WO2012155049A1 - Treatment of lung inflammation - Google Patents

Abstract

Provided is a method of treating an inflammatory lung disorder by administering an effective amount of an S100A9 inhibitor.

Description

Treatment of Lung Inflammation

Field of the Invention

The present invention relates to a method of treating a disorder of the lung marked by inflammation using an inhibitor of SI 00 A9.

Background of the Invention

The SI 00 protein family is the largest subgroup within the superfamily of proteins carrying the Ca²⁺ binding EF hand motif. Twenty-one members of SI 00 protein have been identified in humans and most of the SI 00 proteins form homo- and heterodimers in solution (Heizmann, et al. (2007) Subcell. Biochem. 45:93-138). Although the S100 protein sequences exhibit some variety, the key structural features of all SI 00 proteins are highly conserved. Each SI 00 polypeptide is composed of two EF-hand Ca²⁺ binding domains connected by a central hinge region (Marenholz, et al. (2004) Biochem. Biophys. Res. Commun. 322, 1111-1122). Many vital physiological functions and metabolic processes are regulated by Ca²⁺ signaling. Like other Ca²⁺ binding proteins, SI 00 proteins are involved in the regulation of diverse cellular processes such as cell cycle regulation, cell growth, cell differentiation, transcription, secretion and motility (Heizmann, et al. (2007) Subcell. Biochem. 45:93-138; Marenholz, et al. (2004) Biochem. Biophys. Res. Commun. 322, 1111-1122). In addition to their intracellular functions, several S100 proteins (such as S100B, S100A4, S100A8, S100A9 and S100A12) are secreted and act in a cytokine-like manner (Huttunen, et al. (2000) J. Biol. Chem. 275:40096-40105; Newton, R. A. & Hogg (1998) J. Immunol. 160: 1427-1435; Schmidt-Hansen et al. (2004) Oncogene 23:5487-5495.

Recently, SI 00 proteins have received increasing attention due to their close association with human disease, including cancer, neurodegenerative disorders, heart disease, inflammation and autoimmune disease (Heizmann, et al. (2007) Subcell. Biochem. 45:93-138; Gebhardt, C. et al. (2006) Biochem. Pharmacol. 72, 1622-1631 (2006); Sedaghat, F. & Notopoulos, A. (2008) Hippokratia 12: 198-204). Calgranulins, a subgroup of S100 proteins (including S100A8, S100A9 and S100A12) belong to the damage-associated molecular pattern (DAMP) family and have been identified as important proinflammatory factors of innate immunity as well as pathogenesis of inflammation and autoimmune diseases (Hsu et al. (2009) Anti-Inflamm. & Anti- Allergy Agents Med. Chem. 8:290-305), For example, serum levels of S100A8, S100A9 and S100A12 correlated with RA patients' disease activity, autoantibody level and classical risk markers of joint and vascular damage (Chen, Y.S. et al. (2009) Arthritis Res. Ther. 11 :R39). In addition, serum level of the sl00A8/A9 complex is significantly raised in SLE verse pSS patients and healthy donors, and there is a significant correlation between S100A8/A9 and SLEDAI scores (Soyfoo, M. S. et al. (2009) J. Rheumatol. 36:2190-2194). S100A8 and S100A9 have also been implicated in development of autoreactive CD8+ T cells in CD40L-transgenic autoimmunity in a murine model (Loser K et al. (2010) Nat. Med. 16:713-717). Furthermore, in clinical trials for Q compound (Quinoline-3-Carboxamide) in MS and SLE, it was shown that S100A9 is the target protein of Q compound and that Q compound was able to inhibit the binding of S100A9 to RAGE and TLR4 in vitro (Bjork et al. (2009) PLoS Biol. 2009

7(4):el000097). Taken together, this data suggest calgranulins, particularly S100A9, may play an important role in pathogenesis of respiratory, inflammation and autoimmune disease.

The biology of SlOO proteins is very complicated. In addition to multiple members of SlOO proteins, there are also multiple receptors and co-receptors that have been identified for SlOO proteins, including RAGE (Tsoporis, J. N. et al. (2010) Circ. Res. 106:93-101; Sparvero, L. J. et al. (2009) J. Transl. Med. 7: 17; Boyd, J. H. et al. (2008) Circ. Res. 102: 1239-1246; Foell, D. (2007) J. Leukoc. Biol. 81 :28-37; Hofmann, M. A. et al. (1999) Cell 97:889-901); TLR 4 (Buchau, A. S. et al. (2007) J. Invest. Dermatol. 127:2596-2604; Ehrchen, J. M. et al. (2009) J. Leukoc. Biol. 86:557-566; Halayko, A. J. & Ghavami, S. (2009) Can. J. Physiol. Pharmacol. 87:743-755; Hiratsuka, S. et al. (2008) Nat. Cell. Biol. 10: 1349-1355; Vogl, T. et al. (2007) Nat. Med. 13: 1042-1049); CD36 (Robinson, M. J. (2002) J. Biol. Chem. 277:3658-3665; Kerkhoff, C, (2001) Biochemistry 40:241-248), and MAC I (Lau, W. et al. (1995) J. Clin. Invest. 95: 1957- 1965). Different SlOO members may trigger distinct signaling pathway such as MAPK and PI3 kinase, and lead to different biological consequences, including cell proliferation, cell apoptosis, migration and proinflammatory cytokines release. Recent progress in the study of SI 00s is the finding that some SI 00 members can direct cell migration and proinflammatory cytokine release. Recent work indicates that S100A7 and S100A15 utilize distinct pathways for cell migration and inflammation, either mediated by the RAGE or Gi protein-coupled receptor (Wolf, R. et al. (2008) J. Immunol. 181 :1499-1506. In another recent paper S100A8/9 was shown to be an endogenous TLR4, able to amplify phagocyte activation during sepsis upstream of a TNFa-dependent effect (Vogl, T. et al. (2007) Nat. Med. 13: 1042-1049). However, the exact receptor responsible for S100A9 induced cell migration is still unknown. To date, there are no definitive studies on S100A9's role in inflammation and obstructive airway disease.

2. SUMMARY OF THE INVENTION

The invention provides a method of preventing, inhibiting, treating or managing a lung inflammatory disorder in an animal in need thereof, comprising administering a therapeutically effective amount of a S100A9 inhibitor. The inhibitor may be a small molecule inhibitor, a monoclonal antibody, an S100A9 polypeptide fragment, a mutant S100A9 or an anti-S100A9 antibody. The inflammatory lung disorder may be, for example, inflammation in the lung associated with COPD, asthma, IPF, MS, RA or SLE. The inhibitors of S100A9 are useful in treating, preventing or alleviating the symptoms of an inflammatory disorder of the lung in a subject in need thereof.

The invention also provides a method for inhibiting an S100A9 activity in a cell expressing S100A9, comprising contacting the cell with an S100A9 inhibitor. The inhibitor is effective in inhibiting S100A9 activity in the cell, such as induction of cell migration, induction of cytokine release, binding to TLR4 and combinations thereof.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows that hS100A8, hS100A9 or hS100A12 induction of proinflammatory cytokines is TLR4 dependent and RAGE independent. Human PBMC were stimulated with human S100A8, S100A9 or S100A12 (1 ug/ml respectively) for 16 hrs with or without anti- TLR4 (10 ug/ml) or anti-RAGE Abs (10 ug/ml). IFNy, IL-Ιβ, IL-6 and TNF-a levels in supernatant was determined by 4plex human proinflammatory cytokines detection kit (Meso Scale Discovery Technology). *, P<0.05, **, P<0.01, ***P<0.001, when compared with induction (black bar).

Figure 2 show that hS100A8, hS100A9 or hS100A12 induction of THP-1 cell migration is RAGE dependent but TLR4 independent. THP-1 cells migration in response to hS100A8 (A), hS100A9 (C), hS100A12 (E). Shown are results (mean±SD of triplicate wells) of one experiment representative of three independent experiments. *, P<0.05, **, P<0.01,

***P<0.001, when compared with background migration (open bar). Effect of anti-RAGE and anti-TLR4 ab on hS100A8(B), hS100A9 (D) and hS100A12 (F) induced THP-1 cell migration:

Serial dilution of Anti-RAGE, anti-TLR4 or isotype-matched control Ab was added together with THP-1 cells in the upper wells of chemotaxis chamber, hS100A8 (1 ng/ml), hS100A9 (1 ng/ml) and hS100A12 (100 ng/ml) were added in the lower wells. The chemotactic activities of

S100A4, S100A7, S100A8/A9, and to a lesser extent S100A6 mediated migration of THP1 cells were also dependent on RAGE, whereas migration of S100A1, and S100A10 were not affected by RAGE or TLR4 blockade (Fig. 2G). Data represent here is the percentage inhibition compared to no Ab treatment.

Figure 3 shows that hS100A9-induced lymphocyte and monocyte migration is RAGE dependent but TLR4 independent. Panels (A) and (B): Chemotactic response of indicated leukocyte subsets from peripheral blood of human volunteers. Shown is the average (mean ± SD) migration index of three wells per group. The concentration used for S100A9 is 1 ng/ml. Similar results were obtained from three separate experiments. *, P<0.05, **, P<0.01, when compared with background migration. Panels (C),(D) and (E): hS100A9 induced lymphocyte, granulocytes and monocyte migration is RAGE dependent but TLR4 independent. 10 ug/ml of Anti-RAGE, anti-TLR4 or isotype-matched control Ab was added together with lymphocytes (C), granulocytes (D) or monocytes (E) in the upper wells of chemotaxis chambers. The concentration used for S100A9 was 1 ng/ml. *, P<0.05, ***P<0.001, when compared to background migration.

Figure 4 shows that hS100A9-induced THP-1 cell migration occurs via MEK/ERK, PI3K but not P38. Chemotactic response of THP-1 toward hS100A9 in the absence or presence of specific inhibitors target the MEK/ERK pathway (Panel A,B); the P38 MAPK pathway (Panel C); or the PI3 kinase pathway (Panel D,E). Shown is the average (mean ± SD) cell migration of three wells per group. The concentration of inhibitors is indicated and the concentration used for S100A9 was 1 ng/ml (representative of three independent experiments).

Figure 5 shows the effect of anti-RAGE and anti-TLR4 Abs on mS100A9-induced raw cell migration and proinflammatory cytokine induction. Panel A: Effect of anti-RAGE and anti- mouse TLR4/Md2 Ab on murine S100A9 induced Raw cell migration. Serial dilution of anti- RAGE, anti-mouse TLR4/Md2 or isotype-matched control Ab was added together with Raw cells in the upper wells of a chemotaxis chamber. Murine S100A9 (1 ng/ml) was added in the lower wells. Data represents the percentage inhibition compared to no Ab treatment. Panel B: Raw cells were stimulated with mS100A9 (1 ug/ml) or LPS (1 ng/ml) for 16 hrs with or without anti-mouse TLR4/MD2 (10 ug/ml) or anti-RAGE Abs(10 ug/ml). mIL-6 and mTNF-a levels in the supernatant was determined by the mouse proinflammatory cytokines detection kit (Meso Scale Discovery Technology). *, P<0.05; **; P<0.01; ***, PO.001.

Figure 6: shows the comparison of S100A9 induced lung inflammation in wild type (C57BL/6) and RAGE KO mice. Wild type or RAGE KO mice were intranasally inoculated with PBS, Adenovirus null or Adenovirus-expressing S100A9. After 8 days, mice were sacrificed and BALF were collected for further analysis. (Panel A, B and C): Total and differential cell counts in BALF in wild type and RAGE KO mice; (Panel D and E): mIFNg and mIL-6 expression in BALF in wild type and RAGE KO mice; (Panel F): Western blot analysis of S100A9 expression in BALF in wild type and RAGE KO mice; (Panel H): Hematoxylin and eosin (H&E) staining of lung tissue for wild type C57/B16 mice (upper panel) and RAGE KO (lower panel); (Panel I)Lung pathology scores for wild-type and RAGE KO mice.

Figure 7 shows a comparison of S100A9 induced lung inflammation in wild type (C3H/HeOuJ) and TLR4 deficient (C3H/Hej) mice. Wild type or TLR4 deficient mice were intranasally inoculated with PBS, Adenovirus null or Adenovirus-expressing S100A9. After 10 days, mice were sacrificed and BALF were collected for further analysis. Panel A: Total and differential cell counts in BALF in wild type and TLR4 deficient mice; Panel B: mIFNg and mIL-6 expression in BALF in wild type and TLR4 deficient mice; Panel C: Western blot analysis of S100A9 expression in BALF in wild type and TLR4 deficient mice. (Panel H): Hematoxylin and eosin (H&E) staining of lung tissue for wild type mice C3H/HeOuJ mice (upper panel) and TLR4-defective C3H/HeJ (lower panel); (Panel I) Lung pathology scores for wild-type and RAGE KO mice.

DETAILED DESCRIPTION

The present invention provides methods of treating preventing or inhibiting inflammation in the lung comprising administering an inhibitor of SI 00 A9. The inhibitor may be, for example, an anti-S100A9 antibody (including human, humanized and/or chimeric forms, as well as S100A9-binding fragments, derivatives/conjugates) and compositions thereof that bind to S100A9; small molecule inhibitors of S100A9, mutant S100A9 polypeptides and fragments of S100A9 that inhibit endogenous S100A9 from binding to its receptor. The methods are useful in the prevention, inhibition and treatment of inflammatory lung disorders, such as those associated with Chronic Obstructive Pulmonary Disease (COPD), asthma, Idiopathic Pulmonary Fibrosis (IPF), rheumatoid arthritis (RA), Systemic Lupus Erythematosus (SLE), and Multiple Sclerosis (MS).

As used herein, the terms "antibody" and "antibodies", also known as immunoglobulins, encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two different epitope binding fragments (e.g., bispecific antibodies), human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments that exhibit the desired biological activity (e.g. the antigen binding portion), disulfide-linked Fvs (dsFv), and anti- idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intrabodies, and epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain at least one antigen-binding site. Immunoglobulin molecules can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), subisotype (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or allotype (e.g., Gm, e.g., Glm(f, z, a or x), G2m(n), G3m(g, b, or c), Am, Em, and Km(l, 2 or 3)). Antibodies may be derived from any mammal, including, but not limited to, humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc., or other animals such as birds (e.g. chickens).

Native antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains (CH). Each light chain has a variable domain at one end (VL) and a constant domain (CL) at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Light chains are classified as either lambda chains or kappa chains based on the amino acid sequence of the light chain constant region. The variable domain of a kappa light chain may also be denoted herein as VK.

