. The sequences of SEQ ID NOs: 160. the heavy and light chain CDRs 161, 162, 163, 90, and 164 are grafted to a human framework, such as the framework described below. The binding of the antibody to ST2L, its ability to block the IL-33/ST2L interaction, and other features such as affinity to human ST2L and/or cyno ST2L, and inhibition of mast cell response may be analyzed using the methods described herein.

In one embodiment, an isolated antibody antagonist that specifically binds human ST2L as described herein comprises:

SEQ ID NO: 78 or 95-108 HCDR 1;

SEQ ID NO: 81, 109, 118 or 120, 129 HCDR 2;

SEQ ID NO: 84 or 165-185 HCDR 3;

SEQ ID NO: LCDR1 of 87 or 130;

SEQ ID NO: LCDR2 of 90; and

SEQ ID NO: 92 or 131 and 134.

In another embodiment, an isolated antibody antagonist that specifically binds human ST2L comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as shown in fig. 10, 11, and 12 and described herein.

In another embodiment, an isolated antibody antagonist that specifically binds human ST2L as described herein comprises:

SEQ ID NO: HCDR1 of 23 or 24;

SEQ ID NO: HCDR2 of 27 or 28;

SEQ ID NO: HCDR3 of 31 or 32;

SEQ ID NO: LCDR1 of 35 or 36;

SEQ ID NO: LCDR2 of 39 or 40; and

SEQ ID NO: LCDR3 of 43 or 44.

In another embodiment, an isolated antibody antagonist that specifically binds human ST2L as described herein comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences:

are respectively SEQ ID NO: 23. 27, 31, 35, 39 and 43;

are respectively SEQ ID NO: 24. 28, 32, 36, 40, and 44; (HFA CDR);

are respectively SEQ ID NO: 24. 28, 146, 36, 40, and 147; or

Are respectively SEQ ID NO: 24. 28, 146, 36, 40 and 44.

Another embodiment of the invention is an isolated human or human adaptive antibody antagonist or fragment thereof that specifically binds to human ST2L (SEQ ID NO: 1) as described herein, comprising a heavy chain variable region (VH) having a VH framework derived from human IGHV3-23(SEQ ID NO: 158), IGHV1-24 (SEQ ID NO: 148) or IGHV1-f 01(SEQ ID NO: 149) framework sequences, and a heavy chain variable region (VH) having a VH framework derived from human IGKV3-11(L6) (SEQ ID NO: 159), IGKV3-15 (L2) (SEQ ID NO: 150), IGKV1-9 (L8) (SEQ ID NO: 151), IGKV1-5 (L12) (SEQ ID NO: 152), IGKV 588-12 (L5) (SEQ ID NO: 1-153) (SEQ ID NO: 12), IGKV 3527 (IGKV 27) (SEQ ID NO: 3527) or fragment thereof, comprising IGHV 9636-7 (SEQ ID NO: 43) (SEQ ID NO: 46) framework sequences), IGKV1, IGKV, 18-5, IGKV, Ig5962, Ig27, SEQ ID NO: 43, SEQ ID NO: 1, SEQ ID NO The light chain variable region (VL) of the VL framework of the framework sequence.

In another embodiment, an isolated antibody that specifically binds to domain I of human ST2L as described herein comprises a VH having a VH framework derived from the human VH3-23 framework sequence (SEQ ID NO: 158); and a light chain variable region (VL) having a VL framework derived from the human V.kappa.L 6 framework sequence (SEQ ID NO: 159). Human framework sequences are well known and typically include human immunoglobulin germline variable region sequences joined to a junction (J) sequence. SEQ ID NO: 158 comprises a human germline VH3-23 sequence joined to IGHJ4, and seq id NO: 159 includes the human Vk L6 germline sequence joined to IGKJ1, as shown by Shi et al, J Mol Biol 397: 385-96, 2010; international patent publication WO 2009/085462; and U.S. patent publication US 2010/0021477. Exemplary antibodies having a VH sequence derived from human VH3-23 and a VL sequence derived from human vkl 6 are those shown in fig. 12 and 13.

A human or human adaptive antibody comprising a heavy or light chain variable region "derived from" a particular framework or germline sequence refers to an antibody obtained from a system using human germline immunoglobulin genes, such as from a transgenic mouse or from a phage display library, as discussed below. An antibody "derived" from a particular framework or germline sequence may contain amino acid differences compared to the sequence from which it was derived due to, for example, naturally occurring somatic mutations or deliberate substitutions.

In another embodiment, an isolated human or human adaptive antibody antagonist that specifically binds domain I (SEQ ID NO: 9) of human ST2L as described herein, or a fragment thereof, is complementary to a human or human adaptive antibody antagonist comprising the amino acid sequence of SEQ ID NO: 47 and SEQ id no: 51 (antibody C2244), competes for binding to human ST2L (SEQ ID NO: 1).

In another embodiment, an isolated antibody of the invention as described herein is represented in SEQ ID NO: 1 at amino acid residues 35-48 (RCPRQGKPSYTVDW; SEQ ID NO: 210) binds to human ST 2L. The antibodies as described herein may also be found in SEQ ID NO: 1, at amino acid residues T93 and F94, binds to human ST 2L.

Competition between antibodies of the invention comprising certain HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 amino acid sequences, or comprising certain VH and VL sequences, as described herein, for specific binding to human ST2L can be determined in vitro using well-known methods. For example, MSD Sulfo-Tag^TMBinding of NHS ester-labeled antibody to human ST2L in the presence of unlabeled antibody can be assessed by ELISA, or competition with the antibodies of the invention can be confirmed using Biacore analysis or flow cytometry. The ability of the test antibody to inhibit C2244 binding to human ST2L demonstrates that the test antibody can compete with these antibodies for binding to human ST 2L. Such exemplary antibodies are C2494, STLM208 and STLM213 shown in table 3 and figure 13.

Antibodies that compete with C2244 for binding to domain I of ST2L, as described herein, block IL-33/ST2L interactions and inhibit a range of ST2L biological activities, including IL-33-induced mast cell responses. A non-neutralizing (i.e., non-inhibitory) epitope also exists on ST2L domain I as a second antibody competitor group (represented by antibody C2240 that binds to domain I of ST2L, does not compete with C2244, and does not inhibit ST2L signaling).

Antibodies of the invention that bind to a particular ST2L domain or epitope, as described herein, can be prepared by immunizing a mouse expressing a human immunoglobulin locus (Lonberg et al, Nature 368: 856-9, 1994; Fishwild et al, Nature Biotechnology 14: 845-51, 1996; Mendez et al, Nature Genetics 15: 146-56, 1997; U.S. Pat. Nos. 5,770,429, 7,041,870 and 5,939,598) or a Balb/c mouse with a peptide encoding the epitope, such as a peptide having the amino acid sequence of domain I of human ST 2L: KFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQLLKFLPAAVADSGIYTCIVRSPTFNRTGYANVTIYKKQSDCNVPDYLM YSTV (SEQ ID NO: 9), or a peptide having the amino acid sequence RCPRQGKPSYTVDW (SEQ ID NO: 210), and was synthesized using Kohler et al, Nature 256: 495-97, 1975. The resulting antibodies were tested for binding to the epitope using standard methods. For example, (in silico) protein-protein docking on a computer chip can be performed to identify compatible sites of interaction when the structure of each component is known. The antigen and antibody complexes can be used for hydrogen-deuterium (H/D) exchange to map the regions on the antigen that are likely to be bound by the antibody. Fragments of the antigen and point mutagenesis can be used to locate amino acids important for antibody binding. The monoclonal antibodies identified may be further identified by methods such as Queen et al, Proc Natl Acad Sci (USA) 86: 10029-32, 1989 and Hodgson et al, Bio/Technology 9: 421, 1991, modified to retain binding affinity in combination with altered framework support residues.

The antibodies of the invention as described herein may be human antibodies or human adaptive antibodies. The antibodies of the invention as described herein may be of the IgA, IgD, IgE, IgG or IgM type.

Antibodies whose antigen binding site amino acid sequences are substantially the same as those shown in fig. 10, fig. 11, fig. 12, fig. 13, fig. 15, table 3, table 9, and table 12 are encompassed within the scope of the present invention. Typically, this involves one or more amino acid substitutions with amino acids having similar charge or hydrophobic or stereochemical characteristics, and the aim is to improve antibody properties, such as stability or affinity. For example, a conservative substitution may involve a substitution of a native amino acid residue with a non-native residue such that the polarity or charge of the amino acid residue at that position has little or no effect. In addition, any natural residues in the polypeptide may also be replaced with alanine, as described previously for alanine scanning mutagenesis (MacLennan et al, Acta physiol Scan and Suppl 643: 55-67, 1998; Sasaki et al, Adv Biophys 35: 1-24, 1998). The desired amino acid substitutions (whether conservative or non-conservative) may be determined by those skilled in the art when such substitutions are required. For example, amino acid substitutions may be used to identify residues that are important to the function of the antibody, such as residues that affect affinity, or residues that confer undesirable properties such as aggregation. Exemplary amino acid substitutions are shown in fig. 12 and 13.

Substitutions within the framework regions may also be made in contrast to the antigen binding site, provided they do not adversely affect the properties of the antibody. Framework substitutions may be made, for example, at Vernier Zone residues (us patent 6,649,055) to improve antibody affinity or stability. Substitutions may also be made in the antibody at positions in the framework that differ in sequence when compared to the homologous human germline gene sequence to reduce possible immunogenicity. These modifications can be made to antibodies derived, for example, from de novo antibody libraries such as pIX libraries.

Conservative amino acid substitutions also encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. Amino acid substitutions can be made, for example, by PCR mutagenesis (U.S. Pat. No. 4,683,195). Libraries of variants can be generated using well known methods, e.g., using random (NNK) or non-random codons (e.g., DVK codons, which encode 11 amino Acids (ACDEGKNRSYW)), and screening the libraries or variants for desired properties.

While the embodiments shown in the examples include paired variable regions, paired full-length antibody chains, or paired CDR1, CDR2, and CDR3 regions, one from the heavy chain and one from the light chain, the skilled artisan will recognize that alternative embodiments may include a single heavy chain variable region or a single light chain variable region, a single full-length antibody chain, or CDR1, CDR2, and CDR3 regions, from either the heavy or light chain of one antibody chain. The corresponding domains in one chain can be screened for using a single variable region of the other chain, the full length antibody chain or the CDR1, CDR2, and CDR3 regions, both of which are capable of forming an antibody that specifically binds ST 2L. The screening can be accomplished by phage display screening methods using, for example, the hierarchical dual combinatorial approach disclosed in PCT publication WO 1992/01047. In this method, a single colony comprising either an H chain clone or an L chain clone is used to infect a complete library of clones encoding the other chain (L or H), and the resulting double-stranded specific antigen-binding domain is selected according to the phage display technique described.

The invention provides isolated VH and VL domains of an antibody of the invention as described herein, as well as antibodies comprising particular VH and VL domains. The VH and VL variable regions of certain antibodies of the invention as described herein are shown in figure 13 and table 12.

One embodiment of the invention is an isolated human or human adaptive antibody antagonist or fragment thereof that specifically binds to domain I of human ST2L (SEQ ID NO: 9), comprising an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 191 the VH has a VH of at least 90% identity.

Another embodiment of the invention is an isolated human or human adaptive antibody antagonist or fragment thereof that specifically binds to Domain I of human ST2L (SEQ ID NO: 9), comprising an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: the VL of 209 has a VL of at least 94% identity.

In some embodiments described herein, the invention provides an antibody comprising SEQ ID NO: 143. 144, 145, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204 or 205.

In some embodiments described herein, the invention provides an antibody comprising SEQ ID NO: 135. 136, 137, 138, 139, 140, 141, 142, 206, 207, 208, or 209.

In some embodiments described herein, the invention provides an antibody comprising

SEQ ID NO: 186. 187, 197, 198, 199, 200, 201, 202, 203, 204, or 205 and the VH of SEQ id no: 206 VL;

SEQ ID NO: 195 or 196 and SEQ ID NO: 207 VL;

SEQ ID NO: 188. 189 or 190 and SEQ ID NO: 208 VL; or

SEQ ID NO: 187. 191, 192, 193 or 194 and SEQ ID NO: VL of 209.

Another embodiment of the invention is an isolated human or human adaptive antibody antagonist or fragment thereof that specifically binds to Domain I of human ST2L (SEQ ID NO: 9), comprising:

SEQ ID NO:97 HCDR 1;

SEQ ID NO: 114 HCDR 2;

SEQ ID NO: HCDR3 of 84;

SEQ ID NO: 130 LCDR 1;

SEQ ID NO: LCDR2 of 90;

SEQ ID NO: LCDR3 of 134; or

SEQ ID NO: 191 and SEQ ID NO: VL of 209.

Human monoclonal antibodies lacking any non-human sequence can be generated by, for example, Knappik et al, J Mol Biol 296: 57-86, 2000; and Krebs et al, J Immunol Meth 254: 67-842001 were prepared and optimized from phage display libraries. In an exemplary method, the antibodies of the invention are isolated from a library of antibody heavy and light chain variable regions expressed as fusion proteins with phage pIX coat proteins. Antibody libraries were screened for those that bound to human ST2L-ECD and the resulting positive clones were further characterized, and the Fabs were isolated from the clone lysates and expressed as full-length IgG. Exemplary antibody libraries and screening methods are described in Shi et al, J Mol Biol 397: 385-96, 2010; international patent publication WO2009/085462 and U.S. patent No. 12/546850; U.S. Pat. nos. 5,223,409, 5,969,108 and 5,885,793).

The resulting monoclonal antibody may be further modified in its framework regions to alter certain framework residues to residues found in the matching human germline.

The immune effector properties of the antibodies of the invention can be enhanced or silenced via Fc modification by techniques known to those skilled in the art. For example, Fc effector functions such as C1q binding, Complement Dependent Cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors; BCR), and the like, can be provided and/or controlled by modifying residues in the Fc that contribute to these activities. Pharmacokinetic properties may also be enhanced by mutating residues in the Fc domain that prolong the half-life of the antibody (Strohl Curr Opin Biotechnol 20: 685-91, 2009). Exemplary Fc modifications are IgG4S228P/L234A/L235A, IgG2M252Y/S254T/T256E (Dall' Acqua et al, J Biol Chem 281: 23514-24, 2006; or IgG2V234A/G237A/P238S, V234A/G237A/H268Q, H268A/V309L/A330S/P331 or V234/G237A/P238S/H268A/V309L/A330S/P331S on IgG2 (International patent application WO2011/066501) (numbering according to EU numbering).

In addition, the antibodies of the invention as described herein can be post-translationally modified by processes such as glycosylation, isomerization, deglycosylation, or non-naturally occurring covalent modifications such as the addition of polyethylene glycol moieties (pegylation) and lipidation. Such modifications can be made in vivo or in vitro. For example, an antibody of the invention as described herein may be conjugated to polyethylene glycol (pegylated) to improve its pharmacokinetic properties. Conjugation can be performed by techniques known to those skilled in the art. Conjugation of therapeutic antibodies to PEG has been shown to enhance pharmacodynamics while not interfering with function (Knigh et al, Plateleles 15: 409-18, 2004; Leong et al, Cytokine 16: 106-19, 2001; Yang et al, Protein Eng 16: 761-70, 2003).

