INTERLEUKIN.13 BINDING PROTEINS
Cross-Reference to Related Application
This application claims the benefit of priority to US provisional application no. 60/843,249 filed September 8,2006.
Field of the Invention
The present invention relates to IL-13 binding proteins, and specifically to their uses in the prevention and/or treatment of various diseases including asthma, allergy, COPD, fibrosis, and cancer.
Reference to joint research agreement
Contents of this application are under a joint research agreement entered into by and between Protein Design Labs, Inc. and Abbott Laboratories on December 14,2005, and directed to recombinantly engineered antibodies to IL-13.
Background of the Invention
Human IL-13 is a 17-kDa glycoprotein c10ned from activated T cells (Zurawski and de Vries 1994 Immunol Today 15 19-26), and is produced by activated T cells of the Th2 lineage, although ThO and Thl CD4+ T cells, CD8+ T cells, and several non-T cell populations such as mast cells also produce IL-13 (Zurawski and de Vries 1994 Immunol 10day ii 19-26). The function of IL-13 includes immunog10bulin isotype switching to IgE in human B cells (Punnonen, Aversa et al. 1993 Proc Natl Acad Sci U S A 90 3730-4) and suppressing inflammatory cytokine production in both human and mouse (de Waal Malefyt, Figdor et al. 1993 J Immunol 151 6370-81; Doherty, Kastelein et al. 1993 J Inmunol 151 7151-60). IL-13 binds to its cell surface receptors, IL-13Ralphal and IL-13Ralpha2. The IL-13Ralphal interacts with IL-13 with a 10w affinity (KD -10nM), fol10wed by recruitment of IL-4Ra to form the high affinity (KD - 0.4 nM) signaling heterodimeric receptor complex (Aman, Tayebi et al. 1996 J Biol Chem 271 29265-70; Hilton, Zhang et al. 1996 Proc Natl Acad Sci U S A 93 497-501). The IL-4R/IL-13Ralphal complex is expressed on many cell types such as B cells, monocyte/macrophages, dendritic cells, eosinophils, basophils, fibroblasts, endothelial cells, airway epithelial cells, and airway smooth muscle cells (Graber, Gretener et al. 1998 Eur J Immunol 28 4286-98; Murata, Husain et al. 1998 Int Immunol 10 1103-10; Akaiwa, Yu et al. 2001 Cytokine 13 75-84). Ligation of the IL-
13Ralphal/IL-4R receptor complex results in activation of a variety of signal-transduction pathways including signal transducer and activator of transcription (STAT6) and the insulin receptor substrate-2 (IRS-2) pathways {Wang, Michieli et al. 1995 B10od 86 4218-27; Takeda, Kamanaka et al. 1996 J Immunol 157 3220-2). The IL-13Ralpha2 chain a10ne has a high affinity (KD ~ 0.25-0.4 nM) for IL-13, and functions as both a decoy receptor negatively regulating IL-13 binding (Donaldson, Whitters et al. 1998 J Immunol 161 2317-24), and a signaling receptor that induces TGF-b synthesis and fibrosis via AP-1 pathway in macrophages and possibly other cell types (Fichtner-Feigl, Strober et al. 2006 Nat Med 12 99-106).
Several studies conducted in preclinical animal models for asthma indicate that IL-13 plays an important role in asthma. These data include resistance to asthma in the IL-13 knockout mice as well as inhibition of the asthma phenotype with IL-13 antagonists (soluble IL-13 receptors, anti-IL-13 mAbs, etc.) in various mouse models (Sela 1999 Harefuah 137 317-9; Wills-Karp and Chiaramonte 2003 Curr Opin Pulm Med 9 21-7; Wills-Karp 2004 Immunol Rev 202 175-90). Multiple studies have demonstrated that pharmaco10gic administration of recombinant IL-13 to the lungs of mice as well as guinea pigs induces airway mucus hyper-secretion, eosinophilia and AHR (Grunig, Wamock et al. 1998 Science 282 2261-3; Wills-Karp, Luyimbazi et al. 1998 Science 282 2258-61; Kibe, Inoue et al. 2003 Am J Respir Crit Care Med 167 50-6; Vargaftig and Singer 2003 Am J Physiol Lung Cell Mol Physiol 284 L260-9; Vargaftig and Singer 2003 Am J Respir Cell Mol Biol 28 410-9). These effects of IL-13 are reproduced in transgenic mouse systems with either constitutive or inducible expression of IL-13 (Zhu, Homer et al. 1999 J Clin Invest 103 779-88; Zhu, Lee et al. 2001 Am J Respir Crit Care Med 164 S67-70; Lanone, Zheng et al. 2002 J Clin Invest 110 463-74). Chronic transgenic over-expression of IL-13 also induces subepithelial fibrosis and emphysema. Mice deficient in the IL-13 (and IL-4) signaling molecule STAT6 fail to deve10p allergen-induced AHR and mucus overproduction (Kuperman, Huang et al. 2002 Nat Med 8 885-9). Studies using soluble IL-13 receptor fusion protein (sIL-13Ralpha2Fc) have demonstrated the pivotal role of this cytokine in experimental allergen ovalbumin (OVA)-induced airway disease (Grunig, Wamock et al. 1998 Science 282 2261-3; Wills-Karp, Luyimbazi et al. 1998 Science 282 2258-61; Taube, Duez et al. 2002 J Immunol 169 6482-9). Efficacy of anti-IL-13 treatment was also demonstrated in a chronic model of murine asthma. In addition to exhibiting features of mucus hyper-secretion and AHR, this model of chronic asthma demonstrates several hallmarks of human disease that are lacking in the more acute models. These include eosinophilia of the lung tissue 10cated in inter-epithelial spaces as well as smooth muscle fibrosis as measured by increases in collagen deposition. The chronic asthma model is induced with repeated aerosol challenges with OVA in OVA-sensitized mice Ix/week for a total of 4 weeks. Anti-IL-13 antibody administered for the final 2 weeks of OVA challenges (from day 36 with efficacy readouts assessed on day 53 of study) significantly
inhibited AHR, pulmonary inflammation, goblet cell hyperplasia, mucus hypersecretion, and airway fibrosis (Yang, Li et al. 2005 J Pharmacol Exp Ther). Moreover, therapeutic effect of IL-13 antagonist was also demonstrated to inhibit AHR in a primate model of asthma [Abstract, American Thoracic Society 2005].
IL-13 is implicated in the pathogenesis of human asthma as elevated levels of IL-13 mRNA and protein have been detected in lungs of asthmatic patients, which correlate with severity of the disease (Huang, X1ao et al. 1995 J Immunol 155 2688-94). In addition, human IL-13 genetic polymorphisms, which lead to elevated IL-13 levels, have been identified and are associated with asthma and atopy (Heinzmann, Mao et al. 20(X) Hum Mol Genet 9 549-59; Hoerauf, Kruse et al. 2002 Microbes Infect 4 37-42; Vercelli 2002 Curr Opin Allergy Clin Immunol 2 389-93; Heinzmann, Jerkic et al. 2003 J Allergy Clin bnmunol 112 735-9; Chen, Ericksen et al. 2004 J Allergy Clin Immunol 114 553-60; Vladich, Brazille et al. 2005 J Clin Invest), and elevated IL-13 levels have been detected in the lung of asthma patients (Huang, X1ao et al. 1995 J Immunol 155 2688-94; Arima, Umeshita-Suyama et al. 2002 J Allergy Clin Immunol 109 980-7; Berry, Parker et al. 2004 J Allergy Clin Immunol 114 1106-9). A genetic linkage between IL-13 and asthma has also been demonstrated as individuals with a polymorphism in the IL-13 gene which causes higher plasma IL-13 levels have an increased risk for atopy and asthma (Wills-Karp 2000 Respir Res 1 19-23).
Due to the role of human IL-13 in a variety of human disorders, therapeutic strategies have been designed to inhibit or counteract IL-13 activity. In particular, antibodies that bind to, and neutralize, IL-13 have been sought as a means to inhibit IL-13 activity. However, there eX1sts a need in the art for improved antibodies capable of binding IL-13. Preferably the antibodies bind human IL-13. Preferably the antibodies are capable of neutralizing human IL-13. The present invention provides a novel family of binding proteins, CDR grafted antibodies, humanized antibodies, and fragments thereof, capable binding human IL-13, binding with high affinity, and binding and neutralizing human IL-13.
SunMnarv of the Invention
This invention pertains to IL-13 binding proteins. Binding proteins of the inventions include, but are not limited to antibodies, antigen binding portions, and other antigen binding proteins capable of binding the human IL-13. Further, the invention provides methods of making and using IL-13 binding proteins.
One aspect of the invention pertains to a binding protein capable of binding IL-13. In a preferred embodiment, the binding protein binds human IL-13. Preferably the binding protein is
capable of modulating a bio10gical function of IL-13. More preferably the binding protein is capable of neutralizing IL-13.
In one aspect of the invention, the binding protein is capable of binding IL-13, and preventing the binding of IL-13 to the IL-13al receptor. In another aspect of the invention, the binding protein is capable of binding IL-13, and preventing the binding of IL-13 to the IL-13(x2 receptor. In a preferred embodiment, the binding protein is capable of binding IL-13, and preventing the binding of IL-13 to both the IL-13al receptor and the IL-13a2.
One embodiment of the invention provides an isolated antibody, or antigen binding fragment thereof, wherein said antibody, or antigen binding fragment thereof binds human IL-13 and inhibits the binding of said IL-13 to the IL-13cx2 receptor in a cell surface-based receptor binding assay with an IC50 selected from the group consisting of about 1.5x10* to 1x10* M, 1x10* to Ix10' M, 10"' to 10'M and 10'° to 10"" M or in an ELISA-based receptor binding assay with an with an IC50 selected from the group consisting of about 1.8x10"* to 1x10* M, 1x10" * to 1x10"' M, 10"' to 10""*M and 10"'° to 10"" M. Preferably the antibody binds human IL-13 and inhibits the binding of said IL-13 to the IL-13a2 receptor in a cell surface-based receptor binding assay with an IC50 of 2.7 X1O'M and in an ELISA-based receptor binding assay with an with an IC50 of 1.1x10"' M. Preferably the antibody, or antigen binding fragment thereof binds human IL-13 and inhibits the binding of said IL-13 to the IL-13(x2 receptor in a cell surface-based receptor binding assay or in an ELISA-based receptor binding assay by about 70-100% at a concentration of 10OnM. Preferably the antibody is 13C5.5. More preferably the antibody is not BAK502G9, mAbl3.2orMJ2-7.
In another aspect, the invention provides an isolated antibody, or antigen binding ' fragment thereof, wherein said antibody, or antigen binding fragment thereof binds human IL-13 and inhibits AHR by about 50 %, 60%, 70%, 80%, 90% or 100% in a human IL-13 induced asthma model. Preferably, the antibody inhibits AHR by greater than 86% in a human IL-13 induced asthma model. In another embodiment, the isolated antibody, or antigen binding fragment thereof, binds human IL-13 and inhibits AHR by about 50 %. 60%, 70%, 80% 90% or 100% and inhibits mucus production by about 40%, 50 %, 60%, 70%, 80% 90% or 100% a human IL-13 induced asthma model. Preferably the antibody is 13C5.5. More preferably the antibody is not BAK502G9, mAbl3.2 or MJ2-7.
In one embodiment, the binding protein of the invention has an on rate constant (kon) to IL-13 of at least about 10^M's''; at least about IO'M'S"'; at least about 10*M"'s'; at least about IO'M'S"'; or at least about 10*M"'s"', as measured by surface plasmon resonance. Preferably, the binding protein of the invention has an on rate constant (ko„) to IL-13 between IO^M'S"' to IO'M"
's'; between 10'M's''to 10M''S; between 10Vs to 10^M's'; or between 10'M'S to 10*M 's', as measured by surface plasmon resonance.
In another embodiment, the binding protein of the invention has an off rate constant (kos) to IL-13 of at most about 10s'; at most about 10s'; at most about 10's''; or at most about 10' *s, as measured by surface plasmon resonance. Preferably, the binding protein of the invention has an off rate constant (koff) to IL-13of 10" V to 10"*s; of 10s to 10s; or of 10V to 10s', as measured by surface plasmon resonance.
In another embodiment, the binding protein of the invention has a dissociation constant (KD) to IL-13of at most about 10 M; at most about 10"* M; at most about 10"' M; at most about 10"•" M; at most about 10'" M; at most about 10^ M; or at most 10 M. Preferably, the binding protein of the invention has a dissociation constant (KD) to IL-13of 10"' M to 10"* M; of 10"' M to 10"' M; of 10"' M to 10"'" M; of 10"'" to 10 M; of 10 M to 10"'^ M; or of 10"'^ to M 10M.
Preferably the antibody or antigen binding fragment thereof, binds IL-13 with binding characteristics selected from the group consisting of: a) an on rate constant (kon) between about 10^M-'s to 10*M's or about 10*M's to 10'M's-',or b) an off rate constant (Ks) of about 10s" ' to 10"'s''; or of about 10"'s'' to 10'*s'', as measured by surface plasmon resonance; ore) a dissociation constant (KD) of about l.Sx10"'" to 1x10* M or about 10"'° to 10'" M. Preferably the antibody, or antigen binding fragment thereof has an on rate constant (ko„) to IL-13 selected from the group consisting of: 6.68X10'M'S, 7.86X10'M'S'', 8.35X10'M'S, 8.69X10'M'S', 9.15X10'M-'S, 1.26X10M-'S, 1.7x10'M's-',and2.51x10M-'s'. Preferably the antibody, or antigen binding fragment thereof has an off rate constant (koir) to IL-13 selected from the group consisting of: 1.23x10-*s; 1.76xl0s; 4.74xl0s; 1.91x10"V'; 2.14x10"V', 3.82x10"V'; 8.81x10's'\and 9.65xl0''s, as measured by surface plasmon resonance. Preferably the antibody, or antigen binding fragment thereof has a dissociation constant (Kp) to IL-13 selected from the group consisting of: 1.05x10* M; 7.10x10"'° M; 1x10 M; 2.20x10 M; 2.72x10'" M; 4.17x10" M; 5.68x10" M; 7.01x10 M; 7.10x10 M; and9.79x10 M.
One aspect of the invention pertains to binding proteins capable of a specific epitope on IL-13. Preferably the specific epitope comprises the C-terminal Helix D region of human IL-13. More preferably, the specific epitope comprises the amino acid sequence VRDTK BEVAQ FVKDL LL HLK KLFRE GR, corresponding to amino acid 104-130 of SEQ ID NO. 1. In another aspect the antibody or antigen binding portion, binds an epitope comprising C-terminal Helix D region and N-terminal Helix A region of human IL-13. Preferably the antibody, or antigen binding fragment thereof binds human IL-13 such that IL-13 with said antibody, or antigen binding fragment thereof, bound to the epitope defined by the topographic regions Ser26-Thr27-Ala28-Leu29-Arg30-Glu31-Leu32-Ile33-Glu34-Glu35-Leu36-Val37-Asn38andLysl23-Lysl24-Leul25-Phel26-Argl27-Glu-128-GIy 129-Argl30 of SEQ ID No. 1 is inhibited from
binding to the E.-13 receptor. Preferably the antibody, or antigen binding fragment thereof binds human IL-13 such that IL-13 with said antibody, or antigen binding fragment thereof, bound to the epitope defined by the topographic regions Arg30-Glu31-Leu32-Ile33-Glu34-Glu35-Leu36-Val37-Asn38 and Lysl23-Lysl24-Leul25-Phel26-Argl27 of SEQ ID No. 1 is inhibited from binding to the IL-13 receptor. Preferably the antibody, or antigen binding fragment thereof binds human IL-13 such that IL-13 with said antibody, or antigen binding fragment thereof, bound to the epitope defined by the topographic regions Ser26-Thr27-Ala28-Leu29-Arg30-Glu31-Leu32-Ile33-Glu34-Glu35-Leu36-Val37-Asn38andLysl23-Lysl24-Leul25-Phel26-Argl27-Glu-128-Glyl29-Argl30 of SEQ ED No. 1 is inhibited from binding to the IL-13a2 receptor. More preferably the antibody, or antigen binding fragment thereof binds human IL-13 such that IL-13 with said antibody, or antigen binding fragment thereof, bound to the epitope defined by the topographic regions Ser26-Thr27-Ala28-Leu29-Arg30-Glu31-Leu32-Ile33-Glu34-Glu35-Leu36-Val37-Asn38 and Lysl23-Lysl24-Leul25-Phel26-Argl27-Glu-128-Glyl29-Argl30 of SEQ ID No. 1 is inhibited from binding to the IL-13a2 receptor, provided said antibody is not BAK502G9 or MJ2-7. Most preferably the antibody is 13C5.5.
In one aspect the isolated antibody, or antigen binding fragment thereof, binds IL-13 and prevents binding of IL-13 to the IL-13a2 receptor with binding characteristics selected from the group consisting of binding to an epitope on IL-13 including Helix A and D; an on rate constant (kon) between about 10MS to 10*M'S' or about 10*M's to IO^M'S; an off rate constant (koff) of about 10'V to 10s'; or of about 10s'' to 10s'', as measured by surface plasmon resonance; and a dissociation constant (KD) of about 1.5x10"'° to Ix10'" M or about 10'** to 10'" M. In another aspect the isolated antibody, or antigen binding fragment thereof, binds variant IL-13 and prevents binding of variant IL-13 to the IL-13a2 receptor with binding characteristics selected from the group consisting of binding to an epitope on IL-13 including Helix A and D; an on rate constant (kon) between about 10^'s to 10MS or about 10MS to IO'M'S; an off rate constant (kos) of about 10V to 10's'; or of about 10's' to 10V, as measured by surface plasmon resonance; and a dissociation constant (KD) of about l.Sx10'" to 1x10° M or about 10' "'to10"M.
In one aspect the invention binding protein capable of binding IL-13, said antigen binding domain comprising at least one CDR comprising an amino acid sequence selected firom the group consisting of:
CDR-Hl. X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 64), wherein;
X1 is T, D, G, or S;
X2 is S;
X3 is D;
X4 is M, S, Y, L, or H;
X5 is G, W, Y, A, S, or N;
Xe is V, I, or M; and
X, is D, H, S, Y, N, or G;
CDR-H2 . X1-X2-X3-X4-X5-X6-X7-Xg-X9-X1o-X1i-X12-X13-X14-X15-X15-X1,7
{SEQ ID NO: 65), wherein; X1 is M, E, H, R, S, G, or L; X2 is I or not present; X3 is H, Y, A, D, S, or W; X4 is P, S, W, or G; X5 is S, G, E, or D; Xe is D, G, S, E, or N; X7 is S, Y or G; Xg is E, N, Y, V, or R; Xj is T, I, or K; X1o is R, Y, I, D, or A; Xn is L, Y, D, or F; X12 is N, P, S, or D; X13 is Q, E, D, P, or S; X14 is K, M, S, T, A, or V; X1s is F, L, V, or M; X16 is K, R, or Q; and X17 is D, G, or S; CDR-H3. X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 (SEQ ID NO: 66), wherein;
X1 is W, T, G, Y, D, or I; X2 is R, A, S, G, or V; X3 is T, F, Y, or S; X4 is S, T, or Y; X5 is Y, F, or G; Xg is F, or Y; X7 is S, Y, I, or F; Xg is D, L, Y, or P; X9 is Y; X1o is G;
Xn is Y, A, P, or E; X12 is F, M, S, L, or I; Xn is D, V, N, or K; and X14 is Y, or F;
CDR-Ll.CDR-L2,
and CDR-L3
X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15 X16-X17
(SEQ ID NO: 67), wherein;
X1 is K, or R;
X2 is S, or A;
X3 is S or T;
X4 is Q, K, or I;
X5 is N, S, T, G, or E;
Xe is L, T, or S;
X7 is L, Q, or V;
XB is Y, N, H, D, or T;
X9 is S, I, or T;
X1o is S, D, N, H, or Y;
X1i is N, or G;
X12 is Q;
X13 is K, F, N, E, or S;
X14 is N, T, or S;
X15 is Y, or F;
X16 is L, A, or M; and
X17 is A, D, E, H, or N X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 68), wherein;
X1 is L, S, K, T, W, or Y;
X2 is V, T, or A;
X3 is S, or N;
X4 is N, K, T, M, or R;
X5 is R, K, or, L;
X5 is F, D, E, H, P, or A; and
X7 is S, R, or P;
X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 69), wherein;
X1 is F, W, Q or A;
X2 is Q or L;
X3 is H, G, Y, W, or N;
X4 is N, S, T, L, or Y;
X5 is Y, T, S, E, or H;
Xe is L, V, F, Y, N, G, P, or D;
X7 is P, or, H;
Xa is L, F, Y, W, or R; and
X9 is T, or V.
Preferably, the antigen binding domain comprises at least one CDR comprising an amino acid sequence selected from the group consisting of:

residues 31-35 of SEQ ID NO.:32; residues
residues 50-66 of SEQ ID NO.:32; residues
residues 99-105 of SEQ ID NO.:32; residues
residues 24-39 of SEQ ID NO.:33;residues
residues 55-61 of SEQ ID NO.:33; residues
residues 94-102 of SEQ ID NO.:33; residues
residues 31-35 of SEQ ID NO.:34; residues
residues 50-66 of SEQ ID NO.:34; residues
residues 99-105 of SEQ ID NO.:34; residues
residues 24-39 of SEQ ID NO.:35; residues
residues 55-61 of SEQ ID NO.:35; residues
residues 94-102 of SEQ ID NO.:35; residues
residues 31-35 of SEQ ID NO.:36; residues
residues 50-66 of SEQ ID NO.:36; residues
residues 99-109 of SEQ ID NO.:36; residues
residues 24-39 of SEQ ID NO.:37; residues
residues 55-61 of SEQ ID NO.:37; residues
residues 94-102 of SEQ ID NO.:37; residues
residues 31-35 of SEQ ID NO.:38; residues
residues 50-66 of SEQ ID NO.:38; residues
residues 99-109 of SEQ ID NO.:38; residues
residues 31-35 of SEQ ID NO.:39; residues
residues 50-66 of SEQ ID NO.:39; residues
residues 99-112 of SEQ ID NO.:39; residues
residues 24-39 of SEQ ID NO.:40; residues
residues 55-61 of SEQ ID NO.:40; residues
residues 94-102 of SEQ ID NO.:40; residues
residues 31-35 of SEQ ID N0.:41; residues
residues 50-66 of SEQ ID NO.:41; residues
residues 99-112 of SEQ ID NO.:41; residues
residues 31-35 of SEQ ID NO.:42; residues
residues 50-66 of SEQ ID NO.:42; residues
residues 99-100 of SEQ ID NO.:42; residues
residues 24-39 of SEQ ID NO.:43; residues
residues 55-61 of SEQ ID NO.:43; residues
residues 94-102 of SEQ ID NO.:43; residues
residues 31-35 of SEQ ID NO.:44; residues
residues 50-65 of SEQ ID NO.:44; residues
residues 98-106 of SEQ ID NO.:44; residues
residues 24-40 of SEQ ID NO.:45; residues
residues 56-62 of SEQ ID NO.:45; residues
residues 95-103 of SEQ ID NO.:45; residues
residues 32-38 of SEQ ID NO.:46; residues
residues 52-67 of SEQ ID NO.:46; residues
residues 100-112 of SEQ ID NO.:46; residues