The antibodies of the invention include full length or intact antibody, antibody fragments, native sequence antibody or amino acid variants, human, humanized, post-translationally modified, chimeric or fusion antibodies, immunoconjugates, and functional fragments thereof. The antibodies can be modified in the Fc region to provide desired effector functions or serum half-life. As discussed in more detail below, where it is desirable to eliminate or reduce effector function, so as to minimize side effects or therapeutic complications, certain Fc regions may be used. The Fc region of the antibodies of the invention can be modified to increase the binding affinity for FcRn and thus increase serum half-life. Alternatively, the Fc region can be conjugated to PEG or albumin to increase the serum half-life, or some other conjugation that results in the desired effect.

The present anti-S100A9 antibodies are useful for diagnosing and/or treating and/or alleviating one or more symptoms of the S100A9 associated diseases or disorders in a mammal. Such diseases include Chronic pulmonary obstructive disease (COPD), asthma, Idiopathic Pulmonary Fibrosis (IPF) and lung inflammation due to systemic lupus erythematosus (SLE), Multiple Sclerosis, and rheumatoid arthritis. The invention provides a method of treating the lung inflammatory disorders with a composition comprising an inhibitor of S100A9, such as a small molecule inhibitor, an anti- Si 00A9 antibody, an S100A9 polypeptide fragment, a mutant S100A9 or a combination thereof.

The anti-S100A9 antibody for use in the invention may be supplied with a

pharmaceutically-acceptable carrier. For the purposes of treating S100A9 associated disease, compositions can be administered to the patient in need of such treatment, wherein the composition can comprise one or more anti-S100A9 antibodies present as an immunoconjugate or as the naked antibody. In a further embodiment, the compositions can comprise these antibodies in combination with other therapeutic agents such as cytotoxic or growth inhibitory agents, including chemotherapeutic agents. The invention also provides formulations comprising an anti-S100A9 antibody of the invention and a carrier. In one embodiment, the formulation is a therapeutic formulation comprising a pharmaceutically acceptable carrier.

In certain embodiments the invention provides methods useful for treating a S100A9 associated disease/condition and/or preventing and/or alleviating one or more symptoms of the disease in a mammal, comprising administering a therapeutically effective amount of the anti- S100A9 antibody to the mammal. The antibody therapeutic compositions can be administered short term (acute) or chronic, or intermittently as directed by physician.

Terminology

Before describing the present invention in detail, it is to be understood that this invention is not limited to specific compositions or process steps, as such may vary. It must be noted that, as used in this specification and the appended claims, the singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

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 is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this invention. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical

Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

The numbering of amino acids in the variable domain, complementarity determining region (CDRs) and framework regions (FR), of an antibody follow, unless otherwise indicated, the Kabat definition as set forth in Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence. Maximal alignment of framework residues frequently requires the insertion of "spacer" residues in the numbering system, to be used for the Fv region. In addition, the identity of certain individual residues at any given Kabat site number may vary from antibody chain to antibody chain due to interspecies or allelic divergence.

Anti-S100A9 Antibodies

In certain embodiments, the anti-S100A9 antibodies are isolated and/or purified and/or pyrogen free antibodies. The term "purified" as used herein, refers to other molecules, e.g. polypeptide, nucleic acid molecule that have been identified and separated and/or recovered from a component of its natural environment. Thus, in one embodiment the antibodies of the invention are purified antibodies wherein they have been separated from one or more components of their natural environment. The term "isolated antibody" as used herein refers to an antibody which is substantially free of other antibody molecules having different antigenic specificities (e.g., an isolated antibody that specifically binds to S100A9 is substantially free of antibodies that specifically bind antigens other than S100A9; however a bi- or multi-specific antibody molecule is an isolated antibody when substantially free of other antibody molecules). Thus, in one embodiment the antibodies of the invention are isolated antibodies wherein they have been separated from antibodies with a different specificity. Typically an isolated antibody is a monoclonal antibody. An isolated antibody that specifically binds to an epitope, isoform or variant of human S100A9 may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., S100A9 species homologs). Moreover, an isolated antibody of the invention may be substantially free of one or more other cellular materials and/or chemicals and is herein referred to an isolated and purified antibody. In one embodiment of the invention, a combination of "isolated" monoclonal antibodies relates to antibodies having different specificities and being combined in a well defined composition. Methods of production and purification/isolation of the anti-S100A9 antibodies are described below in more detail.

The isolated antibodies of the present invention comprise antibody amino acid sequences disclosed herein encoded by any suitable polynucleotide, or any isolated or formulated antibody. In one embodiment, the anti-S100A9 antibody binds human S100A9 and, thereby partially or substantially alters at least one biological activity of the S100A9 (e.g. receptor binding, catalytic activity, etc.).

The anti-S100A9 antibodies of the invention immunospecifically bind at least one specified epitope specific to the S100A9 protein, peptide, subunit, fragment, portion or any combination thereof and do not specifically bind to other polypeptides. The at least one epitope can comprise at least one antibody binding region that comprises at least one portion of the S100A9 protein. The term "epitope" as used herein refers to a protein determinant capable of binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non- conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

Thus, in a specific embodiment the isolated/purified anti-S100A9 antibodies of the invention immunospecifically bind to S100A9 having the amino acid sequence according to SEQ ID.NO:l . In certain embodiments, the anti-S100A9 antibodies of the invention also bind S100A9 homologs or orthologs from different species. The species cross-reactive characteristics of the present antibodies are described below in more detail.

Table 1 : Amino acid and Nucleotide sequences of S100A9

SEQ ID NO: l

TCKMSQLER NIETIINTFH QYSVKLGHPD TLNQGEFKEL VRKDLQNFLK KENKNEKVIE HI EDLDTNA DKQLSFEEFI LMARLTWAS HEK HEGDEG

PGHHHKPGLG EGTP

SEQ ID NO:2

atgacttgca aaatgtcgca gctggaacgc aacatagaga

ccatcatcaa caccttccac caatactctg tgaagctggg

gcacccagac accctgaacc agggggaatt caaagagctg

gtgcgaaaag atctgcaaaa ttttctcaag aaggagaata

agaatgaaaa ggtcatagaa cacatcatgg aggacctgga

cacaaatgca gacaagcagc tgagcttcga ggagttcatc

atgctgatgg cgaggctaac ctgggcctcc cacgagaaga

tgcacgaggg tgacgagggc cctggccacc accataagcc

aggcctcggg gagggcaccc cc

The antigen-binding portion of an antibody comprises one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., S100A9). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full- length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

In certain embodiments, the anti-S100A9 antibodies immunospecifically bind to human S100A9 and antigenic fragments thereof. In one embodiment, the anti-S100A9 antibodies immunospecifically bind to SEQ ID NO: 1 , or at least any three contiguous amino acids of SEQ ID NO: 1. In another embodiment, the epitope is at least 4 amino acid residues, at least 5 amino acid residues, at least 6 amino acid residues, at least 7 amino acid residues, at least 8 amino acid residues or at least 9 amino acid residues to the entire specified portion of contiguous amino acids of SEQ ID NO: l . In one embodiment, the anti-S100A9 antibodies immunospecifically bind a S100A9 polypeptide or antigenic fragments thereof, having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or having at least 100% identity to the amino acid sequence of SEQ ID NO: 1. In a further embodiment, the anti-S100A9 antibodies immunospecifically bind to a S100A9 polypeptide or antigenic fragments thereof, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or having at least 100% identity to the amino acid sequence of SEQ ID NO: 1.

In certain embodiments, the antibodies of the invention may bind epitopes conserved across species. In one embodiment, antibodies of the invention bind murine, non-human primate, rat, bovine, pig or other mammalian S100A9 and antigenic fragments thereof. In one embodiment the antibodies of the invention may bind to one or more S100A9 orthologs and or iso forms. In a specific embodiment, antibodies of the invention bind to S100A9 and antigenic fragments thereof from one or more species, including, but not limited to, mouse, rat, monkey, primate, and human. In certain embodiments, the antibodies of the invention may bind an epitope within humans across S100A9 homo logs and/or iso forms and/or conformational variants and/or subtypes.

The interactions between antigens and antibodies are the same as for other non-covalent protein-protein interactions. In general, four types of binding interactions exist between antigens and antibodies: (i) hydrogen bonds, (ii) dispersion forces, (iii) electrostatic forces between Lewis acids and Lewis bases, and (iv) hydrophobic interactions. Hydrophobic interactions are a major driving force for the antibody-antigen interaction, and are based on repulsion of water by non- polar groups rather than attraction of molecules (Tanford, 1978). However, certain physical forces also contribute to antigen-antibody binding, for example, the fit or complimentary of epitope shapes with different antibody binding sites. Moreover, other materials and antigens may cross-react with an antibody, thereby competing for available free antibody.

Measurement of the affinity constant and specificity of binding between antigen and antibody is a pivotal element in determining the efficacy of therapeutic, diagnostic and research methods using the anti-S100A9 antibodies. "Binding affinity" generally refers to the strength of the sum total of the noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (Kd), which is calculated as the ratio k_off/k_on. See, e.g., Chen, Y., et al, (1999) J. Mol Biol 293:865-881.

Affinity can be measured by common methods known in the art, including those described and exemplified herein, such as BiaCore. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.

The anti-S100A9 antibodies preferably will have binding affinities for a S100A9 epitope that include a dissociation constant (IQ) of less than l l(T²M, l l(T³M, l l(T⁴M, l l(T⁵M, l x l(T⁶M, l x l(T⁷M, l x l(T⁸M, 1 ^χ 1(Γ⁹Μ, 1 ^χ 1(Γ¹⁰Μ, 1 ^χ ΚΓ^πΜ, 1 ^χ 1(Γ¹²Μ, 1 ^χ 1(Γ¹³Μ, 1 ^χ 1(Γ¹⁴Μ or less than 1 x 10 ¹⁵M. In one embodiment, the anti-S100A9 antibodies have a IQ of less than 10^"7M, less than 5xlO^"8M, less than 10^"8M, less than 5xlO^~9M, less than 10^"9M, less than 5x10^{" 10}M, less than 10^"10M, less than 5xlO^"uM, less than 10^"UM, less than 5xlO^~12M, less than 10^~12M, less than 5xlO^"13M, less than 10^"13M, less than 5xlO^"14M, less than 10^"14M, less than 5xlO^"15M, or less than 10^"15 M.

In certain embodiments, the anti-S100A9 antibodies for use in the invention are high- affinity antibodies. By "high-affinity antibody" is meant an antibody which binds to a S100A9 epitope with an affinity less than 10^~8M (e.g., 10^~9M, 10^~10 M, etc.).

The anti-S100A9 antibodies may be described as having a binding affinity of a specific molarity or better. "Or better" when used herein refers to a stronger binding, represented by a smaller numerical Kd value. For example, an antibody which has an affinity for an antigen of "0.6 nM or better", the antibody's affinity for the antigen is <0.6 nM, i.e. 0.59 nM, 0.58 nM, 0.57 nM etc. or any value less than 0.6 nM.

The affinity of the anti-S100A9 antibodies may alternatively be described in terms of the association constant (K_a), which is calculated as the ratio k_on/k_0ff. In this instance the present anti-S100A9 antibodies have binding affinities for a S100A9 epitope that include an association constant (K_a) of at least l x l0²M^_1, l l0³M^_1, l x l0⁴M^_1, l x l0⁵M^_1, l x l0⁶M^_1, l x

1 X 10⁸M^" l x l0⁹M^_1, l x l0¹⁰M^_1 l x

l x l0¹⁴M^_1 or at least l x l0¹⁵M^_1. In one embodiment, the anti-S100A9 antibodies have a K_a of at least 10⁷ M^"1, at least 5 X 10⁷ M^"1, at least 10⁸ M^"1, at least 5 X 10⁸ M^"1, at least 10⁹ M^"1, at least 5 X 10⁹ M^"1, at least 10¹⁰ M^"1, at least 5 X 10¹⁰ M^"1, at least 10¹¹ M^"1, at least 5 X 10¹¹ M^"1, at least 10¹² M^"1, at least 5 X 10¹² M^"1, at least 10¹³ M^_1, at least 5 X 10¹³ M¹, at least 10¹⁴ M^"1, at least 5 X 10¹⁴ M^"1, at least 10¹⁵ M^"1, or at least 5 X 10¹⁵ M^_1.

The rate at which the anti- S100A9 antibodies dissociates from a S100A9 epitope may be more relevant than the value of the Kj or the K_a. In this instance the anti-S100A9 antibodies may bind to S100A9 with a k_0ff of less than 10^"3 s^"1, less than 5xl0^"3 s^"1, less than 10^"4 s^"1, less than 5x10^~4 s^"1, less than 10^"5 s^"1, less than 5x10^~5 s^"1, less than 10^"6 s^"1, less than 5x10^~6 s^"1, less than 10^"7 s^"1, less than 5xl0^"7 s^"1, less than 10^"8 s^"1, less than 5xl0^"8 s^"1, less than 10^"9 s^"1, less than 5x10^~9 s^"1, or less than 10^"10 s^"1. In other embodiments, the rate at which the anti-S100A9 antibodies associate with a S100A9 epitope may be more relevant than the value of the Kj or the K_a. In this instance the anti-S100A9 antibodies may bind to S100A9 with a k_on rate of at least 10⁵ M^_1s^_1, at least 5χ10⁵

at least 5 x 10⁷ M^_1s^_1, or at least 10⁸ M^_1s^_1, or at least 10⁹ M^_1s^_1.

Determination of binding affinity can be measured using the specific techniques described further in the Example section, See Example X and methods well known in the art. One such method includes measuring the disassociation constant "Kd" by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay that measures solution binding affinity of Fabs for antigen by equilibrating Fab with a minimal concentration of (¹²⁵I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody- coated plate (Chen, et al, (1999) J. Mol Biol 293:865-881). To establish conditions for the assay, microtiter plates (Dynex) are coated overnight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (H 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23° C). In a non-adsorbant plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of an anti-VEGF antibody, Fab-12, in Presta et al, (1997) Cancer Res. 57:4593-4599). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., 65 hours) to insure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% Tween-20 in PBS. When the plates have dried, 150 μΐ/well of scintillant (MicroScint-20; Packard) is added, and the plates are counted on a Topcount gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.

In another instance the Kd value may be measured by using surface plasmon resonance assays using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C with immobilized antigen CM5 chips at ~10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N'-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 110 mM sodium acetate, pH 4.8, into 5 ug/ml (~0.2 uM) before injection at a flow rate of 5 ul/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, IM ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25 ul/min. Association rates (k_on) and dissociation rates (k_0ff) are calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgram.