Antibodies or fragments thereof of the invention modified as described herein to improve stability, selectivity, cross-reactivity, affinity, immunogenicity, or other desired biological or biophysical properties are within the scope of the invention. The stability of antibodies is affected by many factors, including (1) the core stack of individual domains that affect their intrinsic stability, (2) protein/protein interface interactions that have an effect on HC and LC pairing, (3) the entrapment of polar and charged residues, (4) the H-bond network of polar and charged residues; and (5) surface charge and polar residue distribution in other intramolecular and intermolecular forces (Worn et al, J MolBiol 305: 989-1010, 2001). Potential structurally unstable residues can be identified based on the crystal structure of the antibody or, in some cases, by molecular modeling, and the effect of the residues on antibody stability can be tested by generating and evaluating variants carrying mutations in the identified residues. One of the methods for increasing the stability of an antibody is to increase the thermal transition midpoint (Tm) as determined by Differential Scanning Calorimetry (DSC). Generally, the Tm of a protein is linked to its stability and inversely related to its susceptibility to unfolding and denaturation in solution and degradation processes that depend on the unfolding tendency of the protein (Remmele et al, Biopharm 13: 36-46, 2000). A number of studies have found a correlation between the level of formulation physical stability as measured by DSC in terms of thermal stability and physical stability as measured by other methods (Gupta et al, AAPS PharmSci 5E8, 2003; Zhang et al, J Pharm Sci 93: 3076-89, 2004; Maa et al, Int JPharm 140: 155-68, 1996: Bedu-Addo et al, Pharm Res 21: 1353-61, 2004; Remmele et al, Pharm Res 15: 200-8, 1997). Formulation studies suggest that the Tm of Fab is involved in the long-term physical stability of the corresponding monoclonal antibody. Amino acid differences within the framework or CDRs can have a significant impact on the thermal stability of the Fab domain (Yasui et al, FEBS Lett 353: 143-6, 1994).

As described hereinThe antibodies of the invention that specifically bind to human ST2L domain I can be engineered into bispecific antibodies, which are also encompassed within the scope of the invention. The VL and/or VH domains of the antibodies of the invention can be engineered into structures such asSingle chain bispecific antibodies designed (international patent publication WO 1999/57150; U.S. patent publication US2011/0206672), or engineered to have structures as disclosed in U.S. patent nos. US 5869620; bispecific scFV of the structure in international patent publication WO1995/15388A, international patent publication WO1997/14719 or international patent publication WO 2011/036460.

The VL region and/or VH region of an antibody of the invention, as described herein, may be engineered into a bispecific full length antibody, wherein each antibody arm binds a different antigen or epitope. Such bispecific antibodies are typically prepared by modulating the CH3 interaction between the heavy chains of these two antibodies, using techniques such as those described in US 7695936; international patent publications WO 04/111233; U.S. patent publication US 2010/0015133; U.S. patent publication US 2007/0287170; international patent publication WO 2008/119353; U.S. patent publication US 2009/0182127; U.S. patent publication US 2010/0286374; U.S. patent publication US 2011/0123532; international patent publication WO 2011/131746; international patent publication WO 2011/143545; or those described in US patent publication US2012/0149876 to form bispecific antibodies. Further bispecific structures in which the VL region and/or VH region of an antibody of the invention can be bound are, for example, double variable domain immunoglobulins (international patent publication WO2009/134776), or structures comprising multiple dimeric domains to link two antibody arms with different specificities, such as leucine zippers or collagen dimeric domains (international patent publication WO 2012/022811; U.S. patent US 5932448; U.S. patent US 6833441).

Another aspect of the invention is an isolated polynucleotide encoding any of the antibody heavy chain variable regions or antibody light chain variable regions or fragments thereof of the invention, or their complements. Certain exemplary polynucleotides are disclosed herein, however, other polynucleotides encoding the antibody antagonists of the invention in view of the degeneracy or codon bias of a given genetic code in a given expression system are also within the scope of the invention. Exemplary polynucleotides of the invention are those set forth in SEQ ID NO: 211. 212, 213 and 214.

Another embodiment of the invention is a vector comprising a polynucleotide of the invention. Such vectors may be plasmid vectors, viral vectors, baculovirus expression vectors, transposon-based vectors, or any other suitable vector for introducing the polynucleotides of the invention into a given organism or genetic background by any means.

Another embodiment of the invention is a host cell comprising a polynucleotide of the invention. Such host cells may be eukaryotic cells, bacterial cells, plant cells, or archaeal cells. Exemplary eukaryotic cells can be of mammalian, insect, avian, or other animal origin. Mammalian eukaryotic cells include immortalized cell lines such as hybridoma or myeloma cell lines such as SP2/0 (American type culture Collection (ATCC), Manassas, VA, CRL-1581), NS0 (European cell culture Collection (ECACC), Salisbury, Wiltshire, UK, ECACC No.85110503), FO (ATCC CRL-1646), and Ag (ATCC CRL-1580) murine cell lines. An exemplary human myeloma cell line is U266(ATTC CRL-TIB-196). Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells, such as CHO-KlSV (Lonza Biologics, Walkersville, Md.), CHO-K1(ATCC CRL-61) or DG 44.

Another embodiment of the invention is a method for producing an antibody that specifically binds to domain I of ST2L, said method comprising culturing a host cell of the invention and recovering the antibody produced by the host cell. Methods for making and purifying antibodies are well known in the art.

Another embodiment of the invention is a method of inhibiting the interaction of ST2L with IL-33 in a subject, comprising administering to the subject an antibody that specifically binds domain I of ST2L in an amount sufficient to inhibit the interaction of ST2L and IL-33.

Method of treatment

An ST2L antagonist of the invention as described herein, e.g., an ST2L antibody antagonist that blocks IL-33/ST2L interaction and binds ST2L domain I, an antagonist of an antibody that specifically binds to a peptide comprising SEQ ID NO: 47 and SEQ ID NO: 51 competes for binding to the antibody of human ST2L (SEQ ID NO: 1), or the light chain variable region of SEQ ID NO: 1 (RCPRQGKPSYTVDW; SEQ ID NO: 210) that binds to human ST2L may be used to modulate the immune system. The antibodies of the invention as described herein may be more effective in antagonizing ST2L biological activity when compared to antibodies that bind other domains and/or regions of ST2L, e.g., the antibodies of the invention are able to more effectively reduce IL-33-induced mast cell responses. Any antibody of the invention can be used in the methods of the invention. Exemplary antibodies that can be used in the methods of the invention are antibodies STLM62, STLM15, STLM103, STLM107, STLM108, STLM123, STLM124, STLM206, STLM207, STLM208, STLM209, STLM210, STLM211, STLM212, STLM 213. Without wishing to be bound by any theory, it is theorized that antibody antagonists that bind domain I and block the IL-33/ST2L interaction may inhibit IL-1RAcP/IL-33/ST2L/cKit complex formation or downstream signaling on mast cells, whereas domain III binding antibodies, while capable of inhibiting IL-1RAcP recruitment to the ST2L/IL-33 complex, may not be able to disrupt the formation of larger IL-1RAcP/IL-33/ST2L/cKit complexes that are specifically present on mast cells. The chip analysis performed supports the inference, as the chip analysis demonstrated that anti-ST 2L domain I binding antibody inhibits most mast cell signaling pathways induced by IL-33, while anti-ST 2L domain III binding antibody inhibits only a small portion of these signaling pathways. It is possible that, since IL-33 binds to ST2L before the helper proteins IL-1RAcP associate, blocking IL-33 binding to ST2L by domain I binding antibodies could avoid any other association of helper proteins, including cKit or yet unidentified helper receptors. Domain III binding antibodies that do not interfere with IL-33 binding to ST2L theoretically block IL-1RAcP association, but do not block the association of other co-receptors, including co-receptors not yet identified. A number of models have been proposed to explain how IL-1RAcP interacts with the IL-1/IL-1R or ST2L/IL-33 complex (Lingel et al, Structure 17: 1398-. These models indicate that IL-1RAcP can bind to one side of the complex, but it is not yet known which side binds. Thus, it is possible that the "other side" or "free side" of the complex may be available for association with alternative co-receptors that are not blocked by the domain III antibody, and off-target effects such as increased recruitment of another co-receptor may occur, resulting in increased signaling.

In the methods of the invention, any antibody antagonist that specifically binds to human ST2L domain I, an antibody antagonist that blocks IL-33/ST2L interaction and binds to human ST2L domain I, an antibody that specifically binds to a polypeptide comprising SEQ ID NO: 47 and seq id NO: 51 competes for binding to the antibody of human ST2L (SEQ ID NO: 1), or the light chain variable region of SEQ ID NO: 1 (RCPRQGKPSYTVDW; SEQ ID NO: 210) and human ST 2L. Additional features of such antibodies include the ability of the antibodies to block the IL-33/ST2L interaction and inhibit the human mast cell response.

Thus, the antibodies of the invention are useful for treating a range of ST 2L-mediated disorders, ST 2L-mediated inflammatory disorders, and disorders requiring inhibition of mast cell responses.

The methods of the invention can be used to treat animal patients belonging to any classification. Examples of such animals include mammals such as humans, rodents, canines, felines and farm animals. For example, the antibodies of the invention may be used to prevent and treat ST 2L-mediated disorders, such as inflammatory diseases, including asthma, airway hyperreactivity, sarcoidosis, Chronic Obstructive Pulmonary Disease (COPD), Idiopathic Pulmonary Fibrosis (IPF), cystic fibrosis, Inflammatory Bowel Disease (IBD), rheumatoid arthritis, eosinophilic esophagitis, scleroderma, atopic dermatitis, allergic rhinitis, bullous pemphigoid, chronic urticaria, diabetic nephropathy, interstitial cystitis, or graft-versus-host disease (GVHD). The antibodies of the invention are useful for the prevention and treatment of immune diseases mediated at least in part by mast cells, such as asthma, eczema, pruritus, allergic rhinitis, allergic conjunctivitis, and autoimmune diseases, such as rheumatoid arthritis, bullous pemphigoid, and multiple sclerosis.

The antibodies of the invention may also be used in the preparation of a medicament for such treatment, wherein the medicament is prepared for administration at a dose as defined herein.

Mast cells play a central role in allergic inflammation and asthma by releasing a variety of mediators (reviewed in Amin, Respir Med 106: 9-14, 2012). ST2L is highly expressed on mast cells, and its activation leads to the expression of a variety of pro-inflammatory cytokines and other mediators. Inhibition of ST2L activity is presumed to interfere with mast cell mediated inflammatory cell recruitment and to modulate chronic inflammation.

Mast cells respond rapidly to stimuli, including allergens, cold air, pathogens; injury to epithelial cells by these stimuli can result in the release of IL-33 (reviewed in Zhao and Hu, Cell & Molec Immunol 7: 260-2, 2012). Mast cells release leukotrienes, histamine, prostaglandins, and cytokines to increase vascular permeability and bronchoconstriction, and recruit other immune cells, such as neutrophils, eosinophils, and T lymphocytes, to this site (Henderson et al, JEM 184: 1483-94, 1996; White et al, JACI 86: 599-. In addition, they enhance immune responses by inducing upregulation of adhesion molecules on endothelial cells to increase immune cell trafficking (Meng et al, Jcell Physiol 165: 40-53, 1995). Mast cells play an important role in airway remodeling; in asthmatic patients, increased numbers of mast cells are found within the Airway Smooth Muscle (ASM) cell layer, which secrete mediators to promote ASM proliferation (reviewed in Okayama et al, Curr Opin Immunol 19: 687-93, 2007).

Inflammatory pulmonary disorders are one example of inflammatory disorders. Exemplary inflammatory pulmonary disorders include infection-induced pulmonary disorders, including pulmonary disorders associated with viral, bacterial, fungal, parasitic, or prion infections; allergen-induced pulmonary disorders; pollutant-induced lung disorders such as asbestosis, silicosis or berylliosis; pulmonary disorders induced by inhalation of gastric contents, immune disorders, inflammatory disorders with genetic predisposition such as cystic fibrosis, and pulmonary disorders induced by physical trauma such as ventilator lung injury. These inflammatory conditions also include asthma, emphysema, bronchitis, Chronic Obstructive Pulmonary Disease (COPD), sarcoidosis, histiocytosis, lymphangiosarcoidosis, acute lung injury, acute respiratory distress syndrome, chronic lung disease, bronchopulmonary dysplasia, community-acquired pneumonia, nosocomial pneumonia, ventilator-associated pneumonia, septicemia, viral pneumonia, influenza infection, variant influenza virus infection, rotavirus infection, human metapneumovirus infection, respiratory syncytial virus infection, and Aspergillus (Aspergillus) or other fungal infections. Exemplary infection-related inflammatory diseases may include viral or bacterial pneumonia, including severe pneumonia, cystic fibrosis, bronchitis, acute exacerbation of the respiratory tract, and Acute Respiratory Distress Syndrome (ARDS). Such infection-related disorders may involve multiple infections, such as primary viral infections and secondary bacterial infections. Dysregulated ST2L signaling may play a role in the pathogenesis of pulmonary diseases such as asthma and Chronic Obstructive Pulmonary Disease (COPD) (reviewed in Alcorn et al, Annu Rev Physiol 72: 495-516, 2010). Commonly used animal models of asthma and airway inflammation include ovalbumin challenge model, methacholine sensitization model, and sensitization with Aspergillus fumigatus (Hessel et al, Eur J Pharmacol 293: 401-12, 1995). Inhibition of cytokine and chemokine production from cultured human bronchial epithelial cells, bronchial fibroblasts, or airway smooth muscle cells can also be used as an in vitro model. Administration of the antagonists of the invention to any of these models can be used to assess the use of these antagonists to improve symptoms and alter the course of asthma, airway inflammation, COPD, and the like.

Asthma is an inflammatory disease of the lung characterized by airway hyperresponsiveness ("AHR"), bronchoconstriction, wheezing, eosinophilic or neutrophilic inflammation, mucus hypersecretion, subepithelial fibrosis, and elevated IgE levels. Patients with asthma experience "acute exacerbations" (exacerbations of symptoms), most commonly due to microbial infections of the respiratory tract (e.g., rhinovirus, influenza virus, haemophilus influenzae). Asthma attacks can be triggered by environmental factors (e.g., roundworms, insects, animals (e.g., cats, dogs, rabbits, mice, rats, hamsters, guinea pigs, and birds), fungi, atmospheric pollutants (e.g., tobacco smoke), irritant gases, smoke, vapors, aerosols, chemicals, pollen, active or cold air in addition to asthma, a variety of chronic inflammatory diseases affecting the lungs are characterized by neutrophilic infiltration into the airways, such as Chronic Obstructive Pulmonary Disease (COPD), bacterial pneumonia, and cystic fibrosis (Linden et al, Eur Respir J15: 973-7, 2000; Rahman et al, Clin Immunol 115: 268-76, 2005), and diseases such as COPD, allergic rhinitis, and cystic fibrosis are characterized by airway hyperresponsiveness (Fahy and O' Byrne, Am J Respir Med cre 163: 822-3, 2001). Commonly used animal models of asthma and airway inflammation include a model of methacholine challenge following ovalbumin sensitization and challenge (Hessel et al, Eur JPharmacol 293: 401-12, 1995). Inhibition of cytokine and chemokine production from cultured human bronchial epithelial cells, bronchial fibroblasts, or airway smooth muscle cells can also be used as an in vitro model. Administration of the antibody antagonists of the invention to any of these models can be used to assess the use of these antagonists to improve symptoms and alter the course of asthma, airway inflammation, COPD, and the like.