52-67 of SEQ ID NO.:48; 100-112 of SEQ ID NO.:48; 24-34 of SEQ ID NO.:49; 50-56 of SEQ ID NO.:49; 89-97 of SEQ ID NO.:49; 31-37 of SEQ ID NO.:50; 52-67 of SEQ ID NO.:50; 100-112 of SEQ ID NO.:50; 24-34 of SEQ ID NO.:51; 60-66 of SEQ ID NO.:51; 89-97 of SEQ ID N0.:51; 31-35 of SEQ ID NO.:52; 50-66 of SEQ ID NO.:52; 99-107 of SEQ^ ID NO.:52; 23-36 of SEQ ID NO.:53; 52-58 of SEQ ID NO.:53; 91-99 of SEQ ID NO.:53; 31-35 of SEQ ID NO.:54; 50-65 of SEQ ID NO.:54; 98-107 of SEQ ID NO.:54; 24-38 of SEQ ID NO.:55; 54-60 of SEQ ID NO.:55; 93-101 of SEQ ID NO.:55; 31-35 of SEQ ID NO.:56; 50-65 Of SEQ IDNO.:56; 98-107 of SEQ ID NO.:56; 24-38 of SEQ ID NO.:57; 54-60 of SEQ ID NO.:57; 93-101 of SEQ ID NO.:57; 31-35 of SEQ ID NO.:58; 50-65 of SEQ ID NO.:58; 98-107 of SEQ ID NO.:58; 24-38 of SEQ ID NO.:59; 54-60 of SEQ ID NO.:59; 93-101 of SEQ ID NO.:59; 31-35 of SEQ ID NO.:60; 50-65 of SEQ ID NO.:60; 98-107 of SEQ ID NO.:60; 24-38 of SEQ IDN0.:61; 54-60 of SEQ ID N0.:61; 93-101 of SEQ ID N0.:61; 31-35 of SEQ ID NO.:62; 50-65 of SEQ ID NO.:62; 98-107 of SEQ ID NO.:62; 24-38 of SEQ ID NO.:63;

residues 24-34 of SEQ ID NO.:47,- residues 54-60 of SEQ ID NO.:63; residues 50-56 of SEQ ID NO.:47; and
residues 89-97 of SEQ ID NO.:47; residues 93-101 of SEQ ID NO.:63. residues 31-37 of SEQ ID NO.:48;
In a preferred embodiment, the binding protein comprises at least 3 CDRs are selected from a variable domain CDR set consisting of:
(TABLE REMOVED)
Preferably the binding protein comprising at least two variable domain CDR sets. Preferably at least two variable domain CDR sets are selected from a group consisting of:
VH 25C8 CDR Set & VL 25C8 CDR Set;
VH 9C11 CDR Set & VL 9C11 CDR Set;
VH 21D9 CDR Set & VL 21D9 CDR Set;
VH 22D10 CDR Set & VL 22D10 CDR Set;
VH 5F1 CDR Set & VL 5F1 CDR Set;
VH 5G1 CDR Set & VL 5G1 CDR Set;
VH 3H7 CDR Set & VL 3H7 CDR Set;
VH 14B2 CDR Set & VL 14B2 CDR Set;
VH 13C5 CDR Set & VL 13C5 CDR Set;
VH 29G5 CDR Set & VL 2905 CDR Set;
VH 33C3 CDR Set & VL 33C3 CDR Set;
VH 4A8 CDR Set & VL 4A8 CDR Set;
VH 1B6 CDR Set & VL 136 CDR Set;
VH 3E5CDR Set & VL 3E5 CDR Set;
VH 6C8 CDR Set & VL 6C8 CDR Set;
VH 5D3 CDR Set & VL 5D3 CDR Set; and VH 8B6 CDR Set & VL 8B6 CDR Set.
In another embodiment the binding protein disc10sed above further comprises a human acceptor framework. Preferably the human acceptor framework comprises an amino acid sequence selected from the group consisting of:
SEQ ID N0.:6 SEQ ID N0.:16 SEQ ID NO.:26
SEQ ID N0.:7 SEQ ID N0.:17 SEQ ID NO.:27
SEQ ID NO.-.8 SEQ ID NO.-.18 SEQ ID NO.-.28
SEQ ID N0.:9 SEQ ID N0.:19 SEQ ID NO.:29
SEQ ID NO.:10 SEQ ID NO.:20 SEQ ID NO.:30
SEQ ID N0.:11 SEQ ID N0.:21 AND
SEQ ID N0.:12 SEQ ID NO.:22 SEQ ID N0.:31
SEQ ID N0.:13 SEQ ID NO.:23
SEQ ID N0.:14 SEQ ID NO.:24
SEQ ID N0.:15 SEQ ID NO.:25
In a preferred embodiment the binding protein is a CDR grafted antibody or antigen binding portion thereof capable of binding IL-I3. Preferably the CDR grafted antibody or antigen binding portion thereof comprise one or more CDRs disc10sed above. Preferably the CDR grafted antibody or antigen binding portion thereof comprises a human acceptor framework. More preferably the human acceptor framework is any one of the human acceptor frameworks disc10sed above.
In a preferred embodiment the binding protein is a humanized antibody or antigen binding portion thereof capable of binding IL-13. Preferably the humanized antibody or antigen binding portion thereof comprise one or more CDRs disc10sed above incorporated into a human antibody variable domain of a human acceptor framework. Preferably the human antibody variable domain is a consensus human variable domain. More preferably the human acceptor framework comprises at least one Framework Region amino acid substitution at a key residue, wherein the key residue is selected from the group consisting of a residue adjacent to a CDR; a glycosylation site residue; a rare residue; a residue capable of interacting with human IL-13; a residue capable of interacting with a CDR; a canonical residue; a contact residue between heavy chain variable region and light chain variable region; a residue within a Vernier zone; and a residue in a region that overlaps between a Chothia-defined variable heavy chain CDRl and a Kabat-defined first heavy chain framework. Preferably the human acceptor framework human acceptor framework comprises at least one Framework Region amino acid substitution, wherein the amino acid sequence of the framework is at least 65% identical to the sequence of said human
acceptor framework and comprises at least 70 amino acid residues identical to said human acceptor framework. Preferably the Framework Region amino acid substitution at a key residue is selected from the group consisting of 2L, 15L, 22L, 41L, 42L, 44L, 49L, SOL, 51L, 62L, 71L, 73L. 10H, 44H, 46H. 48H, 67H, 68H, 70H, 72H, 74H, 76H, 83H, 84H, 86H, 87H, and 97H.
In a preferred embodiment the binding protein is a humanized antibody or antigen binding portion thereof capable of binding IL-13. Preferably the humanized antibody, or antigen binding portion, thereof comprises one or more CDRs disc10sed above. More preferably the humanized antibody, or antigen binding portion, thereof comprises three or more CDRs disc10sed above. Most preferably the humanized antibody, or antigen binding portion, thereof comprises six CDRs disc10sed above.
In another embodiment of the claimed invention, the humanized antibody or antigen binding portion thereof comprises at least one variable domain having an amino acid sequence selected from the group selected from the group consisting of;
SEQ ID N0.:70 SEQ ID NO.:77 SEQ ID NO.:84
SEQ ID N0.:71 SEQ ID NO.:78 SEQ ID NO.:85
SEQ ID NO.:72 SEQ ID NO.:79 SEQ ID NO.:92
SEQ ID NO.:73 SEQ ID NO.:80 SEQ ID NO.:93
SEQ ID NO.:74 SEQ ID N0.:81 and
SEQ ID NO.:75 SEQ ID NO.:82 SEQ ID NO.:94.
SEQ ID NO.:76 SEQ ID NO.:83
More preferably the humanized antibody or antigen binding portion thereof comprises two variable domains selected from the group disc10sed above. More preferably binding protein comprises two variable domains, wherein said two variable domains have amino acid sequences selected from the group consisting of;
SEQ ID NO.:70 & SEQ ID N0.:71, SEQ ID NO.:72 & SEQ ID NO.:73, SEQ ID NO.:74 & SEQ ID NO.:75, SEQ ID NO.:76 & SEQ ID NO.:77, SEQ ID NO.:78 & SEQ ID NO.:79, SEQ ID NO.:80 & SEQ ID NO.:81, SEQ ID NO.:82 & SEQ ID NO.:83, SEQ ID NO.:84 & SEQ ID NO.:85 SEQ ID NO.:80 & SEQ ID NO.:92, SEQ ID NO.:80 & SEQ ID NO.:93, AND SEQ ID NO.:80 & SEQ ID NO.:94.
One embodiment of the invention provides an antibody construct comprising any one of the binding proteins disc10sed above and a linker polypeptide or an immunog10bulin. In a preferred embodiment the antibody construct is selected from the group consisting of an immunog10bulin molecule, a monoc10nal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab', a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody, a diabody, a multispecific antibody, a dual specific antibody, and a bispecific antibody. In a preferred embodiment the antibody construct comprises a heavy chain immunog10bulin constant domain selected from the group consisting of a human IgM constant domain, a human IgOl constant domain, a human IgG2 constant domain, a human IgG3 constant domain, a human IgG4 constant domain, a human IgE constant domain, and a human IgA constant domain. More preferably, the antibody construct comprises SEQ ID N0.:2; SEQ ID N0.:3; SEQ ID N0.:4; and SEQ ID N0.:5. In another embodiment the invention provides an antibody conjugate comprising an the antibody construct disc10sed above and an agent an agent selected from the group consisting of; an immunoadhension molecule, an imaging agent, a therapeutic agent, and a cytotoX1c agent. In a preferred embodiment the imaging agent selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. More preferably the imaging agent is a radiolabel selected from the group consisting of: 'H. '"C, '^S, Y, C, "'in, ','l"LU, '"HO, and "'Sm. In a preferred embodiment the therapeutic or cytotoX1c agent is selected from the group consisting of; an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, toX1n, and an apoptotic agent.
In another embodiment the antibody construct is glycosylated. Preferably the glycosylation is a human glycosylation pattern.
In another embodiment binding protein, antibody construct or antibody conjugate disc10sed above eX1sts as a crystal. Preferably the crystal is a carrier-fi'ee pharmaceutical controlled release crystal. In a preferred embodiment the crystallized binding protein, crystallized antibody construct or crystallized antibody conjugate has a greater half life in vivo than its soluble counterpart. In another preferred embodiment the crystallized binding protein, crystallized antibody construct or crystallized antibody conjugate retains bio10gical activity after crystallization.
One aspect of the invention pertains to a DVD binding protein comprising binding proteins capable of binding IL-13. Preferably the DVD binding protein is capable of binding IL-13 and a second target. The second target is selected from the group consisting of CSFl (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FGF2, IFNal, IFNPI, IFNy, histamine and histamine receptors.
BL-la, IL-ip, IL-2, IL-3, BL^, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-11, IL-12ot, IL-12P, IL-14, IL-15, E.-16, IL-n, IL-18, IL-19, KITLG, PDGFB, IL-2Ra, IL-4R, IL-5Ra, lL-8Ra, IL-8RP, IL-12RP1, IL-12RP2, IL-13Ral, IL-13Ra2, IL-18R1, TSLP, CCLl, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL17, CCL18, CCL19, CCL20, CCL22, CCL24,CX3CL1, CXCLl, CXCL2, CXCL3, XCLl, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1, GPR2, XCRl, FOS, GATA3, JAKl, JAK3, STAT6, TBX21, TGFBl, TNFSF6, YYl, CYSLTRl, FCERl A, FCER2, LTB4R, TB4R2, LTBR, and Chitinase. More preferably, the DVD protein is capable of recognizing IL-13 and IL-ip, IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-p; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; or IL-13 and ADAMS. Most preferably, the DVD protein is capable of binding IL-13 and TNFa.
One aspect of the invention pertains to an isolated nucleic acid encoding any one of the binding protein, antibody construct or antibody conjugate disc10sed above. A further embodiment provides a vector comprising the isolated nucleic acid disc10sed above wherein said vector is selected from the group consisting of pcDNA; pTT (Durocher et al., Nucleic Acids Research 2002, Vol 30, No.2); pTT3 (pTT with additional multiple c10ning site; pEFBOS (Mizushima, S. and Nagata, S., (1990) Nucleic acids Research Vol 18, No. 17); pBV; pJV; and pBJ.
In another aspect a host cell is transformed with the vector disc10sed above. Preferably the host cell is a prokaryotic cell. More preferably the host cell is E.Coli. In a related embodiment the host cell is an eukaryotic cell. Preferably the eukaryotic cell is selected from the group consisting of protist cell, animal cell, plant cell and fungal ceil. More preferably the host cell is a mammalian cell including, but not limited to, CHO and COS; or a fungal cell such as Saccharomyces cerevisiae; or an insect cell such as Sf9.
Another aspect of the invention provides a method of producing a binding protein that binds IL-13, comprising culturing any one of the host cells disc10sed above in a culture medium under conditions sufficient to produce a binding protein that binds IL-13. Another embodiment provides a binding protein produced according to the method disc10sed above.
One embodiment provides a composition for the release of a binding protein wherein the composition comprises a formulation which in turn comprises a crystallized binding protein, crystallized antibody construct or crystallized antibody conjugate as disc10sed above and an ingredient; and at least one polymeric carrier. Preferably the polymeric carrier is a polymer selected from one or more of the group consisting of: poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate), poly (caprolactone), poly
(dioxanone); poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide, poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride- alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellu10se and cellu10se derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polyeaccharides, blends and copolymers thereof. Preferably the ingredient is selected from the group consisting of albumin, sucrose, treha10se, lactitol, gelatin, hydroxypropyl-P" cyc10dextrin, methoxypolyethylene glycol and polyethylene glycol. Another embodiment provides a method for treating a mammal comprising the step of administering to the mammal an effective amount of the composition disc10sed above.
The invention also provides a pharmaceutical composition comprising a binding protein, antibody construct or antibody conjugate as disc10sed above and a pharmaceutically acceptable carrier. In a further embodiment the pharmaceutical composition comprises at least one additional therapeutic agent for treating a disorder in which IL-13 activity is detrimental. Preferably the additional agent is selected from the group consisting of: Therapeutic agent, imaging agent, cytotoX1c agent, angiogenesis inhibitors (including but not limited to anti-VEGF antibodies or VEGF-trap); kinase inhibitors (including but not limited to KDR and TIE-2 inhibitors); co-stimulation molecule b10ckers (including but not limited to anti-B7.1, anti-B7.2, CTLA4-Ig, anti-CD20); adhesion molecule b10ckers (including but not limited to anti-LPA-1 Abs, anti-E/L selectin Abs, small molecule inhibitors); anti-cytokine antibody or functional fragment thereof (including but not limited to anti-IL-18, anti-TNF, anti-IL-6/cytokine receptor antibodies); methotrexate; cyc10sporin; rapamycin; FK506; detectable label or reporter; a TNF antagonist; an antirheumatic; a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a 10cal anesthetic, a neuromuscular b10cker, an antimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin, an immunization, an immunog10bulin, an immunosuppressive, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an epinephrine or ana10g, a cytokine, and a cytokine antagonist.
In another aspect, the invention provides a method for inhibiting human IL-13 activity comprising contacting human IL-13 with a binding protein disc10sed above such that human IL-13 activity is inhibited. In a related aspect the invention provides a method for inhibiting human IL-13 activity in a human subject suffering from a disorder in which IL-13 activity is detrimental, comprising administering to the human subject a binding protein disc10sed above such that human IL-13 activity in the human subject is inhibited and treatment is achieved.
In another aspect, the invention provides a method of treating (e.g., curing, suppressing, ameliorating, delaying or preventing the onset of, or preventing recurrence or relapse of) or preventing an IL-13-associated disorder, in a subject. The method includes: administering to the
subject an JL-13 binding agent (particularly an antagonist); e:g., an anti-IL-13 antibody or fragment thereof as described herein, in an amount sufficient to treat or prevent the IL-13-associated disorder. The IL-13 antagonist, e.g., the anti-IL-13 antibody or fragment thereof, can be administered to the subject, a10ne or in combination with other therapeutic modalities as described herein.
In one embodiment, the subject is a mammal, e.g., a human suffering from one or more IL-13-associated disorders, including, e.g., respiratory disorders (e.g., asthma (e.g., allergic and nonallergic asthma), chronic obstructive pulmonary disease (COPD), and other conditions involving airway inflammation, eosinophilia, fibrosis and excess mucus production; atopic disorders (e.g., atopic dermatitis and allergic rhinitis); inflammatory and/or autoimmune conditions of, the skin, gastrointestinal organs (e.g., inflammatory bowel diseases (EBD), such as ulcerative colitis and/or Crohn's disease), and liver (e.g., cirrhosis, fibrosis); scleroderma; tumors or cancers, e.g., Hodgkin's lymphoma as described herein. Accordingly, the disc10sure includes the use of an IL-13 binding agent (such as an anti-IL-13 antibody or fragment thereof described herein) for a treatment described herein and the use of an IL-13 binding agent (such as an anti-IL-13 antibody or fragment thereof described herein) for preparing a medicament for a treatment described herein. Examples of IL-13-associated disorders include, but are not limited to, a disorder chosen from one or more of: respiratory disorders, e.g., asthma (e.g., allergic and nonallergic asthma (e.g., asthma due to infection with, e.g., respiratory syncytial virus (RSV), e.g., in younger children)), chronic obstructive pulmonary disease (COPD), and other conditions involving airway inflammation, eosinophilia, fibrosis and excess mucus production, e.g., cystic fibrosis and pulmonary flbrosis; atopic disorders, e.g., resulting from an increased sensitivity to IL-13 (e.g., atopic dermatitis, urticaria, eczema, allergic rhinitis, and allergic enterogastritis); inflammatoyv and/or autoimmune conditions of, the skin (e.g., atopic dermatitis), gastrointestinal organs (e.g., inflammatory bowel diseases (IBD), such as ulcerative colitis and/or Crohn's disease), liver (e.g., cirrhosis, hepatocellular carcinoma), and scleroderma; tumors or cancers (e.g., soft tissue or solid tumors), such as leukemia, glioblastoma, and lymphoma, e.g., Hodgkin's lymphoma; viral infections (e.g., from HTLV-1); fibrosis of other organs, e.g., fibrosis of the liver, (e.g., fibrosis caused by a hepatitis B and/or C virus); and suppression of expression of protective type 1 immune responses, (e.g., during vaccination), as described herein.
In other embodiments, this application provides a method of treating (e.g., reducing, ameliorating) or preventing one or more symptoms associated with a respiratory disorder, e.g., asthma (e.g., allergic and nonallergic asthma); allergies; chronic obstructive pulmonary disease (COPD); a condition involving airway inflammation, eosinophilia, fibrosis and excess mucus production, e.g., cystic fibrosis and pulmonary fibrosis. For example, symptoms of asthma include, but are not limited to, wheezing, shortness of breath, bronchoconstriction, airway