If the on-rate exceeds 10⁶ IVT¹ S ¹ by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red cuvette. An "on-rate" or "rate of association" or "association rate" or "k_on" according to this invention can also be determined with the same surface plasmon resonance technique described above using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) as described above.

Methods and reagents suitable for determination of binding characteristics of an antibody of the present invention, or an altered/mutant derivative thereof (discussed below), are known in the art and/or are commercially available (U.S. Patent Nos. 6,849,425; 6,632,926; 6,294,391 ; 6, 143,574). Moreover, equipment and software designed for such kinetic analyses are commercially available (e.g. Biacore® A100, and Biacore® 2000 instruments; Biacore

International AB, Uppsala, Sweden).

A binding assay may be performed either as direct binding assays or as competition- binding assays. Binding can be detected using standard ELISA or standard Flow Cytometry assays. In a direct binding assay, a candidate antibody is tested for binding to S100A9 antigen. Competition-binding assay, on the other hand, assess the ability of a candidate antibody to compete with a known anti-S 100A9 antibody or other compound that binds S 100A9. In general any method that permits the binding of an antibody with a S 100A9 that can be detected is encompassed with the scope of the present invention for detecting and measuring the binding characteristics of the antibodies. One of skill in the art will recognize these well known methods and for this reason are not provided in detail here. These methods are also utilized to screen a panel of antibodies for those providing the desired characteristics.

In certain embodiments an antibody of the invention immunospecifically binds to S 100A9 and has one or more of the characteristics selected from the group consisting of: (a) affecting cell migration, binding to TLR4, and inducing cells to secrete cytokines.

The anti-S 100A9 antibodies may be described as inhibiting one or more biological activities of S 100A9. The term "inhibition" as used herein, refers to any statistically significant decrease in biological activity, including full blocking of the activity. For example, "inhibition" can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in biological activity. In certain embodiments, the anti-S 100A9 antibodies inhibit one or more biological activities of S 100A9 by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In a specific embodiment, the anti-S100A9 antibodies inhibit cytokine secretion. In one embodiment, an anti-S100A9 antibody of the invention may achieve at least about 20%, at least about 30%), at least about 40%>, at least about 50%>, at least about 60%>, at least about 70%>, at least about 80%, at least about 90%, at least about 95%, or about 100% inhibition.

In certain embodiments, the anti-S100A9 antibodies of the invention may inhibit the growth of a cell expressing S100A9 receptor. As used herein, an antibody that "inhibits the growth of cells expressing S100A9 receptor" or a "growth inhibitory" antibody is one which results in measurable growth inhibition of cells expressing or overexpressing the S100A9 receptor. In one embodiment, growth inhibitory anti-S100A9 antibodies inhibit growth of S100A9 receptor-expressing cells by greater than 20%>, 30%>, or greater than 50%> (e.g., from about 50% to about 100%) as compared to the appropriate control, the control typically being cells not treated with the antibody being tested.

Production of Anti-S100A9 Antibodies

The following describes exemplary techniques for the production of the antibodies useful in the present invention. The S100A9 antigen to be used for production of antibodies may be SEQ ID NO: l or an antigenic fragment thereof. Alternatively, cells expressing S100A9 at their cell surface or membranes prepared from such cells can be used to generate antibodies. The nucleotide and amino acid sequences of S100A9 are available as SEQ ID NO:2 and SEQ ID NO: l, respectively. S100A9 can be produced recombinantly in an isolated from, bacterial or eukaryotic cells using standard recombinant DNA methodology. S100A9 can be expressed as a tagged (e.g., epitope tag) or other fusion protein to facilitate isolation as well as identification in various assays. Antibodies or binding proteins that bind to various tags and fusion sequences are available as elaborated below. Other forms of S100A9 useful for generating antibodies will be apparent to those skilled in the art.

Various tag polypeptides and their respective antibodies are well known in the art.

Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al, Mol. Cell. Biol, 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al, Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al, Protein Engineering, 3(6):547-553 (1990)]. The FLAG-peptide [Hopp et al, BioTechnology, 6:1204-1210 (1988)] is recognized by an anti-FLAG M2 monoclonal antibody (Eastman Kodak Co., New Haven, Conn.). Purification of a protein containing the FLAG peptide can be performed by immunoaffinity chromatography using an affinity matrix comprising the anti-FLAG M2 monoclonal antibody covalently attached to agarose (Eastman Kodak Co., New Haven, Conn.). Other tag polypeptides include the KT3 epitope peptide [Martin et al, Science, 255:192-194 (1992)]; an a-tubulin epitope peptide

[Skinner et al, J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al, Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].

Polyclonal Antibodies

Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art. For example, a S100A9 polypeptide or immunogenic fragment thereof can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.

Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.

Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen (especially when synthetic peptides are used) to a protein that is immunogenic in the species to be immunized. For example, the antigen can be conjugated to keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent (reactive group), e.g., activated ester (conjugation through cysteine or lysine residues), glutaraldehyde, succinic anhydride, SOCl₂, or R^! =C=NR, where R and R¹ are different alkyl groups. Conjugates also can be made in recombinant cell culture as fusion proteins.

Typically animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining an appropriate concentration of antigen or conjugate with adjuvant and injecting the solution at multiple sites. One month later, the animals are boosted with 1/5 to 1/10 the original amount of antigen or conjugate in adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. In addition, aggregating agents such as alum are suitably used to enhance the immune response.

Monoclonal Antibodies

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma (Kohler et al, Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);

Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981), recombinant, and phage display technologies, or a combination thereof. The term

"monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous or isolated antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or multiple antigenic sites in the case of multispecific engineered antibodies.

Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against the same determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. Following is a description of representative methods for producing monoclonal antibodies which is not intended to be limiting and may be used to produce, for example, monoclonal mammalian, chimeric, humanized, human, domain, diabodies, vaccibodies, linear and multispecific antibodies.

Hybridoma Techniques

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In the hybridoma method, mice or other appropriate host animals, such as hamster, are immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent or fusion partner, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). In certain

embodiments, the selected myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells. In one aspect, the myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol, 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and

Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987)).

Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal e.g., by i.p. injection of the cells into mice.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion- exchange chromatography, affinity tags, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc. Exemplary purification methods are described in more detail below.

Recombinant DNA Techniques

Methods for producing and screening for specific antibodies using recombinant DNA technology are routine and well known in the art (e.g. US Patent No. 4,816,567). DNA encoding the monoclonal antibodies may be readily isolated and/or sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs., 130: 151-188 (1992). As described below for antibodies generated by phage display and humanization of antibodies, DNA or genetic material for recombinant antibodies can be obtained from source(s) other than hybridomas to generate antibodies of the invention.

Recombinant expression of an antibody or variant thereof generally requires construction of an expression vector containing a polynucleotide that encodes the antibody. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody or a portion thereof, or a heavy or light chain CDR, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., US. Patent Nos. 5,981,216; 5,591,639; 5,658,759 and 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.

Once the expression vector is transferred to a host cell by conventional techniques, the transfected cells are then cultured by conventional techniques to produce an antibody. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention or fragments thereof, or a heavy or light chain thereof, or portion thereof, or a single-chain antibody of the invention, operably linked to a heterologous promoter. In certain embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

Mammalian cell lines available as hosts for expression of recombinant antibodies are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human

hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and a number of other cell lines. Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the antibody or portion thereof expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (a murine myeloma cell line that does not endogenously produce any functional

immunoglobulin chains), SP20, CRL7030 and HsS78Bst cells. In one embodiment, human cell lines developed by immortalizing human lymphocytes can be used to recombinantly produce monoclonal antibodies. In one embodiment, the human cell line PER.C6. (Crucell, Netherlands) can be used to recombinantly produce monoclonal antibodies.

Additional cell lines which may be used as hosts for expression of recombinant antibodies include, but are not limited to, insect cells (e.g. Sf21/Sf9, Trichoplusia ni Bti-Tn5bl- 4) or yeast cells (e.g. S. cerevisiae, Pichia, US7326681; etc), plants cells (US20080066200); and chicken cells (WO2008142124)

In certain embodiments, antibodies of the invention are expressed in a cell line with stable expression of the antibody. Stable expression can be used for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express the antibody molecule may be generated. Host cells can be transformed with an appropriately engineered vector comprising expression control elements {e.g. , promoter, enhancer, transcription terminators, polyadenylation sites, etc.), and a selectable marker gene. Following the

introduction of the foreign DNA, cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells that stably integrated the plasmid into their chromosomes to grow and form foci which in turn can be cloned and expanded into cell lines. Methods for producing stable cell lines with a high yield are well known in the art and reagents are generally available commercially.

In certain embodiments, antibodies of the invention are expressed in a cell line with transient expression of the antibody. Transient transfection is a process in which the nucleic acid introduced into a cell does not integrate into the genome or chromosomal DNA of that cell. It is in fact maintained as an extrachromosomal element, e.g. as an episome, in the cell. Transcription processes of the nucleic acid of the episome are not affected and a protein encoded by the nucleic acid of the episome is produced.

The cell line, either stable or transiently transfected, is maintained in cell culture medium and conditions well known in the art resulting in the expression and production of monoclonal antibodies. In certain embodiments, the mammalian cell culture media is based on commercially available media formulations, including, for example, DMEM or Ham's F12. In other embodiments, the cell culture media is modified to support increases in both cell growth and biologic protein expression. As used herein, the terms "cell culture medium," "culture medium," and "medium formulation" refer to a nutritive solution for the maintenance, growth, propagation, or expansion of cells in an artificial in vitro environment outside of a multicellular organism or tissue. Cell culture medium may be optimized for a specific cell culture use, including, for example, cell culture growth medium which is formulated to promote cellular growth, or cell culture production medium which is formulated to promote recombinant protein production. The terms nutrient, ingredient, and component are used interchangeably herein to refer to the constituents that make up a cell culture medium.

In one embodiment, the cell lines are maintained using a fed batch method. As used herein, "fed batch method," refers to a method by which a fed batch cell culture is supplied with additional nutrients after first being incubated with a basal medium. For example, a fed batch method may comprise adding supplemental media according to a determined feeding schedule within a given time period. Thus, a "fed batch cell culture" refers to a cell culture wherein the cells, typically mammalian, and culture medium are supplied to the culturing vessel initially and additional culture nutrients are fed, continuously or in discrete increments, to the culture during culturing, with or without periodic cell and/or product harvest before termination of culture.

The cell culture medium used and the nutrients contained therein are known to one of skill in the art. In one embodiment, the cell culture medium comprises a basal medium and at least one hydrolysate, e.g., soy-based, hydrolysate, a yeast-based hydrolysate, or a combination of the two types of hydro lysates resulting in a modified basal medium. In another embodiment, the additional nutrients may include only a basal medium, such as a concentrated basal medium, or may include only hydrolysates, or concentrated hydrolysates. Suitable basal media include, but are not limited to Dulbecco's Modified Eagle's Medium (DMEM), DME/F12, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, a-Minimal Essential Medium (a-MEM), Glasgow's Minimal Essential Medium (G-MEM), PF CHO (see, e.g., CHO protein free medium (Sigma) or EX-CELL™ 325 PF CHO Serum-Free Medium for CHO Cells Protein-Free (SAFC Bioscience), and Iscove's Modified Dulbecco's Medium. Other examples of basal media which may be used in the invention include BME Basal Medium (Gibco-Invitrogen; see also Eagle, H (1965) Proc. Soc. Exp. Biol. Med. 89, 36); Dulbecco's Modified Eagle Medium (DMEM, powder) (Gibco-Invitrogen (# 31600); see also Dulbecco and Freeman (1959) Virology 8, 396; Smith et al. (1960) Virology 12, 185. Tissue Culture Standards Committee, In Vitro 6:2, 93); CMRL 1066 Medium (Gibco-Invitrogen (#11530); see also Parker R. C. et al (1957) Special Publications, N.Y. Academy of Sciences, 5, 303).

In certain embodiments, the basal medium may be is serum-free, meaning that the medium contains no serum (e.g., fetal bovine serum (FBS), horse serum, goat serum, or any other animal-derived serum known to one skilled in the art) or animal protein free media or chemically defined media.

The basal medium may be modified in order to remove certain non-nutritional components found in standard basal medium, such as various inorganic and organic buffers, surfactant(s), and sodium chloride. Removing such components from basal cell medium allows an increased concentration of the remaining nutritional components, and may improve overall cell growth and protein expression. In addition, omitted components may be added back into the cell culture medium containing the modified basal cell medium according to the requirements of the cell culture conditions. In certain embodiments, the cell culture medium contains a modified basal cell medium, and at least one of the following nutrients, an iron source, a recombinant growth factor; a buffer; a surfactant; an osmolality regulator; an energy source; and non-animal hydrolysates. In addition, the modified basal cell medium may optionally contain amino acids, vitamins, or a combination of both amino acids and vitamins. In another embodiment, the modified basal medium further contains glutamine, e.g, L-glutamine, and/or methotrexate.

In certain embodiments, antibody production is conducted in large quantity by a bioreactor process using fed-batch, batch, perfusion or continuous feed bioreactor methods known in the art. Large-scale bioreactors have at least 1000 liters of capacity, preferably about 1,000 to 100,000 liters of capacity. These bioreactors may use agitator impellers to distribute oxygen and nutrients. Small scale bioreactors refers generally to cell culturing in no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters. Alternatively, single-use bioreactors (SUB) may be used for either large-scale or small scale culturing.

Temperature, pH, agitation, aeration and inoculum density will vary depending upon the host cells used and the recombinant protein to be expressed. For example, a recombinant protein cell culture may be maintained at a temperature between 30 and 45 degrees Celsius. The pH of the culture medium may be monitored during the culture process such that the pH stays at an optimum level, which may be for certain host cells, within a pH range of 6.0 to 8.0. An impellor driven mixing may be used for such culture methods for agitation. The rotational speed of the impellor may be approximately 50 to 200 cm/sec tip speed, but other airlift or other

mixing/aeration systems known in the art may be used, depending on the type of host cell being cultured. Sufficient aeration is provided to maintain a dissolved oxygen concentration of approximately 20% to 80%> air saturation in the culture, again, depending upon the selected host cell being cultured. Alternatively, a bioreactor may sparge air or oxygen directly into the culture medium. Other methods of oxygen supply exist, including bubble-free aeration systems employing hollow fiber membrane aerators.