IL-5 and IL-13 (type 2 cytokine) secretion is caused by IL-33 signaling through ST2L receptors on TH2 cells, basophils, mast cells and newly discovered innate type 2 lymphocytes (ILC, reviewed in Spits et al, Nature Reviews Immunology 13: 145-149, 2013). The correlation of these pathways is demonstrated by the beneficial effects of therapies targeting IL-5 or IL-13 in asthma. IL-5 activates eosinophils and a subset of severe asthma patients with hypersial eosinophilia are treated with monoclonal antibodies that neutralize IL-5, with the result that exacerbation events are reduced (Nair et al, N Engl J Med.2009; 360 (10): 985-93). IL-13 has been reported to contribute to IgE synthesis, mucus secretion and fibrosis. Treatment of severe asthma patients with anti-IL-13 monoclonal antibodies resulted in improved lung function, with a subset showing greater improvement (coren et al, n.engl.j.med., 365: 1088-. Other mediators of the differential immune pathway are also involved in asthma pathogenesis, and blocking these mediators other than ST2L may provide additional therapeutic benefits. Therapies that target multiple type 2 cytokines, or upstream pathways of type 2 cytokine production, may have beneficial effects in severe disease.

The VH and VL domains of the ST2L antibodies of the invention can be incorporated into bispecific antibodies and molecules described herein, wherein the bispecific antibody specifically binds domain I of ST2L and a second antigen, such as TSLP (thymic stromal lymphopoietin), IL-25, IL-17RB, or TSLPR.

IL-25 and TSLP (e.g., IL-33) trigger type 2 cytokine release through different signaling complexes: IL-25(IL-17E) is a member of the IL-17 family and signals through IL-17RA/IL-17RB, while TSLP is a member of the IL-7 family and signals through the TSLPR/IL-7R α heterodimer (reviewed in Koyasu et al, Immunol 132: 475-. Animals deficient in IL-33, ST2L, IL-25, IL-17RB, TSLP, or TSLPR demonstrated less severe airway inflammation in at least one of a number of different types of mouse models of asthma; in most of these animal models, however, protection against airway inflammation may be lacking, thereby increasing the likelihood that exposure of epithelial cells to various allergens or pathogens may concomitantly trigger release of IL-33, IL-25 and TSLP. Hammat et al reported that administration of house dust mite extracts to mice resulted in the release of IL-25, TSLP, and IL-33 (as well as IL-5 and IL-13 downstream of IL-33) into the airways (Hammat et al, NatMed 15: 210-. This suggests that blocking ST2L and TSLP and/or IL-25 may have beneficial effects, particularly in severe airway disease.

In another embodiment of the invention, antibody antagonists that specifically bind to human ST2L domain I can be used to generate bispecific molecules that bind ST2L and TSLP, ST2L and IL-25, ST2L and TSLPR, ST2L and IL-17RA, or ST2L and IL-17 RB.

In another embodiment of the invention, the antibody antagonist that specifically binds human ST2L domain I is a bispecific antibody, wherein the antibody further binds TSLP, IL-25, TSLPR, IL-17RA, or IL-17 RB.

TSLP, IL-25, TSLPR, IL-17RA and IL-17RB binding antibodies can be generated using the methods described herein, such as immunizing mice expressing human immunoglobulin loci (Lonberg et al, Nature 368: 856-9, 1994; Fishwild et al, Nature Biotechnology 14: 845-51, 1996; Mendez et al, Nature Genetics 15: 146-56, 1997; U.S. Pat. Nos. 5,770,429, 7,041,870 and 5,939,598) or Balb/c mice with the corresponding protein or the extracellular domain of the protein or using a phage display library as described herein. Alternatively, bispecific molecules can be generated using existing antibodies to TSLP, IL-25, TSLPR, IL-17RA, and IL-17 RB. Exemplary IL-25 antibodies that can be used are those described in, for example, international patent publication WO 2011/123507.

Arthritis (including osteoarthritis, rheumatoid arthritis, arthritic joints caused by injury, etc.) is a common inflammatory condition that may benefit from the therapeutic use of anti-inflammatory proteins such as the antagonists of the present invention. Activation of ST2L signaling may sustain inflammation and further tissue damage in inflamed joints. Various animal models of rheumatoid arthritis are known. For example, in the collagen-induced arthritis (CIA) model, mice develop chronic inflammatory arthritis that is highly similar to human rheumatoid arthritis. ST2L deficient (ST2KO) mice developed mild disease in this model, and the lesions in this model were dependent on mast cell ST2L expression (Xu et al, PNAS 105: 10913-8, 2008). In this model, infiltration of monocytes and polymorphonuclear cells and synovial hyperplasia in the joints of the ST2KO mouse were reduced. ST2KO mice cocultured with Collagen (CII) drain lymph nodes showing significantly reduced IL-17, IFN γ and TNF α production. ST2L deficient mice adoptively transferred Wild Type (WT) bone marrow-derived mast cells (BMMC) prior to induction of CIA developed more severe CIA than these ST2KOBMMC transplanted mice. Thus, mast cell ST2L signaling is critical to the development of arthritis in a mouse model resembling human rheumatoid arthritis. Administration of the ST2L antibodies of the invention, which inhibit mast cell responses, to CIA model mice can be used to assess the utility of these antagonists in improving symptoms and altering the course of the disease.

Exemplary gastrointestinal inflammatory disorders are Inflammatory Bowel Disease (IBD), Ulcerative Colitis (UC) and Crohn's Disease (CD), environmental damage-induced colitis (e.g., gastrointestinal inflammation (e.g., colitis) caused or associated (e.g., as a side effect) by treatment regimens such as administration of chemotherapy, radiation therapy, etc.), infectious colitis, ischemic colitis, collagenous or lymphatic colitis, necrotizing enterocolitis, colitis under conditions such as chronic granulomatous disease or celiac disease, food allergies, gastritis, infectious gastritis or enterocolitis (e.g., Helicobacter pylori infectious chronic active gastritis), and other forms of gastrointestinal inflammation due to infectious agents. There are several animal models for gastrointestinal inflammatory disorders. Some of the most widely used models are the 2, 4, 6-trinitrobenzenesulfonic acid/ethanol (TNBS) -induced colitis model orThe azolone model, which induces chronic inflammation and ulceration in the colon (Neurath et al, Intern Rev Immunol 19: 51-62, 2000). Another model used Dextran Sodium Sulfate (DSS), which induced acute colitis manifested by bloody diarrhea, weight loss, colon shortening and mucosal ulceration with neutrophil infiltration. Another model involves the initial CD45RB^{Height of}Adoptive transfer of CD4T cells to RAG or SCID mice. In this model, donor naive T cells attack the recipient intestine causing chronic intestinal inflammation and symptoms similar to human inflammatory bowel disease (Read and powre, Curr Protoc Immunol, chapter 15, unit 15.13, 2001). Administration of the antagonists of the invention in any of these models can be used to assess that these antagonists improve symptoms and changes and the gutPotential efficacy of inflammatory diseases such as inflammatory bowel disease-related processes.

Renal fibrosis can develop from acute lesions such as graft ischemia/reperfusion (Freese et al, Nephrol Dialtransplant 16: 2401-6, 2001) or chronic disorders such as diabetes (Ritz et al, Nephrol Dialtransplant 11Suppl 9: 38-44, 1996). The pathogenic mechanism is usually characterized by an initial inflammatory response followed by persistent fibrogenesis in the glomerular filter and tubulointerstitium (Liu, Kidney Int 69: 213-7, 2006). Tubulointerstitial fibrosis has been shown to play a key role in the pathogenesis of renal injury to end-stage renal failure, and proximal tubule cells have been demonstrated to be the central mediator (Phillips and Steadman, Histol Histopathol 17: 247-52, 2002; Phillips, ChangGung Med J30: 2-6, 2007). Fibrogenesis in the tubulointerstitial compartment is mediated in part by the activation of resident fibroblasts that secrete proinflammatory cytokines, stimulating proximal tubulo-epithelial cells to secrete local inflammatory and fibrogenic mediators. In addition, chemotactic cytokines are secreted by fibroblasts and epithelial cells and provide a directional gradient that directs infiltration of monocytes/macrophages and T cells into the tubulointerstitium. Inflammatory infiltration produces additional fibrogenic and inflammatory cytokines that further activate fibroblast and epithelial cytokine release, while also stimulating epithelial cells to phenotypically transform, wherein the cells deposit excess extracellular matrix components (Simonson, Kidney Int 71: 846-54, 2007).

Other exemplary fibrotic disorders may include liver fibrosis (including but not limited to alcohol-induced sclerosis, virus-induced sclerosis, autoimmune-induced hepatitis); pulmonary fibrosis (including but not limited to scleroderma, idiopathic pulmonary fibrosis); kidney fibrosis (including but not limited to scleroderma, diabetic nephritis, glomerulonephritis, lupus nephritis); epidermal fibrosis (including but not limited to scleroderma, hypertrophic and keloid scars, burns); myelofibrosis; neurofibroma; fibroids; intestinal fibrosis; and surgically induced fibrous adhesions. The fibrosis may be organ specific fibrosis or systemic fibrosis. The organ-specific fibrosis may be associated with pulmonary fibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, vascular fibrosis, skin fibrosis, ocular fibrosis or bone marrow fibrosis. The pulmonary fibrosis may be associated with idiopathic pulmonary fibrosis, drug-induced pulmonary fibrosis, asthma, sarcoidosis, or chronic obstructive pulmonary disease. The liver fibrosis may be associated with cirrhosis, schistosomiasis, or cholangitis. The cirrhosis can be alcoholic cirrhosis, posthepatitis C cirrhosis, primary biliary cirrhosis. The cholangitis may be sclerosing cholangitis. The renal fibrosis may be associated with diabetic nephropathy or lupus glomerulosclerosis. The cardiac fibrosis may be associated with myocardial infarction. The vascular fibrosis may be associated with arterial restenosis or atherosclerosis following angioplasty. The skin fibrosis may be associated with burn scars, hypertrophic scars, keloids, or nephrogenic fibrotic skin diseases. The ocular fibrosis may be associated with posterior ocular fibrosis, cataract surgery or proliferative vitreoretinopathy. The myelofibrosis may be associated with primary myelofibrosis or drug-induced myelofibrosis. The systemic fibrosis may be systemic sclerosis or graft versus host disease.

Other inflammatory conditions and neuropathies that may be prevented or treated by the methods of the present invention are those resulting from autoimmune diseases. These conditions and neuropathies include multiple sclerosis, systemic lupus erythematosus, and neurodegenerative and Central Nervous System (CNS) disorders including Alzheimer's disease, Parkinson's disease, Huntington's disease, bipolar disorder, and Amyotrophic Lateral Sclerosis (ALS), liver diseases including primary biliary cirrhosis, primary sclerosing cholangitis, non-alcoholic fatty liver disease/steatohepatitis, fibrosis, Hepatitis C Virus (HCV) and Hepatitis B Virus (HBV), diabetes, and insulin resistance, cardiovascular diseases including atherosclerosis, cerebral hemorrhage, stroke, and myocardial infarction, arthritis, rheumatoid arthritis, psoriatic arthritis, and Juvenile Rheumatoid Arthritis (JRA), osteoporosis, osteoarthritis, pancreatitis, fibrosis, encephalitis, psoriasis, giant cell arteritis, osteoporosis, and Alzheimer's disease, Ankylosing spondylitis, autoimmune hepatitis, Human Immunodeficiency Virus (HIV), inflammatory skin disorders, transplantation, cancer, allergy, endocrine diseases, wound repair, other autoimmune diseases, airway hyperreactivity, and cell, virus or prion-mediated infections or disorders.

One embodiment of the invention is a method of treating or preventing a ST 2L-mediated disorder comprising administering a therapeutically effective amount of a peptide that specifically binds to human ST2L domain I (SEQ ID NO: 9), blocks IL-33/ST2L interaction, binds to a polypeptide comprising SEQ ID NO: 47 and SEQ ID NO: 51 (VL) competes for binding to human ST2L (SEQ ID NO: 1) and/or an amino acid sequence set forth in SEQ ID NO: 1 (RCPRQGKPSYTVDW; SEQ ID NO: 210) to human ST2L for a time sufficient to treat or prevent an ST 2L-mediated disorder.

Another embodiment of the invention is a method of inhibiting mast cell response in a patient comprising administering a therapeutically effective amount of a peptide that specifically binds human ST2L domain I (SEQ ID NO: 9), blocks IL-33/ST2L interaction, binds to a polypeptide comprising SEQ ID NO: 47 and SEQ ID NO: 51 (VL) competes for binding to human ST2L (SEQ ID NO: 1) and/or an amino acid sequence set forth in SEQ ID NO: 1 (RCPRQGKPSYTVDW; SEQ id no: 210) to human ST2L for a time sufficient to inhibit mast cell response in a patient in need thereof.

Another embodiment of the invention is a method of inhibiting the interaction of IL-33 and ST2L in a subject, comprising administering to the subject a peptide that specifically binds to human ST2L domain I (SEQ ID NO: 9), blocks IL-33/ST2L interaction, binds to a peptide comprising the amino acid sequence of SEQ ID NO: 47 and SEQ id no: 51 (VL) competes for binding to human ST2L (SEQ ID NO: 1) and/or an amino acid sequence set forth in SEQ ID NO: 1 amino acid residues 35-48 (RCPRQGKPSYTVDW; SEQ ID NO: 210) binds to human ST 2L.

In another embodiment, the ST 2L-mediated disorder is asthma, airway hyperreactivity, sarcoidosis, Chronic Obstructive Pulmonary Disease (COPD), Idiopathic Pulmonary Fibrosis (IPF), cystic fibrosis, Inflammatory Bowel Disease (IBD), eosinophilic esophagitis, scleroderma, atopic dermatitis, allergic rhinitis, bullous pemphigoid, chronic urticaria, diabetic nephropathy, rheumatoid arthritis, interstitial cystitis, or Graft Versus Host Disease (GVHD).

In another embodiment, the ST 2L-mediated disorder is associated with inflammatory cell recruitment, goblet cell proliferation, or increased mucus secretion in the lung.

In another embodiment, the ST 2L-mediated condition is associated with a mast cell response.

In another embodiment, the inhibiting a mast cell response comprises inhibiting the level of GM-CSF, IL-5, IL-8, IL-10, or IL-13 released from human cord blood-derived mast cells with 50 μ g/ml antibody by at least 50%.

In another embodiment, the antibody antagonist administered to a patient in need thereof is a peptide that specifically binds human ST2L domain I (SEQ ID NO: 9), blocks IL-33/ST2L interaction, binds to a peptide comprising SEQ ID NO: 47 and SEQ ID NO: 51 (VL) competes for binding to human ST2L (SEQ ID NO: 1) and/or an amino acid sequence set forth in SEQ ID NO: 1 (RCPRQGKPSYTVDW; SEQ ID NO: 210) and also binds TSLP, IL-25, TSLPR, IL-17RA or IL-17 RB.

Administration/pharmaceutical composition

A "therapeutically effective amount" of an anti-ST 2L antibody effective to treat a disorder in which modulation of the biological activity of ST2L is desired can be determined by standard research techniques. For example, the dose of anti-ST 2L antibody that will be effective in treating an inflammatory disorder, such as asthma or rheumatoid arthritis, can be determined by administering the anti-ST 2L antibody to a relevant animal model, such as the model described herein.