hyperreactivity, decreased lung capacity, fibrosis, airway inflanunation, and mucus production. The method comprises administering to the subject an IL-13 antagonist, e.g., an IL-13 antibody or a fragment thereof, in an amount sufficient to treat (e.g., reduce, ameliorate) or prevent one or more symptoms. The IL-13 antibody can be administered therapeutically or prophytactically, or both. The IL-13 antagonist, e.g., the anti-IL-13 antibody, or fragment thereof, can be administered to the subject, a10ne or in combination with other therapeutic modalities as described herein. Preferably, the subject is a mammal, e.g., a human suffering from an IL-13- associated disorder as described herein.
In another aspect, this application provides a method for detecting the presence of IL-13 in a sample in vitro (e.g., a bio10gical sample, such as serum, plasma, tissue, biopsy). The subject method can be used to diagnose a disorder, e.g., an immune cell-associated disorder. The method includes: (i) contacting the sample or a control sample with the anti-IL-13 antibody or fragment thereof as described herein; and (ii) detecting formation of a complex between the anti-IL-13 antibody or fragment thereof, and the sample or the control sample, wherein a statistically significant change in the formation of the complex in the sample relative to the control sample is indicative of the presence of the IL-13 in the sample.
In yet another aspect, this application provides a method for detecting the presence of IL-13 in vivo (e.g., in viva imaging in a subject). The subject method can be used to diagnose a disorder, e.g., an IL-13- associated disorder. The method includes: (i) administering the anti-IL-13 antibody or fragment thereof as described herein to a subject or a control subject under conditions that al10w binding of the antibody or fragment to IL-13; and (ii) detecting formation of a complex between the antibody or fragment and IL-13, wherein a statistically significant change in the formation of the complex in the subject relative to the control 'Gubject is indicative of the presence of IL-13.
In another aspect, the binding proteins of the invention are useful for treating a disorder selected from the group consisting of arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondy10arthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, insulin dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitis scleroderma, graft versus host disease, organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease. Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granu10matosis, Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toX1c shock syndrome, sepsis syndrome, cacheX1a, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary
cirrhosis, hemolytic anemia, malignancies, heart failure, myocardial infarction, Addison's disease, sporadic, polyglandular deficiency type I and polyglandular deficiency type H, Schmidt's syndrome, adult (acute) respiratory distress syndrome, a10pecia, a10pecia areata, seronegative arthopathy, arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic synovitis, chlamydia, yersinia and salmonella associated arthropathy, spondy10arthopathy, atheromatous disease/arteriosclerosis, atopic allergy, autoimmune bul10us disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia. Coombs positive haemolytic anaemia, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic encephalitis/Royal Free Disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis. Acquired Immunodeficiency Disease Syndrome, Acquired Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C, common varied immunodeficiency (common variable hypogammag10bulinaemia), dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian failure, fibrotic lung disease, cryptogenic fibrosing alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonitis, connective tissue disease associated interstitial lung disease, mixed connective tissue disease associated lung disease, systemic sclerosis associated interstitial lung disease, rheumatoid arthritis associated interstitial lung disease, systemic lupus erythematosus associated lung disease, dermatomyositis/polymyositis associated lung disease, Sjogren's disease associated lung disease, anky10sing spondylitis associated lung disease, vasculitic diffuse lung disease, haemosiderosis associated lung disease, drug-induced interstitial lung disease, fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia, autoimmune neutropaenia, renal disease NOS, g10meru10nephritides, microscopic vasulitis of the kidneys, lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis. Still's disease, systemic sclerosis, Sjorgren's syndrome, Takayasu's disease/arteritis, autoimmune thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic
uveitis, primary vasculitis, vitiligo acute liver disease, chronic liver diseases, alcoholic cirrhosis, alcohol-induced liver injury, choleosatatis, idiosyncratic liver disease, Drug-Induced hepatitis. Non-alcoholic Steatohepatitis, allergy and asthma, group B streptococci (GBS) infection, mental disorders (e.g., depression and schizophrenia), Th2 Type and Thl Type mediated diseases, acute and chronic pain (different forms of pain), and cancers such as lung, breast, stomach, bladder, co10n, pancreas, ovarian, prostate and rectal cancer and hematopoietic malignancies (leukemia and lymphoma), Abetalipoprotemia, Acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute mye10id leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, adenocarcinomas, aerial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, al10graft rejection, alpha-l- antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, angina pectoris, anterior horn cell degeneration, anti cd3 therapy, antiphospholipid syndrome, anti-receptor hypersensitivity reactions, aordic and peripheral aneuryisms, aortic dissection, arterial hypertension, arteriosclerosis, arteriovenous fistula, ataX1a, atrial fibrillation (sustained or paroxysmal), atrial flutter, atrioventricular b10ck, B cell lymphoma, bone graft rejection, bone marrow transplant (BMT) rejection, bundle branch b10ck, Burkitt's lymphoma. Bums, cardiac arrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation response, cartilage transplant rejection, cerebellar cortical degenerations, cerebellar disorders, chaotic or multifocal atrial tachycardia, chemotherapy associated disorders, chromic mye10cytic leukemia (CML), chronic alcoholism, chronic inflammatory patho10gies, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate intoX1cation, co10rectal carcinoma, congestive heart failure;.conjunctivitis, contact dermatitis, cor pulmon^cle, coronarj^tutery disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic fibrosis, cytokine therapy associated disorders. Dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever, dermatitis, dermato10gic conditions, diabetes, diabetes mellitus, diabetic ateriosclerotic disease, Diffuse Lewy body disease, dilated congestive cardiomyopathy, disorders of the basal ganglia, Down's Syndrome in middle age, drug- induced movement disorders induced by drugs which b10ck CNS dopamine receptors, drug sensitivity, eczema, encepha10myelitis, endocarditis, endocrinopathy, epig10ttitis, epstein-barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial hematophagocytic lymphohistiocytosis, fetal thymus implant rejection, Friedreich's ataX1a, functional peripheral arterial disorders, fungal sepsis, gas gangrene, gastric ulcer, g10merular nephritis, graft rejection of any organ or tissue, gram negative sepsis, gram positive sepsis, granu10mas due to intracellular organisms, hairy cell leukemia, Hallerrorden-Spatz disease, hashimoto's thyroiditis, hay fever, heart transplant rejection, hemachromatosis, hemodialysis, hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, hemorrhage.
hepatitis (A), His bundle arrythmias, HIV infection/HIV neuropathy, Hodgkin's disease, hyperkinetic movement disorders, hypersensitity reactions, hypersensitivity pneumonitis, hypertension, hypokinetic movement disorders, hypothalamic-pituitary-adrenal aX1s evaluation, idiopathic Addison's disease, idiopathic pulmonary fibrosis, antibody mediated cytotoX1city. Asthenia, infantile spinal muscular atrophy, inflammation of the aorta, influenza a, ionizing radiation exposure, iridocyclitis/uveitis/optic neuritis, ischemia- reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, kidney transplant rejection, legionella, leishmaniasis, leprosy, lesions of the corticospinal system, lipedema, liver transplant rejection, lymphederma, malaria, malignamt Lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic/idiopathic, migraine headache, mitochondrial multi-system disorder, mixed connective tissue disease, monoc10nal gammopathy, multiple mye10ma, multiple systems degenerations (Mencel Dejerine- Thomas Shi-Drager and Machado-Joseph), myasthenia gravis, mycobacterium avium intracellulare, mycobacterium tubercu10sis, mye10dyplastic syndrome, myocardial infarction, myocardial ischemic disorders, nasopharyngeal carcinoma, neonatal chronic lung disease, nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscular atrophies, neutropenic fever, non-hodgkins lymphoma, occlusion of the abdominal aorta and its branches, occulsive arterial disorders, okt3 therapy, orchitis/epidydimitis, orchitis/vasectomy reversal procedures, organomegaly, osteoporosis, pancreas transplant rejection, pancreatic carcinoma, paraneoplastic syndrome/hypercalcemia of malignancy, parathyroid transplant rejection, pelvic inflanunatory disease, perennial rhinitis, pericardial disease, peripheral ather10sclerotic disease, peripheral vascular disorders, peritonitis, pernicious anemia, Pneumocystis carinii pneumonia, pneumonia, POEMS syndrome (polyneuropathy; organomegaly, endocrinopathy, n&>n6c10nul gaiflmopathy, and skin changes syndrome), post perfusion syndrome, post pump syndrome, post-MI cardiotomy syndrome, preeclampsia. Progressive supranucleo Palsy, primary pulmonary hypertension, radiation therapy, Raynaud's phenomenon and disease, Raynoud's disease, Refsum's disease, regular narrow QRS tachycardia, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea. Senile Dementia of Lewy body type, seronegative arthropathies, shock, sickle cell anemia, skin al10graft rejection, skin changes syndrome, small bowel transplant rejection, solid tumors, specific arrythmias, spinal ataX1a, spinocerebellar degenerations, streptococcal myositis, structural lesions of the cerebellum, Subacute sclerosing panencephalitis. Syncope, syphilis of the cardiovascular system, systemic anaphalaX1s, systemic inflammatory response syndrome, systemic onset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia, thromboangitis obliterans, thrombocytopenia, toX1city, transplants, trauma/hemorrhage, type HI hypersensitivity reactions, type IV hypersensitivity, unstable angina, uremia, urosepsis, urticaria, valvular heart diseases, varicose
veins, ,vasculitis, venous diseases, venous thrombosis, ventricular fibrillation, viral and fungal infections, vital encephalitis/aseptic meningitis, vital-associated hemaphagocytic syndrome, Wernicke- Korsakoff syndrome, Wilson's disease, xenograft rejection of any organ or tissue, Acute coronary syndromes. Acute Idiopathic Polyneuritis, Acute Inflammatory Demyelinating Polyradicu10neuropathy, Acute ischemia, Adult Still's Disease, A10pecia areata, AnaphylaX1s, Anti-Phospholipid Antibody Syndrome, Aplastic anemia. Arteriosclerosis, Atopic eczema. Atopic dermatitis. Autoimmune dermatitis. Autoimmune disorder associated with Streptococcus infection, Autoinunune Enteropathy, Autoimmune hearing10ss. Autoimmune Lymphoproliferative Syndrome (ALPS), Autoimmune myocarditis, Autoimmune premature ovarian failure. Blepharitis, Bronchiectasis, Bul10us pemphigoid. Cardiovascular Disease, Catastrophic Antiphospholipid Syndrome, Celiac Disease, Cervical Spondy10sis, Chronic ischemia. Cicatricial pemphigoid, Clinically isolated Syndrome (CIS) with Risk for Multiple Sclerosis, Conjunctivitis, Childhood Onset Psychiatric Disorder, Chronic obstructive pulmonary disease (COPD), Dacryocystitis, dermatomyositis. Diabetic retinopathy. Diabetes meilitus, Disk herniation. Disk prolaps. Drug induced immune hemolytic anemia. Endocarditis, Endometriosis, endophthalmitis. Episcleritis, Erythema multiforme, erythema multiforme major, Gestational pemphigoid, Guillain-Barre Syndrome (GBS), Hay Fever, Hughes Syndrome, Idiopathic Parkinson's Disease, idiopathic interstitial pneumonia, IgE-mediated Allergy, Immune hemolytic anemia. Inclusion Body Myositis, Infectious ocular inflammatory disease , Inflammatory demyelinating disease. Inflammatory heart disease. Inflammatory kidney disease, IPF/UIP, Iritis, Keratitis, Keratojuntivitis sicca, Kussmaul disease or Kussmaul-Meier Disease, Landry's Paralysis, Langerhan's Cell Histiocytosis, Livedo reticularis. Macular Degeneration, Microscopic Polyangiitis, Morbus Bechterev; Motor Neuron-Disorders, Mucous membrane pemphigoid, Multiple Organ failure. Myasthenia Gravis, Mye10dysplastic Syndrome, Myocarditis, Nerve Root Disorders, Neuropathy, Non-A Non-B Hepatitis, Optic Neuritis, Osteolysis, Pauciarticular JRA , peripheral artery occlusive disease (PAOD), peripheral vascular disease (PVD), peripheral artery disease (PAD), Phlebitis, Polyarteritis nodosa (or periarteritis nodosa), Polychondritis, Polymyalgia Rheumatica, Poliosis, Polyarticular JRA, Polyendocrine Deficiency Syndrome, Polymyositis, polymyalgia rheumatica (PMR), Post-Pump Syndrome, primary parkinsonism. Prostatitis, Pure red cell aplasia. Primary Adrenal Insufficiency, Recurrent Neuromyelitis Optica, Restenosis, Rheumatic heart disease, SAPHO (synovitis, acne, pustu10sis, hyperostosis, and osteitis). Scleroderma, Secondary Amy10idosis, Shock lung, Scleritis, Sciatica, Secondary Adrenal Insufficiency, Silicone associated connective tissue disease, Sneddon-Wilkinson Dermatosis, spondilitis anky10sans, Stevens-Johnson Syndrome (SJS), Systemic inflammatory response syndrome. Temporal arteritis, toxoplasmic retinitis, toX1c epidermal necrolysis, Transverse myelitis, TRAPS (Tumor Necrosis Factor Receptor, Type 1 allergic reaction. Type 11
Diabetes, Urticaria, Usual interstitial pneumonia (UIP), Vasculitis, Vernal conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome). Wet macular degeneration, and Wound healing.
In another aspect, the binding proteins of the invention are useful for treating a disorder selected from the group consisting of Acute Lymphoblastic Leukemia, Acute Mye10id Leukemia, Adrenocortical Carcinoma, Anal Cancer, Appendix Cancer, Cerebellar Astrocytoma, Cerebral Astrocytoma, Basal Cell Carcinoma, Bile Duct Cancer, Extrahepatic, Bladder Cancer, Bone Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma Brain Stem Glioma, Brain Tumor, Brain Stem Glioma, Cerebral strocytoma/Malignant Glioma, Ependymoma, Medul10blastoma, Supratentorial Primitive Neuroectodermal Tumors, Visual Pathway and Hypothalamic Glioma, Breast Cancer, Bronchial Adenomas/Carcinoids, Carcinoid Tumor, Carcinoid Tumor,Gastrointestinal Carcinoma of Unknown Primary, Central Nervous System Lymphoma, Primary Cerebellar Astrocytoma, Cervical Cancer, Chronic Lymphocytic Leukemia, Chronic Mye10genous Leukemia Chronic Mye10proliferative Disorders, Co10n Cancer, Co10rectal Cancer, Cutaneous T-Cell Lymphoma,, Endometrial Cancer, Ependymoma, Esophageal Cancer, Ewing Family of Tumors, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Intraocular Melanoma Retinoblastoma, Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor (GIST), Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Ovarian Germ Cell Tumor, Gestational Trophoblastic Tumor, Glioma, Brain Stem Glioma, Cerebral Astrocytoma Glioma, Childhood Visual Pathway and Hypothalamic Glioma, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular (Liver) Cancer, Hodgkin Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Carcinoma (Endocrine Pancreas), Kaposi Sarcoma, Kidney (Renal Cell) Cancer, Laryngeal Cancer, Acute Lymphoblastic Leukemia, Acute Mye10id Leukemia, Chronic Lymphocytic Leukemia, Chronic Mye10genous Leukemia, Hairy Cell Leukemia, Lip and Oral Cavity Cancer, Liver Cancer, Non-Small Cell Lung Cancer, Small Cell Lung Cancer, AIDS-Related Lymphoma, Burkitt Lymphoma, Cutaneous T-Cell Lymphoma, Hodgkin Lymphoma, Non-Hodgkin Lymphoma, Primary Central Nervous System Lynq)honia, WaldenstrSm Macrog10bulinemia, Malignant Fibrous Histiocytoma of Bone/Osteosarcoma, Medul10blastoma, Melanoma, Intraocular (Eye) Melanoma, Merkel Cell Carcinoma, Malignant Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Mouth Cancer, Multiple Endocrine Neoplasia Syndrome, Multiple Mye10ma/Plasma Cell Neoplasm, Mycosis Fungoides, Mye10dysplastic Syndromes, Mye10dysplastic/Mye10proliferative Diseases, Mye10genous Leukemia, Chronic Mye10id Leukemia, Multiple Mye10ma, Mye10proliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Oral Cancer, Oral Cavity Cancer, Lip and Oropharyngeal Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma of
Bone, Ovarian Cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian 10w Malignant Potential Tumor, Pancreatic Cancer, Islet Cell Pancreatic Cancer, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pineoblastoma and Supratentorial Primitive Neuroectodermal Tumors, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Mye10ma, Pleuropulmonary Blastoma, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional Cell Cancer, Retinoblastoma, Salivary Gland Cancer, Sarcoma, Ewing Family of Tumors, Kaposi Sarcoma, Soft Tissue Sarcoma, Uterine Sarcoma, S6zary Syndrome, Skin Cancer (Nonmelanoma), Skin Cancer (Melanoma), Merkel Cell Skin Carcinoma, Small Intestine Cancer, Squamous Cell Carcinoma, Metastatic Squamous Neck Cancer with Occult Primary, Stomach (Gastric) Cancer, Supratentorial Primitive Neuroectodermal Tumors, Cutaneous T-Cell Lymphoma, Testicular Cancer, Throat Cancer, Thymoma, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Gestational Trophoblastic Tumor, Ureter and Renal Pelvis, Transitional Cell Cancer, Urethral Cancer, Uterine Cancer, Endometrial Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom Macrog10bulinemia, Wilms Tumor.
In another aspect the invention provides a method of treating a patient suffering from a disorder in which human IL-13 is detrimental comprising the step of administering any one of the binding proteins disc10sed above before, concurrent, or after the administration of a second agent, as discussed above. In a preferred embodiment the additional therapeutic agent that can be coadministered and/or coformulated with one or more IL-13 antagonists, (e. g., anti- IL-13 antibodies or fragments thereof,) include, but are not limited to, one or more of: inhaled steroids; oral steroids; beta-agonistS, e.g., short-acting or 10ng acting beta-agonists; antagonists of leukotrienes or leukotriene receptors; combination drugs such as ADVAIR; IgE inhibitors, e.g., anti-IgE antibodies (e. g., XOLAIR); phosphodiesterase inhibitors (e. g., PDE4 inhibitors); xanthines; anticholinergic drugs; mast cell-stabilizing agents such as cromolyn; IL-4 inhibitors; IL-5 inhibitors; eotaX1n/CCR3 inhibitors; antagonists of histamine or its receptors including HI, H2, H3, and H4, and antagonists of prostaglandin D or its receptors (DPI and CRTH2) . Such combinations can be used to treat asthma and other respiratory disorders. Additional examples of therapeutic agents that can be coadministered and/or coformulated with one or more anti-IL-13 antibodies or fragments thereof include one or more of: TNF antagonists (e.g., a soluble fragment of a TNF receptor, e.g., p55 or p75 human TNF receptor or derivatives thereof, e.g., 75 kD TNFR-IgG (75 kD TNF receptor-IgG fusion protein, ENBREL)); TNF enzyme antagonists, e.g., TNF converting enzyme (TACE) inhibitors; muscarinic receptor antagonists; TGF-beta antagonists; interferon gamma; perfenidone; chemotherapeutic agents, e.g., methotrexate, leflunomide, or a sirolimus (rapamycin) or an ana10g thereof, e.g., CCI-779; C0X2 and cPLA2
inhibitors; NSADDs; immunomodulators; p38 inhibitors, TPL-2, MK-2 and NFkB inhibitors, among others. Additional second agent is selected from the group consisting of budenoside, epidermal growth factor, corticosteroids, cyc10sporin, sulfasalazine, aminosalicylates, 6-mercaptopurine, azathioprine, metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine, balsalazide, antioX1dants, thromboxane inhibitors, IL-1 receptor antagonists, anti-IL-ip monoc10nal antibodies, anti-BL-6 monoc10nal antibodies, growth factors, elastase inhibitors, pyridinyl-imidazole compounds, antibodies or agonists of TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-n, GM-CSF, FGF, and PDGF, antibodies of CD2, CD3, CD4, CDS, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands, methotrexate, cyc10sporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, ibuprofen, corticosteroids, predniso10ne, phosphodiesterase inhibitors, adensosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, IRAK, NIK, IKK, p38, MAP kinase inhibitors, EL-ip converting enzyme inhibitors, TNFoconverting enzyme inhibitors, T-cell signalling inhibitors, metal10proteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors, soluble p55 TNF receptor, soluble p75 TNF receptor, sIL-IRI, sIL-IRH, sIL-6R, antiinflammatory cytokines, IL-4, IL-IO, IL-11, and TGFp.
In a preferred embodiment the pharmaceutical compositions disc10sed above are administered to the subject by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenaU^intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.
One aspect of the invention provides at least one IL-13 anti-idiotype antibody to at least one IL-13 binding protein of the present invention. The anti-idiotype antibody includes any protein or peptide containing molecule that comprises at least a portion of an immunog10bulin molecule such as, but not limited to, at least one complementarily determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or; any portion thereof, that can be incorporated into a binding protein of the present invention.
Detailed Description of the Invention
This invention pertains to human IL-13 binding proteins, particularly anti-IL-13 antibodies, or antigen-binding portions thereof, that bind IL-13. Various aspects of the invention
relate to antibodies and antibody fragments, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such antibodies and fragments. Methods of using the antibodies of the invention to detect human IL-13, to inhibit human IL-13 activity, either in vitro or in vivo; and to regulate gene expression are also encompassed by the invention.
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, deHnitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that con:q>rise more than one subimit unless specifically stated otherwise.
Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular bio10gy, immuno10gy, microbio10gy, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional nvethods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art of a^ described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
That the present invention may be more readily understood, select terms are defined be10w.
The term "Polypeptide" as used herein, refers to any polymeric chain of amino acids. The terms "peptide" and "protein" are used interchangeably with the term polypeptide and also refer to a polymeric chain of amino acids. The term "polypeptide" encompasses native or artificial proteins, protein fragments and polypeptide ana10gs of a protein sequence. A polypeptide may be monomeric or polymeric.
The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally associated components that accompany it in its native state; is substantially free of other proteins from the same species; is expressed by a cell from a different species; or does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
The term "recovering" as used herein, refers to the process of rendering a chemical species such as a polypeptide substantially free of naturally associated components by isolation, e.g., using protein purification techniques well known in the art.
The terms "human 0.-13" and "human E.-13 wild type" (abbreviated herein as h IL-13, h IL-13wt), as used herein, includes a human cytokine that is secreted primarily by T helper 2 cells. The term includes a monomeric protein of 13 kDa polypeptide. The structure of human lL-13 is described further in, for example, (Moy, Diblasio et al. 2001 J Mol Biol 310 219-30). The term human IL-13 is intended to include recombinant human IL-13 (rh IL-13), which can be prepared by standard recombinant expression methods. Table 1 shows the amino acid sequence of human IL-13, SEQ ID No. 1, which is known in the art.
Table 1: Sequence of human IL-13

(TABLE REMOVED)
The term "human IL-13 variant" (abbreviated herein as h IL-13v), as used herein, includes a variant of human IL-13 wherein amino acid residue 130 of SEQ ID NO. 1 is changed from Arginine to Glutamine (R130Q).
"Bio10gical activity " as used herein, refers to all inherent bio10gical properties of the cytokine. Bio10gical properties of IL-13 include but are not limited to binding IL-13 receptor; (other examples include immunog10bulin isotype switching to IgE in human B cells and suppressing inflammatory cytokine production).
The terms "specific binding" or "specifically binding", as used herein, in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific
protein structure rather than to proteins generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody.
The term "antibody", as used herein, broadly refers to any immunog10bulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative anitbody formats are known in the art. Nonlimiting embodiments of which are discussed be10w.
In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the fol10wing order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4. Immunog10bulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgAl and IgA2) or subclass.
The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., hIL-13). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens. 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, Winter et al., PCT publication WO 90/05144 Al herein incorporated by reference), which comprises a single variable 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. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to al10w for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., HoUiger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).
The term "antibody construct" as used herein refers to a polypeptide comprising one or more the antigen binding portions of the invention linked to a linker polypeptide or an immunog10bulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Such linker polypeptides are well known in the art (see e.g., HoUiger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123). An inmiunog10bulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art and represented in Table 2.
Table 2: Sequence of human IgG heavy chain constant domain and light chain constant
domain
(TABLE REMOVED)
Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecules, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S.M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S.M., et al (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.
An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities {e.g., an isolated antibody that specifically binds hIL-13 is substantially free of antibodies that specifically_bind antigens other than hIL-13). An isolated antibody that specifically binds hIL-13 may, however.
have cross-reactivity to other antigens, such as EL-13 molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.
The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunog10bulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunog10bulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CORs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further in Section IIC, be10w), antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom H.R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W.E., (2(X)2) Clin. Biochem. 35:425-445; Gavi10ndo J.V., and Larrick J.W. (2(X)2) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (20(X)) Immuno10gy Today 21:371-378), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunog10bulin genes (see e.g., Tay10r, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L.L. (2002) Current Opinion in Biotechno10gy 13:593-597; Little M. et al (2(XK)) Immuno10gy Today 21:364-370) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunog10bulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunog10bulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally eX1st within the human antibody germline repertoire in vivo. One embodiment provides fully human antibodies capable of binding human IL-13 which can be generated using techniques well known in the art, such as, but not limited to, using human Ig phage libraries such as those disc10sed in Jermutus et al., PCT publication No.WO 2005/007699 A2.
The term "chimeric antibody" refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.
The term "CDR-grafted antibody" refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.
The term "humanized antibody" refers to antibodies which comprise heavy and light chain variable region sequences firom a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more "human-like", i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences. In one embodiment, humanized anti human IL-13 antibodies and antigen binding portions are provided. Such antibodies were generated by obtaining murine anti-hIL-13 monoc10nal antibodies using traditional hybridoma techno10gy fol10wed by humanization using in vitro genetic engineering, such as those disc10sed in Kasaian et al PCT publication No. WO 2005/123126 A2.
The terms "Kabat numbering", "Kabat definitions and "Kabat labeling" are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat etal. (1971) Ann. NY Acad. Sci. 190:382-391 and, Kabat, E.A., etal. (1991) Sequences of Proteins of Immuno10gical Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDRl, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 for CDRl, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.
As used herein, the terms "acceptor" and "acceptor antibody" refer to the antibody or nucleic acid sequence providing or encoding at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% of the amino acid sequences of one or more of the framework regions. In some embodiments, the term "acceptor" refers to the antibody amino acid or nucleic acid sequence providing or encoding the constant region(s). In yet another embodiment, the term "acceptor" refers to the antibody amino acid or nucleic acid sequence providing or encoding one or more of the framework regions and the constant region(s). In a specific embodiment, the term "acceptor" refers to a human antibody amino acid or nucleic acid sequence that provides or encodes at least 80%, preferably, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequences of one or more of the framework regions. In accordance with this
embodiment, an acceptor may contain at least 1, at least 2, at least 3, least 4, at least 3, or at least 10 amino acid residues that does (do) not occur at one or more specific positions of a human antibody. An acceptor framework region and/or acceptor constant region(s) may be, e.g., derived or obtained from a germline antibody gene, a mature antibody gene, a functional antibody (e.g., antibodies well-known in the art, antibodies in deve10pment, or antibodies commercially available).
As used herein, the term "CDR" refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDRl, CDR2 and CDR3, for each of the variable regions. The term "CDR set" as used herein refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immuno10gical Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia &Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al.. Nature 342:877-883 (1989)) found that certain sub- portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as LI, L2 and L3 or HI, H2 and H3 where the "L" and the "H" designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRis overlapping with the Kabat CDRs have been described by Pcdlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundary definitions may not strictly fol10w one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use Kabat or Chothia defined CDRs.
As used herein, the term "canonical" residue refers to a residue in a CDR or fi^mework that defines a particular canonical CDR structure as defined by Chothia et al. (J. Mol. Biol. 196:901-907 (1987); Chothia et al., J. Mol. Biol. 227:799 (1992), both are incorporated herein by reference). According to Chothia et al., critical portions of the CDRs of many antibodies have nearly identical peptide backbone confirmations despite great diversity at the level of amino acid
sequence. Each canonical structure specifies primarily a set of peptide backbone torsion angles for a contiguous segment of amino acid residues forming a 10op.
As used herein, the terms "donor" and "donor antibody" refer to an antibody providing one or more CDRs. In a preferred embodiment, the donor antibody is an antibody from a species different from the antibody from which the framework regions are obtained or derived. In the context of a humanized antibody, the term "donor antibody" refers to a non-human antibody providing one or more CDRs.
As used herein, the term "framework" or "framework sequence" refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-Ll, CDR-L2, and CDR-L3 of light chain and CDR-Hl, CDR-H2, and CDR-H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FRl, FR2, FR3 and FR4) on each chain, in which CDRl is positioned between FRl and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FRl, FR2, FR3 or FR4, a framework region, as referred by others, represents the combined PR's within the variable region of a single, naturally occurring immunog10bulin chain. As used herein, a FR represents one of the four sub- regions, and FRs represents two or more of the four sub- regions constituting a framework region.
Human heavy chain and light chain acceptor sequences are known in the art. In one embodiment of the invention the human heavy chain and light chain acceptor sequences are selected from the sequences described in Table 3 and Table 4.