Phage Display Techniques

In another embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol., 222:581-597 (1991). In such methods antibodies of the invention can be isolated by screening of a recombinant combinatorial antibody library, preferably a scFv phage display library, prepared using human VL and VH cDNAs prepared from mRNA derived from human lymphocytes. Methodologies for preparing and screening such libraries are known in the art. In addition to commercially available kits for generating phage display libraries (e.g., the

Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SurfZAP.TM. phage display kit, catalog no. 240612), examples of methods and reagents particularly amenable for use in generating and screening antibody display libraries can be found in, for example, US Patent Nos. 6,248,516; US 6,545,142; 6,291,158; 6,291,1591; 6,291,160; 6,291,161; 6,680,192; 5,969,108; 6,172,197; 6,806,079; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,593,081; 6,582,915; 7,195,866. Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for generation and isolation of monoclonal antibodies.

In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, humanized antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al, BioTechniques 12(6):864-869 (1992);; and Better et al, Science 240: 1041-1043 (1988).

Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498. Thus, techniques described above and those well known in the art can be used to generate recombinant antibodies wherein the binding domain, e.g. ScFv, was isolated from a phage display library.

Antibody Purification and Isolation

Once an antibody molecule has been produced by recombinant or hybridoma expression, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigens Protein A or Protein G, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the antibodies of the present invention or fragments thereof may be fused to heterologous polypeptide sequences (referred to herein as "tags") described above or otherwise known in the art to facilitate purification.

When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced

intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology, 10: 163- 167 (1992) describe a procedure for isolating antibodies which are secreted into the periplasmic space of E. coli. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, hydrophobic interaction chromatography, ion exchange chromatography, gel electrophoresis, dialysis, and/or affinity chromatography either alone or in combination with other purification steps. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody and will be understood by one of skill in the art. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH₃ domain, the Bakerbond ABX resin (J.T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, and performed at low salt concentrations (e.g., from about 0-0.25 M salt).

Thus, in certain embodiments is provided antibodies of the invention that are

substantially purified/isolated. In one embodiment, these isolated/purified recombinantly expressed antibodies may be administered to a patient to mediate a prophylactic or therapeutic effect. In another embodiment these isolated/purified antibodies may be used to diagnose a S100A9 mediated disease.

Humanized Antibodies

In certain embodiments, the antibodies of the invention are humanized antibodies, which are generated using methods well known in the art. Humanized antibodies are antibody molecules derived from a non-human species antibody (also referred to herein as a donor antibody) that bind the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule (also referred to herein as an acceptor antibody). Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding and/or reduce immunogenicity. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g,, Riechmann et al., Nature 332:323 (1988)). In practice, and in certain embodiments, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. In alternative embodiments, the FR residues are fully human residues.

Humanization can be essentially performed following the method of Winter and coworkers (Jones et al, Nature, 321 :522-525 (1986); Reichmann et al, Supra; Verhoeyen et al, Science, 239: 1534-1536 (1988)), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Specifically, humanized antibodies may be prepared by methods well known in the art including CDR grafting approaches (see, e.g., US Patent No. 6,548,640), veneering or resurfacing (US Patent Nos. 5,639,641 and 6,797,492; Studnicka et al, Protein Engineering 7(6):805-814 (1994); Roguska. et al, PNAS 91 :969-973 (1994)), chain shuffling strategies (see e.g., U.S. Patent No. 5,565,332; Rader et al, Proc. Natl. Acad. Sci. USA (1998) 95:8910-8915), molecular modeling strategies (U.S. Patent No.

5,639,641), and the like. These general approaches may be combined with standard mutagenesis and recombinant synthesis techniques to produce anti-S100A9 antibodies with desired properties.

CDR grafting is performed by replacing one or more CDRs of an acceptor antibody (e.g., a human antibody) with one or more CDRs of a donor antibody (e.g., a non-human antibody). Acceptor antibodies may be selected based on similarity of framework residues between a candidate acceptor antibody and a donor antibody and may be further modified to introduce similar residues. Following CDR grafting, additional changes may be made in the donor and/or acceptor sequences to optimize antibody binding and functionality.

Grafting of abbreviated CDR regions is a related approach. Abbreviated CDR regions include the specificity-determining residues and adjacent amino acids, including those at positions 27d-34, 50-55 and 89-96 in the light chain, and at positions 31-35b, 50-58, and 95-101 in the heavy chain. See (Padlan et al. (1995) FASEB J. 9: 133-9). Grafting of specificity- determining residues (SDRs) is premised on the understanding that the binding specificity and affinity of an antibody combining site is determined by the most highly variable residues within each of the CDR regions. Analysis of the three-dimensional structures of antibody-antigen complexes, combined with analysis of the available amino acid sequence data was used to model sequence variability based on structural dissimilarity of amino acid residues that occur at each position within the CDR. Minimally immunogenic polypeptide sequences consisting of contact residues, which are referred to as SDRs, are identified and grafted onto human framework regions.

Veneering or resurfacing is based on the concept of reducing potentially immunogenic amino acid sequences in a rodent or other non-human antibody by resurfacing the solvent accessible exterior of the antibody with human amino acid sequences. Thus, veneered antibodies appear less foreign to human cells. A non-human antibody is veneered by (1) identifying exposed exterior framework region residues in the non-human antibody, which are different from those at the same positions in framework regions of a human antibody, and (2) replacing the identified residues with amino acids that typically occupy these same positions in human antibodies.

By definition, humanized antibodies are chimeric antibodies. Chimeric antibodies are antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while another portion of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (e.g., Morrison et al., Proc. Natl. Acad. Sci. USA,

81 :6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a nonhuman primate {e.g. , Old World Monkey, such as baboon, rhesus or cynomolgus monkey) and human constant region sequences (U.S. Patent No. 5,693,780).

Human Antibodies

As an alternative to humanization, human antibodies can be generated using methods well known in the art. Human antibodies avoid some of the problems associated with antibodies that possess murine or rat variable and/or constant regions. The presence of such murine or rat derived proteins can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by a patient. In order to avoid the utilization of murine or rat derived antibodies, fully human antibodies can be generated through the introduction of functional human antibody loci into a rodent, other mammal or animal so that the rodent, other mammal or animal produces fully human antibodies.

For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the

homozygous deletion of the antibody heavy-chain joining region (J_H) gene in chimeric and germ- line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al, Nature, 362:255-258 (1993); Bruggemann et al, Year in Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825,

5,591,669 (all of GenPharm); U.S. Pat. No. 5,545,807; and WO 97/17852. In practice, the use of XenoMouse® strains of mice that have been engineered to contain up to but less than 1000 kb-sized germline configured fragments of the human heavy chain locus and kappa light chain locus. See Mendez et al. Nature Genetics 15: 146-156 (1997) and Green and Jakobovits J. Exp. Med. 188:483-495 (1998). The XenoMouse® strains are available from Amgen, Inc. (Fremont, Calif).

The production of the XenoMouse® strains of mice and antibodies produced in those mice is further discussed and delineated in U.S. Patent Nos. 6,673,986; 7,049,426; 6,833,268; 6,162,963, 6,150,584, 6,114,598, 6,075,181, 6,657,103; 6,713,610 and 5,939,598; US

Publication Nos. 2004/0010810; 2003/0229905; 2004/0093622; 2005/0054055; 2005/0076395; and 2006/0040363.

Essentially, XenoMouse® lines of mice are immunized with an antigen of interest (e.g. S100A9), lymphatic cells (such as B-cells) are recovered from the hyper-immunized mice, and the recovered lymphocytes are fused with a myeloid-type cell line to prepare immortal hybridoma cell lines using techniques described above an well known in the art. These hybridoma cell lines are screened and selected to identify hybridoma cell lines that produced antibodies specific to the antigen of interest.

In an alternative approach, others, including GenPharm International, Inc., have utilized a "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more V_H genes, one or more D_H genes, one or more ½ genes, a mu constant region, and usually a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal. This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,625,825; 5,625,126;

5,633,425; 5,661,016; 5,770,429; 5,789,650; 5,814,318; 5,877,397; 5,874,299; 6,255,458;

5,591,669; 6,023,010; 5,612,205; 5,721,367; 5,789,215; 5,643,763; and 5,981,175.

Kirin has also demonstrated the generation of human antibodies from mice in which, through microcell fusion, large pieces of chromosomes, or entire chromosomes, have been introduced. See Patent No. 6,632,976. Additionally, KM™— mice, which are the result of crossbreeding of Kirin' s Tc mice with Medarex's minilocus (Humab) mice have been generated. These mice possess the human IgH transchromosome of the Kirin mice and the kappa chain transgene of the Genpharm mice (Ishida et al., Cloning Stem Cells, (2002) 4:91-102).

Human antibodies can also be derived by in vitro methods. Suitable examples include but are not limited to phage display (Medlmmune (formerly CAT), Morphosys, Dyax,

Biosite/Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed) ribosome display (Medlmmune (formerly CAT)), yeast display, and the like. The phage display technology (See e.g., US Patent No. 5,969,108) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as Ml 3 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al, Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from

unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al, J. Mol. Biol. 222:581-597 (1991), or Griffith et al, EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

In certain embodiments, the present antibodies are antibody fragments or antibodies comprising these fragments. The antibody fragment comprises a portion of the full length antibody, which generally is the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')₂, Fd and Fv fragments. Diabodies; linear antibodies (U.S. Pat. No. 5,641,870); single-chain antibody molecules; and multispecific antibodies are antibodies formed from these antibody fragments. Antibody Fragments

Traditionally, these fragments were derived via proteolytic digestion of intact antibodies using techniques well known in the art. However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. In one embodiment, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can also be directly recovered from E. coli and chemically coupled to form F(ab')₂ fragments (Carter et ah, Bio/Technology, 10: 163-167 (1992)). According to another approach, F(ab')₂ fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single- chain Fv fragment (scFv). In certain embodiments, the antibody is not a Fab fragment. Fv and scFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv.

In certain embodiments, the present antibodies are domain antibodies, e.g., antibodies containing the small functional binding units of antibodies, corresponding to the variable regions of the heavy (V_H) or light (V_L) chains of human antibodies. Examples of domain antibodies include, but are not limited to, those available from Domantis that are specific to therapeutic targets (see, for example, WO04/058821; WO04/081026; WO04/003019; WO03/002609; U.S. Patent Nos. 6,291,158; 6,582,915; 6,696,245; and 6,593,081). Commercially available libraries of domain antibodies can be used to identify anti-S100A9 domain antibodies. In certain embodiments, anti-S100A9 antibodies comprise a S100A9 functional binding unit and an Fc gamma receptor functional binding unit.

In certain embodiments of the invention, the present antibodies are linear antibodies. Linear antibodies comprise a pair of tandem Fd segments (V_H-C_HI-V_H-C_HI) which form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific. See, Zapata et ah, Protein Eng., 8(10): 1057-1062 (1995). Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the S100A9 protein. Other such antibodies may combine a S100A9 binding site with a binding site for another protein. Methods for making bispecific antibodies are known in the art. (See, for example, Millstein et al, Nature, 305:537-539 (1983); Traunecker et al, EMBO J., 10:3655- 3659 (1991); Suresh et al, Methods in Enzymology, 121 :210 (1986); Kostelny et al, J.

Immunol, 148(5): 1547-1553 (1992); Hollinger et al, Proc. Natl Acad. Sci. USA, 90:6444-6448 (1993); Gruber et al, J. Immunol, 152:5368 (1994); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; 5,731,168; 4,676,980; 5,897,861; 5,660,827; 5,811,267;

5,849,877; 5,948,647; 5,959,084; 6,106,833; 6,143,873 and 4,676,980, WO 94/04690; and WO 92/20373.)

Traditional production of full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al, EMBO J., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, C_H2, and C_H3 regions. It is preferred to have the first heavy-chain constant region (C_HI) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant affect on the yield of the desired chain combination.

In one embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure may facilitate the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. For further details of generating bispecific antibodies see, for example, Suresh et al, Methods in Enzymology, 121 :210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the C_H3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (US Patent No. 5,897,861).

Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al, J. Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')₂ molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al, J. Immunol, 148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al, Proc. Natl. Acad. Sci USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a V_H connected to a V_Lby a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V_H and V_L domains of one fragment are forced to pair with the complementary V_L and V_H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al, J. Immunol, 152:5368 (1994) and US Patent Nos.

5,591,828; 4,946,778; 5,455,030; and 5,869,620.

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared, Tutt et al. J. Immunol. 147: 60 (1991), and multispecific valencies US Patent No. 5,258,498. Multivalent Antibodies

The antibodies of the present invention can be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g. tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody (See e.g., US Publication No. 2009/0155275. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. In one embodiment dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. In certain embodiments the multivalent antibody herein comprises (or consists of) three to about eight antigen binding sites. The multivalent antibody comprises at least one polypeptide chain wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VDl-(Xl)_n-VD2-(X2)_n- Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, XI and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH-CH1 -flexible linker-VH-CHl-Fc region chain; or VH-CHl-VH-CHl-Fc region chain. The multivalent antibody herein preferably further comprises at least two light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a C_L domain.

Other Amino Acid Sequence Modifications

In addition to the above described human, humanized and/or chimeric antibodies, the present invention also encompasses further modifications and, their variants and fragments thereof, of the anti-S100A9 antibodies that are useful in the method of the invention. Such modifications may comprise one or more amino acid residues and/or polypeptide substitutions, additions and/or deletions in the variable light (V_L) domain and/or variable heavy (V_H) domain and/or Fc region and post translational modifications. Included in these modifications are antibody conjugates wherein an antibody has been covalently attached to a moiety. Moieties suitable for attachment to the antibodies include but are not limited to, proteins, peptides, drugs, labels, and cytotoxins. These changes to the antibodies may be made to alter or fine tune the characteristics (biochemical, binding and/or functional) of the antibodies as is appropriate for treatment of S100A9 mediated diseases.

Methods for forming conjugates, making amino acid and/or polypeptide changes and post-translational modifications are well known in the art, some of which are detailed below. The following description is not intended to be limiting, but instead a non-limiting description of some embodiments, more of which will be obvious to one of skill in the art. It is also understood that some of the following methods were used to develop the human, humanized and/or chimeric antibody sequences described above. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.

In certain embodiments, altered antibodies are generated by one or more amino acid alterations (e.g., substitutions, deletion and/or additions) introduced in one or more of the variable regions of the antibody. In another embodiment, the amino acid alterations are introduced in the framework regions. One or more alterations of framework region residues may result in an improvement in the binding affinity of the antibody for the antigen. This may be especially true when these changes are made to humanized antibodies wherein the framework region may be from a different species than the CDR regions. Examples of framework region residues to modify include those which non-covalently bind antigen directly (Amit et al, Science, 233:747-753 (1986)); interact with/effect the conformation of a CDR (Chothia et al., J. Mol. Biol, 196:901-917 (1987)); and/or participate in the V_L-V_H interface (US Patent Nos. 5,225,539 and 6,548,640). In one embodiment, from about one to about five framework residues may be altered. Sometimes, this may be sufficient to yield an antibody mutant suitable for use in preclinical trials, even where none of the hypervariable region residues have been altered.