In addition, in vitro assays may optionally be used to aid in identifying optimal dosage ranges. The selection of a particular effective dose can be determined by one of skill in the art based on consideration of a variety of factors (e.g., via clinical trials). Such factors include the disease to be treated or prevented, the symptoms involved, the weight of the patient, the immune status of the patient, and other factors known to the skilled artisan. The precise dose to be employed in the formulation will also depend on the route of administration and the severity of the disease and should be determined at the discretion of the practitioner and the condition of the individual patient. Effective doses can be derived from dose response curves from in vitro or animal model test systems.

The mode of administration for therapeutic use of the antibodies of the invention may be any suitable route of delivery of the agent to the host. Pharmaceutical compositions of these antibodies are particularly useful for parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal administration.

The antibodies of the invention can be prepared as pharmaceutical compositions containing an effective amount of the agent as the active ingredient in a pharmaceutically acceptable carrier. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the active compound is administered. Such pharmaceutical vehicles can be liquids, such as water and oils, including those derived from petroleum, animal, vegetable or synthetic sources, such as peanut oil, soybean oil, mineral oil, and sesame oil, and the like. For example, 0.4% saline solution and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional known sterilization techniques, such as filtration. The composition may contain pharmaceutically acceptable auxiliary substances as necessary to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickening agents, lubricants, and coloring agents, and the like. The concentration of the antibody of the invention in such pharmaceutical formulations can vary widely, i.e., less than about 0.5%, typically at or at least about 1% up to 15 or 20% by weight, and will be selected based primarily on the desired dose, fluid volume, viscosity, etc., depending on the particular mode of administration selected.

Thus, a pharmaceutical composition of the invention for intramuscular injection may be prepared to contain 1ml of sterile buffered water, and between about 1ng to about 100mg of an anti-ST 2L antibody of the invention, e.g., about 50ng to about 30mg, or more preferably about 5mg to about 25 mg. Similarly, a pharmaceutical composition of the invention for intravenous infusion may be prepared to contain about 250ml of sterile ringer's solution, and about 1mg to about 30mg, and preferably 5mg to about 25mg, of an antagonist of the invention. The actual methods for preparing compositions for parenteral administration are well known and are described in more detail, for example, in "Remington's pharmaceutical Science", 15 th edition, Mack Publishing Company, Easton, Pa.

The antibodies of the invention can be lyophilized for storage and reconstituted in a suitable vehicle prior to use. This technique has proven effective for conventional immunoglobulin and protein preparation, and lyophilization and reconstitution techniques known in the art can be employed.

The invention will now be described with reference to the following specific, non-limiting examples.

Materials and methods (in general)

Human and cynomolgus macaques (Macaca fascicularis, cyno) receptor-ligand binding inhibition assay (RLB assay)

With 50. mu.l of His with C-terminus in bicarbonate buffer₆Tag 4. mu.g/ml human ST2L-ECD (amino acids 19-328 of SEQ ID NO: 1) or 2. mu.g/ml cyno ST2L-ECD (amino acids 19-321 of SEQ ID NO: 2) were coated on 96-well plates for 16 hours at 4 ℃. All subsequent steps were performed at room temperature. The plate was blocked with 200. mu.l blocking buffer and washed 3 times with 300. mu.l wash buffer containing PBS + 0.05% Tween. Mu.l of each dilution of anti-ST 2L monoclonal antibody was added to the wells and incubated for 1 hour. For the human receptor-ligand binding assay, 20. mu.l of biotinylated human IL-33 (residue 112-270 of SEQ ID NO: 3) was added at a final concentration of 100ng/ml and incubated for 30 minutes. For cynoReceptor-ligand binding assay 20. mu.l of biotinylated cyno IL-33 (residue 112-269 of SEQ ID NO: 4) was added at a final concentration of 200ng/ml and incubated for 30 minutes. The plate was washed 3 times with 300. mu.l of wash buffer. Add 50. mu.l of 0.2. mu.g/ml streptavidin-HRP (Jackson Immunoresearch) and incubate for 30 min. The plate was washed 3 times with 300. mu.l of washing buffer containing PBS + 0.05% Tween. To each well 50. mu.l of TMB substrate (EMBBIOSCEMENTS) was added. The reaction was terminated by adding 100. mu.l of 0.2N sulfuric acid. OD450 was measured using an Envision microplate reader (Perkin Elmer).

Generation of chimeric ST2L constructs

Various constructs characterized by human and mouse ST2L domains I, II and III crossovers were designed and generated using standard molecular biology techniques. The constructs are listed in table 1. The amino acid numbering corresponds to the human ST2L (hST2L) (SEQ ID NO: 1; NP-057316) and mouse ST2L (mST2L) (SEQ ID NO: 5; NP-001020773) proteins.

Table 1.

hST 2L: human ST2L SEQ ID NO: 1

mST 2L: mouse ST2L SEQ ID NO: 5

Domain binding assay。

Antibodies binding to ST2L domains I, II and III were determined using a standard capture ELISA assay using an electrochemiluminescence detection format (Meso-Scale Discovery (MSD) technique). 10 μ g/mL of each antibody was coated on each well of the MSD HighBind plate (5 μ L/well) at room temperature for 2 hours. The plates were blocked with 150. mu.L of 5% MSD blocking buffer for 2 hours at room temperature and washed 3 times with HEPES wash buffer, then 25. mu.L of sulfonic acid-tagged huST2L-ECD or mouse ST2L-ECD (amino acids 28-326 of SEQ ID NO: 5) or HHM-ST2L (SEQ ID NO: 6) or HMH-ST2L (SEQ ID NO: 8) chimeras or HH-ST2L (residues 19-205 of SEQ ID NO: 1) were added to the plates at increasing concentrations of 5nM to 40 nM. The plates were covered with aluminum foil and incubated at room temperature for 1 hour with gentle shaking. The plates were then washed 3 times with HEPES wash buffer. MSD read buffer (150 μ l) was added to each well, and the plates were then read using msdsactor Imager 6000.

These antibodies, which bind to human ST2L-ECD, HHM-ST2L, and HMH-ST2L but not mouse ST2L-ECD, recognize domain I of human ST 2L-ECD. Antibodies that bind to human ST2L-ECD and HMH-ST2L but not HHM-ST2L and mouse ST2L-ECD recognize domain III of human ST 2L-ECD. Antibodies that bind to human and mouse ST2L-ECD but not HH-ST2L recognize domain III of ST2L-ECD in humans and mice.

Affinity measurement of anti-ST 2L monoclonal antibody。

The anti-ST 2L monoclonal antibody, huST2L-ECD and cynoST2L-ECD were expressed using standard methods. Goat anti-human IgG Fc gamma fragment-specific antibodies (catalog number 109. sup. 005. sup. 098) were purchased from Jackson ImmunoResearch laboratories (West Grove, Pa.). GLC sensor chips (Bio-Rad Cat No. 176-.

anti-ST 2L antibody and His-carrying₆Tagged human ST2L-ECD and His₆The interaction of the tagged cyno ST2L-ECD was studied by ProteOn using ProteOn XPR36 at 25 ℃. Biosensor surfaces were prepared by coupling goat anti-human IgG Fc γ fragment specific antibodies (Ab) to the surface of a GLC sensor chip using the manufacturer's instructions for amine coupling chemistry. The coupling buffer was 10mM sodium acetate, pH 4.5. Goat anti-human IgG Fc γ (about 4500 response units) was immobilized in a horizontal orientation. The anti-ST 2L antibody was provided in the form of a purified or crude supernatant. In either case, these antibodies were diluted to a concentration of about 0.5 μ g/mL in PRB (PBS pH74 supplemented with 3mM EDTA and 0.005% tween 20). Antibody Capture in vertical orientation (60-130 RU)) On anti-human Fc γ antibody modified GLC chips. Following capture of the anti-ST 2L monoclonal antibody, either the hunst 2L ECD in solution (0.024 to 15nM, 5-fold dilution) or the cynoST2LECD in solution (0.020-5nM, 4-fold dilution) was injected in a horizontal orientation. Association was monitored for 4 minutes in all experiments (200 μ Ι _ injected at 50 μ Ι/min). Dissociation was monitored for 30 min. Regeneration of the sensor surface was obtained with three 15 second pulses of 10mM glycine (pH 1.5). The data were fitted using ProteOn software and using a 1: 1 binding model with mass transfer.

The Biacore experiments were performed using a Biacore2000 or Biacore 3000 optical biosensor (Biacore AB). All experiments were run in BRB (PBS pH7.4, supplemented with 3mMEDTA and 0.005% tween 20) with or without 0.1% BSA at 25 ℃.

Biacore bioreactor surfaces were prepared by coupling goat anti-human IgG Fc γ fragment specific antibodies to carboxymethylated dextran surfaces of CM-5 chips using the manufacturer's instructions for amine coupled chemistry. The coupling buffer was 10mM sodium acetate, pH 4.5. An average of 6000 Response Units (RU) of the antibody was immobilized in each of the four flow-through cells. The anti-ST 2L monoclonal antibody was captured (approximately 33RU) on the anti-human Fc γ antibody modified sensor chip surface. Following capture of the anti-ST 2L monoclonal antibody, either huST2L ECD (0.2 to 15nM, 3-fold dilution) in solution or cynoST2L ECD (0.2 to 15nM or 0.020-5nM, 3-fold dilution) in solution was injected. Association was monitored for 4 or 8 minutes (for C2521 and C2519, 200. mu.L was injected at 50. mu.L/min or 20. mu.L/min). Dissociation was monitored for 10 minutes or up to 2.5 hours. By injection of 50mM NaOH and/or injection of 100mM H₃PO₄Regeneration of the sensor surface is obtained.

Data were processed using the Scrubber software version 1.1g (BioLogic software). The double-reference-difference subtraction of the data is performed as follows: the curve generated by the buffer injection was subtracted from the reference subtraction curve for analyte injection to correct for the contribution of buffer to signal and instrument noise (Myszka, Journal of Mol Recogn 12: 279-84, 1999).

After data processing, the data generated for the kinetic and affinity assays were analyzed using either the Scrubber software or BIAevaluation software version 4.0.1(Biacore, AB). Kinetic data were analyzed using a simple 1: 1 binding model (including terms of mass transfer).

Affinity measurement of anti-mouse ST2L mAb (C1999/CNTO3914) for murine ST2L ECD。

The anti-ST 2LmAb (C1999/CNTO3914) and murine ST2L extracellular domains (muST2L-ECD) were expressed and purified using standard methods. Anti-mouse IgG Fc gamma fragment specific antibodies can be purchased from Jackson ImmunoResearch laboratories (West Grove, Pa.). Sensor chips and reagents for preparing capture surfaces are available from Biacore (GE healthcare, Piscataway, NJ). Experimental Biacore electrophoresis buffer (BRB) contained PBS pH7.4 plus 0.005% Tween 20 and 0.1mg/mL BSA and data were collected at 25 ℃.

The interaction of anti-ST 2L antibody with muST2L-ECD was studied on Biacore2000 at 25 ℃. Biosensor surfaces were prepared by coupling anti-mouse-Fc specific antibodies to the surface of CM4 sensor chips using the manufacturer's instructions for amine coupled chemistry. C1999/CNTO3914 and muST2L-ECD were diluted in BRB. C1999 was captured using an anti-mouse Fc γ antibody (approximately 85 RU). After capture, muST2L ECD (residues 28-326 of SEQ ID NO: 5) was injected as a solution (starting at 15nM, 5 concentrations, 3-fold serial dilution). Association was monitored for 8 minutes. The dissociation was monitored for up to 6000 seconds. Regeneration was performed using 1/100 dilution of phosphoric acid. Data were fitted using a 1: 1 binding model.

Human basophil cell line assay (basophil cytokine release assay)

KU812 cells (human basophilic granulocytic line; ATCC, CRL-2099) were seeded at 25,000 or 50,000 cells/well in a total of 40. mu.l of RPMI1640 growth medium (Invitrogen) supplemented with 10% FBS and penicillin/streptomycin in sterile 96-well U-bottom tissue culture plates. Anti-human ST2L monoclonal antibody and controls were added at various concentrations (50. mu.l/well) and incubated at 37 ℃. After 1 hour of incubation, recombinant "mature" IL-33 (amino acids 111-270 of SEQ ID NO: 3) was added to 10. mu.l of RPMI growth medium at a final concentration of 10 ng/ml. The cells were then incubated at 37 ℃ for 18-24 hours to allow IL-33 mediated induction of IL-5 and IL-6. Following incubation, cells were harvested and cell supernatants collected for subsequent detection of iL-33-induced iL-5 and iL-6 using ELISA (R & D systems) or bead-based multiplex assays (Millipore).

Human mast cell cytokine release assay and PGD ₂ Release assay

Mast cells derived from CD34⁺Human cord blood cells (Lonza) > 1.0 × 10⁶CD34⁺Frozen vials of cord blood cells were quickly thawed and transferred to 50ml conical tubes A few drops of warm or room temperature Stem-Pro 34 media + supplement (25 ml total; Invitrogen) were slowly added to the cells were centrifuged at 1,000rpm for 15 minutes and resuspended in media (10ml of StemPro-34, containing supplements: 30ng/ml IL-3, 100ng/ml IL-6 and 100ng/ml SCF.) the cells were seeded into 2 wells of 6-well plates and cultured for 1 week on day 4 the cells were expanded 1: 3 in supplemented Stem Pro-34 media on day 7 non-adherent cells were removed and cultured at 0.5 × 10⁶Per ml inoculated in StemPro-34 Medium containing 10ng/ml IL-6 and 100ng/ml SCF cells were expanded weekly to maintain 0.5 × 10⁶Cell density per ml until mast cells mature at 6-10 weeks (assessed by FcR1, cKit and tryptase expression).

Mature mast cells were treated at 0.5 × 10⁶The cells were cultured in StemPro-34 at IL-4(10 ng/ml; Peprotech), IL-6(10 ng/ml; R) daily&D Systems) and SCF (100 ng/ml; invitrogen) for 4 days. Prior to assay, cells were harvested, centrifuged at 1,000RPM for 10 minutes, and resuspended in fresh StemPro-34 medium or RPMI (containing 100ng/ml human recombinant SCF without antibiotics) containing 10% FCS. Cells were seeded at a density of 65,000 to 75,000 cells per 0.16 ml/well in flat-bottom tissue culture-treated 96-well plates. 30 minutes before IL-33 addition, anti-ST 2L monoclonal antibody was added to the plates to a final concentration of 50, 10,2. 0.4, 0.08, 0.016 and 0.0032 mu g/ml. Recombinant human "mature" IL-33 (residues 111-270 of SEQ ID NO: 3) was also prepared at 10X (10 or 30ng/ml) in culture +100ng/ml SCF. Mu.l of 10X IL-33 was added to wells to a final concentration of 1 (FIGS. 6 and 7A-7E) or 3ng/ml (FIGS. 8A-8E), and plates were plated at 37 ℃ with 5% CO₂The mixture was incubated overnight. Culture supernatants were harvested 18-24 hours after stimulation. The plates were centrifuged at 1,000RPM for 10 minutes. The supernatant was removed, placed in a U-bottom 96-well plate, and stored at-20 ℃ prior to assay. Using the human cytokine kit from Millipore, Luminex was used^TMThe techniques analyze cytokine levels. The level of PGD2 was measured using prostaglandin D2-MOX EIA kit from Cayman Chemical Company according to the manufacturer's instructions. To enhance the sensitivity of the ELISA, PGD in the mast cell culture supernatant was added₂By treatment with methoxylamine hydrochloride (MOX-HCl), it is converted into non-degradable MOX-PGD₂(Methoxyamine-PGD)₂)。

Mouse receptor-ligand binding inhibition assay (mouse RLB assay)

A96-well transparent plate (VWR) was coated with 50. mu.l of goat anti-human IgG, Fc γ fragment specific (Jackson Immunoresearch) antibody at 4 ℃ for about 16 hours. The remaining steps were completed at room temperature. The wells were incubated with blocking buffer, washed and 50. mu.l of 2. mu.g/ml mouse ST2L-ECD fused to human Fc was added for 1 hour. The plates were washed and 1. mu.g/ml biotinylated mIL-33 with or without anti-mST 2L antibody was added. The plates were washed, detection was accomplished with streptavidin-HRP (Jackson Immune research), and signals were developed with TMB substrate (RDI division of Fitzgerald Industries) according to the manufacturer's instructions.