(TABLE REMOVED)
As used herein, the term "germline antibody gene" or "gene fragment" refers to an immunog10bulin sequence encoded by non- lymphoid cells that have not undergone the maturation process that leads to genetic rearrangement and mutation for expression of a particular immunog10bulin. (See, e.g., Shapiro et al., Grit. Rev. Immunol. 22(3): 183-200 (2002); Marcha10nis et al., Adv Exp Med Biol. 484:13-30 (2001)). One of the advantages provided by various embodiments of the present invention stems from the recognition that germline antibody genes are more likely than mature antibody genes to conserve essential amino acid sequence structures characteristic of individuals in the species, hence less likely to be recognized as from a foreign source when used therapeutically in that species.
As used herein, the term "key" residues refer to certain residues within the variable region that have more impact on the binding specificity and/or affinity of an antibody, in particular a humanized antibody. A key residue includes, but is not limited to, one or more of the fol10wing: a residue that is adjacent to a CDR, a potential glycosylation site (can be either N- or 0-glycosylation site), a rare residue, a residue capable of interacting with the antigen, a residue capable of interacting with a CDR, a canonical residue, a contact residue between heavy chain variable region and light chain variable region, a residue within the Vernier zone, and a residue in
the region that overlaps between the Chothia definition of asvariable heavy chain CDRl and the Kabat definition of the first heavy chain framework.
As used herein, the term "humanized antibody" is an antibody or a variant, derivative, ana10g or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (ODR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab') 2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunog10bulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunog10bulin consensus sequence. Preferably, a humanized antibody also comprises at least a portion of an immunog10bulin constant region (Fc), typically that of a human immunog10bulin. In some embodiments, a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CHI, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only contains a humanized light chain. In some embodiments, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a humanized variable domain of a light chain and/or humanized heavy chain.
The humanized antibody can be selected from any class of immunog10bulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including withbut limitation IgG !. lgG2, IgG3 and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well- known in the art.
The framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework may be mutagenized by substitution, insertion and/or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. In a preferred embodiment, such mutations, however, will not be extensive. Usually, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in the consensus immunog10bulin sequence. As used herein, the term "consensus immunog10bulin sequence" refers to the sequence formed from the most frequently
occurring amino acids (or nucleotides) in a family of related immunog10bulin sequences (See e.g., Winnaker, From Genes to C10nes (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of immunog10bulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.
As used herein, "Vernier" zone refers to a subset of framework residues that may adjust CDR structure and fine-tune the fit to antigen as described by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which is incorporated herein by reference). Vernier zone residues form a layer underlying the CDRs and may impact on the structure of CDRs and the affinity of the antibody.
The term "multivalent binding protein" is used in this specification to denote a binding protein con^rising two or more antigen binding sites. The multivalent binding protein is preferably engineered to have the three or more antigen binding sites, and is generally not a naturally occurring antibody. The term "multispecific binding protein" refers to a binding protein capable of binding two or more related or unrelated targets. Dual variable domain (DVD) binding proteins as used herein, are binding proteins that comprise two or more antigen binding sites and are tetravalent or multivalent binding proteins. Such DVDs may be monospecific, i.e capable of binding one antigen or multispecific, i.e. capable of binding two or more antigens. DVD binding proteins comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides are refered to a DVD Ig. Each half of a DVD Ig comprises a heavy chain DVD polypeptide, and a light chain DVD polypeptide, and two antigen binding sites. Each binding site con^jrises a heavy chain variable domain and a light chain variable domain with a total of 6 CDRs involved in antigen binding per antigen binding site.
As used herein, the term "neutralizing" refers to neutralization of bio10gical activity of a cytokine when a binding protein specifically binds the cytokine. Preferably a neutralizing binding protein is a neutralizing antibody whose binding to hIL-13 and/or hIL-13 results in inhibition of a bio10gical activity of hIL-13 and/or hIL-13. Preferably the neutralizing binding protein binds hIL-13 and/or hIL-13 and reduces a bio10gically activity of IL-13 and/or hIL-13 by at least about 20%, 40%, 60%, 80%, 85% or more. Inhibition of a bio10gical activity of hIL-13 and/or hIL-13 by a neutralizing binding protein can be assessed by measuring one or more indicators of hIL-13 and/or hIL-13 bio10gical activity well known in the art. For example inhibition of human IL-13 induced production of TARC (CCL-17) by A-549 cells (see Example 1.1.C).
The term "activity" includes activities such as the binding specificity/affinity of an antibody for an antigen, for example, an anti-hIL-13 antibody that binds to an IL-13 antigen and/or the neutralizing potency of an antibody, for example, an anti-hIL-13 antibody whose
binding to hIL-13 inhibits the bio10gical activity of hIL-13, e.g For example inhibition of human IL-i3 induced production of TARC (CCL-17) by A-549 cells (see Example l.l.C).
The term "epitope" includes any polypeptide determinant capable of specific binding to an immunog10bulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
The terra "surface plasmon resonance", as used herein, refers to an optical phenomenon that al10ws for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et at. (1991) Biotechniques 11:620-627; Johnsson.B., era/. (1995)7. Mol. Recognit. 8:125-131; and Johnnson,B.,ef a/. (1991) Ana/. Biochem. 198:268-277.
The terM ko„", as used herein, is intended to refer to the on rate constant for association of an antibody to the antigen to form the antibody/antigen complex as is known in the art.
The term " koir", as used herein, is intended to refer to the off rate constant for dissociation of an antibody from the antibody/antigen complex as is known in the art.
The term " KD ", as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction as is known in the art.
The term "labeled binding protein" as used herein, refers to a protein with a label incorporated that provides for the identification of the binding protein. Preferably, the label is a detectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or co10rimetric methods). Examples of labels for polypeptides include, but are not limited to, the fol10wing: radioisotopes or radionuclides (e.g., ^H, '"C, "S, '"Y, '^TC. '"in, '"l, '"l, '"LU, '^O, or '^^Sm); fluorescent labels (e.g., FTTC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroX1dase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates.
The term "antibody conjugate" refers to a binding protein, such as an antibody, chemically linked to a second chemical moiety, such as a therapeutic or cytotoX1c agent. The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a bio10gical macromolecule, or an extract made from bio10gical materials. Preferably the therapeutic or cytotoX1c agents include, but are not limited to, pertussis toX1n, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, proprano10l, and puromycin and ana10gs or homo10gs thereof.
The terms "crystal", and "crystallized" as used herein, refer to an antibody, or antigen binding portion thereof, that eX1sts in the form of a crystal. Crystals are one form of the solid state of matter, which is distinct from other forms such as the amorphous solid state or the liquid crystalline state. Crystals are composed of regular, repeating, three-dimensional arrays of atoms, ions, molecules (e.g., proteins such as antibodies), or molecular assemblies (e.g., antigen/antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well-understood in the field. The fundamental unit, or building b10ck, that is repeated in a crystal is called the asymmetric unit. Repetition of the asymmetric unit in an arrangement that conforms to a given, well-defined crystal10graphic symmetry provides the "unit cell" of the crystal. Repetition of the unit cell by regular translations in all three dimensions provides the crystal. See Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ea., pp. 20 1-16, Oxford University Press, New York, New York, (1999)."
The term "polynucleotide" as referred to herein means a polymeric form of two or more nucleotides, either ribonucleotides or deoxvnucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA but preferably is double-stranded DNA.
The term "isolated polynucleotide" as used herein shall mean a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or some combination thereoO that, by virtue of its origin , the "isolated polynucleotide": is not associated with all or a portion of a polynucleotide with which the "isolated polynucleotide" is found in nature; is operably linked to a polynucleotide that it is not linked to in nature; or does not occur in nature as part of a larger sequence.
The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA 10op into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA
segments may be ligated into the viral genome. Certain yectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated a10ng with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. "Operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term "expression control sequence" as used herein refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that .stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Protein constructs of the present invention may be expressed, and purified using expression vectors and host cells known in the art, including expression cassettes, vectors, recombinant host cells and methods for the recombinant expression and proteolytic processing of recombinant polyproteins and pre-proteins from a single open reading frame (e.g., WO 2007/014162 incorporated herein by reference).
"Transformation", as defined herein, refers.to any process by which exogenous DNA
i enters a host cell. Transformation may occur under natural or artificial conditions using various
methods well known in the art. Transformation may rely on any known method for the insertion
of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is
selected based on the host cell being transformed and may include, but is not limited to, viral
infection, electroporation, lipofection, and particle bombardment. Such "transformed" cells
include stably transformed cells in which the inserted DNA is capable of replication either as an
autonomously replicating plasmid or as part of the host chromosome. They also include cells
which transiently express the inserted DNA or RNA for limited periods of time.
The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which exogenous DNA has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but, to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Preferably host cells include prokaryotic and eukaryotic cells selected firom any of the Kingdoms of life. Preferred eukaryotic cells include protist, fungal, plant and animal cells. Most preferably host cells include but are not limited to the prokaryotic cell line E.Coli; mammalian cell lines CHO, HEK 293 and COS; the insect cell line Sf9; and the fungal cell Saccharomyces cerevisiae.
Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular C10ning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
'Transgenic organisM, as known in the art and as used herein, refers to an organism having cells that contain a transgene, wherein the transgene introduced into the organism (or an ancestor of the organism) expresses a polypeptide not naturally expressed in the organism. A "transgene" is a DNA construct, which is stably and operably integrated into the genome of a cell from which a transgenic organism deve10ps, directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic organism.
The term "regulate"and "modulate" are used interchangeably, and, as used herein, refers to a change or an alteration in the activity of a molecule of interest (e.g., the bio10gical activity of
hIL-13). Modulation may be an increase or a decrease in the magnitude of a certain activity or function of the molecule of interest. Exemplary activities and functions of a molecule include, but are not limited to, binding characteristics, enzymatic activity, cell receptor activation, and signal transduction.
Correspondingly, the term "modulator," as used herein, is a compound capable of changing or altering an activity or function of a molecule of interest (e.g., the bio10gical activity of hIL-13). For example, a modulator may cause an increase or decrease in the magnitude of a certain activity or function of a molecule compared to the magnitude of the activity or function observed in the absence of the modulator. In certain embodiments, a modulator is an inhibitor, which decreases the magnitude of at least one activity or function of a molecule. Exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, carbohydrates or small organic molecules. Peptibodies are described, e.g., in WOOl/83525.
The term "agonist", as used herein, refers to a modulator that, when contacted with a molecule of interest, causes an increase in the magnitude of a certain activity or function of the molecule compared to the magnitude of the activity or function observed in the absence of the agonist. Particular agonists of interest may include, but are not limited to, IL-13 polypeptides or polypeptides, nucleic acids, carbohydrates, or any other molecules that bind to hIL-13.
The term "antagonist" or "inhibitor", as used herein, refer to a modulator that, when contacted with a molecule of interest causes a decrease in the magnitude of a certain activity or function of the molecule compared to the magnitude of the activity or function observed in the absence of the antagonist. Particular antagonists of interest include those that b10ck or modulate the bio10gical or immuno10gical activity of hIL-13 and/or hIL-13. Antagonists and inhibitors of hIL-13 and/or hlL-13 may include, but are not limited to, proteins, nucleic acids,.carbohydrates, or any other molecules, which bind to hIL-13 and/or hIL-13.
The term "inhibit binding to the receptor" refers to the ability of the binding protein to prevent the binding of IL-13 to one or more of its receptors. Such inhibition of binding to the receptor would result in diminishing or abolishing the bio10gical activity mediated by binding of IL-13 to its receptor or receptors.
As used herein, the term "effective amount" refers to the amount of a therapy which is sufficient to reduce or ameliorate the severity and/or duration of a disorder or one or more synq)toms thereof, prevent the advancement of a disorder, cause regression of a disorder, prevent the recurrence, deve10pment, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent).
The term "sample", as used herein, is used in its broadest sense. A "bio10gical sample", as used herein, includes, but is not limited to, any quantity of a substance from a living thing or
formerly living thing. Such living things include, but are not liinited to, humans, mice, rats, monkeys, dogs, rabbits and other animals. Such substances include, but are not limited to, b10od, serum, urine, synovial fluid, cells, organs, tissues, bone marrow, lymph nodes and spleen.
I. Antibodies that Bind Human IL-13.
One aspect of the present invention provides isolated murine monoc10nal antibodies, or antigen-binding portions thereof, that bind to IL-13 with high affinity, a s10w off rate and high neutralizing capacity. A second aspect of the invention provides chimeric antibodies that bind IL-13. A third aspect of the invention provides humanized antibodies, or antigen-binding portions thereof, that bind IL-13. Preferably, the antibodies, or portions thereof, are isolated antibodies. Preferably, the antibodies of the invention are neutralizing human anti-IL-13 and/or human anti-IL-13 antibodies.
A. Method of making anti IL-13 antibodies
Antibodies of the present invention may be made by any of a number of techniques known in the art.
1. Anti-IL-13 monoc10nal antibodies using Hybridoma techno10gy
Monoc10nal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display techno10gies, or a combination thereof For example, monoc10nal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Har10w et al., Antibodies: A Laboratory Manual, (Gold Spring Harbor Laboratory Press, 2nd ed. 1988); v.,-Hanrunerling, et al., in: Monoc10nal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term "monoc10nal antibody" as used herein is not limited to antibodies produced through hybridoma techno10gy. The term "monoc10nal antibody" refers to an antibody that is derived from a single c10ne, including any eukaryotic, prokaryotic, or phage c10ne, and not the method by which it is produced.
Methods for producing and screening for specific antibodies using hybridoma techno10gy are routine and well known in the art. In one embodiment, the present invention provides methods of generating monoc10nal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with mye10ma cells and then screening the hybridomas resulting from the fusion for hybridoma c10nes that secrete an antibody able to bind a
polypeptide of the invention ( See Example 1.2). Briefly, mice can be immunized with an IL-13 antigen. In a preferred embodiment, the IL-13 antigen is administered with an adjuvant to stimulate the immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes). Such adjuvants may protect the polypeptide from rapid dispersal by sequestering it in a 10cal deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. Preferably, if a polypeptide is being administered, the immunization schedule will involve two or more administrations of the polypeptide, spread out over several weeks.
After immunization of an animal with an IL-13 antigen, antibodies and/or antibody-producing cells may be obtained from the animal. An anti- IL-13 antibody-containing serum is obtained from the animal by bleeding or sacrificing the animal. The serum may be used as it is obtained from the animal, an immunog10bulin fraction may be obtained from the serum, or the anti- IL-13 antibodies may be purified from the serum. Serum or immunog10bulins obtained in this manner are polyc10nal, thus having a heterogeneous array of properties.
Once an immune response is detected, e.g., antibodies specific for the antigen IL-13 are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable mye10ma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and c10ned by limited dilution. The hybridoma c10nes are then assayed by methods known in the art for cells that secrete antibodies capable of binding IL-13. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma c10nes.
In tmother embodiment, antibody-producing immortalized hybridoqias may be prepati^ from the immunized animal. After immunization, the animal is sacrificed and the splenic B cells are fused to immortalized mye10ma cells as is well known in the art. See, e.g., Har10w and Lane, supra. In a preferred embodiment, the mye10ma cells do not secrete immunog10bulin polypeptides (a non-secretory cell line). After fusion and antibiotic selection, the hybridomas are screened using IL-13, or a portion thereof, or a cell expressing IL-13. In a preferred embodiment, the initial screening is performed using an enzyme-linked immunoassay (ELISA) or a radioimmunoassay (RIA), preferably an ELISA. An example of ELISA screening is provided in WO 00/37504, herein incorporated by reference.
Anti- IL-13 antibody-producing hybridomas are selected, c10ned and further screened for desirable characteristics, including robust hybridoma growth, high antibody production and desirable antibody characteristics, as discussed further be10w. Hybridomas may be cultured and expanded in vivo in syngeneic animals, in animals that lack an immune system, e.g., nude mice, or
in cell culture in vitro. Methods of selecting, c10ning and expanding hybridomas are well known
to those of
ordinary skill in the art.
In a preferred embodiment, the hybridomas are mouse hybridomas, as described above. In another preferred embodiment, the hybridomas are produced in a non-human, non-mouse species such as rats, sheep, pigs, goats, cattle or horses. In another embodiment, the hybridomas are human hybridomas, in which a human non-secretory mye10ma is fused with a human cell expressing an anti-IL-13 antibody.
Antibody fragments that recognize specific epitopes may be generated by known techniques. For example. Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunog10bulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain.
2. Anti-IL-13 monoc10nal antibodies using SLAM
In another aspect of the invention, recombinant antibodies are generated from single, isolated lymphocytes using a procedure referred to in the art as the selected lymphocyte antibody method (SLAM), as described in U.S. Patent No. 5,627,052, PCT Publication WO 92/02551 and Babcock, J.S. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. hi this method, single cells secreting antibodies of interest, e.g., lymphocytes derived from any one of the immunized animals described in Section 1, are screened using an antigen-specific hemolytic plaque assay, wherein the antigen IL-13, a subunint of IL-13, or a fragment thereof, is coupled to sheep red b10od cells using a linker, such as biotin, and used to identify single cells that secreto antibodies with specificity for IL-13. Fol10wing identification of antibody-secreting cells of interest, heavy-and light-chain variable region cDNAs are rescued from the cells by reverse transcriptase-PCR and these variable regions can then be expressed, in the context of appropriate immunog10bulin constant regions (e.g., human constant regions), in mammalian host cells, such as COS or CHO cells. The host cells transfected with the amplified immunog10bulin sequences, derived from in vivo selected lymphocytes, can then undergo further analysis and selection in vitro, for example by panning the transfected cells to isolate cells expressing antibodies to IL-13. The amplified immunog10bulin sequences further can be manipulated in vitro, such as by in vitro affinity maturation methods such as those described in PCT Publication WO 97/29131 and PCT Publication WO 00/56772.
3. Anti-IL-13 monoc10nal antibodies using transgenic animals
In another embodiment of the instant invention, antibodies are produced by immunizing a non-human animal comprising some, or all, of the human immunog10bulin 10cus with an EL-13 antigen. In a preferred embodiment, the non-human animal is a XENOMOUSE transgenic mouse, an engineered mouse strain that comprises large fragments of the human immunog10bulin 10ci and is deficient in mouse antibody production. See, e.g.. Green et al. Nature Genetics 7:13-21 (1994) and United States Patents 5,916,771,5,939,598,5,985,615, 5,998,209,6,075.181,6,091,001,6,114,598 and 6,130,364. See also WO 91/10741, published July 25,1991, WO 94/02602, published'February 3,1994, WO 96/34096 and WO 96/33735, both published October 31,1996, WO 98/16654, published April 23,1998, WO 98/24893, published June 11,1998, WO 98/50433, published November 12,1998, WO 99/45031, published September 10,1999, WO 99/53049, published October 21, 1999, WO 00 09560, published February 24,2000 and WO 00/037504, published June 29, 2000. The XENOMOUSE transgenic mouse produces an adult-like human repertoire of fully human antibodies, and generates antigen-specific human Mabs. The XENOMOUSE transgenic mouse contains approX1mately 80% of the human antibody repertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain 10ci and x light chain 10ci. See Mendez et al.. Nature Genetics 15:146-156 (1997), Green and Jakobovits J. Exp. Med. 188:483-495 (1998), the disc10sures of which are hereby incorporated by reference.
4. Anti-IL-13 monocional antibodies using recombinant antibody libraries
In vitro methods also can be used to make the antibodies of the invention, wherein an antibody library is screened to identify an antibody having the desired binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art and include methods described in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al. PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Techno10gy 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; McCafferty et al.. Nature (1990) 348:552-554; Griffiths et al. (1993) EMBOJ 12:725-734; Hawkins et al. (1992) J Mol Biol ^:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PAMS 89=3576-3580; Garrad et al. (1991) Bio/Techno10gy 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PAMS 88:7978-7982, US patent application publication 20030186374, and PCT Publication No. WO 97/29131, the contents of each of which are incorporated herein by reference.
The recombinant antibody library may be from a subject immunized with IL-13 or IL-13, or a portion of IL-13 or IL-13. Alternatively, the recombinant antibody library may be from a naive subject, i.e., one who has not been immunized with IL-13, such as a human antibody library from a human subject who has not been immunized with human IL-13. Antibodies of the invention are selected by screening the recombinant antibody library with the peptide comprising human IL-13 to thereby select those antibodies that recognize IL-13. Methods for conducting such screening and selection are well known in the art, such as described in the references in the preceding paragraph. To select antibodies of the invention having particular binding affinities for hIL-13, such as those that dissociate from human IL-13 with a particular koff rate constant, the art-known method of surface plasmon resonance can be used to select antibodies having the desired koff rate constant. To select antibodies of the invention having a particular neutralizing activity for hIL-13, such as those with a particular an ICjo, standard methods known in the art for assessing the inhibition of hIL-13 activity may be used.
In one aspect, the invention pertains to an isolated antibody, or an antigen-binding portion thereof, that binds human IL-13. Preferably, the antibody is a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or a monoc10nal antibody.
For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. 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, 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 suiface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene IE or gene Vni protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disc10sed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al, Eur. J. Immunol. 24:952-958 (1994); Persic et al.. Gene 187 9-18 (1997); Burton et al.. Advances in Immuno10gy 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5.780,225; 5,658,727; 5,733.743 and 5,969.108; each of which is incorporated herein by reference in its entirety.
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 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 be10w. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be emp10yed using methods known in the art such as those disc10sed in PCX publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.. Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. 4,946,778 and 5,258,498; Huston et al.. Methods in Enzymo10gy 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al.. Science 240:1038-1040 (1988).
Alternative to screening of recombinant antibody libraries by phage display, other methodo10gies known in the art for screening large combinatorial libraries can be applied to the identification of dual specificity antibodies of the invention. One type of alternative expression system is one in which the recombinant antibody library is expressed as RNA-protein fusions, as described in PCT Publication No. WO 98/31700 by Szostak and Roberts, and in Roberts, R.W. and Szostak, J.W. (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302. In this system, a covalent fusion is created between an mRNA and the peptide or protein that it encodes by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3' end. Thus, a specific mRNA can be enriched from a complex mixture of mRNAs (e.g., a combinatorial jibrary) based on the properties of the encoded peptide or protein, e.g., antibody, or portion thereof, such as binding of the antibody, or portion, thereof, to the dual specificity antigen. Nucleic acid sequences encoding antibodies, or portions thereof, recovered from screening of such libraries can be expressed by recombinant means as described above (e.g., in mammalian host cells) and, moreover, can be subjected to further affinity maturation by either additional rounds of screening of mRNA-peptide fusions in which mutations have been introduced into the originally selected sequence(s), or by other methods for affinity maturation in vitro of recombinant antibodies, as described above.
In another approach the antibodies of the present invention can also be generated using yeast display methods known in the art. In yeast display methods, genetic methods are used to tether antibody domains to the yeast cell wall and display them on the surface of yeast. In particular, such yeast can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e. g., human or murine). Examples of yeast display methods that can be used to make the antibodies of the present invention include those disc10sed Wittrup, et al. U.S. Patent No. 6,699,658 incorporated herein by reference.
B. Production of recombinant iL-13 antibodies
Antibodies of the present invention may be produced by any of a number of techniques known in the art. For example, expression from host cells, wherein expression vector(s) encoding the heavy and light chains is (are) transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells is preferable, and most preferable in mammalian host cells, because such eukaryotic cells (and in particular mammalian cells) are more likely than prokaryotic cells to assemble and secrete a properly folded and immuno10gically active antibody.
Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R.J. Kaufinan and P.A. Sharp (1982) Mol. Biol. 159:601-621), NSO mye10ma cells, COS cells and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by cuituring the host cells for a period of time sufTicient to al10w for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure are within the scope of the present invention. For example, it may be desirable to transfect a host cell with DNA encoding functional fragments of either the light chain and/or the heavy chain of an antibody of this invention. Recombinant DNA techno10gy may also be used to remove some, or all, of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigens of interest. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention. In addition, Afunctional antibodies may be produced in which one heavy and one light chain are an antibody of the invention and the other heavy and light chain are specific for an antigen other than the antigens of interest by crosslinking an antibody of the invention to a second antibody by standard chemical crosslinking methods.
In a preferred system for recombinant expression of an antibody, or antigen-binding portion thereof, of the invention, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr- CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which al10ws for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to al10w for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium. Standard molecular bio10gy techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium. Still further the invention provides a method of synthesizing a recombinant antibody of the invention by culturing a host cell of the invention in a suitable culture medium until a recombinant antibody of the invention is synthesized. The method can further comprise isolating the recombinant antibody from the culture medium. 1. Anti IL-13 antibodies
Table 5 is a list of amino acid sequences of VH and VL regions of preferred anti-hIL-13 antibodies of the invention.
Table 5 List of Amino Acid Sequences of VH and VL regions

(TABLE REMOVED)
The foregoing isolated anti-IL-13 antibody CDR sequences establish a novel family of IL-13 binding proteins, isolated in accordance with this invention, and comprising polypeptides that include the CDR sequences listed in Table 6 be10w. To generate and to select CDR's of the invention having preferred IL-13 binding and/or neutralizing activity with respect to hIL-13 and or hIL-13, standard methods known in the art for generating binding proteins of the present invention and assessing the IL-13 and or IL-13 binding and/or neutralizing characteristics of those binding protein may be used, including but not limited to those specifically described herein.