Normally, however, an altered antibody will comprise additional hypervariable region alteration(s). In certain embodiments, the hypervariable region residues may be changed randomly, especially where the starting binding affinity of an anti-S100A9 antibody for the antigen from the second mammalian species is such that such randomly produced antibodies can be readily screened.

One useful procedure for generating altered antibodies is called "alanine scanning mutagenesis" (Cunningham and Wells, Science, 244: 1081-1085 (1989)). In this method, one or more of the hypervariable region residue(s) are replaced by alanine or polyalanine residue(s) to alter the interaction of the amino acids with the S100A9. Those hypervariable region residue(s) demonstrating functional sensitivity to the substitutions then are refined by introducing additional or other mutations at or for the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. The Ala-mutants produced this way are screened for their biological activity as described herein.

In certain embodiments the substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody).

Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A

convenient way for generating such substitutional variants involves affinity maturation using phage display (Hawkins et al., J. Mol. Biol, 254:889-896 (1992) and Lowman et al,

Biochemistry, 30(45): 10832-10837 (1991)). Briefly, several hypervariable region sites {e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody mutants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of Ml 3 packaged within each particle. The phage-displayed mutants are then screened for their biological activity {e.g., binding affinity) as herein disclosed.

Mutations in antibody sequences may include substitutions, deletions, including internal deletions, additions, including additions yielding fusion proteins, or conservative substitutions of amino acid residues within and/or adjacent to the amino acid sequence, but that result in a "silent" change, in that the change produces a functionally equivalent anti-S100A9 antibody. Conservative amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In addition, glycine and proline are residues that can influence chain orientation. Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class. Furthermore, if desired, non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the antibody sequence. Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, a -amino isobutyric acid, 4- aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2- amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,

cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.

In another embodiment, any cysteine residue not involved in maintaining the proper conformation of the anti-S100A9 antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

In certain embodiments of the invention, an antibody can be modified to produce fusion proteins; i.e., the antibody, or a fragment thereof, fused to a heterologous protein, polypeptide or peptide. In certain embodiments, the protein fused to the portion of an antibody is an enzyme component of Antibody-Directed Enzyme Prodrug Therapy (ADEPT). Examples of other proteins or polypeptides that can be engineered as a fusion protein with an antibody include, but are not limited to toxins such as ricin, abrin, ribonuclease, DNase I, Staphylococcal enterotoxin- A, pokeweed anti-viral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and

Pseudomonas endotoxin. See, for example, Pastan et al, Cell, 47:641 (1986), and Goldenberg et al., Cancer Journal for Clinicians, 44:43 (1994). Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232.

Additional fusion proteins may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be employed to alter the characteristics of the antibody or fragments thereof (e.g., an antibody or a fragment thereof with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et ah, 1997, Curr. Opinion BiotechnoL, 8:724-33 ; Harayama, 1998, Trends BiotechnoL 16(2):76-82; Hansson et ah, 1999, J. Mol. Biol., 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308- 313. The antibody can further be a binding-domain immunoglobulin fusion protein as described in U.S. Publication 2003/0118592, and PCT

Publication WO 02/056910.

Variant Fc Regions

It is known that variants of the Fc region (e.g., amino acid substitutions and/or additions and/or deletions) enhance or diminish effector function of the antibody (See e.g., U.S. Patent Nos. 5,624,821; 5,885,573; 6,538,124; 7,317,091; 5,648,260; 6,538,124; WO 03/074679; WO 04/029207; WO 04/099249; WO 99/58572; US Publication No. 2006/0134105; 2004/0132101; 2006/0008883) and may alter the pharmacokinetic properties (e.g. half-life) of the antibody (see, U.S. patents 6,277,375 and 7,083,784). Thus, in certain embodiments, the anti-S100A9 antibodies of the invention comprise an altered Fc region (also referred to herein as "variant Fc region") in which one or more alterations have been made in the Fc region in order to change functional and/or pharmacokinetic properties of the antibodies. Such alterations may result in a decrease or increase of Clq binding and complement dependent cytotoxicity (CDC) or of FcyR binding, for IgG, and antibody-dependent cellular cytotoxicity (ADCC), or antibody dependent cell-mediated phagocytosis (ADCP). The present invention encompasses the antibodies described herein with variant Fc regions wherein changes have been made to fine tune the effector function, enhancing or diminishing, providing a desired effector function. Accordingly, in one embodiment of the invention, the anti-S100A9 antibodies of the invention comprise a variant Fc region (i.e., Fc regions that have been altered as discussed below). Anti-S100A9 antibodies of the invention comprising a variant Fc region are also referred to here as "Fc variant antibodies." As used herein native refers to the unmodified parental sequence and the antibody comprising a native Fc region is herein referred to as a "native Fc antibody." Fc variant antibodies can be generated by numerous methods well known to one skilled in the art. Non- limiting examples include, isolating antibody coding regions (e.g., from hybridoma) and making one or more desired substitutions in the Fc region of the isolated antibody coding region. Alternatively, the antigen-binding portion (e.g., variable regions) of an anti-S100A9 antibody may be subcloned into a vector encoding a variant Fc region. In one embodiment, the variant Fc region exhibits a similar level of inducing effector function as compared to the native Fc region. In another embodiment, the variant Fc region exhibits a higher induction of effector function as compared to the native Fc. In another embodiment, the variant Fc region exhibits lower induction of effector function as compared to the native Fc. Some specific embodiments of variant Fc regions are detailed infra. Methods for measuring effector function are well known in the art.

The effector function of an antibody is modified through changes in the Fc region, including but not limited to, amino acid substitutions, amino acid additions, amino acid deletions and changes in post translational modifications to Fc amino acids (e.g. glycosylation). The methods described below may be used to fine tune the effector function of a present antibody, a ratio of the binding properties of the Fc region for the FcR (e.g., affinity and specificity), resulting in a therapeutic antibody with the desired properties for a particular disease indication and taking into consideration the biology of SI 00 A9.

It is understood that the Fc region as used herein includes the polypeptides comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cy2 and Cy3) and the hinge between Cgammal (Cyl) and Cgamma2 (Cy2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as set forth in Kabat. Fc may refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at a number of different Fc positions, including but not limited to positions 270, 272, 312, 315, 356, and 358 as numbered by the EU index, and thus slight differences between the presented sequence and sequences in the prior art may exist.

In one embodiment, the present invention encompasses Fc variant antibodies which have altered binding properties for an Fc ligand (e.g., an Fc receptor, Clq) relative to a native Fc antibody. Examples of binding properties include but are not limited to, binding specificity, equilibrium dissociation constant (IQ), dissociation and association rates (k_off and k_on respectively), binding affinity and/or avidity. It is known in the art that the equilibrium dissociation constant (IQ) is defined as k₀ff/ko_n. In certain aspects, an antibody comprising an Fc variant region with a low IQ may be more desirable to an antibody with a high IQ. However, in some instances the value of the k_on or k₀ff may be more relevant than the value of the IQ. One skilled in the art can determine which kinetic parameter is most important for a given antibody application. For example, a modification that reduces binding to one or more positive regulator (e.g., FcyRIIIA) and/or enhanced binding to an inhibitory Fc receptor (e.g., FcyRIIB) would be suitable for reducing ADCC activity. Accordingly, the ratio of binding affinities (e.g., the ratio of equilibrium dissociation constants (Kd)) for different receptors can indicate if the ADCC activity of an Fc variant antibody of the invention is enhanced or decreased. Additionally, a modification that reduces binding to Clq would be suitable for reducing or eliminating CDC activity.

In one embodiment, Fc variant antibodies exhibit altered binding affinity for one or more Fc receptors including, but not limited to FcRn, FcyRI (CD64) including isoforms FcyRIA, FcyRIB, and FcyRIC; FcyRII (CD32 including isoforms FcyRIIA, FcyRIIB, and FcyRIIC); and FcyRIII (CD 16, including isoforms FcyRIIIA and FcyRIIIB) as compared to an native Fc antibody.

In one embodiment, an Fc variant antibody has enhanced binding to one or more Fc ligand relative to a native Fc antibody. In another embodiment, the Fc variant antibody exhibits increased or decreased affinity for an Fc ligand that is at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold, or is between 2 fold and 10 fold, or between 5 fold and 50 fold, or between 25 fold and 100 fold, or between 75 fold and 200 fold, or between 100 and 200 fold, more or less than a native Fc antibody. In another embodiment, Fc variant antibodies exhibit affinities for an Fc ligand that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% more or less than an native Fc antibody. In certain embodiments, an Fc variant antibody has increased affinity for an Fc ligand. In other embodiments, an Fc variant antibody has decreased affinity for an Fc ligand.

In a specific embodiment, an Fc variant antibody has enhanced binding to the Fc receptor FcyRIIIA. In another specific embodiment, an Fc variant antibody has enhanced binding to the Fc receptor FcyRIIB. In a further specific embodiment, an Fc variant antibody has enhanced binding to both the Fc receptors FcyRIIIA and FcyRIIB. In certain embodiments, Fc variant antibodies that have enhanced binding to FcyRIIIA do not have a concomitant increase in binding the FcyRIIB receptor as compared to a native Fc antibody. In a specific embodiment, an Fc variant antibody has reduced binding to the Fc receptor FcyRIIIA. In a further specific embodiment, an Fc variant antibody has reduced binding to the Fc receptor FcyRIIB. In still another specific embodiment, an Fc variant antibody exhibiting altered affinity for FcyRIIIA and/or FcyRIIB has enhanced binding to the Fc receptor FcRn. In yet another specific embodiment, an Fc variant antibody exhibiting altered affinity for FcyRIIIA and/or FcyRIIB has altered binding to Clq relative to a native Fc antibody.

In one embodiment, Fc variant antibodies exhibit affinities for FcyRIIIA receptor that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold, or are between 2 fold and 10 fold, or between 5 fold and 50 fold, or between 25 fold and 100 fold, or between 75 fold and 200 fold, or between 100 and 200 fold, more or less than an native Fc antibody. In another embodiment, Fc variant antibodies exhibit affinities for FcyRIIIA that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% more or less than an native Fc antibody.

In one embodiment, Fc variant antibodies exhibit affinities for FcyRIIB receptor that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold, or are between 2 fold and 10 fold, or between 5 fold and 50 fold, or between 25 fold and 100 fold, or between 75 fold and 200 fold, or between 100 and 200 fold, more or less than an native Fc antibody. In another embodiment, Fc variant antibodies exhibit affinities for FcyRIIB that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% more or less than an native Fc antibody.

In one embodiment, Fc variant antibodies exhibit increased or decreased affinities to Clq relative to a native Fc antibody. In another embodiment, Fc variant antibodies exhibit affinities for Clq receptor that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold, or are between 2 fold and 10 fold, or between 5 fold and 50 fold, or between 25 fold and 100 fold, or between 75 fold and 200 fold, or between 100 and 200 fold, more or less than an native Fc antibody. In another embodiment, Fc variant antibodies exhibit affinities for Clq that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% more or less than an native Fc antibody. In still another specific embodiment, an Fc variant antibody exhibiting altered affinity for Ciq has enhanced binding to the Fc receptor FcRn. In yet another specific embodiment, an Fc variant antibody exhibiting altered affinity for Clq has altered binding to FcyRIIIA and/or FcyRIIB relative to a native Fc antibody.

It is contemplated that Fc variant antibodies are characterized by in vitro functional assays for determining one or more FcyR mediated effector cell functions. In certain

embodiments, Fc variant antibodies have similar binding properties and effector cell functions in in vivo models (such as those described and disclosed herein) as those in in vitro based assays. However, the present invention does not exclude Fc variant antibodies that do not exhibit the desired phenotype in in vitro based assays but do exhibit the desired phenotype in vivo.

Antibody Conjugates

In certain embodiments, the antibodies of the invention are conjugated or covalently attached to a substance using methods well known in the art. In one embodiment, the attached substance is a therapeutic agent, a detectable label (also referred to herein as a reporter molecule) or a solid support. Suitable substances for attachment to antibodies include, but are not limited to, an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell, a virus, a fluorophore, a chromophore, a dye, a toxin, a hapten, an enzyme, an antibody, an antibody fragment, a radioisotope, solid matrixes, semi-solid matrixes and combinations thereof. Methods for conjugation or covalently attaching another substance to an antibody are well known in the art.

In certain embodiments, the antibodies of the invention are conjugated to a solid support. Antibodies may be conjugated to a solid support as part of the screening and/or purification and/or manufacturing process. Alternatively antibodies of the invention may be conjugated to a solid support as part of a diagnostic method or composition. A solid support suitable for use in the present invention is typically substantially insoluble in liquid phases. A large number of supports are available and are known to one of ordinary skill in the art. Thus, solid supports include solid and semi-solid matrixes, such as aerogels and hydrogels, resins, beads, biochips (including thin film coated biochips), microfluidic chip, a silicon chip, multi-well plates (also referred to as microtitre plates or microplates), membranes, conducting and nonconducting metals, glass (including microscope slides) and magnetic supports. More specific examples of solid supports include silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides such as Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose,

polyvinylchloride, polypropylene, polyethylene (including poly(ethylene glycol)), nylon, latex bead, magnetic bead, paramagnetic bead, superparamagnetic bead, starch and the like.

In some embodiments, the solid support may include a reactive functional group, including, but not limited to, hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide, etc., for attaching the antibodies of the invention.

A suitable solid phase support can be selected on the basis of desired end use and suitability for various synthetic protocols. For example, where amide bond formation is desirable to attach the antibodies of the invention to the solid support, resins generally useful in peptide synthesis may be employed, such as polystyrene (e.g. , PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE™ resin (obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TentaGel™, Rapp Polymere, Tubingen, Germany), polydimethyl- acrylamide resin (available from Milligen/Biosearch, California), or PEGA beads (obtained from Polymer Laboratories).

In certain embodiments, the antibodies of the invention are conjugated to labels for purposes of diagnostics and other assays wherein the antibody and/or its associated ligand may be detected. A label conjugated to an antibody and used in the present methods and

compositions described herein, is any chemical moiety, organic or inorganic, that exhibits an absorption maximum at wavelengths greater than 280 nm, and retains its spectral properties when covalently attached to an antibody. Labels include, without limitation, a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.