Mouse and human reporter assays (human or mouse RGA assays)

HEK293 cells were seeded at 50,000 cells/well in DMEM, 10% FBS in white clear-bottomed tissue culture-treated 96-well plates (NUNC) and at 37 ℃ with 5% CO₂The cells were incubated in a humidified incubator for 24 hours. Opti-MEM Medium (Invitrogen) was usedLipofectamine in n)^TM2000, cells were co-transfected with a vector encoding human or mouse ST2L-ECD cDNA, NF-. kappa.B-luciferase vector (Stratagene, Agilent Technologies, Santa Clara, Calif.), using standard protocols. At 37 deg.C, 5% CO₂After 24 hours of incubation, the transfected cells were used in mice (R) with or without anti-ST 2L antibody&D Systems, SEQ ID NO: residue 109-266 of 5) or human IL-33(SEQ ID NO: residue 112-270 of 3) at 37 ℃ with 5% CO₂The following treatment was carried out for 16 hours. Use ofReagents (Promega) luciferase activity was measured according to the manufacturer's instructions.

Mouse T cell proliferation assay

Mouse Th2 cells (D10.G4.1, ATCC) were cultured in complete growth Medium RPMI1640 medium with 2 mML-Glutamine adjusted to contain 1.5g/L sodium bicarbonate, 4.5g/L glucose, 10mM HEPES and 1.0mM sodium pyruvate, supplemented with 0.05mM 2-mercaptoethanol, 10pg/ml IL-1 α (R10. G4.1, ATCC)&D Systems), 10% fetal bovine serum, 10% rat T-STIM factor containing ConA (rat IL-2 culture supplement available from Becton Dickinson). Cells were washed twice with assay medium (RPMI, 10% FBS, IL-1 free, T-STIM free) at 1.25X10⁵Cells/ml were resuspended and plated in 80. mu.l of medium in tissue culture treated 96-well plates (NUNC, Rochester, NY) with white clear bottoms. Various amounts of mouse IL-33 (residues 109-266 of SEQ ID NO: 5) were added to the cells to a final assay volume of 100. mu.l. When the test antibody was neutralized, the control antibody (added to the used hybridoma culture medium) or hybridoma supernatant was added to the cells and incubated for 1 hour, followed by the addition of 20pg/ml mIL-33. The plates were incubated at 37 ℃ with 5% CO₂The cells were then incubated in a humidified incubator for 24 hours. Use ofReagents (Promega, Madison, WI) enable quantification of living cells; the protocol was performed according to the manufacturer's instructions.

Mouse bone marrow-derived mast cell assay

Mouse mast cells were derived from bone marrow of Balb/c mice (6 weeks). Cells were seeded at 300,000 cells/well in RPMI medium (endotoxin free), 10% FBS, 10% WEHI cell line conditioned medium, 10ng/ml IL-3(Peprotech), 0.1mM essential amino acids, 1% penicillin/streptomycin (Invitrogen). anti-ST 2L monoclonal antibody (100, 10, 1, 0.1 or 0.01. mu.g/ml) was incubated with the cells for 1 hour, after which recombinant mouse "mature" IL-33 (residues 109-266 of SEQ ID NO: 215 (10 ng/ml; R)&D Systems). After about 24h, the supernatant was harvested and frozen until Luminex was used^TMThe Millipore mouse 22-plex kit of (5) was analyzed according to the manufacturer's instructions.

Cyno endothelial cell assay

Will be cultured inCynomolgus monkey aortic endothelial cells in endothelial cell growth medium-2 (Lonza) were seeded at 10,000 or 20,000 cells/well in 96-well tissue culture plates. Mu.l of anti-ST 2L antibody was added to the cells, starting at 100. mu.g/ml and followed by a 4-or 5-fold dilution, and incubated at 37 ℃ for 1 hour, after which recombinant cyno "mature" IL-33(SEQ ID NO: 4) was added. Fifty microliters of 20ng/ml cynomolgus IL-33 was then added to the cells and incubated at 37 ℃ for 24 hours. To assess IL-33 induced cytokine response, supernatants were harvested and passed through Luminex^TMThe non-human primate IL-8 kit (Millipore) of (1) cytokine levels were assessed according to the manufacturer's instructions.

Mouse peritoneal lavage assay

The peritoneum of 6 Balb/c mice was washed with a total of 3ml PBS to collect peritoneal cells. Most of these cells were found to be lymphocytes and macrophages as determined by B220 and F4/80 expression (FACS analysis). About 1% is cKit⁺(CD117⁺) Mast cells.The cells were centrifuged and the pellet was resuspended in Alpha MEM medium + 10% FBS +100U/ml penicillin + 100. mu.g/ml streptomycin (Invitrogen) to 1 × 10⁶Cells/ml. The cells were seeded at 200. mu.l/well in a 96-well plate and allowed to stand at 37 ℃ for 2 h. anti-ST 2L monoclonal antibody was added to the cells for 30 minutes, followed by the addition of 10ng/ml mouse "mature" IL-33 (R)&D Systems; SEQ ID NO: residue 109-266 of 215). Supernatants were collected 24h after IL-33 addition, stored at-20 ℃ until analysis, and then used Luminex^TMThe Millipore mouse 22-plex kit of (5) was analyzed according to the manufacturer's instructions.

Example 1: generation of rat anti-mouse ST2L antibody

The resulting hybridomas were inoculated in 96-well plates or methylcellulose and cultured for 10 days.an antigen-specific clone that bound mST2-Fc was identified by a standard capture ELISA and cross-screened for Fc protein alone. murine ST 2-specific hybridomas were further tested in ELISA for inhibition of IL-33 binding to ST2 and for inhibition of IL-33-induced proliferation of D10.G4.1 mouse Th2 cells.hybridomas that exhibited neutralization in both receptor-ligand binding and cell-based proliferation assays were selected by limiting dilution in a clonal manner.hybridoma V regions were sequenced and cloned into a mouse IgG1 background.ST 2L-ECD domain specificity was detected by standard immunoadsorption assay for electrochemiluminescence Assays were treated with various human-mouse domain exchange constructs.

The antibody secreted by hybridoma C1999 was cloned into a mouse IgG1 background and named CNTO 3914. The sequences of the CNTO3914 variable regions and CDRs are shown in table 2. CNTO3914 does not cross-react with human ST2L and binds to domain I of mouse ST 2L-ECD.

Table 2.

Example 2: generation of mouse anti-human ST2L antibody

Two different immunization protocols were performed to generate the mouse anti-human ST2 monoclonal antibody.

With soluble ST2-Fc (R)&D Systems, SEQ ID NO: 157) BALB/c was immunized intraperitoneally and specific IgG titers were evaluated. Once sufficient titers were obtained, splenocytes were isolated and fused with FO cells. The resulting hybridomas were inoculated in 96-well plates and cultured for 10 days. Identification of binding to His 6-tagged huST2L-ECD at the C-terminus and His6 by Standard Capture ELISA₆The labeled cyno ST2L-ECD cross-reactive antigen-specific clone. Human ST 2L-specific hybridomas cross-reactive with cyno ST2L were further tested for inhibition of IL-33 binding to huST2L in an ELISA assay and for inhibition of NF- κ B activation in a reporter assay. Clones inhibited in either the reporter assay or both ELISA and reporter assay were selected for further study.

Antibodies from hybridomas C2494, C2519A, and C2521A were selected for further analysis. C2519A and C2521A bind to human ST2L in domain III and C2494 binds to human ST2L in domain I. Antibody C2494 was cloned into a human IgG2 background and the full length antibody was named STLM 62.

Anti-human ST2L monoclonal antibodies were generated by proprietary DNA immunization techniques at Genovac Gmbh using a full-length ST2L construct and boosting with cells transfected to express human ST 2L-ECD. Hybridomas were screened for binding to human ST2L-ECD by flow cytometry. Clones exhibiting binding in this assay were confirmed to bind to hST2L-ECD and further characterized for binding to cyno ST2L-ECD by standard capture ELISA. Selected clones were characterized in a receptor-ligand binding inhibition ELISA and reporter assay. Clones inhibited in either the reporter assay or both ELISA and reporter assay were selected for further study.

Antibodies from Genovac hybridoma C2244 were selected for further analysis and cloned into a human IgG2 background. The full length antibody was designated STLM 15. STLM15 binds to human ST2L in domain I.

The sequences of the VH, VL and CDR domains of the mouse anti-human antibodies are shown in table 3.

Table 3.

Example 3: generation of fully human ST2L antibody

Human ST2L binding Fab was selected from de novo synthetic pIX phage display libraries such as Shi et al, J Mol Biol 397: 385-96, 2010; international patent publication WO 2009/085462; US patent publication US 2010/0021477). Briefly, libraries were generated by diversifying human scaffolds in which germline VH genes IGHV1-69 x 01, IGHV3-23 x 01 and IGHV5-51 x 01 were recombined with human IGHJ-4 minigenes via the H3 loop, and human germline VLkappa genes O12(IGKV1-39 x 01), L6(IGKV3-11 x 01), a27(IGKV3-20 x 01) and B3(IGKV4-1 x 01) were recombined with IGKJ-1 minigenes to assemble complete VH and VL domains. Positions in the heavy and light chain variable regions around the H1, H2, L1, L2, and L3 loops, which correspond to positions identified as frequently contacted with protein and peptide antigens, were selected for diversification. Sequence diversity at selected positions is limited to the residues occurring at each position in the IGHV or IGLV germline gene families of the respective IGHV or IGLV genes. Diversity at the H3 loop was created by using short to medium sized synthetic loops 7-14 amino acids in length. The amino acid profile at H3 was designed to mimic the amino acid variation observed in human antibodies. Library design is detailed in Shi et al, J MolBiol 397: 385-96, 2010. Scaffolds used to generate libraries were named according to their human VH and VL germline gene origin. Combining the 3 heavy chain libraries with 4 germline light chains or a germline light chain library to generate 24 unique VH: VL combinations were used for screening. All 24 VH: VL library combinations were used in phage panning experiments on huST 2L-ECD-Fc.

The library was panned using Fc fusion of huST2L-ECD (residues 19-328 of SEQ ID NO: 1). Panning was done with antigen (Ag) in 2 different formats, i.e. in solution, and displayed Ag. For Ag in solution form, streptavidin coated magnetic beads were blocked in PBS containing 3% skim milk powder. Biotinylated (Bt) antigen with 10x higher concentration of human Fc protein huST2L-ECD human Fc fusion (Bt-huST2L-ECD-Fc) was mixed as competitor with the Fab-pIX phage library. Fab-pIX phage bound to Bt-huST2L-ECD-Fc were captured on blocked Streptavidin (SA) -coated magnetic beads. Three rounds of phage selection were performed in which the concentration of huST2L-ECD-Fc was varied from round 1 to round 3 to 100nM, 10nM, respectively. For Ag display, Bt-huST2L-ECD-Fc was coated on SA-coated magnetic beads. The Fab-pIX phage library and 10 Xexcess human Fc protein were added simultaneously to the Bt-huST2L-ECD-Fc displayed SA magnetic beads. The concentrations of Bt-Ag used in rounds 1 to3 were 100nM, 10nM and 10nM, respectively. Both panning formats accomplished screening by ELISA binding of Fab to the huST2L-ECD-Fc protein. From these selections a total of 79 fabs bound to hST2L-Fc were isolated. Fab HuT2SU-39 with overall optimal binding activity was determined by sequencing ELISA (ranking ELISA).

An ELISA-based IL-33 binding inhibition assay was performed on 79 Fab. A total of 32 Fab's were shown to inhibit IL-33 binding to hug 2L-ECD-Fc. From the pIX de novo synthesis screen (pIX de novo campaign) 46 fabs were selected for affinity maturation.

Example 4: affinity maturation of fully human ST2L antibody

Selected antibodies were selected using Shi et al, J Mol Biol 397: 385-96, 2010 and WO09085462A1 describe a "simultaneous" maturation process for affinity maturation. In this technique, the VH regions of the Fab clones obtained in the first selection are combined with a library of corresponding VL scaffolds. All VH genes from 46 fabs identified in example 3 were cloned into the appropriate VL maturation library as a library from its original VL gene family. The VL scaffold library used and its diversification scheme are shown in table 4. Human VL scaffolds were as follows: IGKV1-39 × 01(O12), IGKV3-11(L6), IGKV3-20(a27), IGKV4-1 × 01(B3), and are described in, for example, U.S. patent publication US 2012/0108795. For affinity maturation panning, the phage library was first added to Bt-huST 2-ECD-Fc. After incubation, the mature library phage/Bt-hST 2L-ECD-Fc complex was added to SA-coated magnetic beads. The Bt-huST2-Fc concentration was varied from R1 to R3 to 10nM, 1nM and 0.1nM, respectively. A3 rd round of final washes was performed overnight at room temperature in the presence of 10nM unlabeled huST2L-ECD-Fc to further promote affinity improvement.

Table 4.

A total of 161 sequence-unique fabs were obtained from the maturation panning. Fab which showed the highest binding to huST2L-ECD was converted to IgG for further characterization.

Monoclonal antibodies ST2M48, ST2M49, ST2M50 and ST2M51 were selected for further characterization and their VH, VL and CDR sequences are shown in table 5. Monoclonal antibodies ST2M48, ST2M49, ST2M50 and ST2M51 bound to domain III with human ST2L and cross-reacted with mouse ST 2L.

Table 5.

Example 5: characterization of anti-ST 2L antibody。

Antibodies derived from the various screens described above (campaign) were further characterized for their ability to block IL-33/ST2L interaction, their ability to inhibit IL-33 induced signaling as measured by NF- κ B reporter gene assay, their ability to inhibit mast cell response, their affinity for human and cyno ST2L, and cross-reactivity with mouse ST 2L. Epitope mapping was done using the human/mouse ST2L domain-swapping chimeric construct as described in materials and methods. The results of the experiments are shown in tables 6, 7 and 8. In tables 7 and 8, "+" indicates that the antibody blocks the IL-33/ST2L interaction, and "-" indicates that it does not block the IL-33/ST2L interaction. Due to the lack of cross-reactivity to humans, experiments with CNTO3914 were completed using mouse cells and reagents. Human cells and human reagents were used in the assay for all other antibodies.