(TABLE REMOVED)
2. Anti IL-13 Chimeric antibodies
A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoc10nal antibody and a human immunog10bulin constant region. Methods for producing chimeric antibodies are known in the art and discussed in detail in Example 2.1. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816.567; and 4,816,397, which are incorporated herein by reference in their entireties. In addition, techniques deve10ped for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452454 which are incorporated herein by reference in their entireties) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate bio10gical activity can be used.
In one embodiment, the chimeric antibodies of the invention are produced by replacing the heavy chain constant region of the murine monoc10nal anti human IL-13 antibodies described in section 1 with a human IgGl constant region. In a specific embodiment the chimeric antibody
of the invention comprises a heavy chain variable region (VH ) comprising the amino acid sequence of SEQ ID NO: 34; SEQ ID NO: 36; SEQ ID NO: 41; SEQ ID NO: 42; SEQ ID NO: 46 and a light chain variable region (VL ) comprising the amino acid sequence of SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 40; SEQ ID NO: 43;0T SEQ ID NO: 47.
3. Anti IL-13 Humanized antibodies
Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunog10bulin molecule. Known human Ig sequences are disc10sed, e.g., www.ncbi.nlm.nih.gov/entrez- /query.fcgi; www.atcc.org/phage/hdb.html; www.sciquest.com/; www.abcam.com/; www.antibodyresource.com/onlinecomp.html;
www.public.iastate.edu/.about.pedro/research_tools.html; www.mgen.uni-heidelberg.de/SD/IT/IT.html; www.whfreeman.com/inununoIogy/CH- 05/kuby05 .htm; www.library.thinkquest.org/12429/Immune/Antibody.html;
www.hhmi.org/grants/lectures/1996/vlab/; www.path.cam.ac.uk/.about.mrc7/m-ikeimages.html; www.antibodyresource.com/; mcb.harvard.edu/BioLinks/Immuno-10gy.html.www.immuno10gylink.com/; pathbox.wustl.edu/.about.hcenter/index.-html; www.biotech.ufl.edu/.about.hcl/; www.pebio.com/pa/340913/340913.html-; www.nal.usda.gov/awic/pubs/antibody/; www.m.ehime-u.acjp/.about.yasuhito-/Elisa.html; www.biodesign.com/table.asp; www.icnet.uk/axp/facs/davies/lin-ks.html; www.biotech.ufl.edu/.about.fccl/protocol.html; www.isac-net.org/sites_geo.html; aX1mtl.imt.uni-marburg.de/.about.rek/AEP-Start.html;baserv.uci.kun.nl/.about.jraats/linksl.html; www.recab.uni-hd.de/immuno.bme.nwu.edu/; www.mrc-cpe.cam.ac.uk/imt-doc/pu-blic/INTRO.html; www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/; www.biochem.ucl.ac.uk/.about.martin/abs/index.html; antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html; www.unizh.ch/.about.honegger/AHOsem-inar/SlideOl.html; www.cryst.bbk.ac.uk/.about.ubcg07s/;
www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm; www.path.cam.ac.uk/.about.mrc7/h-umanisation/TAHHP.html; www.ibt.unam.mx/vir/structure/stat_aim.html; www.biosci.missouri.edu/smithgp/index.html; www.cryst.bioc.cam.ac.uk/.abo-ut.fmolina/Web-pages/Pept/spottech.html; www.jerini.de/fr roducts.htm; www.patents.ibm.com/ibm.html.Kabat et al., Sequences of Proteins of Immuno10gical Interest, U.S. Dept. Health (1983), each entirely incorporated herein by reference. Such imported sequences can be used to reduce
immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic, as known in the art.
Framework residues in the human framework regions may be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. 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.. Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Three-dimensional immunog10bulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunog10bulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunog10bulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunog10bulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. Antibodies can be humanized using a variety of techniques known in the ait, such as but not limited to those described in Jones et al., Nature 321:522 (1986); Verhoeyen et al.. Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immuno10gy 28(:4/5):489-498 (1991); Studnicka et al.. Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCT publication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400, U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5814476, 5763192, 5723323.5,766886,5,714,352,6,204,023,6,180,370,5,693,762,5,530,101,5,585,089, 5,225,539; 4,816,567, each entirely incorporated herein by reference, included references cited therein.
C. Production of Antibodies and Antibody-Producing Cell Lines
Preferrably, anti-IL-13 antibodies of the present invention, exhibit a high capacity to reduce or to neutralize IL-13 activity, e.g.,as assessed by any one of several in vitro and in vivo assays known in the art (e.g., see Example LLC). For example, these antibodies neutralize IL-13-
induced production of TARC by A-549 cells with IC50 values in the range of at least about 10* M, about 10' M, or about 10'" M.
In prefered embodiments, the isolated antibody, or antigen-binding portion thereof, binds human IL-13, wherein the antibody, or antigen-binding portion thereof, dissociates from human IL-13 with a koff rate constant of about 0.1s' or less, as determined by surface plasmon resonance, or which inhibits human IL-13 and/or human IL-13 activity with an IC50 of about 1 x 10"*M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from human IL-13 with a kotr rate constant of about 1 x 10s'or less, as determined by surface plasmon resonance, or may inhibit human IL-13 and/or human IL-13 activity with an ICjo of about 1 X 10M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from human IL-13 with a koft rate constant of about 1 x 10s' or less, as determined by surface plasmon resonance, or may inhibit human IL-13 and/or human IL-13 with an IC50 of about 1 X IO'M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from human IL-13 with a kofrrate constant of about 1 x 10s' or less, as determined by surface plasmon resonance, or may inhibit EL-13 and/or human IL-13 activity with an IC50 of about 1 X IO'M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from human IL-13 with a koirrate constant of about 1 x 10V or less, as determined by surface plasmon resonance, or may inhibit IL-13 and/or human IL-13 activity with an IC50 of about 1 X 10"*M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from human IL-13 with a koir rate constant of about 1 x 10s'or less, as determined by surface plasmon resonance, or may inhibit IL-13 and/or human IL-13 activity with an IC50 of about 1 x 10'"M or less.
IL-13 exerts its actions by binding to the IL-13 receptor (IL-13R) on the celS s.urface, the heterodimer comprised of the IL-13Ral chain (IL-13Ral) and the IL-4R chain (IL-4R). IL-13 binds to IL-13Ral first with 10w affinity (KD = 2-10 nM) and then recruits IL-4R to the complex, generating a high affinity receptor (KD = 0.03-0.4 nM) (Aman, M. J., et al. 1996 J. Biol. Chem. 271,29265-29270; Mi10ux, et al. 1997 FEES Lett. 401,163-166; Andrews, et al 2002 J. Biol. Chem. 277,46073-46078). Heterodimerization of IL-13R causes activation of Janus kinases, TYK2 and JAKl, constitutively associated with lL-13Ral and IL-4R, respectively, fol10wed by activation of the signal transducer and activator of transcription 6 (STAT6) (Izuhara, K., and Arima, K. 2004 Drug News Perspect. 17,91-98). There is another IL-13-binding unit, the IL-13Ra2 chain (IL-13Ra2), which binds to IL-13 with high affinity (0.25-1.2 nM) (Caput, et al 1996 J. Biol. Chem. 271,16921-16926; Donaldson et al 1998 J. Immunol. 161,2317-2324). No other receptor molecule is known to be involved in the IL-13IL-13R2 complex. IL-13R2 is initially thought to act as a nonsignaling "decoy" receptor. However, it was later discovered that it can bind to IL-13 and signals through AP-1 pathway, leading to TNF-beta production in certain
cell types including macrophages, which in tern leads to lung fibrosis (Fichtner-Feigl, 2006 Nat Med 12:99-106). Therefore both IL-13Ral/IL-4Ra and IL-13Ra2 pathways contribute to the overall pathophysio10gy of asthma and other pulmonary inflammatory conditions. Therefore, a therapeutic anti-E^-13 antibody that b10cks IL-13 binding to both receptors will be more effective that those that b10cks only one receptor.
We have isolated monoc10nal antibodies that b10ck IL-13 binding to both IL-13Ral and IL-13Ra2. Both ELISA-based receptor binding assay and 125-I-labeled IL-13 binding assay on cell surface demonstrated that 13C5, both murine version and humanized version (i.e. 13C5.5), were able to effective b10ck IL-13 binding to both receptors. Antibodies in the same lineage as 13C5, including 25C8 and 33C3, were also able to b10ck IL-13 binding to both receptors. Epitope mapping of 13C5 indicated that its binding site(s) included the C-terminal Helix D region of human IL-13 (residues VRDTK lEVAQ FVKDL LLHLK KLFRE GR, corresponding to amino acid 104-130 of SEQ ED NO. 1). The c-terminal helix D region has been proposed to be involved in interactions with the IL-13 receptor (Zuegg et al 2001 Immunol Cell Biol. 79:332-9). Crystal structure of human IL-13 complexed with the Fab portion of 13C5.5 antibody indicated that 13C5.5 binds the C-terminal Helix D region as well as the N-terminal Helix A region of human IL-13. Preferably the antibody, or antigen binding fragment thereof binds human IL-13 such that IL-13 with said antibody, or antigen binding fragment thereof, bound to the epitope defined by the topographic regions Ser26-Thr27-Ala28-Leu29-Arg30-Glu31-Leu32-Ile33-Glu34-Glu35-Leu36-Val37-Asn38andLysl23-Lysl24-Leul25-Phel26-Argl27-Glu-128-Glyl29-Argl30 of SEQ ID No. 1 is inhibited from binding to the IL-13 receptor. Preferably the antibody, or antigen binding fragment thereof binds human IL-13 such that IL-13 with said antibody, or antigen binding fragment thereof, bound to the epitope defined by the topographic fSgions ArgSO* Glu31-Leu32-Ile33-Glu34-Glu35-Leu36-Val37-Asn38andLysl23-Lysl24-Leul25-Phel26-Argl27 of SEQ ID No. 1 is inhibited from binding to the IL-13a2 receptor.
In certain embodiments, the antibody comprises a heavy chain constant region, such as an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. Preferably, the heavy chain constant region is an IgGl heavy chain constant region or an IgG4 heavy chain constant region. Furthermore, the antibody can comprise a light chain constant region, either a kappa light chain constant region or a lambda light chain constant region. Preferably, the antibody comprises a kappa light chain constant region. Alternatively, the antibody portion can be, for example, a Fab fragment or a single chain Fv fragment.
Replacements of amino acid residues in the Fc portion to alter antibody effector function are known in the art (Winter, et al. US PAT NOS 5,648,260; 5624821). The Fc portion of an antibody mediates several important effector functions e.g. cytokine induction, ADCC, phagocytosis, complement dependent cytotoX1city (CDC) and half-life/ clearance rate of antibody
and antigen-antibody complexes. In some cases tHese effector functions are desirable for therapeutic antibody but in other cases might be unnecessary or even deleterious, depending on the therapeutic objectives. Certain human IgG isotypes, particularly IgGl and IgG3, mediate ADCC and CDC via binding to FcyRs and complement Clq, respectively. Neonatal Fc receptors (FcRn) are the critical components determining the circulating half-life of antibodies. In still another embodiment at least one amino acid residue is replaced in the constant region of the antibody, for example the Fc region of the antibody, such that effector functions of the antibody are altered.
One embodiment provides a labeled binding protein wherein an antibody or antibody portion of the invention is derivatized or linked to another functional molecule (e.g., another peptide or protein). For example, a labeled binding protein of the invention can be derived by functionally linking an antibody or antibody portion of the invention (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoX1c agent, a pharmaceutical agent, and/or a protein or peptide that can mediate associate of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
Useful detectable agents with which an antibody or antibody portion of the invention may be derivatized include fluorescent compounds. Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl ch10ride, phycoerythrin and the like. An antibody may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroX1dase, glucose oX1dase and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroX1dase is present, the addition of hydrogen peroX1de and diaminobenzidine leads to a co10red reaction product, which is detectable. An antibody may also be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding.
Another embodiment of the invention provides a crystallized binding protein. Preferably the invention relates to crystals of whole anti-IL-13 antibodies and fragments thereof as disc10sed herein, and formulations and compositions comprising such crystals. In one embodiment the crystallized binding protein has a greater half-life in vivo than the soluble counterpart of the binding protein. In another embodiment the binding protein retains bio10gical activity after crystallization.
Crystallized binding protein of the invention may be produced according methods known in the art and as disc10sed in WO 02072636, incorporated herein by reference.
Another embodiment of the invention provides a glycosylated binding protein wherein the antibody or antigen-binding portion thereof comprises one or more carbohydrate residues. Nascent in vivo protein production may undergo further processing, known as post-translational modification. In particular, sugar (glycosyl) residues may be added enzymatically, a process known as glycosylation. The resulting proteins bearing covalently linked oligosaccharide side chains are known as glycosylated proteins or glycoproteins. Antibodies are glycoproteins with one or more carbohydrate residues in the Fc domain, as well as the variable domain. Carbohydrate residues in the Fc domain have important effect on the effector function of the Fc domain, with minimal effect on antigen binding or half-life of the antibody (R. Jefferis, Biotechnol. Prog. 21 (2005), pp. 11-16). In contrast, glycosylation of the variable domain may have an effect on the antigen binding activity of the antibody. Glycosylation in the variable domain may have a negative effect on antibody binding affinity, likely due to steric hindrance (Co, M.S., et al., Mol. Immunol. (1993) 30:1361-1367), or result in increased affinity for the antigen (Wallick, S.C, et al, Exp. Med. (1988) 168:1099-1109; Wright, A., et al., EMBO J. (1991)10:2717 2723).
One aspect of the present invention is directed to generating glycosylation site mutants in which the O- or N-linked glycosylation site of the binding protein has been mutated. One skilled in the art can generate such mutants using standard well-known techno10gies. Glycosylation site mutants that retain the bio10gical activity, but have increased or decreased binding activity, are another object of the present invention.
In still another embodiment, the glycosylation of the antibody or antigen-binding portion of the invention is modified. For example, an aglycoslated antibody can be made (i.e., the antible with an antibody, or antibody portion, of the invention and detecting either the antibody (or antibody portion) bound to human IL-13 or unbound antibody (or antibody portion),, to thereby detect human IL-13 in the bio10gical sample. The antibody is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroX1dase, alkaline phosphatase, 3-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dich10rotriazinylamine fluorescein, dansyl ch10ride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include ^, '*C. "S. *"¥, "^Tc, '"In, '"I, "'I, '"Lu, '**Ho. or '"Sm.
Alternative to labeling the antibody, human IL-13 can be assayed in bio10gical fluids by a competition immunoassay utilizing rhIL-13 standards labeled with a detectable substance and an unlabeled ami- human IL-13 antibody. In this assay, the bio10gical sample, the labeled rhIL-13
standards and the anti- human IL-13 antibody are combined and the amount of labeled rhIL-13 standard bound to the unlabeled antibody is determined. The amount of human IL-13 in the bio10gical sample is inversely proportional to the amount of labeled rhIL-13 standard bound to the anti-IL-13 antibody. Similarly, human IL-13 can also be assayed in bio10gical fluids by a competition immunoassay utilizing rhIL-13 standards labeled with a detectable substance and an unlabeled anti-human IL-13 antibody.
The antibodies and antibody portions of the invention preferably are capable of neutralizing human IL-13 activity both in vitro and in vivo. Accordingly, such antibodies and antibody portions of the invention can be used to inhibit hIL-13 activity, e.g., in a cell culture containing hIL-13, in human subjects or in other mammalian subjects having IL-13 with which an antibody of the invention cross-reacts. In one embodiment, the invention provides a method for inhibiting hIL-13 activity comprising contacting hIL-13 with an antibody or antibody portion of the invention such that hIL-I3 activity is inhibited. For example, in a cell culture containing, or suspected of containing hIL-13, an antibody or antibody portion of the invention can be added to the culture medium to inhibit hIL-13 activity in the culture.
In another embodiment, the invention provides a method for reducing hIL-13 activity in a subject, advantageously from a subject suffering from a disease or disorder in which IL-13 activity is detrimental. The invention provides methods for reducing IL-13 activity in a subject suffering hrom such a disease or disorder, which method comprises administering to the subject an antibody or antibody portion of the invention such that IL-13 activity in the subject is reduced. Preferably, the IL-13 is human IL-13, and the subject is a human subject. Alternatively, the subject can be a mammal expressing an IL-13 to which an antibody of the invention is capable of binding. Still further the subject can be a manmial into which IL-13 has been introduced (e.g., by administration of IL-13 or by expression of an IL-13 transgene). An antibody of the invention can be administered to a human subject for therapeutic purposes. Moreover, an antibody of the invention can be administered to a non-human mammal expressing an IL-13 with which the antibody is capable of binding for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the invention (e.g., testing of dosages and time courses of administration).
As used herein, the term "a disorder in which IL-13 activity is detrimental" is intended to include diseases and other disorders in which the presence of IL-13 in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysio10gy of the disorder or a factor that contributes to a worsening of the disorder. Accordingly, a disorder in which IL-13 activity is detrimental is a disorder in which reduction of IL-13 activity is expected to alleviate the symptoms and/or progression of the disorder. Such disorders may be evidenced, for example, by an increase in the concentration of IL-13 in a
bio10gical fluid of a subject suffering from the disorder {e.g., an increase in the concentration of IL-13 in serum, plasma, synovial fluid, etc. of the subject), which can be detected, for example, using an anti-IL-13 antibody as described above. Non-limiting examples of disorders that can be treated with the antibodies of the invention include those disorders discussed in the section be10w pertaining to pharmaceutical compositions of the antibodies of the invention.
IL-13 has been implicated as having a pivotal role in causing patho10gical responses associated with asthma. However other mediators of differential immuno10gical pathways are also involved in asthma pathogenesis, and b10cking these mediators, in addition to IL-13, may offer additional therapeutic benefit. Thus, binding proteins of the invention may be incorporated into DVD-Ig proteins where in the DVD is capable of binding target pairs including, but not limited to, IL-13 and a pro-inflammatory cytokine, such as tumor necrosis factor-a (TNF-a). TNF-a may amplify the inflammatory response in asthma and may be linked to disease severity (McDonnell, et al.. Progress in Respiratory Research (2001), 31(New Drugs for Asthma, Allergy and COPD), 247-250.). This suggests that b10cking both IL-13 and TNF-a may have beneficial effects, particularly in severe airway disease. In a preferred embodiment the DVD-Ig of the invention binds the targets IL-13 and TNFa and is used for treating asthma.
In another embodiment binding proteins of the invention can be used to generate DVD-Ig molecules that bind IL-13 and IL-lbeta, IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-P; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; and IL-13 and ADAMS. The present invention also provides DVD-Igs capable of binding IL-13 and one or more targets involved in asthma selected from the group consisting of CSFl (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FGF2, IFNAl.IFNBl; IFNG, histamine and histamine receptors, ILIA, IL1B,'!L2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, ILIO, ILIl, IL12A, IL12B, 1L14, IL15, IL16, IL17, IL18, IL19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, E.-26, IL-27, IL-28, IL-30, IL-31, IL-32, IL-33, KITLG, PDGFB, IL2RA, IL4R, IL5RA, IL8RA, IL8RB, IL12RB1, IL12RB2, IL13RA1,1L13RA2, IL18R1, TSLP, CCLl, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL17, CCL18, CCL19. CCL20, CCL22, CCL24,CX3CL1, CXCLl, CXCL2. CXCL3, XCLl, CCR2. CCR3, CCR4, CCR5. CCR6, CCR7, CCR8, CX3CR1, GPR2, XCRl, FOS, GATA3, JAKl, JAK3, STAT6, TBX21, TGFBl, TNFSF6, YYl, CYSLTRl, FCERIA, FCER2, LTB4R, TB4R2, LTBR, and Chitinase.
D. Pharmaceutical Composition
The invention also provides pharmaceutical compositions comprising an antibody, or antigen-binding portion thereof, of the invention and a pharmaceutically acceptable carrier. The pharmaceutical compositions comprising antibodies of the invention are for use in, but not
limited to, diagnosing, detecting, or monitoring a disorder, in preventing, treating, managing, or ameliorating of a disorder or one or more symptoms thereof, and/or in research. In a specific embodiment, a composition comprises one or more antibodies of the invention. In another embodiment, the pharmaceutical composition comprises one or more antibodies of the invention and one or more prophylactic or therapeutic agents other than antibodies of the invention for treating a disorder in which IL-13 activity is detrimental. Preferably, the prophylactic or therapeutic agents known to be useful for or having been or currently being used in the prevention, treatment, management, or amelioration of a disorder or one or more symptoms thereof. In accordance with these embodiments, the composition may further comprise of a carrier, diluent or excipient.
The antibodies and antibody-portions of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises an antibody or antibody portion of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physio10gically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium ch10ride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auX1liary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion.
Various delivery systems are known and can be used to administer one or more antibodies of the invention or the combination of one or more antibodies of the invention and a prophylactic agent or therapeutic agent useful for preventing, managing, treating, or ameliorating a disorder or one or more symptoms thereof, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or antibody fragment, receptor- mediated endocytosis (see, e. g., Wu and Wu, J. Biol. Chenx 262:4429^M32 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of administering a prophylactic or therapeutic agent of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous) , epidurala administration, intratumoral administration, and mucosal adminsitration (e.g., intranasal and oral routes). In addition, pulmonary administration can be emp10yed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6, 019.968.5.985. 320.5.985.309, 5,934, 272. 5,874,064,5,855.913. 5,290, 540, and 4,880,078; and
PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference their entireties. In one embodinient, an antibody of the invention, combination therapy, or a composition of the invention is administered using Alkermes AIR® pulmonary drug delivery techno10gy (Alkermes, Inc., Cambridge, Mass.). In a specific embodiment, prophylactic or therapeutic agents of the invention are administered intramuscularly, intravenously, intratumorally, orally, intranasally, pulmonary, or subcutaneously. The prophylactic or therapeutic agents may be adnunistered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other bio10gically active agents. Administration can be systemic or 10cal.
In a specific embodiment, it may be desirable to administer the prophylactic or therapeutic agents of the invention 10cally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, 10cal infusion, by injection, or by means of an implant, said implant being of a porous or non-porous material, including membranes and matrices, such as sialastic membranes, polymers, fibrous matrices (e.g., Tissuel®), or collagen matrices. In one embodiment, an effective amount of one or more antibodies of the invention antagonists is administered 10cally to the affected area to a subject to prevent, treat, manage, and/or ameliorate a disorder or a symptom thereof. In another embodiment, an effective amount of one or more antibodies of the invention is adnunistered 10cally to the affected area in combination with an effective amount of one or more therapies (e. g., one or more prophylactic or therapeutic agents) other than an antibody of the invention of a subject to prevent, treat, manage, and/or ameliorate a disorder or one or more symptoms thereof.
In another embodiment, the prophylactic or therapeutic agent of the invention can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the invention (see e.g.. Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No. 5,679,377; U.S. Pat. No. 5, 916,597; U. S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253. Examples of polymers used in sustained release
formulations include, but are not limited to. poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacryIic acid), polyglycolides (PLC), poiyanhydrides, poly(N- vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In a preferred embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In yet another embodiment, a controlled or sustained release system can be placed in proX1mity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
Controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more therapeutic agents of the invention. See, e.g., U. S. Pat. No. 4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698, Ning et al., 1996, "Intratumoral Radioimmunotheraphy of a Human Co10n Cancer Xenograft Using a Sustained-Release Gel," Radiotherapy &Onco10gy 39:179-189, Song et al., 1995, "Antibody Mediated Lung Targeting of 10ng- Circulating Emulsions," PDA Journal of Pharmaceutical Science ATechno10gy 50:372-397, Cleek et al., 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application," Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lamet al., 1997, "Microencapsulation of Recombinant Humanized Monoc10nal Antibody for 10cal Delivery," Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759- 760, each of which is incorporated herein by reference in their entireties.
In a speciflc embodiment, where the composition of the invention is a nucleic acid encoding a prophylactic or therapeutic agent, the nucleic acid can be administered in vivo to promote expression of its encoded prophylactic or therapeutic agent, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U. S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868). Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression by homo10gous recombination.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a
pharmaceutical composition adapted for intravenous, subciitarieous, intramuscular, oral, intranasal, or topical administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a 10cal anesthetic such as lignocamne to ease pain at the site of the injection.
If the compositions of the invention are to be administered topically, the compositions can be formulated in the form of an ointment, cream, transdermal patch, 10tion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). For non- sprayable topical dosage forms, viscous to semisolid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity preferably greater than water are typically emp10yed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auX1liary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or bumectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art.
If the method of the invention comprises intranasal administration of a composition, the composition can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dich10rodifluoromethane, trich10rofluoromethane, dich10rotetrafluoroethane, carbon dioX1de or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
[0257] If the method of the invention comprises oral administration, compositions can be formulated orally in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions, and the like. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellu10se); fillers (e.g., lactose, microcrystalline
cellu10se, or calcium hydrogen phosphate); lubricants (e.g.', magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well-known in the art. Liquid preparations for oral administration may take the form of, but not limited to, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellu10se derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, co10ring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for s10w release, controlled release, or sustained release of a prophylactic or therapeutic agent(s).
[0258] The method of the invention may comprise pulmonary administration, e.g., by use of an inhaler or nebulizer, of a composition formulated with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968,5,985, 320, 5,985,309, 5,934,272,5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference their entireties. In a specific embodiment, an antibody of the invention, combination therapy, and/or composition of the invention is administered using Alkermes AIR® pulmonary drug delivery techno10gy (Alkermes, Inc., Cambridge, Mass.).
The method of the invention may comprise administration of a composition formulated for parenteral administration by injection (e. g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.
[0260] The methods of the invention may additionally comprise of administration of compositions formulated as depot preparations. Such 10ng acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compositions may be formulateld with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).
The methods of the invention encompasses administration of compositions formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydroch10ric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroX1des, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc. [0262] Generally, the ingredients of compositions are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the mode of administration is infusion, composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the mode of administration is by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
In particular, the invention also provides that one or more of the prophylactic or therapeutic agents, or pharmaceutical compositions of the invention is packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the agent. In one embodiment, one or more of the prophylactic or therapeutic agents, or pharmaceutical compositions of the invention is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. Preferably, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions of the invention is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg. The lyophilized prophylactic or therapeutic c. agents or pharmaceutical compositions of the invention should be stored at between 2° C. and 8° C. in its original container and the prophylactic or therapeutic agents, or pharmaceutical compositions of the invention should be administered within 1 week, preferably within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted. In an alternative embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions of the invention is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the agent. Preferably, the liquid form of the administered composition is supplied in a hermetically sealed container at least 0.25 mg/ml, more preferably at least O.S mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml. The liquid form should be stored at between 2° C. and 8° C. in its original container.
The antibodies and antibody-portions of the invention can be incorporated into a pharmaceutical composition suitable for parenteral administration. Preferably, the antibody or antibody-portions will be prepared as an injectable solution containing 0.1-250 mg/ml antibody. The injectable solution can be composed of either a liquid or lyophilized dosage form in a flint or amber vial, ampule or pre-fiUed syringe. The buffer can be L-histidine (1-50 mM), optimally 5-10mM, at pH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium ch10ride can be used to modify the toX1city of the solution at a concentration of 0-300 mM (optimally 150 mM for a liquid dosage form). Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include treha10se and lactose. Bulking agents can be included for a lyophilized dosage form, principally 1-10% mannitol (optimally 2-4%). Stabilizers can be used in both liquid and lyophilized dosage forms, principally 1-50 mM L-Methionine (optimally 5-10 mM). Other suitable bulking agents include glycine, arginine, can be included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRU surfactants. The pharmaceutical composition comprising the antibodies and antibody-portions of the invention prepared as an injectable solution for parenteral administration, can further comprise an agent useful as an adjuvant, such as those used to increase the absorption, or dispersion of a therapeutic protein (e.g., antibody). A particularly useful adjuvant is hyaluronidase, such as Hylenex® (recombinant human hyaluronidase). Addition of hyaluronidase in the injectable solution improves human bioavailability fol10wing parenteral administration, particularly subcutaneous administration. It also al10ws for greater injection site volumes (i.e. greater than 1 ml) with less pain and discomfort; and minimum incidence of injection site reactions, (sets WO2004078140, US2006104968 incorporated herein by reference).
The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions {e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion.
dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, fol10wed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and spray-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Pro10nged absorption of injectable compositions can be brought about by including, in the composition, an agent that delays absorption, for example, monostearate salts and gelatin.
The antibodies and antibody-portions of the present invention can be administered by a variety of methods knovm in the art, although for many therapeutic applications, the preferred route/mode of administration is subcutaneous injection, intravenous injection or infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Li certain embodiments, the active compound may be prepared with a carrier that will protect the conq>ound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for thfe preparation of such formulations are patented or generally known40 those skilltal in the art. See, e.g.. Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed.. Marcel Dekker, Inc., New York, 1978.
In certain embodiments, an antibody or antibody portion of the invention may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enc10sed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, eliX1rs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
Supplementary active compounds can also be incorporated into the compositions. In certain embodiments, an antibody or antibody portion of the invention is coformulated with
and/or coadministered with one or more additional therapeutic agents that are useful for treating disorders in which IL-13 activity is detrimental. For example, an anti-hIL-13 antibody or antibody portion of the invention may be coformulated and/or coadministered with one or more additional antibodies that bind other targets {e.g., antibodies that bind other cytokines or that bind cell surface molecules). Furthermore, one or more antibodies of the invention may be used in combination with two or more of the foregoing therapeutic agents. Such combination therapies may advantageously utilize 10wer dosages of the administered therapeutic agents, thus avoiding possible toX1cities or complications associated with the various monotherapies.
In certain embodiments, an antibody to IL-13 or fragment thereof is linked to a half-life extending vehicle known in the art. Such vehicles include, but are not limited to, the Fc domain, polyethylene glycol, and dextran. Such vehicles are described, e.g., in U.S. Application Serial No. 09/428,082 and published PCT Application No. WO 99/25044, which are hereby incorporated by reference for any purpose.
In a specific embodiment, nucleic acid sequences comprising nucleotide sequences encoding an antibody of the invention or another prophylactic or therapeutic agent of the invention are administered to treat, prevent, manage, or ameliorate a disorder or one or more symptoms thereof by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded antibody or prophylactic or therapeutic agent of the invention that mediates a prophylactic or therapeutic effect.
Any of the methods for gene therapy available in the art can be used according to the present invention. For general reviews of the methods of gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993. Ann. Rev. Pharmacol. ToX1col. 32:573-596; Mulligan, Science 260:926- 932 (1993); and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5): 155-215. Methods conunonly known in the art of recombinant DNA techno10gy which can be used are described in Ausubel et al. (eds.). Current Protocols in Molecular Bio10gy, John Wiley &Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990). Detailed description of various methods of gene therapy are disc10sed in US2(X)5(X)42664 Al which is incorporated herein by reference.
In another aspect, this application features a method of treating (e.g., curing, suppressing, ameliorating, delaying or preventing the onset of, or preventing recurrence or relapse oO or preventing an IL-13-associated disorder, in a subject. The method includes: administering to the subject an IL- 13 binding agent (particularly an antagonist), e.g., an anti-IL-13 antibody or fragment thereof as described herein, in an amount sufficient to treat or prevent the IL-I3-associated disorder. The IL-13 antagonist, e.g., the anti-IL-13 antibody or fragment thereof, can
be administered to the subject, a10ne or in combination with other therapeutic modalities as described herein.
In one embodiment, the subject is a mammal, e.g., a human suffering from one or more IL-13-associated disorders, including, e.g., respiratory disorders (e.g., asthma (e.g., allergic and nonallergic asthma), chronic obstructive pulmonary disease (COPD), and other conditions involving airway inflammation, eosinophilia, fibrosis and excess mucus production; atopic disorders (e.g., atopic dermatitis and allergic rhinitis); inflammatory and/or autoimmune conditions of, the skin, gastrointestinal organs (e.g., inflammatory bowel diseases (IBD), such as ulcerative colitis and/or Crohn's disease), and liver (e.g., cirrhosis, fibrosis); scleroderma; tumors or cancers, e.g., Hodgkin's lymphoma as described herein. Accordingly, the disc10sure includes the use of an EL-13 binding agent (such as an anti-E.-B antibody or fragment thereof described herein) for a treatment described herein and the use of an IL-13 binding agent (such as an anti-IL-13 antibody or fragment thereof described herein) for preparing a medicament for a treatment described herein.
Examples of IL-13-associated disorders include, but are not limited to, a disorder chosen from one or more of: respiratory disorders, e.g., asthma (e.g., allergic and nonallergic asthma (e.g., asthma due to infection with, e.g., respiratory syncytial virus (RSV), e.g., in younger children)), chronic obstructive pulmonary disease (COPD), and other conditions involving airway inflammation, eosinophilia, fibrosis and excess mucus production, e.g., cystic fibrosis and pulmonary fibrosis; atopic disorders, e.g., resulting from an increased sensitivity to IL-13 (e.g., atopic dermatitis, urticaria, eczema, allergic rhinitis, and allergic enterogastritis); inflammatoyv and/or autoimmune conditions of, the skin (e.g., atopic dermatitis), gastrointestinal organs (e.g., tnflammator)' bowel diseases (IBD), such as ulcerative colitis and/or Crohn's disease), liver (e.g., cirrhosis, hepatocellular carcinoma), and scleroderma; tumors or cancers (e.g., soft tissue or solid tumors), such as leukemia, glioblastoma, and lymphoma, e.g., Hodgkin's lymphoma; viral infections (e.g., from HTLV-1); fibrosis of other organs, e.g., fibrosis of the liver, (e.g., fibrosis caused by a hepatitis B and/or C virus); and suppression of expression of protective type 1 immune responses, (e.g., during vaccination), as described herein.
In other embodiments, this application provides a method of treating (e.g., reducing, ameliorating) or preventing one or more symptoms associated with a respiratory disorder, e.g., asthma (e.g., allergic and nonallergic asthma); allergies; chronic obstructive pulmonary disease (COPD); a condition involving airway inflammation, eosinophilia, fibrosis and excess mucus production, e.g., cystic fibrosiVand pulmonary fibrosis. For example, symptoms of asthma include, but are not limited to, wheezing, shortness of breath, bronchoconstriction, airway hyperreactivity, decreased lung capacity, fibrosis, airway inflammation, and mucus production. The method comprises administering to the subject an IL-13 antagonist, e.g., an IL-13 antibody or
a fragment thereof, in an amount sufficient to treat (e.g., reduce, ameliorate) or prevent one or more symptoms. The IL-13 antibody can be administered therapeutically or prophylactically, or both. The IL-13 antagonist, e.g., the anti-IL-13 antibody, or fragment thereof, can be administered to the subject, a10ne or in combination with other therapeutic modalities as described herein. Preferably, the subject is a mammal, e.g., a human suffering from an IL-13- associated disorder as described herein.
In another aspect, this application provides a method for detecting the presence of IL-13 in a sample in vitro (e.g., a bio10gical sample, such as serum, plasma, tissue, biopsy). The subject method can be used to diagnose a disorder, e.g., an immune cell-associated disorder. The method includes: (i) contacting the sample or a control sample with the anti-IL-13 antibody or fragment thereof as described herein; and (ii) detecting formation of a complex between the anti-IL-13 antibody or fragment thereof, and the sample or the control sample, wherein a statistically significant change in the formation of the complex in the sample relative to the control sample is indicative of the presence of the IL-13 in the sample.
In yet another aspect, this application provides a method for detecting the presence of IL-13 in vivo (e.g., in viva imaging in a subject). The subject method can be used to diagnose a disorder, e.g., an IL-13- associated disorder. The method includes: (i) administering the anti-IL-13 antibody or fragment thereof as described herein to a subject or a control subject under conditions that al10w binding of the antibody or fragment to IL-13; and (ii) detecting formation of a complex between the antibody or fragment and IL-13, wherein a statistically significant change in the formation of the complex in the subject relative to the control subject is indicative of the presence of IL-13.
Antibodies of the invention, or antigen binding portions thereof can be used a10ne or in combination to treat such diseases. It should be understood that the antibodies of the invention or antigen binding portion thereof can be used a10ne or in combination with an additional agent, e.g., a therapeutic agent, said additional agent being selected by the skilled artisan for its intended purpose. For example, the additional agent can be a therapeutic agent art-recognized as being useful to treat the disease or condition being treated by the antibody of the present invention. The additional agent also can be an agent that imparts a beneficial attribute to the therapeutic composition e.g., an agent which effects the viscosity of the composition.
It should further be understood that the combinations which are to be included within this invention are those combinations useful for their intended purpose. The agents set forth be10w are illustrative for purposes and not intended to be limited. The combinations, which are part of this invention, can be the antibodies of the present invention and at least one additional agent selected from the lists be10w. The combination can also include more than one additional agent.
e.g., two or three additional agents if the combination is siicHthat the formed composition can perform its intended function.
The combination therapy can include one or more IL-13 antagonists, e.g., anti-IL-13 antibodies or fragments thereof, coformulated with, and/or coadministered with, one or more additional therapeutic agents, e. g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti- inflammatory agents (e.g., systemic anti-inflanunatory agents), anti-fibrotic agents, metabolic inhibitors, enzyme inhibitors, and/or cytotoX1c or cytostatic agents, as described in more herein.
Examples of preferred additional therapeutic agents that can be coadministered and/or coformulated with one or more IL-13 antagonists, e. g., anti- IL-13 antibodies or fragments thereof, include, but are not limited to, one or more of: inhaled steroids; beta-agonists, e.g., short-acting or 10ng- acting beta-agonists; antagonists of leukotrienes or leukotriene receptors; combination drugs such as ADVAIR; IgE inhibitors, e.g., anti-IgE antibodies (e. g., XOLAIR); phosphodiesterase inhibitors (e. g., PDE4 inhibitors); xanthines; anticholinergic drugs; mast cell-stabilizing agents such as cromolyn; IL-4 inhibitors; IL-5 inhibitors; eotaX1n/CC3l3 inhibitors; antagonists of histamine or its receptors including Hi, H2, H3, and H4, and antagonists of prostaglandin D or its receptors (DPI and CRTH2). Such combinations can be used to treat asthma and other respiratory disorders. Additional exanq)les of therapeutic agents that can be coadministered and/or coformulated with one or more anti-IL-13 antibodies or fragments thereof include one or more of: TNF antagonists (e.g., a soluble fragment of a TNF receptor, e.g., p55 or p75 human TNF receptor or derivatives thereof, e.g., 75 kD TNFR-IgG (75 kD TNF receptor-IgG fusion protein, ENBREL)); TNF enzyme antagonists, e.g., TNF converting enzyme (TACE) inhibitors; muscarinic receptor antagonists; TGF-beta antagonists; iiiterferon gamma; perfenidone; chemotherapeutic agents, e.g., methotrexate, leflunomide, or a sirolimus (rapamycin) or an ana10g thereof, e.g., CCI-779; C0X2 and cPLA2 inhibitors; NSAIDs; immunomodulators; p38 inhibitors, TPL-2, MK-2 and NFkB inhibitors, among others.
Other preferred combinations are cytokine suppressive anti-inflammatory drug(s) (CSAIDs); antibodies to or antagonists of other human cytokines or growth factors, for example, EL-l, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, IL-31, interferons, EMAP-n, GM-CSF, FGF, EGF, PDGF, and edothelin-1, as well as the receptors of these cytokines and growth factors. Antibodies of the invention, or antigen binding portions thereof, can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CDS, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA or their ligands including CD154 (gp39 or CD40L).
Preferred combinations of therapeutic agents may interfere at different points in the inflammatory cascade; preferred exan^les include TNF antagonists like chimeric, humanized or
human TNF antibodies, D2E7, (PCX Publication No. WO 97/29131), CA2 (RemicadeTM). CDP 571, and soluble p55 or p75 TNF receptors, derivatives, thereof, (p75TNFRlgG (EnbrelTM) or p55TNFRlgG (Lenercept), and also TNF converting enzyme (TACE) inhibitors; similarly E.-1 inhibitors (Interleukin-1-converting enzyme inhibitors, IL-IRA etc.) may be effective for the same reason. Other preferred combinations include Interleukin 4. Yet another preferred combination are other key players of the asthmatic response which may act parallel to, dependent on or in concert with lL-13 function; especially preferred are IL-9 antagonists including IL-9 antibodies. It has been shown that IL-13 and IL-9 have overlapping but distinct functions and a combination of antagonists to both may be most effective. Yet another preferred combination are anti-IL-5 antibodies. Yet other preferred combinations include antagonists of chemokines including MCP-1, MCPA, eotaX1ns, RANTES, MDC, CCL-12 and CCL-17 (TARC) and chemokine receptors including CCR2, CCR3, CCR4, and CXCR4. Yet combinations can include antagonists to asthma mediators including acid mammalian chitinase, CRHT2, chymase, SlPl, S1P2, Tyk2, ROCKII, Stat6, p38, NFkB, phosphodiesterase 4 (PDE-4), mast cell trytase, NO, adenosine, IKK2, GATA3, ICAM-1, VCAM-1, and ICOS.
The pharmaceutical compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antibody portion of the invention. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the antibody or antibody portion may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toX1c or detrimental effects of the antibody, or antibody portion, are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the eX1gencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in
association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active con:q)ound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the invention is 0.1-20 rag/kg, more preferably 1-10 mg/kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the invention described herein are obvious and may be made using suitable equivalents without departing from the scope of the invention or the embodiments disc10sed herein. Having now described the present invention in detail, the same will be more clearly understood by reference to the fol10wing examples, which are included for purposes of illustration only and are not intended to be limiting of the invention.
Examples
Example 1: Generation and isolation of anti human IL-13 monoc10nal antibodies
Example 1.1: Assays to identify anti human IL-13 antibodies
Throughout Example 1 the fol10wing assays were used to identify and characterize anti human IL-13 antibodies unless otherwise stated.
Example 1.1.A: ELISA
Enzyme Linked Immunosorbent Assays to screen for antibodies that bind human IL-13 were performed as fol10ws.
ELISA plates (Coming Costar, Acton, MA) were coated with 50|xL/well of 5ng/ml goat anti-mouse IgG Fc specific (Pierce # 31170, Rockford, EL.) in Phosphate Buffered Saline (PBS) overnight at 4 degrees Celsius. Plates were washed once with PBS containing 0.05% Tween-20. Plates were b10cked by addition of 200 fiL/well b10cking solution diluted to 2% in PBS (BioRad #170-6404, Hercules, CA.) for 1 hour at room temperature. Plates were washed once after b10cking with PBS containing 0.05% Tween-20.
Fifty microliters per well of mouse sera or hybridoma supematants diluted in PBS containing 0.1% Bovine Serum Albumin (BSA) (Sigma, St. 10uis, MO.) was added to the ELISA plate prepared as described above and incubated for 1 hour at room temperature. Wells were washed three times with PBS containing 0.05% Tween-20. Fifty microliters of biotinylated recombinant purified human IL-13 variant (Rl 10Q) diluted to 10Ong/mL in PBS containing 0.1% BSA was added to each well and incubated for 1 hour at room temperature. Plates were washed 3 times with PBS containing 0.05% Tween-20. Streptavidin HRP (Pierce # 21126, Rockland, IL.) was diluted 1:20000 in PBS containing 0.1% BSA; 50 (xL/well was added and the plates incubated for 1 hour at room temperature. Plates were washed 3 times with PBS containing 0.05% Tween-20. Fifty microliters of TMB solution (Sigma # T0440, St. 10uis, MO.) was added to each well and incubated for 10 minutes at room temperature. The reaction was stopped by addition of IN sulphuric acid. Plates were read spectrophotmetrically at a wavelength of 450 nm.
Example 1.1.B: Affinity Determinations using BIACORE techno10gy
The BIACORE assay (Biacore, Inc, Piscataway, NJ) determines the affinity of antibodies with kinetic measurements of on-, off-rate constants. Binding of antibodies to recombinant purified human IL-13 or recombinant purified human IL-13 variant (Rl 10Q) were determined by surface plasmon resonance-based measurements with a Biacore® 30(X) instrument (Biacore®
AB, Uppsala, Sweden) using running HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25° C. All chemicals were obtained from Biacore® AB (Uppsala, Sweden) or otherwise from a different source as described in the text. ApproX1mately 5000 RU of goat anti-mouse IgG, (Fey), fragment specific polyc10nal antibody (Pierce Biotechno10gy Inc, Rockford, EL) diluted in 10 mM sodium acetate (pH 4.5) was directly immobilized across a CM5 research grade biosensor chip using a standard amine coupling kit according to manufacturer's instructions and procedures at 25 (xg/ml. Unreacted moieties on the biosensor surface were b10cked with ethanolamine. Modified carboxymethyl dextran surface in f10wcell 2 and 4 was used as a reaction surface. Unmodified carboxymethyl dextran without goat anti-mouse IgG in f10w cell 1 and 3 was used as the reference surface. For kinetic analysis, rate equations derived from the 1:1 Langmuir binding model were fitted simultaneously to association and dissociation phases of all eight injections (using g10bal fit analysis) with the use of Biaevaluation 4.0.1 software. Purified antibodies were diluted in HEPES-buffered saline for capture across goat anti-mouse IgG specific reaction surfaces. Mouse antibodies to be captured as a ligand (25 |Xg/ml) were injected over reaction matrices at a f10w rate of 5 ^1/min. The association and dissociation rate constants, kon (unit M'S') and kotr (unit s') were determined under a continuous f10w rate of 25 ^Unun. Rate constants were derived by making kinetic binding measurements at ten different antigen concentrations ranging from 10 - 200 nM. The equilibrium dissociation constant (unit M) of the reaction between mouse antibodies and recombinant purified human IL-13 or recombinant purified human 1L-I3 was then calculated from the kinetic rate constants by the fol10wing formula: KD = kou/kon- Binding is recorded as a function of time and kinetic rate constants are calculated. In this assay, on-rates as fast as 10^M'^s' and off-rates as s10w as 10'** s*' can be measured.
Example l.l.C: Functional Activity of anti human IL-13 antibodies
To examine the functional activity of the anti-human IL-13 antibodies of the invention, the antibodies were used in the fol10wing assays that measure the ability of an antibody to inhibit IL-13 activity.
Example l.l.C 1A-549 bioassav
The ability of anti-human IL-13 antibodies to inhibit the human IL-13 induced production of TARC (CCL-17) by A-549 cells was analyzed as fol10ws. A-549 cells were seeded on day one in 96-well plate (2E5 cells/well) in RPMI growth medium (with 10% FBS). On day two, the medium was replaced with fresh RPMI growth medium containing 400ng/ml rhTNF (10Onl/well). Meanwhile, various concentrations of immunized mouse serum, murine hybridoma supernatant or purified anti-human IL-13 antibodies were preincubated for one hour at 37°C with
10 ng/ml recombinant purified hunian IL-13 or IL-13 variant in 100|xL RPMI complete medium in a microtiter plate (U-bottom, 96-well, Costar). The antibody plus recombinant purified human IL-13 mixture was then added (10Onl/well) to the TNF-traeted A-549 cells, with the final volume of 200nl/well (final IL-13 and TNF concentrations were 5ng/ml and 200ng/ml, respectively), and incubated for 18 hours at 37°C. After incubation, l50\iL of cell-free supernatant was withdrawn from each well and the level of human TARC produced was measured using a human TARC ELISA (R&D Systems Cat#DDNOO).
The A-549 cells also respond to IL-13 of other species, including cynomolgus monkey, mouse, rat, and sheep, with ED50 values similar to that of human IL-13. Therefore it was emp10yed for cross-reactive analysis of anti-hIL-13 mAbs to rL-13 of other species using the same experimental protocol.
Example 1J2: Generation of Anti human IL-13 monoc10nal antibodies
Anti human IL-13 mouse monoc10nal antibodies were obtained as fol10ws:
Example 1.2A: Immunization of mice with human IL-13 antigen
Twenty micrograms of recombinant purified hunian IL-13 variant (Peprotech) mixed with conq)lete Freund's adjuvant or Immunoeasy adjuvant (Qiagen, Valencia, CA) was injected subcutaneously into five 6-8 week-old Balb/C, five C57B/6 mice, and five AJ mice on Day 1. On days 24,38, and 49, twenty micrograms of recombinant purified human IL-13 variant mixed with incomplete Freund's adjuvant or Immunoeasy adjuvant was injected subcutaneously into the same mice. On day 84 or day 112 or day 144, mice were injected intravenously with 1 ug recombinant purified human IL-13 variant.
Example 1^.B: Generation of Hybridoma
Splenocytes obtained from the immunized mice described in Example 1.2.A were fused with SP2/0-Ag-14 cells at a ratio of 5:1 according to the established method described in Kohler, G. and Milstein 1975, Nature, 256:495 to generate hybridomas. Fusion products were plated in selection media containing azaserine and hypoxanthine in 96-well plates at a density of 2.5x10* spleen cells per well. Seven to ten days post fusion, macroscopic hybridoma co10nies were observed. Supernatant from each well containing hybridoma co10nies was tested by ELISA for the presence of antibody to IL-13 variant (as described in Example 1.1.A). Supematants displaying IL-13 variant-specific activity were then tested for the ability to neutralize IL-13 variant and IL-13 wild-type in the A-549 bioassay for TARC (as described in Example l.l.C).
Example 1.2.C: Identification and characterization of anti human lL-13 monoc10nal antibodies
Hybridomas producing antibodies that bound IL-13 variant, generated according to Examples 1.2.B and 1.2.C, and capable of binding IL-13 variant specifically and particularly those with IC50 values in the A-549 bioassay of 5nM or less than 5nM were scaled up and c10ned by limiting dilution.
Hybridoma cells were expanded into media containing 10% 10w IgG fetal bovine serum (Hyc10ne #SH30151, 10gan, UT.). On average, 250 mL of each hybridoma supernatant (derived from a c10nal population) was harvested, concentrated and purified by protein A affinity chromatography, as described in Har10w, E. and Lane, D. 1988 "Antibodies: A Laboratory Manual". The ability of purified mAbs to inhibit IL-13 activity was determined using the A-549 bioassay as described in Examples l.l.C. Table 7 shows IC50 values from the A-549 bioassays for 17 monoc10nal antibodies.
Table 7: Neutralization of IL-13 by anti IL-13 mAbs in A-549 bioassay