In certain embodiments, the anti-S100A9 antibodies are conjugated to a fluorophore. As such, fluorophores used to label antibodies of the invention include, without limitation; a pyrene (including any of the corresponding derivative compounds disclosed in US Patent 5,132,432), an anthracene, a naphthalene, an acridine, a stilbene, an indole or benzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a 4-amino-7-nitrobenz-2-oxa-l, 3-diazole (NBD), a cyanine (including any corresponding compounds in US Patent Nos.6, 977,305 and 6,974,873), a carbocyanine (including any corresponding compounds in US Serial Nos. 09/557,275; U.S.; Patents Nos. 4,981,977; 5,268,486; 5,569,587; 5,569,766; 5,486,616; 5,627,027; 5,808,044; 5,877,310; 6,002,003; 6,004,536; 6,008,373; 6,043,025; 6,127,134; 6,130,094; 6,133,445; and publications WO 02/26891, WO 97/40104, WO 99/51702, WO 01/21624; EP 1 065 250 Al), a carbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, a perylene, a pyridine, a quinoline, a borapolyazaindacene (including any corresponding compounds disclosed in US Patent Nos. 4,774,339; 5,187,288; 5,248,782; 5,274,113; and 5,433,896), a xanthene (including any corresponding compounds disclosed in U.S. Patent No. 6,162,931; 6,130,101; 6,229,055; 6,339,392; 5,451,343; 5,227,487; 5,442,045; 5,798,276; 5,846,737; 4,945,171; US serial Nos. 09/129,015 and 09/922,333), an oxazine (including any corresponding compounds disclosed in US Patent No. 4,714,763) or a benzoxazine, a carbazine (including any corresponding compounds disclosed in US Patent No. 4,810,636), a phenalenone, a coumarin (including an corresponding compounds disclosed in US Patent Nos. 5,696,157; 5,459,276; 5,501,980 and 5,830,912), a benzofuran (including an corresponding compounds disclosed in US Patent Nos. 4,603,209 and 4,849,362) and benzphenalenone (including any corresponding compounds disclosed in US Patent No. 4,812,409) and derivatives thereof. As used herein, oxazines include resorufins (including any corresponding compounds disclosed in 5,242,805), aminooxazinones, diaminooxazines, and their benzo-substituted analogs.

In a specific embodiment, the fluorophores conjugated to the antibodies described herein include xanthene (rhodol, rhodamine, fluorescein and derivatives thereof) coumarin, cyanine, pyrene, oxazine and borapolyazaindacene. In other embodiments, such fluorophores are sulfonated xanthenes, fluorinated xanthenes, sulfonated coumarins, fluorinated coumarins and sulfonated cyanines. Also included are dyes sold under the tradenames, and generally known as, Alexa Fluor, DyLight, Cy Dyes, BODIPY, Oregon Green, Pacific Blue, IRDyes, FAM, FITC, and ROX.

The choice of the fluorophore attached to the anti-S100A9 antibody will determine the absorption and fluorescence emission properties of the conjugated antibody. Physical properties of a fluorophore label that can be used for antibody and antibody bound ligands include, but are not limited to, spectral characteristics (absorption, emission and stokes shift), fluorescence intensity, lifetime, polarization and photo-bleaching rate, or combination thereof. All of these physical properties can be used to distinguish one fluorophore from another, and thereby allow for multiplexed analysis. In certain embodiments, the fluorophore has an absorption maximum at wavelengths greater than 480 nm. In other embodiments, the fluorophore absorbs at or near 488 nm to 514 nm (particularly suitable for excitation by the output of the argon-ion laser excitation source) or near 546 nm (particularly suitable for excitation by a mercury arc lamp). In other embodiment a fluorophore can emit in the NIR (near infra red region) for tissue or whole organism applications. Other desirable properties of the fluorescent label may include cell permeability and low toxicity, for example if labeling of the antibody is to be performed in a cell or an organism (e.g., a living animal).

In certain embodiments, an enzyme is a label and is conjugated to an anti-S100A9 antibody. Enzymes are desirable labels because amplification of the detectable signal can be obtained resulting in increased assay sensitivity. The enzyme itself does not produce a detectable response but functions to break down a substrate when it is contacted by an appropriate substrate such that the converted substrate produces a fluorescent, colorimetric or luminescent signal. Enzymes amplify the detectable signal because one enzyme on a labeling reagent can result in multiple substrates being converted to a detectable signal. The enzyme substrate is selected to yield the preferred measurable product, e.g. colorimetric, fluorescent or chemiluminescence. Such substrates are extensively used in the art and are well known by one skilled in the art.

In one embodiment, colorimetric or fluorogenic substrate and enzyme combination uses oxidoreductases such as horseradish peroxidase and a substrate such as 3,3'-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC), which yield a distinguishing color (brown and red, respectively). Other colorimetric oxidoreductase substrates that yield detectable products include, but are not limited to: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o- phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB), o-dianisidine, 5 -aminosalicylic acid, 4-chloro-l-naphthol. Fluorogenic substrates include, but are not limited to, homovanillic acid or 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reduced

benzothiazines, including Amplex^® Red reagent and its variants (U.S. Pat. No. 4,384,042) and reduced dihydroxanthenes, including dihydrofiuoresceins (U.S. Pat. No. 6,162,931) and dihydrorhodamines including dihydrorhodamine 123. Peroxidase substrates that are tyramides (U.S. Pat. Nos. 5,196,306; 5,583,001 and 5,731,158) represent a unique class of peroxidase substrates in that they can be intrinsically detectable before action of the enzyme but are "fixed in place" by the action of a peroxidase in the process described as tyramide signal amplification (TSA). These substrates are extensively utilized to label targets in samples that are cells, tissues or arrays for their subsequent detection by microscopy, flow cytometry, optical scanning and fluorometry.

In another embodiment, a colorimetric (and in some cases fluorogenic) substrate and enzyme combination uses a phosphatase enzyme such as an acid phosphatase, an alkaline phosphatase or a recombinant version of such a phosphatase in combination with a colorimetric substrate such as 5-bromo-6-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolyl phosphate, 5-bromo-6-chloro-3-indolyl phosphate, /?-nitrophenyl phosphate, or o-nitrophenyl phosphate or with a fluorogenic substrate such as 4-methylumbelliferyl phosphate, 6,8-difluoro-7-hydroxy-4- methylcoumarinyl phosphate (DiFMUP, U.S. Pat. No. 5,830,912) fluorescein diphosphate, 3-0- methylfluorescein phosphate, resorufm phosphate, H-(l,3-dichloro-9,9-dimethylacridin-2-one- 7-yl) phosphate (DDAO phosphate), or ELF 97, ELF 39 or related phosphates (U.S. Pat. Nos. 5,316,906 and 5,443,986). Glycosidases, in particular beta-galactosidase, beta-glucuronidase and beta-glucosidase, are additional suitable enzymes. Appropriate colorimetric substrates include, but are not limited to, 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) and similar indolyl

galactosides, glucosides, and glucuronides, o-nitrophenyl beta-D-galactopyranoside (ONPG) and p-nitrophenyl beta-D-galactopyranoside. In one embodiment, fluorogenic substrates include resorufm beta-D-galactopyranoside, fluorescein digalactoside (FDG), fluorescein diglucuronide and their structural variants (U.S. Pat. Nos. 5,208,148; 5,242,805; 5,362,628; 5,576,424 and 5,773,236), 4-methylumbelliferyl beta-D-galactopyranoside, carboxyumbelliferyl beta-D- galactopyranoside and f uorinated coumarin beta-D-galactopyranosides (U.S. Pat. No.

5,830,912).

Additional enzymes include, but are not limited to, hydrolases such as cholinesterases and peptidases, oxidases such as glucose oxidase and cytochrome oxidases, and reductases for which suitable substrates are known.

Enzymes and their appropriate substrates that produce chemiluminescence are preferred for some assays. These include, but are not limited to, natural and recombinant forms of luciferases and aequorins. Chemiluminescence-producing substrates for phosphatases, glycosidases and oxidases such as those containing stable dioxetanes, luminol, isoluminol and acridinium esters are additionally useful.

In another embodiment, haptens such as biotin, are also utilized as labels. Biotin is useful because it can function in an enzyme system to further amplify the detectable signal, and it can function as a tag to be used in affinity chromatography for isolation purposes. For detection purposes, an enzyme conjugate that has affinity for biotin is used, such as avidin-HRP.

Subsequently a peroxidase substrate is added to produce a detectable signal.

Haptens also include hormones, naturally occurring and synthetic drugs, pollutants, allergens, affector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides and the like.

In certain embodiments, fluorescent proteins may be conjugated to the antibodies as a label. Examples of fluorescent proteins include green fluorescent protein (GFP) and the phycobiliproteins and the derivatives thereof. The fluorescent proteins, especially

phycobiliprotein, are particularly useful for creating tandem dye labeled labeling reagents. These tandem dyes comprise a fluorescent protein and a fluorophore for the purposes of obtaining a larger stokes shift wherein the emission spectra is farther shifted from the wavelength of the fluorescent protein's absorption spectra. This is particularly advantageous for detecting a low quantity of a target in a sample wherein the emitted fluorescent light is maximally optimized, in other words little to none of the emitted light is reabsorbed by the fluorescent protein. For this to work, the fluorescent protein and fluorophore function as an energy transfer pair wherein the fluorescent protein emits at the wavelength that the fluorophore absorbs at and the fluorphore then emits at a wavelength farther from the fluorescent proteins than could have been obtained with only the fluorescent protein. A particularly useful combination is the phycobiliproteins disclosed in US Patent Nos. 4,520,110; 4,859,582; 5,055,556 and the sulforhodamine fluorophores disclosed in US Patent No. 5,798,276, or the sulfonated cyanine fluorophores disclosed in US Patent Nos. 6,977,305 and 6,974,873; or the sulfonated xanthene derivatives disclosed in US Patent No. 6,130,101 and those combinations disclosed in US Patent No.

4,542,104. Alternatively, the fluorophore functions as the energy donor and the fluorescent protein is the energy acceptor.

In certain embodiments, the label is a radioactive isotope. Examples of suitable radioactive materials include, but are not limited to, iodine (.sup.1211, .sup.1231, .sup.1251, .sup.1311), carbon (.sup.l4C), sulfur (.sup.35S), tritium (.sup.3H), indium (.sup.l 1 lln,

.sup. H2In, .sup. l l3mln, .sup.l 15mln), technetium (.sup.99Tc, .sup.99mTc), thallium

(.sup.201Ti), gallium (.sup.68Ga, .sup.67Ga), palladium (.sup.l03Pd), molybdenum

(.sup.99Mo), xenon (.sup.l 35Xe), fluorine (.sup. l8F), .sup. l53Sm, .sup. l77Lu, .sup. l59Gd, .sup.l49Pm, .sup.l40La, .sup. l75Yb, .sup.l66Ho, .sup.90Y, .sup.47Sc, .sup. l86Re, .sup. l88Re, .sup.l42Pr, .sup.l05Rh, and .sup.97Ru

Therapeutic Methods of Uses

In certain embodiments, the anti-S100A9 antibodies and compositions thereof of the invention may be administered for prevention and/or treatment of diseases marked by lung inflammation such as COPD, asthma, IPF, SLE, RA, and MS. The invention encompasses methods of preventing, treating, maintaining, ameliorating, or inhibiting a S100A9-mediated disease or disorder, wherein the methods comprise administering anti-S100A9 antibodies of the invention. Pharmaceutical Formulations

In certain embodiments, the anti-S100A9 antibodies of the invention may be formulated with a pharmaceutically acceptable carrier as pharmaceutical (therapeutic) compositions, and may be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. As used herein, the pharmaceutical formulations comprising the anti-S100A9 antibodies are referred to as formulations of the invention. The term "pharmaceutically acceptable carrier" means one or more nontoxic materials that do not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable preparations may also routinely contain compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being comingled with the antibodies of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.

EXAMPLES

The examples below are given so as to illustrate the practice of this invention. They are not intended to limit or define the entire scope of this invention.

Materials and Methods

Reagents:

Cell migration was assessed using a 96 well ChemoTX system (Neuro Probe, Gaithersburg, MD). The chemoattractors hSIOO proteins or hMCP-1 (R&D Systems, Minneapolis, MN) were diluted in RPMI 1640 containing 1% BSA and placed in the lower 25 μΐ chamber. Cells were washed and suspended by the above medium. THP-1 cells, human PBMC or human granulocytes were incubated with or without antibodies or cell signaling inhibitors for 30 mins at 37 °C before the cells were added to the upper chamber. Cells were allowed to migrate for 3 h for PBMC (8 μιη filter) and RAW cells (5 μιη filter), 1.5 h for THP-1 (5 um filter) or 1 h for granulocytes (5 μηι filter). Migration of cells to the human SI 00 proteins or human MCP-1 in the lower chamber was enumerated by flow cytometry. All antibodies used for flow cytometry analysis were purchased from BD/PharMingen (San Diego, CA). PBMCs were incubated with mouse anti- human CD3-PeCy7, CD4-Pacific orange, CD8-Alexa Fluor® 488, CD14-PE, CD1 IB- Alexa Fluor® 700, CD19-APC-Cy7 and CD56-APC antibodies 4 degree for 30 min, after wash with FACS buffer (PBS, 1% FBS, pH 7.4) , cells were analyzed with a BD LSR II Flow Cytometer (Becton Dickinson, San Jose,CA). Granulocytes were gated by forward and side scatter. For some chemotaxis experiments cells were incubated with small molecule inhibitors PD98059, Uol26, Sb203580, wortmannin and ly294002 (Cell signaling Technology, Danvers, MA). THP- 1 cells (10⁶/sample) were treated with small molecule inhibitors for 6 h, cell viability was measured by using Vybrant Apoptosis Assay kit #2 (Invitrogen, CA) using the protocols provided by the manufacturer.

Generation of mS100A9 adeno-virus

Full-length murine S100A9 was synthesized by GeneArt and cloned into the adenoviral shuttle plasmid pShuttleCMV (AdEasy system, Agilent). The plasmid containing murine S100A9 and the adenoviral genome, pAdmS100A9, was generated by recombination in BJ5183-Ad cells (Agilent). pAdmS100A9 was linearized with Pad and trans fected into Ad293 cells (Agilent). After 7 days, the crude viral lysate (CVL) was harvested and amplified on Ad293 cells. Several days later, cytopathic effect (CPE) was seen and the CVL harvested. The CVL was used to infect a large scale culture of 293F cells (Invitrogen). Forty-eight hours post infection, the virus was harvested and purified on two cesium chloride gradients (one step and one continuous). Expression of murine S100A9 protein was confirmed in the supernatants of MLE infected cells by Western Blot. For mammalian expression of murine S100A9, a 6X His tag was added to the C-terminus of the murine S100A9 in the pShuttleCMV vector. The vector was used for transient expression of HEK293F cells using standard lipid transfection methods, and protein was purified from the supernatant using Nickel columns. Bacterially expressed low endotoxin recombinant human S100A8, S100A9 and S100A12, along with preparations of S100A1, S100A4, S100A6, S100A7, S100A10, S100A14, S100B and SI OOP were purchased from MBL International (Woburn, MA). S100A8/A9 heterodimers were purified directly from human neutrophils.