The antibodies characterized were grouped into antibodies that blocked the IL-33/ST2L interaction (monoclonal antibodies STLM15, STLM62 and CNTO3914) and antibodies that did not block the IL-33/ST2L interaction (monoclonal antibodies C2519, C2521, ST2M48, ST2M49, ST2M50 and ST2M 51). Antibodies that block the IL-33/ST2L interaction bind to ST2L domain I, while non-blocking antibodies bind to ST2L domain III. The tested antibodies inhibited ST2L downstream signaling as assessed by NF- κ B reporter gene assay and IL-33 induced cytokine release by KU812 human basophil cell line, or in the case of CNTO3914, by mouse Th2 cell proliferation. As assessed by cytokine and chemokine secretion, the antibody bound to ST2L domain I inhibited human mast cell responses at higher levels when compared to anti-ST 2L antibody that binds ST2L domain III. CNTO3914, which binds to mouse ST2L domain I and does not cross-react with humans, is also able to inhibit IL-33-induced mouse mast cell responses.

Table 6.

Table 7.

Inhibition of receptor-ligand binding

# reporter Gene assay

hD 1-human ST2L D1 domain

mD 1-mouse ST2L D1 domain

hD 3-human ST2L D3 Domain

h/mD 3-human and mouse ST2L D1 and D3 domains

not tested

Table 8.

Inhibition of receptor-ligand binding

# reporter Gene assay

Bone marrow derived material

Example 7: ST2L Domain I binding antibody CNTO3914 inhibits intranasal IL-33-induced Airway Hyperresponse (AHR), Airway inflammation and mouse mast cell response。

Female BALB/c mice were administered four consecutive intranasal doses of 2 μ g/mouse "mature" IL-33(R & D Systems) (residues 109-266 of SEQ ID NO: 215). The anti-mouse ST2L antibody CNTO3914 was administered prophylactically by subcutaneous injection at 20mg/kg (or 2mg/kg or 0.2mg/kg) 24 hours prior to the first intranasal administration of IL-33. Control mice received either isotype control CNTO5516 or PBS 24 hours prior to the first intranasal administration of IL-33. Airway Hyperresponsiveness (AHR) to increasing methacholine doses was measured using forced manipulation with the Flexivent system (Scireq, Montreal, Quebec, Canada). To measure Airway Hyperresponse (AHR), mice were anesthetized with 100mg/kg pentobarbital and 13mg/kg phenytoin and the trachea was dissected prior to attachment to FlexiVent. Mice were sprayed with saline to give a baseline reading, followed by two doses (10 and 20mg/mL) of methacholine. Resistance (R) values were collected for approximately 2 minutes using "snap shot" perturbation for saline and each methacholine dose. The peak resistance value was calculated using only those values having a COD (determination coefficient) of 0.9 or more.

A separate group of mice was treated and analyzed for cellular responses in the lungs. Finally twenty IL-33 isoforms or PBS administrationAfter four hours, the reaction mixture is passed throughMice were sacrificed i.p. Lungs of mice were lavaged with 0.7ml of cold PBS containing 0.1% BSA. The resulting Bronchoalveolar (BAL) fluid was centrifuged at 1200rpm for 10 minutes and the cell-free supernatant was stored at-80 ℃ until cytokine/chemokine analysis. BAL samples were used for total counts using a hemocytometer. For categorical BAL counts, approximately 200 cells were counted from cytospin smear (cytospin smear) under light microscope after staining with giemsa rapi (right giemsa).

Cell-free supernatants were collected and stored at-80 ℃ until used for Luminex protein analysis. Lung tissue was removed and then perfused through the right ventricle using 5ml of cold sterile PBS until the appropriate perfusion volume. The lung lobes were then placed in Fast containing 1ml of PBS + protease inhibitorIn tubes, and frozen at-80 ℃ for cytokine/chemokine expression profiling. Cytokine/chemokine multiplex assays were performed according to the manufacturer's protocol of murine Millipore 22-plex. Mouse mast cell protease-1 (mcp-1) in BAL fluid was analyzed by elisa (moredden scientific).

Airway hyper-response

CNTO3914 significantly inhibited airway hyperresponse in a model of pneumonia induced by intranasal administration of IL-33 (fig. 1). CNTO3914 was administered subcutaneously 24 hours prior to four days of continuous intranasal administration of 2 μ g/mouse mIL-33. The peak airway resistance as determined by Flexivent decreased significantly at CNTO3914 doses of 20 mg/kg. Each error bar represents the mean ± SEM of three (CNTO5516, isotype control antibody) to six mice per group. These results have been repeated in two separate studies. Significance was determined using two-factor analysis of variance with Bonferroni post-hoc testing, CNTO3914/IL-33 vs CNTO5516/IL-33, # p < 0.05; and p < 0.001 relative to the PBS group treated with IL-33.

Inflammation of the airways

In the model used, CNTO3914 significantly inhibited bronchoalveolar lavage (BAL) cell recruitment (fig. 2). CNTO3914 was administered subcutaneously 24 hours prior to four days of continuous intranasal administration of 2 mg/mouse mIL-33. BAL leukocytes increased significantly with IL-33 administration and were significantly inhibited by 20mg/kg CNTO 3914. Each error bar represents the mean ± SEM of three (CNTO5516, isotype control antibody) to six mice per group. These results have been repeated in two separate studies. Significance was determined using two-way anova with Bonferroni post test, p < 0.001.

Mast cell response in vivo

Mast cells store proteases (including tryptase and chymase) in their granules, which are rapidly released upon activation of the mast cells. Mouse mast cell protease 1(mMCP-1) is a β -chymase released by activated mast cells and is known to be important in the control of parasitic infections (Knight et al, J Exp Med 192: 1849-56, 2000; Huntley et al, Parasite Immunol 12: 85-95, 1990). measurement of mcp-1 can be used as a marker for mast cell activation, and has been shown to be induced in mast cell dependent models of airway inflammation: house dust mites (Yu and Chen, JImmunol 171: 3808-15, 2003). MMCP-1 was significantly increased in BAL fluid from IL-33 administered mice as determined by elisa (moredun scientific) and dose-dependently inhibited by CNTO3914 (fig. 3). Significance was determined using single-factor analysis of variance and Tukey post hoc testing, with p < 0.01 and p < 0.001 relative to IL-33 treatment.

Example 8: anti-ST 2L Domain I binding antibodies inhibit mast cell response in vitro

Chemokines and cytokines released by mouse and human mast cells and prostaglandin D in human mast cells₂To assess mast cell response.

anti-ST 2L domain I binding antibody CNTO3914 inhibits IL-33-induced release of cytokines from mouse bone marrow-derived mast cells, including GM-CSF (FIG. 4A), IL-5 (FIG. 4B), and TNF α (FIG. 4C).

Anti-human ST2L domain I binding monoclonal antibody C2494(STLM62) inhibited IL-33-induced PGD in human umbilical cord blood-derived mast cells induced by 3ng/ml IL-33 at antibody concentrations of 2, 10 and 50. mu.g/ml₂Released (fig. 5).

anti-ST 2L domain I binding antibodies C2494 and C2244 inhibited IL-33-induced release of GM-CSF, IL-5, IL-8, IL-13, and IL-10 by human cord blood-derived mast cells at antibody concentrations of 50. mu.g/ml, 10. mu.g/ml, and 2. mu.g/ml (FIGS. 6 and 8A-8E). The extent of inhibition depends on the cytokine/chemokine measured, the antibody and antibody concentration tested and the medium used. The mean percent (%) inhibition calculated in all assays performed at an antibody concentration of 2 μ g/ml was between 50.6-100% and between 62-100% at an antibody concentration of 50 μ g/ml (figure 9).

At antibody concentrations of 50. mu.g/ml and 10. mu.g/ml, anti-ST 2L domain III bound to antibodies C2521, C2519, ST2M48, ST2M49, ST2M50 and ST2M51 exhibited slight or no inhibition of IL-33-induced mast cell cytokine release, or stimulated IL-33-induced mast cell cytokine release (FIGS. 7A-7E and 8A-8E). The extent of inhibition depends on the cytokine/chemokine measured, the antibody tested and the medium used. The mean percent (%) inhibition calculated in all assays performed at an antibody concentration of 2. mu.g/ml was between-594.4-31.9% and between-481.5-36% at an antibody concentration of 50. mu.g/ml (FIG. 9). In some assays, antibody ST2M50 inhibited GM-CSF, IL-5, IL-10, and IL-13 secretion at an antibody concentration of 10. mu.g/ml (FIGS. 8A-8E).

The average% inhibition was calculated using the formula: (1- (concentration of cytokine released in the presence of monoclonal antibody)/(concentration of the same cytokine released in response to IL-33 in the absence of monoclonal antibody)). times.100. Cytokine concentrations are in pg/ml. In some cases, the% inhibition is negative, indicating that cytokine release is actually higher in the presence of the monoclonal antibody than in the absence of the monoclonal antibody. The potency of monoclonal antibodies may vary slightly depending on the concentration of IL-33 used to induce cytokine release in mast cells. Similarly, the activity of monoclonal antibodies may vary slightly depending on the assay medium used (StemPro-34 vs. RPMI/10% FCS). All tested ST2L domain I binding antibodies inhibited all measured cytokine and chemokine release by at least 50%, as measured by mean% inhibition, at a concentration of 2 μ g/ml, 10 μ g/ml, or 50 μ g/ml.

Example 9: ST2L Domain I binding antibodies inhibit intranasal IL-33-induced airway remodeling。

On days D1, D3, D5, D7 and D9, C57BL/6 mice were intranasally administered with 1. mu.g/mouse "mature" IL-33 (or PBS) (residues 109 and 266 of SEQ ID NO: 215) and the lungs were analyzed on day 10 or day 20. The anti-mouse ST2L antibody CNTO3914 or isotype control (CNTO5516) was administered subcutaneously at 2mg/kg 6 hours prior to the first intranasal administration of IL-33. Control mice received either isotype control CNTO5516 or PBS 6 hours prior to the first intranasal administration of IL-33. The inflated lungs were fixed in 10% buffered formalin for histological analysis; dyes used for analysis included hematoxylin-eosin (H & E), Masson's Trichrome (Masson Trichrome), and PAS.

IL-33 treatment induced moderate to marked hypertrophy and hyperplasia of the bronchiolar epithelium with goblet cell hyperplasia and peribronchiolar infiltrates mixed primarily with eosinophils. Hypertrophy and hyperplasia of the bronchiolar epithelium was not evident in animals receiving CNTO 3914. Masson's trichrome dye was used to determine the amount of collagen present; this staining method showed goblet cell hypertrophy in IL-33 treated animals. Infiltration in the alveolar and peribronchiolar regions was absent in animals treated with CNTO 3914.

Example 10: generation of fully human ST2L antibody

Additional human ST2L binding Fab were selected from de novo synthesized pIX phage display libraries essentially as described in example 3, except that the library was panned using a chimeric HHM-ST2L construct (SEQ ID NO: 6, Table 1) in which biotinylated antigen was captured on streptavidin-coated magnetic beads. The phage library was blocked in PBS-T containing 3% skim milk powder. The competitor protein MHM-ST2L chimera (SEQ ID NO: 7, Table 1) was added to the blocking solution to drive the phage selection towards Fab that would specifically bind to the human ST2L domain I amino acid sequence. Three rounds of phage selection were performed followed by screening for Fab binding to hST2L-Fc protein by ELISA.

Nineteen fabs binding to hST2L-Fc were isolated from these selections and further screened for binding to the chimeric ST2L construct (table 1) and to the mouse ST2L and human ST2L proteins to localize specific domains and characterize their ability to block IL-33/hST2L interactions. Fab ST2F1, ST2F4 and ST2F6 blocked the hIL-33/ST2L interaction and bound to domain I of ST2L and progressed forward to affinity maturation.

Table 9.

Example 11: affinity maturation of human ST2L binding Fab

ST2F1, ST2F4, and ST2F6 were measured using Shi et al, j Mol Biol 397: 385-396, 2010 and the "simultaneous" maturation process described in international patent publication WO2009/085462 and example 4. Affinity maturation libraries for ST2F1, ST2F4 and ST2F6 were made by diversifying the corresponding light chain libraries (B3, L6 and L6, respectively) and combining these libraries with Fab VH regions. The diversification scheme of the light chain residues for the L6 and B3 affinity maturation libraries is shown in table 10. Position numbering is according to Kabat. For affinity maturation panning, biotinylated huST2-ECD-Fc was captured on Streptavidin (SA) -coated magnetic beads at concentrations of 10nM for round 1, 1nM for round 2, and 0.1nM for round 3. Final 3-round washes were performed overnight at room temperature in the presence of 10nM unlabeled huST 2L-ECD-Fc.

Table 10.

ST2F6 light chain maturation library selection yielded improved binders (ST2F14, ST2F17, ST2F31 and ST2F41) (fig. 10 and 11). These selections as Fab were examined using ProteOn and demonstrated modest affinity improvement from 2nM to 400 pM.

To further improve the affinity of ST2F14, ST2F17, ST2F31 and ST2F41, the heavy chain ST2H41, common in ST2F14, ST2F17, ST2F31 and ST2F41, was randomized at HCDR1 and HCDR2Kabat positions 31, 32, 33, 35, 50, 52, 53, 56 and 58 using the diversification scheme shown in table 11. The resulting heavy chain library was paired with four affinity-improved light chains ST2L32, ST2L35, ST2L49 and ST2L59, and the library was panned and screened as described for the light chain maturation library. Fab with improved binding relative to ST2F14 was isolated and converted to IgG for further characterization. The resulting antibodies (STLM103, STLM107, STLM108, STLM123, STLM124, STLM206, STLM207, STLM208, STLM209, STLM210, STLM211, STLM212, STLM213, STLM214, STLM215, STLM216, STLM217, STLM218, STLM219, STLM220, STLM221, STLM222) (FIGS. 10 and 11) had a framework derived from VH3-23 or V κ -L6. All antibodies bound to ST2L domain I and blocked the IL-33/ST2L interaction.

Table 11.

Position of	Amino acids
		31	SDNTAY
32	SDAY
		33	SDAY
35	SN
		50	SDNTAY
52	SANTKDEGR
		53	SANEY
56	SANTKDEGR
		58	SDNTAY

An additional variant of STLM208VH ST2L257 was designed and expressed to replace the DP motif at the beginning of HCDR 3. The sequence of the variants is shown in figure 12.

Example 11: c2494 Human Frame Adaptation (HFA)

The frame adaptation process was substantially as described in U.S. patent publication US2009/0118127 and Fransson et al, JMol Biol 398: 214, 231, 2010. Briefly, the heavy and light chain sequences were compared to human germline sequences (only the "01" allele since 10.1 of 2007) using BLAST searches against the IMGT database (Kaas et al, Nucl. acids. Res.32, D208-D210, 2004; Lefranc et al, Nucl. acids Res., 33, D593-D597, 2005). From the set of human germline genesRedundant genes (100% identical at the amino acid level) were removed as well as those with unpaired cysteine residues. The remaining closest matching human germline genes were selected as acceptor human frameworks in both framework and CDR regions. A total of 9 VL and 7 VH germline human frameworks were selected based on overall sequence homology and CDR length and CDR similarity. FR-4, JK2 for VL chain and JH1 for VH chain were selected based on sequence similarity of IGHJ/IGJK germline genes (Kaas et al, Nucl. acid Res.32, D208-D210, 2004; Lefranc M. -P et al, Nucl. acid Res., 33, D593-D597, 2005) and having C2494 sequence). The CDRs of C2494 (underlined in fig. 14) were then transferred to the selected acceptor human framework to generate HFA variants, except at the corresponding V_HIs outside the region of CDR-H1. For this region, a combination of CDRs and HV, or shorter HCDR2 (referred to as Kabat-7, see U.S. patent publication US2009/0118127), was transferred from a non-human antibody to human FR, since the HCDR2 residues highlighted in gray in fig. 14 have not been found to be in contact with antigen-antibody complexes of known structure (Almagro, J Mol recognit.17, 132, 2004).