(TABLE REMOVED)
The binding affinities of the monoc10nal antibodies to recombinant purified human IL-13 variant and wild-type were determined using surface plasmon resonance (Biacore®) measurenient as described in Example l.l.B. Table 8 shows the affinity of the 18 monoc10nal antibodies described above for human IL-13.
Table 8: Affinity of anti IL-13 mAbs for human wild-type and variant IL-13

(TABLE REMOVED)
Example 1.2.C.1: Species Specificity of murine monoc10nal anti-human IL-13 antibodies
To determine whether the 17 monoc10nal antibodies described above recognize murine IL-13, an indirect ELISA was set up by coating ELISA plates with Sug/ml of goat anti-mouse IgG, Fc fragment specific antibody (Pierce # 31170, Rockland, EL). Murine anti-human IL-13 mAbs were prepared at various concentrations ranging from 0.1 to 100 ng/ml in PBS containing 0.1% BSA: 50 ui of eacli antibody dilution was added to the coated ELISA plate and incubated for I hour at room temperature. Wells were washed 3 times with PBS containing 0.05% Tween-20. Recombinant biotinylated mouse IL-13 (R&D Systems) was diluted at 0.1 ug/ml in PBS containing 0.1% BSA; 50 ul/well was added and the plates incubated for 1 hour at room temperature. Wells were washed 3 times with PBS containing 0.05% Tween-20. Streptavidin HRP (Pierce # 21126, Rockland, IL.) was diluted 1:20000 in PBS containing 0.1% BSA; 50 mL/well was added and the plates incubated for 1 hour at room temperature. Plates were washed 3 times with PBS containing 0.05% Tween-20. Fifty microliters of TMB solution (Sigma # T0440, St. 10uis, MO.) was added to each well and incubated for 10 nninutes at room temperature. The reaction was stopped by addition of IN sulphuric acid. Plates were read spectrophotmetrically at a wavelength of 450 nm. Results from the indirect ELISA indicated that mAb 3H7 was able to bind mIL-13. In subsequent bioassay it was shown that 3H7 could inhibit mIL-13-stimulated TARC production in a dose-dependent mannor, with an ICjo of 2.4nM.
Biacore analysis also demonstrated positive binding of 3H7 to mIL-13, with a KD of 12nM. All other mAbs in table 8 did not show any positive binding to mouse IL-13.
Neutralisation potency of anti-hIL-13 mAbs against non-human primate (cynomolgus) IL-13 and sheep IL-13 were also measured in the A-548 bioassay. To generate cyno and sheep IL-13, cDNA for each protein was obtained by PCR on genomic DNA template using degenerate primers based on the human IL-13 sequence. Recombinant cyno and sheep IL-13 proteins were subsequently expressed in transiently transfected COS cells. Wild-type human IL-13 was also generated in parallel as a control in all functional studies. A-549 cells responded to both cyno and sheep IL-13 with a similar ED50 to that of human IL-13. Most of the mAbs neutralized activity of cyno IL-13, demonstrating cross-reactivity to cyno IL-13 (Table 7). However none of the antibodies showed significant neutralization of sheep IL-13.
Example 1.2.C.2: Murine monoc10nal anti-human IL-13 antibodies b10ck IL-13 binding to IL-13 receptors aL-13Ral and IL-13Rot2)
IL-13 activity is mediated through a receptor complex consisting of the IL-13Ral and IL-4Ra chains. The cytokine first undergoes a relatively 10w affinity interaction with IL-13Ral on the surface of cells. The lL-13/IL-13Ral complex then recruits IL-4Ra to form the complete IL-13 receptor, which is bound to its ligand (IL-13) with high affinity (Zurawski et al. (1993) EMBO J. 12:2663; Zurawski et al. (1995) J. Biol. Chem. 270:23869). The binding of IL-13 with the high affinity receptor then sends downstream signals through the IL-4Ra chain involving the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, e.g., via phosphorylation of STAT6, which can be monitored as one of the earliest cellular responses to IL-13 (Murata et al., supra).
There is another IL-13-binding receptor, the IL-13Ra2 chain (IL-13Ra2), which binds to IL-13 with high affinity (0.25-1.2 nM) (Caput, et al 1996 J. Biol. Chem. 271,16921-16926; Donaldson et al 1998 J. Immunol. 161,2317-2324). No other receptor molecule is known to be involved in the IL-13/IL-13Ra2 complex. IL-13R(X2 was initially thought to act as a nonsignaling "decoy" receptor. However, it was later discovered that it can bind to IL-13, and signals through AP-1 pathway, leading to TGF-beta production in certain cell types including macrophages, which in turn leads to lung fibrosis (Fichmer-Feigl. 2006 Nat Med 12:99-106). Therefore, both IL-13Ral/IL-4R complex and IL-13Ra2 pathways contribute to the overall pathophysio10gy of asthma and other IL-13 mediated diseases. Several approaches, such as epitope mapping, receptor binding assays, size exclusion chromatography (SEC), and further BIACORE analysis, were used to elucidate the interaction between the anti-D^-lB antibodies of the invention and human IL-13.
To determine whether the monoc10nal antibodies described above are able to b10ck lL-13 binding to lL-13 receptors (IL-13Ral and IL-13Ra2), a receptor binding ELISA was deve10ped as fol10ws. High-binding 96-well ELISA plates were coated with 4 ug/ml of recombinant IL-13Ral/Fc or E.-I3R(x2/Fc (R&D Systems) in 10Oul/well coating buffer (Carbonate-bicarbonate buffer. Pierce) at 4"*C. After 16hr, coating solution was removed by flicking plate contents in sink, and plates were washed and b10cked 4 times with Superb10ck B10cking Buffer (240ul/well) (Pierce). Anti-IL13 mAbs (1:4 serially diluted from 40ug/ml, 50ul/well) and Biotin-IL-13 (50ul/well, final concentrations of 5nM for hIL-13Ral/Fc, and O.SnM for hBL-13R(x2/Fc) were added and incubated for 2hr at room temperature (RT). Plates were washed 5 times with 300ul 0.1%PBST, and then 10Oul of 1:5000 diluted mouse anti-Biotin MAb (Jackson Immunosciences) was added and incubated at RT for 45 min. The plates were washed again 5 times with 300ul 0.1%PBST, fol10wed by addition of TMB substrate reagent (10Oul/well, Pharmingen); deve10ped for 5 min, and stopped by adding 50ul of 2M H2S04 (VWR). ODs at 450nm were determined by spectrophotometry.
Additionally, the receptor b10cking properties of the mAbs were also assessed by receptor binding assay using IL-13Ra2-transfected COS cells. Recombinant human IL-13 was labeled with '"l (Amersham, Arlington Heights, IL), using 10DO-GEN reagent (Pierce, Rockford, IL) as previously described (Obiri NI et al., (1995) J Biol Chem. 270:8797-8804). The specific activity of the radiolabeled IL-13 was estimated to be 158 \^Ci/^lg protein. The labeled IL-13 exhibited similar bioactivity as unlabeled IL-13, as assessed by the A-549 bioassay. For binding experiments, COS cells were transiently transfected with human IL-I3RG(2 by Lipofectamine 2000 (Invitrogen), and incubated for 48 hr. Transfected COS cells (5 x 105 cells in 100 (iL binding buffer: RPMI1640 containing 0.2% human serum atlbumin and 10 mmol HEPES) were incubated with 1.0 nM '"l-IL-13 with or without 1 uM unlabeled IL-13 at 4''C for 2 hours. Cell-bound '^I-IL-13 was separated from unbound '^I-IL-13 by centrifugation through a phthalate oil gradient, and radioactivity was determined with a gamma counter (Wallac, Gaithersburg, MD). For antibody displacement assay, transfected COS cells were incubated with 125I-IL-13 (1.0 nM) with or without increasing concentrations (up to 50 ug/ml) of anti-IL-13 antibodies, as described above. Both forms of receptor binding assay demonstrated the fol10wing: First, 13C5 and 9C11 b10cked IL-13 binding to IL-13Ral; second, 13C5 strongly b10cked IL-13 binding to IL-13Ra2 (ICM ~ 1-3 nM in both cell surface RBA and RB ELISA), whereas 9C11 b10cked IL-13 binding of IL-13Ra2 with a 10wer potency (ICM > 10 nM); and third, 5G1 and 3E5 failed to b10ck IL-13 binding to either IL-13Ral or IL-13Ra2. Three other anti-IL-13 antibodies, BAK502G9 (CAT PCT WO 2005/007699), mAbl3.2 (Wyeth PCT WO 2005/123126A2) and MJ2-7 (Wyeth PCT WO 2006/0073148A1) were also analyzed for their
ability to b10ck human IL-13 binding to human IL-13 Ra2 on both receptor binding ELISA and cell surface RBA. Antibody mAbl3.2 did not b10ck IL-13 binding to eith IL-13Ral or IL-13Ra2. BAK502G9 and MJ2-7 was able to b10ck IL-13 binding to IL-13Ral; however they exhibited 10w potency in b10cking IL-13 binding to IL-13Ra2 with antibody concentrations up to 50tig/ml(330nM).
The interaction between IL-13 and IL-13Ral/a2 in the presense of anti-IL-13 mAbs was also analyzed by BIACORE. This analysis was done in several formats. First, IL-13Ral/Fc was bound to the Biacore chip and IL-13 was f10wed over the chip, in the presence and absence of anti-IL-13 mAbs. MAbs 13C5 and 9C11, among others, were able to b10ck IL-13 binding to IL-13Ral, whereas 5G1 and 3E5 failed to inhibit IL-13 binding to IL-13Ral, consistent with the receptor binding assays. Second, IL-4R was bound to the BIACORE chip, and a complex of IL-13 prebound to IL-13Rol was f10wed over the chip. In absence of anti-IL-13 mAbs, formation of a trimolecular complex was demonstrated. However, addition of anti-IL-13 antibody 5G1 to the mixture of IL-13 prebound to IL-13Ral prevented binding to IL-4R on the chip. This indicated that, even though 5G1 could not b10ck IL-13 binding to IL-13Ral, it could b10ck binding of IL-13 binding to IL-4R, providing a mechanistic basis for its IL-13-neutralizing activity. These observations were further confirmed by size exclusion chromatography (SEC), where hetero-trimeric complexes (niAb-IL-13-IL-13Ral/Fc) were observed for 5G1, but not for 13C5. Subsequent epitope mapping studies using proteinase processing of mAb-IL-13 con:q)lex fol10wed by mass spec analysis indicated the fol10wing: First, 5G1 binds to IL-13 residues including the N-terminal 11-aa peptide (GPVPPSTALRE), covering part of the Helix-A region that has been shown to interact with IL-4R (Moy et al 2001J Mol Biol. 310:219 and Horita et al., (2001) J Mol Biol. 310:231); second, antibody 9C11 interacts with a region between Helix C and Helix D (VSAGQFSSLHVR); and third, antibody 13C5 interacts with IL-13 residues including a region covering Helix D (VRDTK EVAQ FVKDL LLHLK KLFRE GR corresponding to amino acid 104-130 of SEQ ID NO. 1). Helix D has been shown to interact with IL-13 receptors (Moy et al 2001J Mol Biol. 310:219; Horita et al 2001 J Mol Biol. 310:231; and Madhankumar et al 2002 JBC 277:43194). Since 13C5 binds human IL-13 variant (KD=50 pM) much more strongly than cynomolgus IL-13 (KD=1800 pM), and the only sequence difference between human IL-13 variant and cynomolgus IL-13 within this potential 13C5 epitope region is L in human but V in cyno IL-13 at position 120, we generated a V120L mutant cyno IL-13 and tested whether this mutation will have increased binding affinity to 13C5 over wild type cyno IL-13. Based on Biacore and bioassay results, the binding affinity as well as neutralization potency of 13CS for the V120L mutant cyno IL-13 were equivalent to that for the wild type cyno IL-13, indicating that this V/L difference at position 120 within the C-terminal region does not contribute to the
13C5 affinity difference for human IL-13 variant versus cyno IL-13, and that there must be other residues outside the C-terminal region that contribute to the differential binding affinity of 13C5 to human and cyno IL-13. This is consistent with the observation that 13C5 does not recognize denatured human IL-13 by Western b10t analysis, indicating that the binding epitope of 13CS on human IL-13 is strongly conformational.
The binding and epitope mapping studies indicated that SGI did not inhibit IL-13 interaction with IL-13Rol but disrupted the interaction of IL-13/IL-13Rol with IL-4Ra. This disruption is thought to interfere with the formation of a functional IL-13 signaling complex. These observations provide a theoretical model for the neutralizing activity of this antibody in an IL-13Ral/IL-4R-mediated system such as A-549 cells. In contrast, 13C5 b10cked binding of IL-13 to both IL-13Ral and IL-13Ra2. Interestingly, even though 9C11 was able to b10ck IL-13 binding to IL-13Ral, it only showed partial (or 10w potency) inhibition of IL-13 binding to IL-13R(x2. Although IL-13Ral and Ra2 adopt similar 3-dimentional fold and IL-13 binding orientation, they have a 10w sequence identity and therefore specific residues responsible for IL-13 binding may vary (Arima 2005 JBC 280:24915; and Madhankumar et al 2002 JBC 277:43194). Consequently, specific residues on IL-13 for binding to IL-13Ral and IL-13Ra2 may differ, which could explain the differential receptor b10cking properties of 9C11.
The receptor binding assays, epitope mapping, Biacore, and bioassay described above collectively indicate that a neutralizing anti-IL-13 antibody can inhibit IL-13-associated activity through the fol10wing mechanisms:
1) Inhibit IL-13 binding to both IL-13Ral and IL-13Rcx2 by interacting with IL-13 in the region involved in receptor binding to both IL-13Ral and IL-13R(x2. An example of such an antibody is I3C5. Such antibodies will inhibit IL-13 signalling through both IL-13Ral/IL-4R complex and IL-13Ra2.
2) Does not inhibit IL-13 binding to either IL-13Ral or IL-13Rcx2. However the antibody inhibits interaction with IL-4 receptor, therefore, inhibits IL-13 signalling through IL-I3Ral/IL-4R complex. Such antibody may not inhibit IL-13R(x2 signalling. Exan^les of such antibodies are 5G1 and mAbl3.2 (Wyeth PCT WO 2005/123126).
3) Inhibits IL-13 binding to IL-13Ral but not effectively inhibit IL-13 binding to IL-13Ra2. This could occur for the fol10wing reasons: a) epitope: a region that involved in IL-13Ral binding but not in IL-13Ra2 binding or not as strongly involved in IL-13Ra2 binding. An example is 9C11; b) affinity: since IL-13Ra2 has much higher affinity than IL-13Ral for IL-13, a 10w affinity antibody may be able to b10ck IL-13 binding to IL-13Ral but not to IL-13R(x2 at physio10gical concenttations of a therapeutic antibody. An example is BAK502G9, which displayed a 2.11nM affinity to recombinant wild-type human IL-13 as assessed by Biacore (CAT
PCT WO 2005/007699). Another example is MJ2-7, which displayed a 1.4 nM affinity to recombinant wild-type human IL-13 and a higher affinity (43 pM) for monkey IL-13 as assessed by Biacore (Wyeth PCT WO 2006/0073148A1). Due to the affinity difference, this mAb can inhibit monkey IL-13 binding to IL-13Ra2 effectively; however it inhibits human IL-13 binding to the same receptor with much less potency.
The mAbs BAK502G9 and MJ2-7 have similar epitopes (CAT PCT WO 2005/007699 and Wyeth PCT WO 2006/0073148A1) and they compete for binding to IL-13 as assessed by competition ELISA. Briefly, BAK502G9 was immobilized on ELISA plate fol10wed by wash and b10cking. Then biotinylated human IL-13 (10ng/ml) was added to the plate in the presence of various concentrations of MJ2-7 (0.2ng/ml to 20ug/inl), fol10wed by wash and detection using HRP-conjugated anti-biotin antibody. This study demonstrated that MJ2-7 dose-dependently competed with BAK502G9 for binding to human IL-13. A negative control IgG did not show competition with BAK502G9.
Example 1.2.D Determination of the amino acid sequence of the variable region for each murine anti-human IL-13 mAb
For each amino acid sequence determination, approX1mately 10x10hybridoma cells were isolated by centrifugation and processed to isolate total RNA with Trizol (Gibco BRL/Invitrogen, Carlsbad, CA.) fol10wing manufacturer's instructions. Total RNA was subjected to first strand DNA synthesis using the Superscript First-Strand Synthesis System (Invitrogen, Carlsbad, CA) per the manufacturers instructions. Oligo(dT) was used to prime first-strand synthesis to select for poly(A)* RNA. The first-strand cDNA product was then amplified by PCR with primers designed for amplification of murine immunog10bulin variable regions (Ig-Primer Sets, Novagen, Madison, WI). PCR products were resolved on an agarose gel, excised, purified, and then subc10ned with the TOPO C10ning kit into pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA) and transformed into TOPIO chemically con^tent E. coli (Invitrogen, Carlsbad, CA). Co10ny PCR was performed on the transformants to identify c10nes containing insert. Plasmid DNA was isolated from c10nes containing insert using a QIAprep Miniprep kit (Qiagen, Valencia, CA). Inserts in the plasmids were sequenced on both strands to determine the variable heavy or variable light chain DNA sequences using Ml3 forward and M13 reverse primers (Fermentas Life Sciences, Hanover MD). Variable heavy and variable light chain sequences of the 17 monoc10nal antibodies described in Example 1.2.C are described in Table 5.
Example 2: Recombinant anti human IL-13 antibodies
antibodies
The DNA encoding the heavy chain constant region of murine anti-human IL-13 monoc10nal antibodies 5G1,13C5,9C11,21D9, and 3H7 was replaced by a cDNA fragment encoding the human IgGl constant region containing 2 hinge-region amino acid mutations by homo10gous recombination in bacteria. These mutations are a leucine to alanine change at position 234 (EU numbering) and a leucine to alanine change at position 235 (Lund et al., 1991, J. Immunol., 147:2657). The light chain constant region of each of these antibodies was replaced by a human kappa constant region. Full-length chimeric antibodies were transiently expressed in COS cells by co-transfection of chimeric heavy and light chain cDNAs ligated into the pBOS expression plasmid (Mizushima and Nagata, Nucleic Acids Research 1990, Vol 18, pg 5322). Cell supematants containing recombinant chimeric antibody were purified by Protein A Sepharose chromatography and bound antibody was eluted by addition of acid buffer. Antibodies were neutralized and dialyzed into PBS.
The purified chimeric anti-human IL-13 monoc10nal antibodies were then tested for their ability to inhibit the IL-13 induced production of TARC by A-549 cells as described in Exanq>les l.l.C 2 and 1.1.C3. Table 12 shows ICjo values from the A-549 bioassays for three chimeric antibodies. Table 9 Neutralization of rhIL-13 wt by anti IL-13 Chimeric Antibodies in A-S49 bioassay