Purified neutrophils were suspended in a PBS cocktail of protease inhibitors (Sigma Aldrich, St. Louis, MO) and sonicated for three cycles to obtain a cell lysate. Cytoplasmic fractions were isolated by centrifugation and dialyzed against Buffer A (50 mM Tris HC1 (pH8.0) containing 1 mM EDTA, 1 mM DTT, 1 mM CaCl₂, protease inhibitor cocktail), and captured on a the HiTrap Q HP (GE-Healthcare, Pittsburgh, PA). The bound S100A8/A9 protein was eluted with a 0-50 % gradient of Buffer B (Buffer A with 500 mM NaCl), and fractions containing S100A8/A9 proteins were determined by Western blot with anti-S100A8 or S100A9 antibodies (Santa Cruz, CA, USA). Pooled S100A8/A9 fractions were diluted 10-fold with Buffer C (50 mM sodium acetate (pH 4.5), 1 mM EDTA, 1 mM DTT, 1 mM CaCl₂) and then positive fractions were applied to a HiTrap SP-HP column (GE Healthcare, Pittsburgh, PA). The column was washed with Buffer C containing 300 mM NaCl, and the SI 008/9 protein was eluted with an increasing concentration of NaCl (300-500 mM) in Buffer C. The eluted S100A8/A9 fraction was dialyzed against PBS 1 mM CaCl₂. Contaminating endotoxin was removed by Affinity Pak Detoxi-Gel (Thermo Fisher Scientific Inc, Waltham, MA). The purity of the protein > 95% by SDS-PAGE with endotoxin levels < 0.004 EU^g protein.

Chemotaxis Assay

Cell migration was assessed using a 96 well ChemoTX system (Neuro Probe, MD).

Human THP-1, whole blood cells or bone marrow-derived macrophages from wild-type or RAGE k/o mice were washed and loaded on the filter with or without antibodies. Migration of cells to the human SI 00 proteins or human MCP-1 in the lower chamber was enumerated by flow cytometry.

Human and Mouse Cytokine Assay:

Human PBMC were stimulated with SI 00 proteins for 16 hrs with or without anti-TLR4 or anti-RAGE Abs. Supematants were collected and IFN-γ, IL-6, IL-Ιβ and TNFa levels were measured using Meso Scale Discovery Inc's human proinflammation cytokine kit. Cytokines in Mice bal fluid were measured by Meso Scale Discovery Inc's mouse proinflammation cytokine kit.

Mice:

Ager (gene product = RAGE) knockout mice were generated by Taconic Artemis

Pharmaceuticals (Germany) for Medlmmune. C57/B6 mice were purchased from Taconic Farms (Hudson, NY). C3H/HeOuJ (TLR4-sufficient) and C3H/HeJ (TLR4-deficient) mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were housed under specific pathogen-free conditions and were used in experiments at 8-12 weeks of age.

RSV infection/necropsy:

Mice were anesthetized with isofluorane prior to intranasal inoculation with 50μ1 of PBS, or 3xl0⁸ pfu of Adenovirus null or Adenovirus expressing S100A9, in the same volume. At the time of necropsy we collected BALF (obtained by 3 x 0.6ml washes with PBS/lOmM EDTA/20mM Hepes) and lung tissue (for RNA and protein).

Differential Counts:

Cells recovered in the BALF were cytospun onto glass slides and were stained with diff- quik to allow for identification of leukocyte populations. A total of 500 cells was counted for each sample and percentages of eosinophils, macrophages, lymphocytes and neutrophils were calculated. Percentages were used to back-calculate to total cell numbers.

Cell Apoptosis Assay THP-1 cells (10⁶/sample) were treated with inhibitors for 6hrs, cell viability was measured by using Vybrant Apoptosis Assay kit #2 (Invitrogen, CA), following the protocols provided by the manufacturer.

Flow Cytometry

Cells were incubated with various dye-conjugated mouse mAb against human CD3, CD4, CD8, CD14, CD1 IB, CD19 and CD56 at room temperature for 30 min, after wash with FACS buffer (PBS, 1% FBS, pH7.4), cells were analyzed with a FACScan cytometer (Becton

Dickinson, San Jose, CA).

Human and Mouse Cell Lines and Human Blood

Human blood from healthy volunteers was obtained by venous puncture under

Medlmmune's blood donation program. Informed consent was provided according to the Declaration of Helsinki. Human monocytic THP-1 and the murine macrophage RAW cell lines were obtained from the American Type Culture Collection (Manassas, VA).

Histopathology and Pathology Scoring

Lung samples were fixed in 10% buffered formalin for at least 24 hours. For histopathology and immunohistochemistry, 5μιη thick sections were cut from paraffin blocks and mounted on SuperFrost Plus slides. Slides were stained with hematoxylin and eosin (H&E).

The lung pathology was scored as follow:

Scores Pathology Scoring Criteria

0 no to rare inflammatory cells

1 a few cells surrounding few bronchioles and vessels

2 a few cells surrounding multiple bronchioles and vessels

a few clusters of cells scattered in few alveolar spaces

3 a ring of cells 1-2 cells deep affecting multiple bronchioles and vessels

a few clusters of cells scattered in multiple alveolar spaces, minimal increased

cellularity in interstitium

4 a ring of cells 2-4 cells deep affecting multiple bronchioles and vessels

a few clusters of cells scattered in multiple alveolar spaces, mild increased cellularity in interstitium 5 a ring of cells >4 cells deep affecting most bronchioles and vessels

multiple clusters of cells scattered in most of the alveolar spaces, moderate increased cellularity in interstitium

Statistical Analysis

All experiments were performed two to three times, and the data of one representative experiment are shown. The statistical significance of difference between groups was analyzed using unpaired Student's t-test or non-parametric Mann Whitney test.

Example 1:

S100A8, S100A9 or S100A12 induction of proinflammatory cytokines is TLR4 dependent and RAGE independent.

Calgranulins including S100A8, S100A9 and S100A12 are damage-associated molecular pattern (DAMP) proteins and have been shown to play an important role in human disease. Previously, it was believed that S100A8 and S100A9 may function as endogenous TLR4 and be able to amplify phagocyte activation during sepsis upstream of TNF. We first compared the effect of S100A8, S100A9 and S100A12 induction of proinflammatory cytokines in human PBMC, consistent with previous published data. We determined that microgram quantities of S100A8 and S100A9 are able to induce IL6, TNFa, IL-Ιβ and IFNy from human PBMCs which were significantly inhibited by anti-TLR4 antibodies, but not anti-RAGE blocking antibodies (Fig. 1A-C). The endotoxin inhibitor Polymyxin B had no effect on cytokine production from low endotoxin SI 00 preparations, whereas it completely inhibited LPS-induced IL-6 production in human PBMCs (Fig. ID). These data demonstrate that the calgranulins S100A8, S100A9 and S100A12 all induce proinflammatory cytokines in a TLR4-dependent, but RAGE-independent manner.

Recombinant preparations of S100A1, S100A4, S100A6, S100A7, S100A10, S100A14, SI OOP and S100B, as well as S100A8/A9 heterodimer isolated directly from neutrophils, were also examined for their potential to induce proinflammatory cytokines (Fig. IE). In most instances cytokine responses were much greater and the anti-RAGE Ab treatment again had no effect. Anti-TLR4 Ab and Polymyxin B completely inhibited the cytokine response for most preparations (eg. SlOOAl), indicating that contaminating endotoxin was primarily driving the cytokine response. There were notable exceptions however: 10 μg/ml of S100B and the

S100A8/A9 heterodimer failed to generate a cytokine response (Fig. IE).

There are multiple receptors and co-receptors that have been identified for SI 00 proteins. The most important receptors among these are TLR4 and RAGE. We used neutralizing Abs against RAGE and TLR4 to address the question of which receptor is responsible for Calgranulin-associated cytokine induction. Our data indicates that S100A8-, S100A9- and S100A12-associated proinflamatory cytokine induction is TLR4 dependent and RAGE independent (Fig. 1).

Example 2

S100A8, S100A9 and S100A12 induced THP-1 cell migration is RAGE dependent but TLR4 independent.

Several SlOOs have been shown to have chemotactic activities ²⁴'²⁵'²⁹'⁴¹'⁴³ RAGE has been implicated in mediating the migration of S100B, S100A12 and S100A7, whereas S100A15 appears to be mediated by an as yet unidentified Gi protein coupled receptor ²⁴'²⁵'⁴¹'⁴³. Using a 96 well ChemoTx system we initially demonstrated that the calgranulins S100A8, S100A9 and SlOOAl 2 all induced THP-1 cell migration with a characteristic bell-shaped response typical of traditional chemokines (Figure 2A,C,E). This migration was significantly less if the SI 00 proteins were put in the upper well indicating that the migration was chemotactic, rather than a consequence of increased motility (data not shown). Using the optimal concentration for each SI 00 we demonstrate that migration of THP1 cells to S100A8, S100A9 and SlOOAl 2 was inhibited in a dose-dependent manner with anti-RAGE Ab, but not an anti-TLR4 Ab (Figure 2B,D,F). Similarly, SlOOAl, S100A4, S100A6, S100A7, SIOOAIO, all induced migration of THP-1 cells. The chemotactic activities of S100A4, S100A7, S100A8/A9, and to a lesser extent S100A6 mediated migration of THPl cells were also dependent on RAGE, whereas migration of S100A1, and S100A10 were not affected by RAGE or TLR4 blockade (Fig. 2G).

Example 3

S100A9-induced lymphocyte and monocyte migration is RAGE dependent but TLR4 independent.

Although the in vitro experiments focused on monocytic THPl cells, SlOO-mediated migration is applicable to other cell types. For example, hS100A9 was used in following assay: granulocytes, lymphocytes and monocytes were isolated from healthy human donors and were shown to be chemotactic in response to hS100A9 (Fig. 3 A). Further characterization revealed that among the lymphocyte fraction, hS100A9 induced migration of CD3+/CD4+ T cells, CD3+/CD8+ T cells, and CD56+ NK cells, but not CD 19+ B cells (Fig. 3B). As we observed with the THPl cells, the S100A9-mediated migration of the granulocytes, monocytes and lymphocytes was RAGE-dependent and TLR4-independent (Fig. 3C, 3D, and 3E). Together, these results indicate that S100A9 induce the migration of multiple cell types, and this effect through RAGE but not TLR4.

Example 4

S100A9 induced THP-1 cell migration occurs via MEK/ERK, and PI3K but not P38.

RAGE is a multi-ligand receptor belonging to the immunoglobulin superfamily. RAGE is expressed in all tissues and a wide variety of cells including monocytes, dendritic cells, and macrophages. It is most abundant in the heart, lung and skeletal muscle. RAGE engagement by a ligand triggers the activation of key signaling pathways involving p42/44, Akt, TNK or P38 MAP kinase as well as NF-kB. In order to test which signaling pathway is involved

S100A9/RAGE triggered cell migration, a couple of specific inhibitors of the MEK/ERK pathway, the p38 pathway and the PI3 kinase pathway were tested in a cell migration assay. The p38 MAPK inhibitor SB203580 failed to block S100A9 induced THP-1 cell migration, but both the PI3 kinase inhibitors (Wortmannin and Ly294002) and the MEK/ERK pathway inhibitors (PD 98059 and U0126) inhibited S100A9 induced THP-1 cell migration in a dose-dependent manner (Fig. 4 A and 4C). The inhibitors tested in the cell migration assay showed no cytotoxic effects even at highest concentrations (data not shown). Thus, S100A9 induced THP-1 cell migration occurs via MEK/ER , and PI3K but not p38.

Example 5

The effect of anti-RAGE and anti-TLR4 Abs on mS100A9 induced Raw cell migration and proinflammatory cytokine induction.

In preparation for in vivo studies, we next investigated whether the activities of S100A9 were similar between human and mouse. To this end, we generated small amounts of endotoxin- free mammalian expressed recombinant murine S100A9. At low concentration (1 ng/ml) mS100A9 induced significant migration of RAW cells, a murine macrophage cell line. This response was completely abrogated by an anti-mRAGE antibody whereas the anti-mTLR4 antibody had no effect (Fig. 5A). At a higher concentration (10 μg/ml), murine S100A9 induced the production of the TNFa and IL-6 (Fig. 5 B, C). Induction of cytokines was significantly inhibited by anti-TLR4 antibody, but not by anti-RAGE antibody. Comparable results were obtained with LPS, which was used as a positive control for cytokine induction. Overall, these data suggest that human and murine S100A9 trigger pro-inflammatory responses in vitro through the same receptors and they appear to be functionally equivalent.

Example 6

In vivo characterization of S100A9-mediated inflammatory responses

Clinical data revealed that S100A8/A9 is increased in the lungs of patients with chronic inflammation and COPD. However, there are no definite studies on the role of S100A8/A9 in inflammation and obstructive airway disease.

To assess the inflammatory role of S100A9 in vivo, a lung inflammation model was developed using adenoviral mS100A9. Dose responses and kinetic analyses were initially performed to establish an SlOO-dependent response. At early time-points (24h and 72h) there was a weak inflammatory response with the adenovirus which was not SlOO-dependent. By day 5, this initial inflammation had subsided and there was an increase in the cellular infiltrate with the high-dose S100A9-adenoviral treated mice, and by day 8, the inflammation was clearly S100A9 dependent. Western blot analysis of the BAL from the mice revealed that S100A9 was only detected in the high-dose mice at this time-point (data not shown).

Based on this model, we first compared the adeno-S100A9 induced lung inflammation between wild type (C57BL/6) and RAGE KO mice. We demonstrated that at day 8, Adeno- S100A9, but not adeno-null was able to induce lung inflammation represented by cell infiltration (total cell counts and differential cell counts) (Fig. 6A, 6B, 6C), and mouse proinflammatory cytokines induction (mIL-6 and mlFNy) (Fig. 6D, 6E). Western blot analysis of the BAL from the mice revealed similar levels of S100A9 in both wild type and RAGE KO mice (Fig. 6F, 6G). Besides increasing the cellular infiltration and cytokines in the BAL, the adeno-S100A9 also induced increased tissue pathology compared to the adeno-null control in the wild type C57/B16 mice (Fig. 6H, 61). There was no difference in the tissue pathology between wild type and RAGE KO mice in the adeno-S100A9 treated groups indicating that the lung tissue pathology was also independent of RAGE (Fig. 6H, 61).