The mature protein sequence of C2494 (VL: SEQ ID NO: 52; VH: SEQ ID NO: 48) is shown in FIG. 14. In this figure, CDR residues (Kabat) are underlined, Chothia HV loops are indicated below the CDRs, and residues transferred into the selected human framework are indicated below HV (hfa). HCDR2 residues highlighted in grey were not transferred in all variants.

The 3D homology model of the Fv fragment of C2494 was constructed using the antibody modeling module of MOE (CCG, Montreal). This model is used to evaluate malleability tendencies (devinophilicities), such as exposed methionine and tryptophan residues, possible N-glycosylation and deamidation motifs. In LCDR3, there is one potentially exposed Met (M94) residue, according to the Fv structural model. To remove this, variants with the M94L mutation (STLL280, O12b) were generated and characterized. For the heavy chain, R residues in the CAR motif just before HCDR3 (residues 92-94 of Chothia, fig. 14) may negatively affect a set of negatively charged residues (Chothia residues D31, D32, D96 and D101a, fig. 14), which may be important for binding. VH with arginine substituted leucine at residue 94 of Chothia (CAR → CAL) was generated and characterized.

Monoclonal antibodies that bind the designed heavy and light chains were expressed with the C2494 parent and assayed for binding to human ST 2L. Among the produced HFA monoclonal antibodies, those having VH chains with heavy chain frameworks (STLH195 and STLH194) of IGHV 1-24X 01(SEQ ID NO: 148) and IGHV1-f 01(SEQ ID NO: 149) expressed the antibody well and bound ST2L when combined with various HFA light chains, the various HFA light chains have frameworks (STLL280, STLL278, STLL277, STLL276, STLL275, STLL274, STLL273, STLL272) IGKV3-15 × 01(L2) (SEQ ID NO: 150), IGKV1-9 × 01(L8) (SEQ ID NO: 151), IGKV1-5 × 01(L12) (SEQ ID NO: 152), IGKV1-12 × 01(L5) (SEQ ID NO: 153), IGKV1-39 × 01(O12) (SEQ ID NO: 154), IGKV1-27 × 01(A20) (SEQ ID NO: 155), or IGKV1-33 × 01(O18) (SEQ ID NO: 156).

The sequences of the HFA VH and VL variants are shown in table 12. The transferred residues are underlined and the additional substitutions above are highlighted in grey. Table 13 shows the SEQ ID NO: as well as unique pDR (plasmid) and CBIS ID. The heavy and light chain combinations of the generated monoclonal antibodies selected for further characterization are shown in table 14.

Table 15 shows the human framework (combined V and J regions) for transfer of the C2494 CDRs.

Table 12.

Framework-adapted VL chains (coupled to JK2 sequence).

The CDRs are underlined.

VL2494 (parent) (SEQ ID NO: 52)

ETTVTQSPASLSVATGEKVTIRCITNTDIDDVIHWYQQKPGEPPKLLISEGNTLRP

GVPSRFSSSGYGTDFVFTIENTLSEDVADYYCLQSDNMLTFGAGTKLELK

＞VL2494-IGKV1-33*01O18(SEQ ID NO：135)

DIQMTQSPSSLSASVGDRVTITCITNTDIDDVIHWYQQKPGKAPKLLIYEGNTLRP

GVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQSDNMLTFGQGTKLEIK

＞VL2494-IGKV1-27*01A20(SEQ ID NO：136)

DIQMTQSPSSLSASVGDRVTITCITNTDIDDVIHWYQQKPGKVPKLLIYEGNTLRP

GVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLQSDNMLTFGQGTKLEIK

＞VL2494-IGKV1-39*01O12(SEQ ID NO：137)

DIQMTQSPSSLSASVGDRVTITCITNTDIDDVIHWYQQKPGKAPKLLIYEGNTLRP

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQSDNMLTFGQGTKLEIK

＞VL2494-IGKV1-12*01L5(SEQ ID NO：138)

DIQMTQSPSSVSASVGDRVTITCITNTDIDDVIHWYQQKPGKAPKLLIYEGNTLRP

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQSDNMLTFGQGTKLEIK

＞VL2494-IGKV1-5*01L12(SEQ ID NO：139)

DIQMTQSPSTLSASVGDRVTITCITNTDIDDVIHWYQQKPGKAPKLLIYEGNTLRP

GVPSRFSGSGSGTEFTLTISSLQPDDFATYYCLQSDNMLTFGQGTKLEIK

＞VL2494-IGKV1-9*01L8(SEQ ID NO：140)

DIQLTQSPSFLSASVGDRVTITCITNTDIDDVIHWYQQKPGKAPKLLIYEGNTLRP

GVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQSDNMLTFGQGTKLEIK

＞VL2494-IGKV3-15*01L2(SEQ ID NO：141)

EIVMTQSPATLSVSPGERATLSCITNTDIDDVIHWYQQKPGQAPRLLIYEGNTLRP

GIPARFSGSGSGTEFTLTISSLQSEDFAVYYCLQSDNMLTFGQGTKLEIK

＞VL2494-IGKV1-39*01O12b(SEQ ID NO：142)

DIQMTQSPSSLSASVGDRVTITCITNTDIDDVIHWYQQKPGKAPKLLIYEGNTLRP

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQSDNLLTFGQGTKLEIK

Framework-adapted VH chain coupled to JH1

VH2494 (parent) (SEQ ID NO: 48)

EVQLQQSVAELVRPGASVKLSCTASAFNIKDDYMHWVKQRPEQGLEWIGRIDPAIGNTEYAPKFQD

KATMTADTSSNTAYLQLSSLTSEDTAVYYCALGDFYAMDYWGQGTSVTVSS

＞VH2494-IGHV1-f*01(SEQ ID NO：143)

EVQLVQSGAEVKKPGATVKISCKVSAFNIKDDYMHWVQQAPGKGLEWMGRIDPAIGNTEYAEKFQG

RVTITADTSTDTAYMELSSLRSEDTAVYYCATGDFYAMDYWGQGTLVTVSS

＞VH2494-IGHV1-24*01(SEQ ID NO：144)

QVQLVQSGAEVKKPGASVKVSCKVSAFNIKDDYMHWVRQAPGKGLEWMGRIDPAIGNTEYAPKFQD

RV^TMTEDTSTDTAYMELSSLRSEDTAVYYCATGDFYAMDYWGQGTLVTVSS

Table 13.

Table 14.

Parent ═ C2494VH and VL

Table 15.

Example 12: design of alanine and human germline mutants for paratope scanning

Site-directed mutagenesis was performed to assess the contribution to binding of individual CDR residues as well as some residues that have potential impact on other antibody properties. According to the molecular model of C2494Fv above, a subset of solvent exposed CDR residues are predicted to be involved in binding antigen. These residues are mutated to alanine and/or the corresponding "human-like" residues, which are the corresponding residues in the most closely matched germline genes. D101aA (Chothia residue) in C2494VH (D104A in SEQ ID NO: 48) substitution makes k_offDecrease by about 4 times from 1.43 × 10^-4To 3.2 × 10^-5。

Reduction of k binding of C2494Fab to ST2L when D101aA is replaced_offIt is expected that the same mutation may also improve the off-rate in the C2494HFA variant. Thus, D101aA (Chothia numbering) was incorporated into the VH of STLH194 (> VH2494-IGHV1-f 01, SEQ ID NO: 143) to generate VH STLH201(SEQ ID NO: 145). STLH201 was paired with 7 light chains STLL280, STLL277, STLL276, STLL275, STLL274, STLL273, and STLL272 (tables 13 and 14) to generate monoclonal antibodies STLM226, STLM227, STLM228, STLM229, STLM230, STLM231, and STLM232, which were further characterized. Monoclonal antibodies STLM226, STLM227, STLM228, STLM229, STLM230, STLM231, and STLM232 therefore had identical LCDR1, LCDR2, LCDR3, HCDR1, and HCDR2 sequences when compared to the parent C2494 antibody and a different HCDR3(SEQ ID NO: 146, GDFYAMAY). In addition, antibody STLM266VL STLM280 has a unique LCDR3：LQSDNLLT(SEQ ID NO：147)

STLH201(SEQ ID NO：145)：

EVQLVQSGAEVKKPGATVKISCKVSAFNIKDDYMHWVQQAPGKGLEWMGRIDPAIGNTEYAEKFQG

RVTITADTSTDTAYMELSSLRSEDTAVYYCATGDFYAMAYWGQGTLVTVSS

HCDR3 incorporates the D101aA (Chothia numbering) substitution:

SEQ ID NO：146：GDFYAMAY

the antibody STLM266VL STLM280 has a unique LCDR 3: LQSDNLLT (SEQ ID NO: 147)

Example 13: characterization of anti-ST 2L antibody

Antibodies obtained by phage display, hybridoma and human frame adaptation screening (human frame adaptation screening) were characterized in various assays including binding to huST2L-ECD, cynoST2L-ECD, affinity measurements, binding to human/mouse chimeras to determine domain binding, receptor-ligand inhibition assays, reporter gene assays and mast cell response assays.

The affinity of antibodies derived from phage display screening (phage display screening) for human and cyno ST2L and their binding specificity for human ST2L are shown in table 16. All antibodies in table 16 bind to domain I of human ST 2L.

Table 16.

The affinity of anti-ST 2L antibodies from HFA screening (HFA campaign) relative to the parent (STLM62, C2494) is shown in table 17. Affinity was analyzed by ProteOn. Experiments were performed at 25 ℃ using ProteOn's PBS-T-E buffer (PBS, 0.005% P20, and 3mM EDTA) as the running buffer. For the experiments, the GLC sensor chip was prepared by covalently immobilizing goat anti-human Fc (about 5800RU), and 122- & 146 Response Units (RU) of monoclonal antibody were captured. Monoclonal antibody capture was followed by injection of ST2L-ECD at 0.024 to 15nM (5 fold dilution) for 4 minutes (200. mu.L at 50. mu.L/min). For all reactions, dissociation was monitored for 30 min. Regeneration was performed using two 15 second pulses of 10mM glycine (pH 1.5). The data were fitted to 1: 1 using a baseline drift model.

The association rate of the samples was fast and langmuir (langmuir) with mass transfer model was used for curve fitting and affinity estimation. All samples had faster off rates than the parental clones and the control monoclonal antibody. The difference in off-rate is the main reason for the lower affinity of the HFA variant when compared to the parent antibody.

Table 17.

STLM62 ═ C2494, parent antibody

Table 18.

Strong inhibition of ++

+ some degree of inhibition

No inhibition

NT not tested

As hybridoma test

RLB ═ receptor-ligand binding inhibition

RGA ═ reporter gene assay

The mast cell response of the selected antibodies was tested to measure inhibition of IL-5, IL-13 and IL-8 release from human cord blood-derived mast cells induced by 3ng/ml IL-33 using 100. mu.g/ml, 10. mu.g/ml, 1. mu.g/ml, 0.1. mu.g/ml or 0.01. mu.g/ml antibody in RPMI + 10% FCS as described. In these assay conditions, all antibodies tested inhibited IL-33-induced cytokine release of IL-5, IL-13, and IL-8 by about 40% -100% at an antibody concentration of 100 μ g/ml when compared to a control sample induced with IL-33.

Example 14: anti-ST 2L antibodies inhibit the downstream signaling pathway in human basophils

anti-ST 2L antibodies were tested for their ability to inhibit p38MAPK signaling in human basophils.

Whole blood was collected in heparinized tubes and left at Room Temperature (RT) before initiation of the assay. 1mL of blood was aliquoted into 50mL conical tubes and anti-ST 2L antibody (STLB252) or isotype control (CNTO8937) diluted in PBS was added to final concentrations of 2, 20 or 200. mu.g/mL. The tubes were gently swirled to mix and placed in an incubator at 37 ℃ for 30 minutes, after 15 minutes gently swirled. The blood was then stained for cell surface antigens with fluorochrome-labeled antibodies (CD123-FITC, CRTH2-PCP-CY5.5, and CD45-APC-C7) and the tubes were incubated at 37 ℃ for 15 minutes. 1mL of warm medium (RPMI-1640/10% FBS/1% penicillin-streptomycin) was added to each tube, followed by IL-33 diluted in warm medium to a final concentration of 10 ng/mL. The samples were incubated at 37 ℃ for 10 minutes and 20mL of pre-warmed BD Phosflow lysis/fixation buffer was added to each tube to simultaneously lyse the red blood cells and fix the samples. The tubes were mixed homogeneously by tumbling 10 times and incubated at 37 ℃ for 10 minutes. The samples were washed with 20mL sterile RT PBS, resuspended in 2mL 1x RT BD rupture/wash buffer, and incubated at room temperature for 30 minutes. The sample was washed once with 2mL of BD rupture/wash buffer and resuspended in 400 μ L of BD rupture/wash buffer. PE-labeled antibody against intracellular p38-MAPK (vCell Signaling, catalog No. 6908S) was added and the samples were incubated at room temperature for 30 minutes, protected from light. The samples were washed once with 5mL of membrane rupture/wash buffer, resuspended in 100 μ L FACS buffer, and transferred to 96-well round bottom plates. Using a BD LSRII flow cytometer with high throughput linesThe System (HTS) collects as many events as possible for each sample to analyze the sample. Data were analyzed using FloJo software. Basophils were identified as CD45⁺CRTH2⁺CD123⁺And the percentage of p38MAPK positive basophils was evaluated for each condition. Preincubation of whole blood with anti-ST 2L monoclonal antibody (STLB252) resulted in dose-dependent inhibition of IL-33-induced p38-MAPK phosphorylation, whereas isotype control (CNTO8937) did not show effect. Anti-human ST2L antibody specifically blocked basophil activation by recombinant human IL-33 in a whole blood background. The results indicate that anti-ST 2L antibody inhibits signaling by endogenous IL-33 in vivo.

Table 19.

Example 15: conjugation of anti-ST 2L antibodies to targets in vivo

Intranasal mIL-336 hour in vivo model of BAL cell recruitment

Male Balb/c mice (6-8 weeks old, Taconnic) were administered a single dose of 1.2. mu.g/mouse mIL-33(R & D systems #3626-ML/CF) or PBS. The rat anti-mouse ST2L antibody CNTO3914 was either 2, 0.2, 0.06, or 0.02mg/kg 24 hours prior to the first intranasal administration of mIL-33. Isotype control (ITC) monoclonal antibody CNTO5516 was administered subcutaneously at 2 mg/kg. Six hours after mIL-33 (or PBS) administration, mice were sacrificed and blood was collected for serum analysis. Bronchoalveolar lavage (BAL) was performed by injecting two volumes of 0.7mL PBS/0.1% BSA into the lungs and removing the effluent. BAL was centrifuged (1200rpm, 10 min) and the cell pellet was resuspended in 200 μ l PBS for total and differential cell counts using a hemocytometer (counting the buff-stained cell preparation).