(TABLE REMOVED)
Example 2.2: Construction and expression of humanized anti human IL-13 antibodies Example 2.2.1: Selection of human antibody frameworks
Each murine variable heavy and variable light chain gene sequence (as described in Table 3) was separately aligned against 44 human immunog10bulin germline variable heavy chain or 46 germline variable light chain sequences (derived fi-om NCBI Ig Blast website at http://www.ncbi.nlm.nih.gov/igblast/retrieveig.html.) using Vector NTI software.
Humanization was based on amino acid sequence homo10gy, CDR cluster analysis, frequency of use among expressed human antibodies, and available information on the crystal structures of human antibodies. Taking into account possible effects on antibody binding, VH-VL pairing, and other factors, murine residues were mutated to human residues where murine and human fi'amework residues were different, with a few exceptions. Additional humanization strategies were designed based on an analysis of human germline antibody sequences, or a
subgroup thereof, that possessed a high degree of homo10gy; i.e., sequence similarity, to the actual amino acid sequence of the murine antibody variable regions.
Homo10gy modeling was used was to identify residues unique to the murine antibody sequences that are predicted to be critical to the structure of the antibody combining site (the CDRs). Homo10gy modeling is a computational method whereby approX1mate three dimensional coordinates are generated for a protein. The source of initial coordinates and guidance for their further refinement is a second protein, the reference protein, for which the three dimensional coordinates are known and the sequence of which is related to the sequence of the first protein. The relationship among the sequences of the two proteins is used to generate a correspondence between the reference protein and the protein for which coordinates are desired, the target protein. The primary sequences of the reference and target proteins are aligned with coordinates of identical portions of the two proteins transferred directly from the reference protein to the target protein. Coordinates for mismatched portions of the two proteins, e.g. from residue mutations, insertions, or deletions, are constructed from generic structural templates and energy refined to insure consistency with the already transferred model coordinates. This computational protein structure may be further refined or emp10yed directly in modeling studies. It should be clear from this description that the quality of the model structure is determined by the accuracy of the contention that the reference and target proteins are related and the precision with which the sequence alignment is constructed.
For the murine sequences 5G1,13C5 and 9C11, a combination of BLAST searching and visual inspection was used to identify suitable reference structures. Sequence identity of 25% between the reference and target amino acid sequences is considered the minimum necessary to attempt a homo10gy modeling exercise. Sequence alignments were.constructed manually and model coordinates were generated with the program Jackal (see Petrey, D., X1ang, Z., Tang, C.L., X1e, L., Gin^lev, M., Mitros, T., Soto, C.S., Goldsmith-Fischman, S., Kemytsky, A., Schlessinger, A., et al. 2(X)3. Using multiple structure alignments, fast model building, and energetic analysis in fold recognition and homo10gy modeling. Proteins 53 (Suppl. 6): 430-435).
The primary sequences of the murine and human framework regions of the selected antibodies share significant identity. Residue positions that differ are candidates for inclusion of the murine residue in the humanized sequence in order to retain the observed binding potency of the murine antibody. A list of framework residues that differ between the human and murine sequences was constructed manually.
The likelihood that a given framework residue would inq>act the binding properties of the antibody depends on its proX1mity to the CDR residues. Therefore, using the model structures, the residues that differ between the murine and human sequences were ranked according to their distance from any atom in the CDRs. Those residues that fell within 4.5 A of any CDR atom
were identified as most important and were recommended'.to be candidates ior retention of the murine residue in the humanized antibody (i.e. back mutation).
For humanization of the 5G1 variable regions, the general approach provided in the present invention was fol10wed. First, a molecular model of the 5G1 variable regions was constructed with the aid of the computer programs ABMOD and ENCAD (Levitt, M., J. Mol. Biol. 168: 595-620 (1983)). Next, based on a homo10gy search against human V and J segment sequences, the VH segment 21/28 (Dersimonian, H., et al., J. Immunol. 139: 2496-2501 (1987)) and the J segment JH4 (Ravetch, J.V., et al.. Cell 27: 583-591 (1981)) were selected to provide the frameworks for the Hu5Gl heavy chain variable region. For the 5G1 light chain variable region, the VL segment HF-21/28 (Chastagner, P., et al.. Gene 101: 305-306 (1991)) and the J segment JK4 (Hieter, P.A., et al., J. Biol. Chem. 257: 1516-1522 (1982)) were used. The identity of the franKwork amino acids between 5G1 VH and the acceptor human 21/28 and JH4 segments was 72%, while the identity between 5G1 VL and the acceptor human HF21/28 and JK4 segments was 83%. At framework positions in which the computer model suggested significant contact with the CDRs, the amino acids from the mouse V regions were substituted for the original human framework amino acids. This was done at residues 48,67, 68,70,72, 74 and 97 of the heavy chain. For the light chain, replacement was made at residue 50. Framework residues that occurred only rarely at their respective positions in the corresponding human V region subgroups were replaced with human consensus amino acids at those positions. This was done at residues 44 and 76 of the heavy chain, and at residues 2,15,41,42,44 and 51 of the light chain.
For humanization of the 13C5 variable regions, the general approach provided in the present invention was fol10wed. First, a molecular model of.the 13C5 variable regions was constructed with the aid of the computer programs ABMOD and ENCAD (Levitt, M., J. Mol. Biol. 168: 595-620 (1983)). Next, based on a homo10gy search against human V and J segment sequences, the VH segment M60 (Schroeder, Jr., H.W. and Wang, J.Y., Proc. Natl. Acad. Sci. USA 87: 6146-6150 (1990)) and the J segment JH4 (Ravetch, LV., et al.. Cell 27: 583-591 (1981)) were selected to provide the frameworks for the Hul3C5 heavy chain variable region. For the Hul3C5 light chain variable region, the VL segment in-3R (Manheimer-10ry, A., et al., J. Exp. Med. 174: 1639-1652 (1991)) and the J segment JK4 (Hieter, P.A., et al., J. Biol. Chem. 257: 1516-1522 (1982)) were used. The identity of the framework amino acids between 13C5 VH and the acceptor human M60 and JH4 segments was 74%, while the identity between 13C5 VL and the acceptor human in-3R and JK4 segments was 75%.
At framework positions in which the computer model suggested significant contact with the CDRs, the amino acids from the mouse V regions were substituted for the original human framework amino acids. This was done at residues 22,49 and 71 for the light chain. Framework
residues that occurred only rarely at their respective positions in the corresponding human V region subgroups were replaced with human consensus amino acids at those positions. This was done at residues 10,46, 83,84, 86 and 87 of the heavy chain, and at residues 62 and 73 of the light chain.
Amino acid sequences of VL and VH of humanized mAbs are shown in Table 10.
Table 10: List of amino acid sequences of humanized mAbs

(TABLE REMOVED)
Example 2.2.2: Construction of humanized antibodies
In silico constructed humanized antibodies described above were constructed de novo using oligonucleotides. For each variable region cDNA, 6 oligonucleotides of 60-80 nucleotides each were designed to overlap each other by 20 nucleotides at the 5' and/or 3' end of each oligonucleotide. In an annealing reaction, all 6 oligos were combined, boiled, and annealed in the presence of dNTPs. Then DNA polymerase I, Large (Klenow) fragment (New England Biolabs #M0210, Beverley, MA.) was added to fill-in the approX1mately 40bp gaps between the overlapping oligonucleotides. PCR was then performed to amplify the entire variable region gene using two outermost primers containing overhanging sequences complementary to the multiple c10ning site in a modified pBOS vector (Mizushima, S. and Nagata, S., (1990) Nucleic acids Research Vol 18, No. 17)). The PCR products derived from each cDNA assembly were separated on an agarose gel and the band corresponding to the predicted variable region cDNA size was excised and purified. The variable heavy region was inserted in-frame onto a cDNA fragment encoding the human IgGl constant region containing 2 hinge-region amino acid mutations by homo10gous recombination in bacteria. These mutations are a leucine to alanine' change at position 234 (EU numbering) and a leucine to alanine change at position 23S (Lund et al., 1991, J. Immunol., 147:2657). The variable light chain region was inserted in-frame with the human kappa constant region by homo10gous recombination. Bacterial co10nies were isolated and plasmid DNA extracted; cDNA inserts were sequenced in their entirety. Correct humanized heavy and light chains corresponding to each antibody were co-transfected into COS cells to transiently produce full-length humanized anti-human IL-13 antibodies. For 13C5, pBOS vectors containing the 13C5 heavy chain grafted cDNA and the 13C5 light chain grafted cDNA were co-transfected into COS cells. Cell supematants containing recombinant chimeric antibody were purified by Protein A Sepharose chromatography and bound antibody was eluted by addition of acid buffer. Antibodies were neutralized and dialyzed into PBS. Several humanized antibodies are described in Table 10.
The ability of purified humanized antibodies to inhibit IL-13 activity was determined using the A-S49 bioassay as described in Examples l.l.C. The binding affinities of the humanized antibodies to recombinant human 0.-13 were determined using surface plasmon resonance (Biacore®) measurement as described in Example I.I.B. Table 11 shows ICjo values from the A-549 bioassays and the affinity of the first six humanized antibodies described in Table 10 for human IL-13wt and variant. Table 11: Neutralization potency and affinity of humanized anti IL-13 mAbs.

(TABLE REMOVED)
The CDR sequences of the humanized antibody 13C5.5 were further mutated using techniques known in the art, and three additional humanized antibodies were generated. The ability of these additional humanized antibodies to inhibit human, cynomolgus and rhesus IL-13 activity was determined using the A-549 bioassay as described in Examples l.l.C. The binding affinities of the additional humanized antibodies to recombinant human, cynomolgus and rhesus IL-13 were determined using surface plasmon resonance (Biacore®) measurement as described in Example 1.1.B. In addition to binding and inhibiting human IL-13, these three additional antibodies showed enhanced affinity for cynomolgus and rhesus IL-13. TaWe 12 shows IC50 values from the A-549 bioassays, and Table 13 shows the affinity of the additional humanized antibodies to human, cynomolgus and rhesus IL-13.
Table 12: Neutralization potency of additional liumanized anti IL-13 mAbs

(TABLE REMOVED)
Example 2.2.3; Characterization of humanized anti IL-13 antibodies
We have isolated monoc10nal antibodies that b10ck IL-13 binding to both IL-13Ral and IL-13R(x2. Both ELISA-based receptor binding assay and 125-I-labeled IL-13 binding assay on cell surface demonstrated that 13C5, both murine version and humanized version (i.e. I3C5.5), were able to effective b10ck IL-13 binding to both receptors. Antibodies in the same lineage as 13C5, including 25C8 and 33C3, were also able to b10ck IL-13 binding to both receptors.
Example 2.23.a;Humanized anti IL-13 antibodies b10ck binding of IL-13 to IL-13 receptor
To determine the ability of humanized antibody 13C5.5 to b10ck IL-13 binding to IL-13 receptors (IL-13Ral and IL-13Ra2), an ELISA-based receptor binding assay was used. High-binding 96-well ELISA plates were coated with 4 ug/ml of recombinant human IL-13Ral/Fc or IL-13Ra2/Fc (R&D Systems) in 10Oul/well coating buffer (Carbonate-bicarbonate buffer, Pierce) at 4°C. After 16hr, coating solution was removed by flicking plate contents in sink, and plates were washed and b10cked 4 times with Superb10ck B10cking Buffer (240ul/well) (Pierce). Humanized anti-IL-13 mAb 13c5.5 and control mAbs (1:4 serially diluted from 40ug/ml, 50ul/well) and Biotin-IL-13 (50ul/well, final concentrations of 5nM for hIL-I3Ral/Fc, and 0.5nM for hIL-13Ra2/Fc) were added and incubated for 2hr at room temperature (RT). Plates were washed 5 times with 300ul 0.1%PBST, and then 10Oul of 1:5000 diluted mouse anti-Biotin MAb (Jackson Immunosciences) was added and incubated at RT for 45 min. The plates were washed again 5 times with 300ul 0.1%PBST, fol10wed by addition of TMB substrate reagent (l(X)ul/well, Pharmingen); deve10ped for 5 min, and stopped by adding 50ul of 2M H2S04 (VWR). ODs at 450nm were determined by spectrophotometry. The results are shown in Table 14.
Additionally, the receptor b10cking properties of the humanized mAbs were also assessed by cell surface-based receptor binding assay using IL-13Ro2-transfected COS cells. Recombinant human IL-13 was labeled with '^I (Amersham, Arlington Heights, IL), using 10DO-GEN reagent (Pierce, Rockford, IL) as previously described (Obiri NI et al., (1995) J Biol Chem. 270:8797-8804). The specific activity of the radiolabeled IL-13 was estimated to be 158 pCi/pg protein. The labeled IL-13 exhibited similar bioactivity as unlabeled IL-13, as assessed by the A-549 bioassay. For binding experiments, COS cells were transiently transfected with human IL-13Ra2 by Lipofectamine 2(X)0 (Invitrogen), and incubated for 48 hr. Transfected COS cells (5 X 105 cells in 100 jiL binding buffer: RPMI 1640 containing 0.2% human serum albumin and 10 mmol HEPES) were incubated with 1.0 nM '"l-IL-13 with or without 1 uM unlabeled IL-13 at 4°C for 2 hours. Cell-bound '"l-IL-13 was separated from unbound '^ML-13 by centrifugation through a phthalate oil gradient, and radioactivity was determined with a gamma
counter (Waliac, Gaithersburg, MD). For antibody displacement assay, transfected COS cells were incubated with 125I-IL-13 (1.0 nM) with or without increasing concentrations (up to 50 ug/ml) of humanized anti-IL-13 antibody 13C5.5, as described above. The results are shown in Table 14.
Table 14: Potency of mAbs in b10cking human IL-13 (wt) binding to human IL-13Ra2 in cell surface-based and ELISA-based receptor binding assays

(TABLE REMOVED)
P.B. Partial b10ckade that does not reach 50% inhibition.
Table 15 shows the binding affinity of the humanized 13C5.5 antibody and other anti-IL-13 antibodies.
Table 15: Binding affinity of anti-IL-13 mAbs as assessed by Biacore