In addition, anti-RAGE Abs also failed to block S100A9 induced cell infiltration and cytokine induction in wild type mice (data not shown). These data suggest, S100A9 induced lung inflammation appears to be completely independent of RAGE in vivo.

Similarly, we also compared the adeno-S100A9 induced lung inflammation between wild type (C3H/HeOuJ) and TLR4 deficient (C3H/HeJ) mice. We demonstrated here that at day 8, Adeno-S100A9 but not adeno-null induced lung inflammation as represented by cell infiltration (total cell counts and differential cell counts) (Fig. 7A, 7B, 7C), and mouse proinflammatory cytokine induction (mIL-6 and mlFNy) (Fig. 7D, 7E). Western blot analysis of the BAL from the mice confirmed that similar levels of S100A9 were present in the wild type and RAGE KO mice (Fig. 7F, 7G). This confirmed our in vitro finding that S100A9-induced cell infiltration is TLR4 independent, while S100A9-induced proinflammatory cytokines are TLR4 dependent. Adeno- S100A9 also induced increased tissue pathology compared to the adeno-null control in the wild type C3H/HeOuJ mice (Fig. 7H, 71). There was no difference in the tissue pathology between wild type and TLR4-defective CH3/HeJ mice in the adeno-S100A9 treated groups indicating that despite the blockade of cytokines, the lung tissue pathology was also independent of TLR4 (Fig. 6H, 61).

Taken together, we demonstrate for the first time that S100A9 is able to induce lung inflammation in vivo, furthermore, our data indicate that S100A9-mediated cellular inflammation appears to be independent of both RAGE and TLR4. Although S100A9-induced proinflammatory cytokines are TLR4 dependent, TLR-4 mediated cytokine induction is not responsible for the cellular inflammation. Thus, S100A9 may mediate pulmonary inflammation by an as yet unidentified receptor/mechanism.

Discussion

Calgranulins, including S100A8 (Calgranulin A), S100A9 (Calgranulin B) and S100A12 (Calgranulin C) are predominantly expressed by neutrophils, monocytes and activated macrophages. Increased levels of these proteins have been found in various autoimmune diseases including cystic fibrosis, chronic obstructive pulmonary disease (COPD), lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), idiopathic pulmonary fibrosis (IPF) and asthma. The role of S100A9 has previous been shown to include chemotaxis and the upregulation of proinflammatory cytokines. In addition, S100A9 is able to bind the receptor for advanced glycation end products (RAGE) and TLR4. It has also has been shown that S100A9- induced cytokines are TLR4 dependent, however, it is unknown which receptor is responsible for S100A9-induced cell migration. Furthermore, there are no definitive studies on S100A9's role in inflammation and obstructive airway disease. TLR4 and RAGE are expressed in lung. TLR4 is linked with innate immunity involved in local airway inflammation and RAGE participates in mediating fibroproliferative remodeling in IPF. This evidence suggests that S100A9 may play an important role in the pathogenesis of asthma. We describe for the first time, that S100A9 is able to induce lung inflammation. Furthermore, our data indicate that S100A9-mediated cellular inflammation appears to be independent of both RAGE and TLR4. Although S100A9 induced proinflammatory cytokines is TLR4 dependent, TLR-4 mediated cytokine induction is not responsible for the cellular inflammation.

Both our in vitro and in vivo cytokine data has confirmed the previous finding that S100A9-induced proinflammatory cytokines are TLR4 dependent, however, our in vivo lung inflammation model also indicates that TLR-4 mediated cytokines induction is not responsible for the cellular inflammation. These data indicate that S100A9 induces cell migration and cytokine release via two distinct pathways: (a) TLR4 dependent (both in vitro and in vivo, and (b) RAGE dependent (in vitro and through an unknown receptor in vivo). As an endogenous TLR4 ligand, blockage of TLR4 alone may not be sufficient to inhibit all biological activity of S100A9.

Our in vitro data indicates that S100A9 is able to induce a variety of immune cell to migrate in a RAGE dependent manner. However, our in vivo model demonstrates that S100A9- induced cell migration is RAGE independent. Since S100A9 is also able to induce cytokine induction, the discrepancy between in vitro and in vivo may be easily explained by proposing that S100A9 induced cytokine contributes to its chemoattractant activity. However, we excluded this possibility because when S100A9-induced cytokines in wild type and TLR4-deficient mice were compared, we saw reduced cytokine induction in TLR4 deficient mice, but no reduction of cell infiltration. Thus, S100A9 may mediate pulmonary inflammation by an as yet unidentified receptor/mechanism.

Previous in vitro studies have indicated that S100A7, SI 008, S100A8/A9, S100A9, S100A12, S100A15 and S100B have chemotactic activities in vitro. We confirmed these findings and extended this list to include S100A1, S100A4, S100A6, and S100A10. Indeed every SI 00 preparation we tested induced significant migration in vitro. An anti-RAGE Ab, that targets the V-Cl domains of RAGE which are responsible for ligand binding, blocked the majority of migration induced by S100A4, S100A7, S100A8, S100A8/A9. These data in part confirm previous findings that have indicated that S100A7, S100A12 and S100B induce migration in a RAGE-dependent manner, and extended the list to include S100A4, and the phagocyte DAMPs S100A8, S100A8/A9 and S100A9. Consistent with these findings, S100A4, SI OOP, S100A8, and S100A9 have all been shown to bind to RAGE. Our analysis of the signaling pathways necessary for S100A9-induced migration of THP1 cells clearly identified a requirement for the MEK/ERK and PI3K pathways, but not for P38. This is consistent with a recent detailed report on the RAGE-dependent signaling induced by S100B on microglia cells which required the recruitment of diaphanous- 1 and activation of Src kinase, Ras, PI3K, MEK/ERK1/2, RhoA/ROCK, Racl/JNK/AP-1, Racl/NFkB, and, to a lesser extent, p38. Interestingly, the S100B mediated migration was also associated with the upregulation of the CCL3 (Mipl ), CCL5 (RANTES) and their cognate receptors CCR1 and CCR5. Further studies will need to elucidate if this RAGE-dependent mechanism is physiologically relevant, and if the mechanism applies to other SI 00s and other cell types.

The partial inhibition of S100A6 with the anti-RAGE Ab is consistent with binding studies that indicate that it can also bind both the V-Cl and C2 domains of RAGE. However, migration of SlOOAl and SlOOAlOwere not inhibited by RAGE blockade. It is plausible that these SI 00s may bind alternate RAGE binding sites not blocked by the antibody. SlOOAl has been shown to bind the V-domain of RAGE and induce neurite outgrowth, but we are not aware of any data that indicates the SlOOAl induced migration is dependent on RAGE. Despite a thorough analysis of the interactions between RAGE and SI 00s there is no data to indicate that SlOOAlOinteracts with RAGE, so it is likely that these SI 00s may induce migration in vitro via an alternate receptor as proposed for SlOOAl 5.

Interestingly, despite a solid body of literature implicating RAGE in the migration of SI 00s, and the potent blockade of migration induced by calgranulins and some other SI 00s with anti-RAGE Ab in vitro described herein, in our in vivo model murine S100A9-mediated airway inflammation is independent of RAGE. Previously it has been shown that polyclonal S100A8 and S100A9 antibodies blocked infiltration of phagocytes into the alveolar space following intranasal challenge with Streptococcus pnuemoniae. This would appear to contradict studies with RAGE-deficient mice that had reduced infiltration into the lung tissue and airspace following challenge with Streptococcus pnuemoniae, Influenza A virus or Respiratory syncytial virus, (Miller, 2012). In these cases the complex mechanism by which RAGE mediates lung inflammation was not resolved, although the later study indicates that the surface expressed and soluble forms of RAGE appear to play opposing roles (Miller, 2012). On the basis of our vitro data and these vivo studies, we predicted that S100A9-mediated inflammation induced in our simple airway model would be mediated through RAGE. However, our data with RAGE- deficient mice and RAGE blockade with ligand blocking antibody in wild-type mice clearly demonstrate that RAGE appears to be redundant in S100A9-induced lung and airway inflammation. Furthermore, the apparent S100A8- and S100A9-dependent inflammation induced by monosodium uric acid crystals in a murine air-pouch model, was also unaffected by RAGE deficiency (data not shown). Although additional experimental confirmation is required, it seems likely that these observations could extend to other tissues. SI 00s have been regularly reported as potent chemoattracts since low concentrations can induce cellular migration in vitro. However, it has been postulated that the concentrations of S100A9 and other calgranulins encountered at inflammatory sites can often be several orders of magnitude higher which would preclude their chemotactic activity. Our in vivo data would support this perspective. Whether or not RAGE plays a physiological role in the migration of other SI 00s remains an open question. The in vitro data reported herein indicate that other SI 00s are active over a different range of concentrations, and in many cases their respective normal or induced levels remain unknown. In any case, the differential in vitro and in vivo S100A9 responses presented here highlight the importance of confirming in vitro observations in vivo. Nevertheless, the S100A9-mediated airway inflammation demonstrated here, and published in vivo studies with S100A8, S100A9 and S100A12 reported elsewhere, demonstrate that calgranulins are likely to play an important role in regulating inflammation, although the receptor mechanism underlying these observations remains unclear.

We considered alternate receptors that could mediate SlOO-mediated inflammation, namely TLR4. Previously, S100A8, but not S100A9 or S100A8/A9, was shown to induce a modest TLR4-mediated cytokine induction. Akin to this study, we also ruled out the potential of endotoxin contamination, and besides S100A8, we showed for the first time that S100A9 and S100A12 also induce similar modest levels of TNFa, IL-6, IL-Ιβ and IFNy in a TLR4-dependent manner. The discrepancy in the induction of cytokines with S100A9 between our data and the aforementioned study is not obviously apparent, but our data is supported by a subsequent study that showed S100A9 binds MD2/TLR4. Moreover, we did show a similar response from mammalian expressed endotoxin- free mS100A9 in vitro, and by adenovirus-mS100A9 infection in vivo, which resulted in the induction of IL-6 and IFNy in a TLR4-dependent fashion. Surprisingly, S100A9-induced cellular infiltration into the airways was unaffected in the TLR4- defective mice demonstrating that the S100A9-mediated cytokine or chemokine induction does not drive the inflammation in this model. Whether S100A9 may promote TLR4 responses in the presence of other proinflammatory mediators to drive pathological inflammatory responses akin to those described with HMGB1 remains to be determined. However, using this simple lung inflammation model we were able to demonstrate that murine S100A9 can drive a robust inflammatory response which is predominately independent of both RAGE and TLR4.

Previously, it has been shown that S100A8/A9 and S100A9 bind to sulfated glycosaminoglycans and carboxylated glycans on endothelial cells, and complexes of S100A8/A9 and arachidonic acid bind to the scavenger receptor CD36 which can also be expressed on endothelial cells. Interactions of this sort could establish an attractive SI 00 substratum for neutrophils and monocyte attachment, activation and transendothelial migration and thereby mediate tissue inflammation. Further investigation is warranted to determine if the SlOO-mediated inflammation is mediated through a diverse set of interactions with exposed sugar moieties or through interactions with specific receptors.

In previous studies, the activity of S100A9 has been investigated using either native or sometimes recombinant proteins, purified by isoelectric focusing or in the presence of a reducing agent such as DTT. These purification protocols could potentially result in partial or total inactivation of the proteins. In addition, endo-toxin contamination of the recombinant protein may provide misleading results. To assess the relative importance of RAGE-dependent cell migration and TLR4-mediated inflammation in vivo we examined lung inflammation following treatment with adenoviral S100A9. This enabled us to ascertain the direct effect of S100A9, rather than relying on an alternative stimulus that could potentially stimulate either a multitude of pathways or stimulate down stream of S100A9. Intranasal inoculation of S100A9 virus but not adeno-null virus induced significant cell infiltration as well as the cytokine release. These data correspond with our in vitro observation and demonstrate that S100A9 is a potent stimulator of neutrophils as well as macrophages, and strongly suggests that it is involved in neutrophil/macrophage migration to inflammatory sites.

In summary, our data indicates that S100A9 is able to induce cell migration and proinflammatory cytokines in vitro and in vivo, we confirmed previous findings that S100A9 induced TLR4 dependent cytokine release both in vitro and in vivo. In vivo, cellular infiltration is independent of RAGE and predominately independent of TLR4-mediated cytokine induction.

The reagents employed in the examples are commercially available or can be prepared using commercially available instrumentation, methods, or reagents known in the art. The foregoing examples illustrate various aspects of the invention and practice of the methods of the invention. The examples are not intended to provide an exhaustive description of the many different embodiments of the invention. Thus, although the forgoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those of ordinary skill in the art will realize readily that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

Claims

What is claimed is:

1. A method of preventing, inhibiting, treating or managing a lung inflammatory disorder in an animal in need thereof, comprising administering to said animal a therapeutically effective amount of a S100A9 inhibitor.

2. The method of claim 1 wherein said S100A9 is selected from the group consisting of a small molecule inhibitor, a monoclonal antibody, an S100A9 polypeptide fragment, a mutant S100A9 and an anti-S100A9 antibody.

3. The method of claim 1 wherein said lung inflammatory disorder is COPD, asthma, IPF, MS, RA or SLE.

4. The method of claims 1-3, wherein said animal is human.

5. A method for treating, preventing or alleviating the symptoms of an inflammatory disorder of the lung in a subject in need thereof comprising administering an effective amount of an S100A9 inhibitor.

6. The method of claim 5 wherein said S100A9 is selected from the group consisting of a small molecule inhibitor, a monoclonal antibody, an S100A9 polypeptide fragment, a mutant S100A9 and an anti-S100A9 antibody.

7. The method of claim 5 wherein said lung inflammatory disorder is COPD, asthma, IPF, MS, RA or SLE.

8. The method of claims 5-7, wherein said animal is human.

9. A method for inhibiting an S100A9 activity in a cell expressing S100A9, comprising contacting the cell with an S100A9 inhibitor, wherein the S100A9 activity in the cell is selected from the group consisting of: (a) induction of cell migration; (b) induction of cytokine release; (c) binding to TLR4; and combinations thereof.

10. The method of claim 9 wherein said S100A9 is selected from the group consisting of a small molecule inhibitor, a monoclonal antibody, an S100A9 polypeptide fragment, a mutant S100A9 and an anti-S100A9 antibody.

11. The method of claim 9 wherein said lung inflammatory disorder is COPD, asthma, IPF, MS, RA or SLE.

12. The method of claims 9-11, wherein said animal is human.