Measurement of CNTO3914 in mouse serum

MSD SA-STD plates were blocked with 50. mu.L/well of assay buffer for 5 min. The plate was inverted to remove assay buffer and tapped on a paper towel. Add 50. mu.l/well of 1.4. mu.g/mL biotinylated recombinant mouse ST2L/IL1R4/Fc chimera (R & D System) in assay buffer and incubate overnight in a refrigerator. Add 150 μ Ι _ of assay buffer to each well of the pre-coated plate without removing the coated reagent and incubate for 30 minutes. The plates were washed three times with wash buffer on a plate washer. The plate was gently tapped on a paper towel to remove residual wash buffer. 50 μ L/well of CNTO3914 sample was added to each well of the plate. The plates were incubated for one hour at ambient temperature with gentle vortex shaking. The plates were washed three times with wash buffer on a plate washer. Ruthenium-labeled mouse anti-mouse IgG1b (BDbiosciences) was added at a titer of 50. mu.L/well to each well of the plate. The plates were incubated for one hour at ambient temperature with gentle vortex shaking. The plates were washed three times with wash buffer on a plate washer. Add 150 μ L of read buffer to each well of the plate. Immediately place the plate on a MSD sector imager 6000 plate reader to read the luminescence level.

Whole blood assay

Blood was mixed at a ratio of 1: 4 in a Sarstedt filter tube in DMEM medium + 1% penicillin + streptomycin solution +/-10ng/ml mouse IL-33. The tubes were incubated overnight at 37 ℃ and then the cytokine and chemokine levels of the supernatant were measured using the Millipore Milliplex mouse cytokine/chemokine kit according to the manufacturer's instructions.

Results

anti-ST 2L antibody was detectable in the serum of mice 24 hours after administration of 0.2 or 2mg/kg CNTO3914 (FIG. 16A).

Intranasal administration of IL-33 induced recruitment of cells to the airways at 6 hours (fig. 16B). Administration of an anti-ST 2L monoclonal antibody reduces BAL cell recruitment; 0.2mg/kg is the minimum dose required to observe significant inhibition of BAL cell recruitment (fig. 16B). Statistical significance was calculated using one-way analysis of variance.

Stimulation of whole blood with mouse IL-33 showed increased levels of cytokines and chemokines, including IL-6 (FIG. 16C) and MCP-1 (FIG. 16D), after 24 hours. In mice administered with 20mg/kg or 2mg/kg of the anti-ST 2L monoclonal antibody CNTO3914, IL-6 and MCP-1 levels were reduced compared to CNTO5516 (isotype control anti-mouse IgG1), indicating conjugation to the target. The minimum dose of 2mg/kg associated with inhibition in the whole blood assay also inhibited BAL cell recruitment (fig. 16B).

Taken together, this data demonstrates that the anti-ST 2L monoclonal antibody reaches the site of action and achieves the desired pharmacological effect (indicating engagement with the target).

Example 16: epitopes of anti-ST 2L antibody

Epitope mapping and competition studies were performed to select anti-ST 2L antibodies.

Competitive binding assays

Competitive binding assays were performed to evaluate the different binding epitope groups of the anti-ST 2L monoclonal antibody. Mu.l (10. mu.g/ml) of ST2L-ECD protein per well was coated on MSD HighBind plates (Meso Scale Discovery, Gaithersburg, Md.) for 2 hours at room temperature. One hundred fifty milliliters of 5% MSD packer a buffer (Meso Scale Discovery, Gaithersburg, MD) was added to each well and incubated at room temperature for 2 hours. The plates were washed three times with 0.1MHEPES buffer (pH 7.4) followed by the addition of a mixture of MSD fluorochrome (sulfonic acid-tagged, NHS ester) labeled anti-ST 2L antibody alone and different competitors. 10 or 30nM labeled antibody was incubated with increasing concentrations of competitor antibody from 1nM to 2 or 5. mu.M, followed by addition to the designated wells in a volume of 25. mu.L of the mixture. After incubation for 2 hours at room temperature with gentle shaking, the plates were washed 3 times with 0.1M HEPES buffer (pH 7.4). MSD read buffer T (4 fold) was diluted with distilled water and dispensed at a volume of 150 μ L/well and analyzed with SECTOR Imager 6000.

The following antibodies were used in the competition assay: ST2L domain I binds to neutralizing antibodies STLM208, STLM213, C2244(STLM15) and C2494(STLM62), ST2L domain III binds to antibody C2539, and non-neutralizing anti-ST 2L antibody C2240 which binds to domain I of human ST 2L. Fig. 17A and 17B show competition experiments. According to this experiment, the epitope bins (epitopbins) identified were: BinA: monoclonal antibodies C2244, C2494, STLM208 or STLM 213; BinB: monoclonal antibody C2240, BinC: C2539. antibodies that block IL33/ST2L interaction and inhibit mast cell response were found in the same epitope bin and cross-compete with each other. A summary of the competition data is shown in table 20.

Table 20.

Epitope mapping: H/D exchange analysis

For H/D exchange, the procedure for analyzing antibody perturbation was similar to the previous procedure (Hamuro, Y. et al, Journal of Biomolecular Techniques, 14: 171-. Recombinant ST2-ECD (expressed by HEK293E with a C-terminal His tag) (residues 18-328 of SEQ ID NO: 157) was incubated in a deuterated aqueous solution for a predetermined time to allow deuterium incorporation at exchangeable hydrogen atoms. The deuterated ST2-ECD was captured on a column containing immobilized anti-ST 2L C2244Fab molecule and then washed with aqueous buffer. The retro-exchanged ST2-ECD protein was eluted from the column and the localization of deuterium containing fragments was determined by protease digestion and mass spectrometry.

FIG. 18 shows a simplified H/D exchange diagram for human ST2-ECD (soluble ST2) complexed with C2244 Fab. SEQ ID NO: residues 18-31 of ST2-ECD of 119 (amino acid residues RCPRQGKPSYTVDW; SEQ ID NO: 210) are protected by Fab (corresponding to residues 35-48 of full length ST2L of SEQ ID NO: 1). The data indicate that C2244 binds to epitope RCPRQGKPSYTVDW; SEQ ID NO: 210) and antibodies that compete with C2244(C2494, STLM208, or STLM213) are likely to bind the same or overlapping epitopes.

Epitope mapping by mutagenesis

Several ST2L mutants were generated with substitutions in ST2L domain I of the corresponding mouse residues. The tested antibodies did not cross-react with mouse ST2L, so ST2L variants with disrupted and/or reduced binding capacity could be expected to represent replacement positions of epitope residues on ST 2L. Variants were made with SEQ ID NO: 1 full length ST2L at residues 19-205 of construct HH-ST 2L. Antibody binding to ST2L variants was tested by ELISA or Proteon.

Surface plasmon resonance

Binding studies were performed using the ProteOn XPR36 protein interaction array system (Bio-Rad) (Bravmant et al, Anal Biochem 358: 281-288, 2006). An anti-human/anti-mouse Fc mixture (Jackson ImmunoResearch, Cat. No. 109-. The anti-ST 2L monoclonal antibody alone was then captured by flowing a solution of the antibody prepared in PBS containing 0.5% Nonidet P-40 and 0.5% sodium deoxycholate (1. mu.g/mL). The signal in the surface reached about 250 resonance units (RU, 1RU ═ 1pg protein/mm) in the anti-Fc coated surface²) These antibodies were confirmed to specifically capture the anti-ST 2L monoclonal antibody. After the liquid system was rotated 90 °, wild-type or variant protein of ST2L-D1D2 (0.5mg/mL PBS containing 0.5% Nonidet P-40 and 0.5% sodium deoxycholate) was injected into parallel flow channels. All these measurements were carried out at 25 ℃. ST2L-D1D 2-dependent signals on the surface were obtained by double referencing, subtracting the response observed on the surface of the immobilized antibody alone and the signal observed by vehicle injection alone (which allowed correction of the binding independent response). The resulting sensorgrams were fitted by the simplest 1: 1 interaction model (ProteOn analysis software) to obtain the corresponding association and dissociation rate constants (k)_aAnd k_d)。

FIG. 19 shows the affinity of the prepared ST2L variant and ST2B206 and ST2B252 anti-ST 2L antibodies for the variant 93NL94 (substitution 93TF94- > 93NL94) reduced the binding affinity of STLM208 and STLB252 by about 5-fold, from about 10.8 × 10^-12M to about 49.5 × 10^-12And M. The lack of significant reduction in binding affinity indicates that the binding energy of the interaction between the antibody and ST2L-D1D2 is the sum of the epitope region (RCPRQGKPSYTVDW; SEQ ID NO: 210) identified by the H/D exchange analysis and the additional contribution from this 93NL94 site. Residue numbering according to SEQ ID NO: 1 full length human ST 2L.

Example 17: ST2L Domain I binding antibodies inhibit Primary human Lung mast cell response in vitro

The ability of ST2L domain I to bind antibodies to inhibit lung mast cell responses was assessed by the release of chemokines and cytokines in primary human lung mast cells.

Isolation of primary human mast cells

Primary human lung mast cells were isolated from normal non-smoker tissue obtained from the International Institute for the Advancement of medicine. Cells were dispersed from the lung parenchyma and small airways by mincing, washing and digesting parenchyma overnight in collagenase and hyaluronidase at 37 ℃. Cells were collected, washed, and an enrichment step was performed using the CD117MicroBead kit (human) from MACS miltnyi Biotec to positively select mast cells from the population. Prior to the experiment, mast cells were cultured in StemPro-34+200ng/ml stem cell factor for 6 weeks. Two weeks after isolation, cells were phenotypically characterized using flow cytometry to determine percent mast cell purity. Cells for subsequent assays for CD117(C-kit or Stem cell factor receptor) and(high affinity IgE receptor) was 89% double positive. Furthermore, they were 94.2% positive for ST 2L; thereby confirming the mast cell phenotype.

Cytokine release assay for primary human lung mast cells

Primary human lung mast cells that had been cultured in StemPro-34+200ng/ml stem cell factor for approximately 6 weeks were harvested and washed by centrifugation in RPMI (10% heat inactivated FCS). Cells were counted and seeded at a density of 65,000 cells in RPMI/10% FCS medium in 96-well plates. anti-ST 2L domain I binding monoclonal antibody was added to primary lung mast cells and allowed to bind at 37 ℃ for 30 minutes prior to stimulation with IL-33. Cells were stimulated with 3ng/ml IL-33 for 24 hours to initiate accumulation of various mediators in the culture supernatant. Culture supernatants were harvested and frozen until assayed in a custom Milliplex 9-plex kit.

anti-ST 2L domain I binding antibody, STLM208, inhibited IL-33-induced release of GM-CSF (FIG. 20A), IL-5 (FIG. 20B), IL-8 (FIG. 20C), and IL-13 (FIG. 20D) in primary human lung mast cells at antibody concentrations of 100. mu.g/ml, 10. mu.g/ml, and 1. mu.g/ml. Similar results were obtained using umbilical cord blood-derived mast cells (data not shown).

Claims (21)

1. An isolated antibody or fragment thereof that specifically binds domain I (SEQ ID NO: 9) of human ST2L, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the sequences: 97, 114, 84, 130, 90 and 134, respectively.

2. The isolated antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof blocks the IL-33/ST2L interaction.

3. The isolated antibody or fragment thereof of claim 2, having between 5 × 10^-12M to 7 × 10^-10Dissociation constant (K) between M for human ST2L_D) Between 2 × 10⁶M^-1s^-1To 1 × 10⁸M^-1s^-1The binding rate constant (K) to human ST2L in between_on) Or between 1 × 10^-6s^-1To 1 × 10^-2s^-1Dissociation rate constant (K) for human ST2L between_off)。

4. The isolated antibody or fragment thereof of claim 3, having between 3 × 10^-12M to 2 × 10^-9For cynomolgus monkey between M (Macaca fascicularis) Dissociation constant (K) of ST2L (SEQ ID NO: 2)_D) Between 4 × 10⁶M^-1s^-1To 1 × 10⁸M^-1s^-1Binding rate constant (K) to cynomolgus monkey ST2L_on) Or between 7 × 10^-5s^-1To 1 × 10^-1s^-1Dissociation rate constant (K) for cynomolgus monkey ST2L_off)。

5. The isolated antibody or fragment thereof of claim 1, comprising: the VH of SEQ ID NO. 191 and the VL of SEQ ID NO. 209.

6. The isolated antibody or fragment thereof of claim 1, which is of the IgG1, IgG2, IgG3, or IgG4 type.

7. The isolated antibody or fragment thereof of claim 6, having a substitution in the Fc region.

8. The isolated antibody or fragment thereof of claim 7, wherein the substitutions comprise substitutions V234A/G237A/P238S/H28A/V309L/A330S/P331S, wherein residue numbering is according to EU numbering.

9. An isolated polynucleotide encoding the VH of SEQ ID NO. 191 or the VL of SEQ ID NO. 209.

10. A vector comprising the isolated polynucleotide of claim 9.

11. A host cell comprising the vector of claim 10.

12. A method of making the isolated antibody or fragment thereof of claim 1, comprising culturing the host cell of claim 11 and recovering the antibody or fragment thereof from the cell.

13. A pharmaceutical composition comprising the isolated antibody or fragment thereof of claim 1 or claim 5 and a pharmaceutically acceptable vehicle.

14. Use of a therapeutically effective amount of the isolated antibody or fragment thereof of claim 1 or claim 5 in the manufacture of a medicament for treating or preventing a disorder mediated by ST 2L.

15. The use of claim 14, wherein the ST 2L-mediated disorder is asthma, airway hyperreactivity, sarcoidosis, Chronic Obstructive Pulmonary Disease (COPD), Idiopathic Pulmonary Fibrosis (IPF), cystic fibrosis, inflammatory bowel disease, eosinophilic esophagitis, scleroderma, atopic dermatitis, allergic rhinitis, bullous pemphigoid, chronic urticaria, diabetic nephropathy, rheumatoid arthritis, interstitial cystitis, or Graft Versus Host Disease (GVHD), or a disorder associated with inflammatory cell recruitment, goblet cell proliferation, increased mucus secretion, or mast cell response in the lung.

16. Use of a therapeutically effective amount of the isolated antibody or fragment thereof of claim 1 in the manufacture of a medicament for inhibiting a mast cell response in a patient.

17. The use of claim 16, wherein inhibiting mast cell response comprises inhibiting the level of GM-CSF, IL-5, IL-8, IL-10, or IL-13 released from human umbilical cord blood-derived mast cells by at least 50% with 50 μ g/ml antibody.

18. Use of a therapeutically effective amount of the isolated antibody or fragment thereof of claim 1 in the manufacture of a medicament for inhibiting the interaction of IL-33 and ST2L in a subject.

19. The use of claim 18, wherein the subject has a ST 2L-mediated disorder.

20. The use of claim 19, wherein the ST 2L-mediated disorder is asthma, airway hyperreactivity, sarcoidosis, Chronic Obstructive Pulmonary Disease (COPD), Idiopathic Pulmonary Fibrosis (IPF), cystic fibrosis, inflammatory bowel disease, eosinophilic esophagitis, scleroderma, atopic dermatitis, allergic rhinitis, bullous pemphigoid, chronic urticaria, diabetic nephropathy, rheumatoid arthritis, interstitial cystitis, or Graft Versus Host Disease (GVHD), or a disorder associated with inflammatory cell recruitment, goblet cell proliferation, or increased mucus secretion, or mast cell response in the lung.

21. The use of claim 19, wherein the isolated antibody or fragment thereof comprises:

a) HCDR1 of SEQ ID NO 97;

b) HCDR2 of SEQ ID NO 114;

c) HCDR3 of SEQ ID NO. 84;

d) LCDR1 of SEQ ID NO. 130;

e) LCDR2 of SEQ ID NO. 90; and

f) LCDR3 of SEQ ID NO. 134; or

g) The VH of SEQ ID NO. 191 and the VL of SEQ ID NO. 209.