(TABLE REMOVED)
In both cell surface-based and ELISA-based receptor binding assays, 13C5.5 exhibits high potency in b10cking human IL-13 binding to human IL-13R(x2, with an ICso between 1 and 3 nM. While both BAK502G9 and MJ2-7 were also able to reduce binding signal, their potencies were much 10wer than that of 13C5.5 (see Table 14), at least partially due to their 10wer affinity for human wt IL-13 (see Table 15). MAbl3.2 was not able to inhibit IL-13 binding to IL-13Ra2, consistent with its epitope. In addition, 13C5.5 could achieve 100% inhibition in both assays at a concentration of 1(X) nM (or 15ug/ml). At the same concentration, BAK502G9 and MJ2-7 exhibited only 40% and 70% inhibition, respectively, in the cell surface-based receptor binding assay, and both exhibited only 60% inhibition in the ELISA-based receptor binding assay.
For a therapeutic mAb with serum half-life between 10 and 20 days in man, the serum concentration is normally between 5-15 ug/ml, with a weekly or bi-weekly IV or SC 3mpk or less dosing regiment. Based on this calculation, 13C5.5 is currently the only anti-IL-13 mAb that is likely to completely (100%) b10ck hunnan IL-13 binding to IL-13Ra2 in vivo as a therapeutic mAb, at a serum concentration of 100 nM (or 15ug/ml), under a conventional dosing regimen of a monoc10nal antibody.
Example 2.23.b: Binding of anti IL-13 antibodies to specific epitope on IL-13
The epitopes on hunnan IL-13 that the anti-IL-13 mAbs 13C5,13C5.5,9C11 and 5G1 bind were mapped using an epitope excision technique, fol10wed by peptide analysis with mass spectrometry (MS). In epitope excision, the protein was first bound to an immobilized mAb and then digested with proteolytic enzymes. Epitope regions on the protein were determined by using MS and MS/MS to identify epitope-containing peptides. CNBr-activated Sepharose beads (Amersham Biosciences, 10mg/reaction) were suspended in S(X)uL of 0.1 M HCl and equilibrated for 15 min. The beads were transferred into compact reaction columns (USB Corporation) and washed with 0.1 M HCl fol10wed by 0.1 M NaHC03 coupling buffer. The mAb (10Oug) was added to the suspension and incubated for 2 h with s10w rotation at room temperature. Beads with the covalently attached mAb were washed with 0.1 M Tris-HCl buffer -pH 8.0. B10cking of unreacted groups on the CNBr Sepharose beads was accomplished by incubation for 2 h with a 0.1 M Tris-HCl buffer ~pH 8.0. Uncoupled mAb was removed by sequential washing with two buffers of different pH; 1) 0.1 M Na acetate, 0.5 M NaCl -pH 4.0 buffer and, 2) 0.1 M Tris-HCl, 0.5M NaCl ~pH 8.0 buffer. The beads were equilibrated in PBS -0.14 M NaCl, 2.7 mM KCl, 4.3 mM Na2HP04, 1.5 mM KH2P04, pH 7.2 and incubated for 2 h at room temperature, with and without IL-13. After washing the beads with PBS -pH 7.2, an aliquot of the suspension was removed for MALDI-TOF analysis.
The affinity bound protein was digested with different proteases (1:1(X)-1:20 enzyme: substrate ratio) for 12 h. Proteases used included: Trypsin, GluC, Chymotrypsin, Carboxypeptidase Y and Aminopeptidase M. Fol10wing proteolysis, the beads were washed with 500uL of digestion buffer. The last 10OuL of wash solution was saved as the control. About 100 uL of 2% TFA was added to the beads and collected. Both the control and .acid wash solutions were first concentrated to about 20uL under vacuum. The peptides were then desalted with C18 ziptips. The samples were analyzed by MALDI-ToF MS, using either a Voyager DE or a Voyager DE-Pro system. Analysis by nano-ESI-LC-MS/MS was performed on an Agilent 1100 Capillary HPLC system interfaced to a Sciex Q-Star Pulsar i MS system.
In studying the epitopes of 13C5, two proteases used in sequential steps gave the best results. With chymotrypsin, a major peptide consisting of amino acid residues 100-130 of SEQ ID NO. 1 was detected, indicating that it may contain the epitope(s). Small amounts of peptides of amino acid residues 103-130 and 104-130 of SEQ ID NO. 1 were also detected. Aminopeptidase M was used after the Chymotrypsin digestion. The major peptide detected was amino acid residues 104-130 of SEQ ID NO. 1, suggesting that the 4 N-terminal amino acid residues (80-83) were not part of the epitope. Further digestion with carboxypeptidase Y resulted in 10ss of affinity. No peptide was observed after digestion and washing. All peptide sequences were confirmed using nano-ESI-LC-MS/MS.
Epitope mapping of 13C5 and 13C5.5 indicated that its binding site(s) included the C-terminal Helix D region of human IL-13 (residues VRDTK lEVAQ FVKDL LL HLK KLFRE GR, corresponding to amino acid 104-130 of SEQ ID NO. 1). The c-terminal helix D region has been proposed to be involved in interactions with the IL-13 receptor (Zuegg et al 2001 Immunol Cell Biol. 79:332-9).
Example 23: Crystallization of anti-IL-13 complexed to IL-13.
The Fab portion of 13C5.5 was complexed with human IL-13 and crystals of the complex were generated as fol10ws. Example 23.1; Preparation and Purification of 13C5.5 Fab Fragment.
To prepare 13C5.5 Fab fragment, 13C5.5 IgG in 0.15 M PBS buffer was first concentrated to 2 mg/ml using an Ultrafree-15 Biomax 10 kDa molecular weight cut-off (MWCO) centrifugal filter device (Millipore). Papain gel slurry (Pierce) was pre-washed and charged in 2-3X with Buffer A (20 mM Na2HP04, 10 mM EDTA, 20 mM cysteine) at a 1:1 volume ratio. The concentrated antibody was then mixed with 50% papain gel slurry and incubated at 37 °C for 24 hours with vigorous shaking. The antibody/slurry mixture was centrifuged (Beckman 6KR) and the supernatant was 10aded onto a PBS pre-equilibrated Superdex 75. A major peak eluted and protein was pooled. A 25 mL Protein A Sepharose 4 Fast F10w affinity column (Amersham Pharmacia) was prepared by washing with 100 mL of PBS. The pooled antibody fragments were applied to the affinity column (2 mL/min f10w rate). Fractions containing 13C5.5 Fab fragments (monitored by UV absorbance at 280 nm) were collected in the f10w-thru. Fractions containing a 13C5.5 Fab fragment concentration greater than 0.-3 mg/mL (determined by UV absorbance at 280 nm) werie pooled and frozen at -80 "C. Sample purity was assessed with SDS-PAGE.
Example 23.2; IL-13/13CS.S Fab Complex Preparation.
Recombinant human IL-13 was expressed in a mammalian expression system and subsequently purified using techniques well known in the art. Recombinant human IL-13 and 13C5.5 Fab protein were mixed at a 1:1 molar ratio and incubated for 1 hour at 4 °C. The complex sample was 10aded onto a pre-equilibrated (20 mM Tris pH 7.5,150 mM NaCI) Superdex 200 column at 0.5 ml/min. Complex was pooled and concentrated to 24 mg/mL using an Ultrafree-15 Biomax 10 kDa molecular weight cut-off (MWCO) centrifugal filter device (Millipore) and frozen at -80°C. Sample purity was assessed with SDS-PAGE.
Example 233; Crystallization of IL-13/13C5.5 Fab Complex
Frozen IL-13/13C5.5 complex stock (-24'mg/mL) was thawed on ice. The complex (1.0 jiL) was mixed with 1.0 \iL of reservoir solution (1.75 M Ammonium Sulfate, 100 mM MES pH 6.5,10 mM CaCI2). The resulting drop was mixed in a sitting drop well (CrysChem sitting-drop plate) over the reservoir at about 18°C. Diamond-like crystals appeared within one week.
Example 23.4; Crvoprotection and Flash Cooling of IL-13/13C5.5 Fab Complex Crystals
Crystals of IL-13/13C5.5 Fab complex were harvested using a fiber 10op in mother liquor + 20% glycerol. The crystals were subsequently flash-cooled by plunging into liquid nitrogen.
Example 23.5: X-rav Diffraction Data Collection of IL-13/13C5 J Fab Complex
X-ray diffraction data from IL-13/13C5.5 Fab crystals were collected at the IMCA beamline at the Advanced Photon Source in Argonne, IL. The crystals were maintained at a temperature of 100 K with an Oxford Cryosystems Cryostream cooler during data collection. A total of 180 frames were collected at an oscillation range of 1.0°. The data were processed with the HKL2(XX) suite of programs (Otwinowski and Minor, 1997). After determining the crystal orientation, the data were integrated with DENZO and scaled and merged with SCALE?ACK, and placed on an absolute scale and reduced to structure factor amplitudes with TRUNCATE (French and Wilson, 1978). Five percent of the unique reflections were assigned, in a random fashion, to the "free" set, for calculation of the free R-factor (Rfree) (BrUnger, 1992); the remaining 95% of the reflections constituted the "working" set, for calculation of the R-factor (R). The x-ray diffraction data are summarized in Table 16. The fol10wing lists indeX1ng for the crystal form: (1) IL-13/13C5.5 Fab: space group P2( 1)2(1)2(1), a = 163.578 A, b = 163.318 A, c = 228.627 A, a = 90.0^,^= 90.0°Y r90.0°. Table 17 lists the xray diffraction statistics for the dataset.
Table 16: Summary of Crystaliographic Unit Cell Information of IL-13/13C53 Fab Complex

(TABLE REMOVED)
Table 17: Summary of X-ray Diffraction Data Statistics for IL-13/13C5.5 Fab Complex,

(TABLE REMOVED)
Highest resolution shell in parentheses.
Example 2.3.6; Molecular Replacement Solution and Refinement of IL-13/13C5.5 Fab Complex Crystal Structure
A maX1mum likelihood molecular replacement solution was determined using the program PHASER (Read, 2001). A total of six 13C5.5 monomers were solved at 3.0 A resolution in the space group P2(l)2(l)2(l). The search model was the crystal structure of Fab reported previously (Protein Data Bank entry IBJl; Muller et al. 1998). Coordinates were generated based on the molecular replacement solution.
The refinement of the IL-13/13C5.5 Fab complex crystal structure began with the molecular replacement solution coordinates, described above, in space group P2(l)2(l)2(l). Refinement began using rigid-body refinement by the program REFMAC available in the CCP4 suite of programs (Murshudov et al., 1997, Collaborative Computational Project, 1994), which resulted in the fol10wing statistics at 2.6 A: R of 40.00% (Rfree 39.00%). De novo lL-13 electron density was observed. Manual building of six IL-13 monomers was guided by the public IL-13 NMR structure lUZ (Moy et al., 2001) using the molecular graphics program O (Jones et al., 1991) and examination of 2Fo-Fc and Fo-Fc electron-density maps. The refinement program REFMAC (Murshudov et al., 1997) was used for iterative rounds of restrained refinement resulting in the fol10wing statistics: R of 25.8% (Rfree 30.5%). Results are shown in Table 18.
Table 18: Summary of Crystal10graphic Refinement Statistics IL-13/13C5.5 Fab Complex.

(TABLE REMOVED)
Example 23.6; IL-13/13C5.5 Fab Complex Structure
Extensive contacts are observed between human IL-13 and multiple I3C5.5 CDRs. The buried surface area at the antibody-antigen interface is 1415.50 A^. The contacts are comprised of critical hydrogen bond and hydrophobic interactions that stabilize the interface. The two minimum sequence segments that comprise the majority of interface contacts are on Q.-13 helices
A and D (for structure of IL-13 see U.S. Patent publication No. 2003-0013851 Al incorporated herein by reference). These contacts engage CDR's.Ll and L3, and H2 and H3. Based on the foregoing, the epitope 13CS.5 binding range comprises the topographical region defined by Ser26-Asn38, Lysl23-Argl30 of SEQ ID NO. 1. More preferably, the epitope 13C5.5 binding range comprises the topographical region defined by Arg30-Asn38, Lysl23-ArgI27 of SEQ ID NO. 1.
Example 2.4: In vivo efficacy of humanized IL-13 antibodies
The in vivo efficacy of anti-hIL-13 antibodies was assessed as fol10ws.
Example 2.4.1; In vivo efficacv of humanized IL-13 antibodies in human IL-13 induced asthma model.
The efficacy of anti-hIL-13 antibodies 5G1,13C5, and 13C5.5 were tested in a human IL-13 induced asthma model in mice. Mice were challenged with recombinant human IL-13 at a dose of 1 ^g in 50 ^1 sterile PBS, delivered into the trachea with a microsprayer using a rodent laryngoscope to visualize the tracheal opening. A total of 2 doses of IL-13 was given on days 1 and 2 of the study and airway hyperresponsiveness (AHR; Hoymann, H.G.; J Pharmacol ToX1col Methods. 2007 Jan-Feb; 55(1): 16-26) as well as mucus, acidic mammalian chitinase (AMCase, Donnelly LE, Barnes PJ., 1: Trends Pharmacol Sci. 2004 Oct;25(10):509-11) and thymus and activation regulated chemokine (TARC; Bisset LR, Schmid-Grendelmeier P., Curr Opin Pulm Med. 2005 Jan;ll(l):35-42) were measured in the broncho-alveolar lavage fluid 24 hr after the final challenge. Antibody doses of 100,3(X), and 1000 ^g were administered by intra-peritoneal injection 1 day prior to the ficsfchallenge with-IL-13 and the. results are summarized in Table 19. 5G1 antibody, which does not b10ck binding of IL-13 to either IL-13Ral or IL-13Ra2, was unable to neutralize IL-13 bioactivity in this in vivo model with comparable levels of AHR, AMCase, and Muc5ac detected in animals treated with 5G1 compared to PBS treated control animals. In contrast, the 13C5 antibody that b10cks binding to both al and (x2 receptors, was effective at reducing all parameters. Treatment with IL-13 increased airways resistance from 3.6 cm H20/ml/sec to 5.7 cm HjO/ml/sec. Treatment with 13C5 (1000 ng) reduced airways resistance to 4.3 cm H20/ml/sec. Mucus hyper-secretion as measured by muc5ac levels were decreased from 356.5 units to a maX1mum 211 U with antibody treatment corresponding to a 40% reduction. Similarly AMCase levels were reduced from 202 U to 68 U corresponding to a 66% reduction with comparable reduction seen in TARC levels (n=10, p < .05, all doses). The recombinant humanized antibody 13C5.5 demonstrated similar results in this model. IL-13 induced an increase in airways resistance fol10wing challenge with 30 µg/ml methacholine from 3.9 to 5.5 cm HiO/mUstc. The
antibody 13C5.5 inhibited airways resistance to 4.1 4.5, and 4.3 cm H20/ml/sec at 100,300 and 1000µg doses respectively." Mucus hyper-secretion as measured by muc5ac levels were reduced from 247 U induced by IL-13 treatment to 154, 30.2, and 11.1 U at 100, 300, and 1000 µg doses of antibody treatment respectively. This represents a 38, 88, and 96% inhibition of mucus production with this antibody. IL-13 treatment induced 130 U AMCase activity that was reduced to 113,98, and 55 U by antibody treatment (100, 300, and 1000 ng doses) representing a 14,24, and 68% inhibition. These data demonstrate that 13C5 and the recombinant humanized antibody 13C5.5 that b10ck binding of IL-13 to both IL-13Ral and a2 can neutralize IL-13 induced responses of AHR, mucus, and AMCase production in the lung . whereas antibodies that do not b10ck binding of IL-13 to al and a2 receptors are not effective at b10cking all of these bio10gical responses.

(TABLE REMOVED)
In another study, the efficacy of anti-hIL-13 antibodies BAK502G9, MJ2-7 and 13C5.5 were compared in the human IL-13 induced asthma model in mice. Mice were challenged with recombinant human IL-13 at a dose of 1 µg in SO µl sterile PBS, delivered intranasally under light sedation. A total of 2 doses of IL-13 was given on days 1 and 2 of the study and airway hyperresponsiveness, as well as mucus, and AMCase, were measured in the broncho-alveolar lavage fluid 24 hr after the final challenge. Antibody doses of 1000 µg were administered by intra-peritoneal injection 1 day prior to the first challenge with IL-13.
Results of the study are summarized in Table 20. The 13C5.5 antibody, which b10cks binding of IL-13 to both IL-13 al and a2 receptors, was effective at significantly reducing all parameters. Treatment with IL-13 increased airways resistance fol10wing challenge with 30 mg/ml methacholine from 4.2 cm U^/mUsec to 7.2 cm UiO/mUsec. Treatment with 13C5.5 (1000 ng) reduced airways resistance by 86.8% to 4.6 cm UiO/mUsec. Mucus hyper-secretion as measured by muc5ac levels were decreased firom 768.2 units to 412.9 U with antibody treatment corresponding to a 58.8% reduction. Similarly AMCase levels were reduced from 316.5 U to 147 U corresponding to a 52% reduction (n=10, p < .001). Both the BAK502G9 and the MJ2-7 antibodies, which inhibit IL-13 binding to IL-13Ral but do not effectively inhibit IL-13 binding to IL-13Ra2 demonstrated comparable ability to neutralize IL-13 induced AHR in this model. The antibodies BAK502G9 and MJ2-7 only inhibited airways resistance from 7.2 to 5.96 cm HzO/niUscc and 5.93 cm HiO/niUsec respectively, representing a 42% and 41.5% reduction in AHR. Mucus hyper-secretion as measured by muc5ac levels were reduced from 768.2 U induced by IL-13 treatment to 627.8 and 380 U at 1000 ng doses of antibody corresponding to a 23% and 64% inhibition by BAK502G9 or the MJ2-7 antibodies respectively. The BAK502G9 antibody was less effective at inhibiting AMCase compared to either 13C5.5 or MJ2-7 antibodies. IL-13 treatment induced 316.5 U AMCase actvity that was reduced to 279, and 169 U by either BAK502G9 or MJ2-7 antibody treatment (1000 ^g dose) representing an 8% and 45% inhibition, respectively. These data demonstrate that the recombinant humanized antibody 13C5.5 that b10cks binding of IL-13 to both IL-13Ral and a2 is most effective at neutralizing IL-13 induced responses of AHR, mucus, and AMCase production in the lung whereas antibodies that b10ck binding of IL-13 to IL-13 a2 receptor with 10wer affinity are not as effective at b10cking these bio10gical responses that contribute to the pathogenesis of asthma.
Table 20: Comparison of 13C5.5, BAK502G9, and MJ2-7 antibodies in the IL-13 induced asthma model.

(TABLE REMOVED)
Example 2.4.2: In vivo efficacy of IL-13 antibodies in an OVA-induced asthma mouse model
To determine if receptor b10ckade properties (particularly regarding to IL-13Ra2) impact the vivo efficacy of the mAbs in asthma mouse models, a panel of rat anti-mouse IL-13 antibodies that exhibited different receptor b10ckade properties, as determined by receptor binding ELISA using mIL-13Ral/Fc and mIL-13Ra2/Fc proteins (R&D Systems) (see Table 21) were generated. Since anti-hIL-13 mAb 3H7 cross-reacts with mouse IL-13, its anti-mIL-13 properties are also included in Table 21. The binding affinities of the antibodies for mouse IL-13 were measured using BIACORE assay against recombinant mouse IL-13 (R&D Systems), and potencies GC50) of antibodies against mouse IL-13 were determined by A-549 bioassay against recombinant mouse IL-13. The variable domain sequences of 51D9 and 48D3 are shown in Table 22.
Table 21. Characterization of anti-mlL-13 monoc10nal antibodies
(TABLE REMOVED)
Table 22. List of amino acid sequences of VH and VL regions of rat anti-mIL-13 mAbs

(TABLE REMOVED)
For the in vivo efficacy study in a murine asthma model, animals (female Balb/c mice) were purchased from Taconic, housed at Abbott Bioresearch Center, and utilized at 8-12 weeks of age. All protocols were approved by the lACUC. Mice were sensitized to OVA (Sigma, endotoX1n was removed from ovalbumin using DetoX1Gel (Pierce) according to manufacturer's protocol and the final material contained less than 0.1 EU/mg protein) on day 0 and 7 with an intra peritoneal injection of 8 ug OVA in 2 mg alum (Pierce). On days 14 and 16, animals received intra-nasal challenge of 0.3 mg OVA in 50 ml sterile PBS. Antibodies 51D9 and 48D3 (purified from the supernatant of hybridoma c10nes S1D9 and 48D3 according to standard procedures, which contained less than 0.1 EU/mg of protein and were negative for rodent pathogens by PCR testing) were administered on day 13 as a single intra peritoneal injection in sterile PBS. Dexamethasone (Sigma) was administered orally once a day on days 13-17 at a dose of 3 mg/kg. All endpoints were analyzed on day 17, 24 hr after the 2nd OVA challenge. Airway hypeiresponsiveness (AHR) was assessed using whole body plethysmography. Briefly, a surgical plane of anesthesia was induced with an intra peritoneal injection of ketamine and xylazine. A tracheal canula was surgically inserted between the 3rd and 4th tracheal rings. Spontaneous breathing was prevented by intra venous jugular injection of pancuronium bromide and animals were placed in a whole body plethysmograph (Buxco) and mechanically ventilated with 0.2 ml room air at ISO breaths per minute with a volume controlled ventilator (Harvard Apparatus). Pressure in the lung and f10w within the plethysmograph were measured using transducers and lung resistance was calculated as pressure/f10w using Biosystem Xa software. Baseline resistance as well.as resistance fol10wing challenge with methacholine (3, 10, & 30 mg/ml) that was delivered with an inline ultrasonic nebulizer were measured. Upon completion of pulmonary function testing, the lungs were lavaged 4 times with 0.5 ml sterile PBS. Lavage fluid was analyzed for TARC, AMCase and cellular infiltrate. Serum was collected for quantification of antibody levels at the conclusion of the study.
Murine TARC levels were determined by ELISA (R&D) according to manufacturer's protocol. AMCase activity was determined in bronchoalveolar lavage (BAL) fluid (1 to 10 dilution with 0.01% BSA, 30 mM sodium citrate, 60 mM sodium phosphate, pH 5.2 in the presence of 80 uM 4-methylumbelliferyl p-D-N,N'-diacetylchitobioside. Reactions were incubated for 15 minutes at room temperature and quenched by addition of 100 uL of 1 M glycine/NaOH pH 10.6. Product formation was determined by fluorescence emission at 460 nm using excitation at 385 nm on a Fluoroskan Ascent fluorometer. To assess goblet cell hyperplasia, lungs were inflated with 10% neutral buffered formalin at 22 cm height for 15 minutes to achieve consistent area of lung surface. Sections were embedded in paraffin, sectioned, and stained with periodic acid schiff (PAS). The area of PAS positive cells a10ng the main bronchus of the left lung was quantitated using ImagePro Plus Software. MucSac levels
were determined by ELISA. A 96 well plate is coated with BAL fluid, dried overnight, and then a biotinylated anti-Muc5ac antibody is added and detected with HRP conjugated streptavidin, fol10wed by cleavage of co10rimetric substrate TMB.
The relative contribution of IL-13Ral and o2 towards the pathogenesis of asthma was tested in a standard model of ovalbumin-induced asthma in mice. Antibodies that b10cked binding of IL-13 to both al and al receptor (51D9,54D1, and 3H7 with potencies of 340,24, and 2430 pM respectively) as well as an antibody that b10cked binding of IL-13 to only al receptor (48D3, potency of 50 pM) were tested by treating animals with the antibodies one day prior to the 10cal challenges with ovalbumin and the results are presented in Table 23. OVA challenge induced increases in lung resistance fol10wing challenge with methacholine, mucus hypersecretion as measured by increased levels of Muc5ac in the BAL fluid as well as increased PAS positive staining of epithelial cells by histo10gical assessment, infiltration of the lung with eosinophils & T cells, and production of asthma-related proteins AMCase and TARC.
Antibodies that b10cked the binding of IL-13 to both IL-13 Ral and cx2 all demonstrated efficacy and the in vivo potency of the reagents shifted in accordance with their measured potency in vitro. 51D9 was tested at doses of 100, 300, and 1000 ug/mouse. OVA treatment caused an increase in airways resistance fol10wing challenge with 30mg/ml methacholine to 6.2 cm H2O /ml/sec compared to 3.6 cm H2O /ml/sec in non-asthmatic animals. Treatment of mice with 51D9 completely prevented the increase in lung resistance with values comparable to that observed in non-asthmatic control animals of 4.1,4.0, and 3.5 cm HiO/ml/sec for doses of 100, 300, and 1000 ng respectively (n=8-10/group; p < .05). The amount of inhibition observed with 51D9 was comparable to that achieved with steroid treatment (3.3 cm H2O /ml/sec). Treatment with 51D9 also dose dependently inhibited mucus hypersecretion from 404 units MucSac down to 55 U in animals treated with 1000 jig of 51D9. Inhibition of mucus hyper-secretion was also observed by histo10gical assessment of PAS reactive epithelial cells. The area of percent positive cells was decreased from 1.0% to 0.6 % and 0.5 % with 300 and 1000 ug dose of 51D9 antibody respectively representing a decrease of 47-65% (n=8, p < .01). 51D9 treatment also inhibited TARC and AMCase. OVA challenge induced 61 pg/ml of TARC that was reduced to 7.8 pg/ml with 1000 ug 51D9 treatment (n=10, p < .05). OVA challenge induced 96 arbitrary units of AMCase activity that was dose dependently reduced with 51D9 to 52,45 and 21 U with 100,300 and 1000 jig of antibody respectively (n=9-10, p < .01). 54D1 which has a 10 fold greater in vitro potency (24 pM) demonstrated inhibition of AHR at the 30 ug dose with reduction of airways resistance from 6.58 cm H2O /ml/sec to 4.4 cm H2O /ml/sec and maX1mal inhibition observed with treatment with 300 ug of antibody to reduce airways resistance to a value of 3.65 cm H2O /ml/sec. Similar potency was observed at inhibition of mucus, AMCase and TARC production. A third antibody 3H7, which has a potency of 2.5 nM, still demonstrated inhibition
of AHR, mucus, and AMCase levels but only at a dose of 1000 ug antibody treatment consistent with a 10 fold shift in potency described in in vitro bioassays.
The efficacy of antibodies that b10ck binding of IL-13 to only IL-13Ral was tested with the antibody 48D3. Animals were treated with of 30, 100, 300, and 1000 ng/mouse. OVA challenge induced a rise in airways resistance to S.69 cm H20/ml/sec compared to 4.1 cm H20/ml/sec in non-asthmatic PBS treated animals. Treatment with 30 ug 48D3 had no effect on OVA-induced lung resistance while treatment with 100, 300 and 1000 ug 48D3 inhibited OVA-induced changes in lung resistance to a maX1mum of 4.4 cm H2O /ml/sec or to -80% of the OVA control levels (n=10, p < .05). In contrast to effects observed with 51D9,48D3 did not inhibit OVA-induced mucus hypersecretion as n»asured by either MucSac ELISA or PAS reactive epithelial cells. A slight but statistical significant reduction in mucus (30%) was observed with 48D3 treatment at the 30 jig dose, whereas all other doses were equivalent to OVA challenged animals.. Histo10gical quantitation of PAS positive staining demonstrated 0.6% in OVA challenged animals vs 0.8 % in animals challenged with OVA and treated with 1000 ug 48D3. In these studies 48D3 inhibited OVA-induced AMCase expression from 196 U detected in OVA-treated animals to 63,90,87, and 96 U at 30,100, 300 and 1000 ug doses respectively. Analysis of antibody levels indicated that comparable levels of 48D3 and S1D9 were detectable in both the serum and BAL fluid of antibody treated mice. Despite the 7 fold greater potency of 48D3 antibody compared to the 51D9 antibody, and equivalent exposure of the two antibodies, the 48D3 antibody was not able to inhibit AHR or AMCase to the same extent as the antibody that b10cked binding of IL-13 to IL-13Ral and a2 and was unable to attenuate mucus production. Together these data suggest that IL-13Ra2 plays a central role in mediating OVA-induced mucus hyper-secretion and that IL-13Ra2 contributes towards regulating the asthmatic phenotype in '
vivo.
Table 23: Efflcacy of anti-mouse IL-13 antibodies in murine model of ovalbumin-induced asthma.

(TABLE REMOVED)

Claim:
1. A binding protein comprising an antigen binding domain, said binding protein capable of binding IL-13, said antigen binding domain comprising six CDRs: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein
CDR-H1 is selected from the group consisting of: X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 64), wherein;
X, is T, D, G, or S;
X2 is S;
X3 is D;
X4 is M, S,Y,L,or H;
X5 is G, W,Y, A, S,or N;
X6 is V, I, or M; and
X7 isD,H, S,Y,N,or G; residues 31-35 of SEQ ID NO:32; residues 31-35 of SEQ ID NO:34; residues 31-35 of SEQ ID NO:36; residues 31-35 of SEQ ID NO:38; residues 31-35 of SEQ ID NO:39; residues 31-35 of SEQ ID NO:41; residues 31-35 of SEQ ID NO:42; residues 31-35 of SEQ ID NO:44; residues 31-35 of SEQ ID NO:52; residues 31-35 of SEQ ID NO:54; residues 31-35 of SEQ ID NO:56; residues 31-35 of SEQ ID NO:58; residues 31-35 of SEQ ID NO:60; and residues 31-35 of SEQ ID NO:62; CDR-H2 is selected from the group consisting of:
X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17 (SEQ ID NO: 65), wherein;
X, is M, E, H, R, S, G, or L;
X2 is I or not present;
X3 is H,Y,A,D, S,orW;
X, is P, S, W, or G;
X5 is S, G, E, or D;
X6 is D.G, S,E,or N;
X7 is S, Y or G;
X8 is E,N,Y,V,or R;

X9 is T,I,or K;
X10 is R,Y, I, D, or A;
X11 is L,Y,D, orF;
X12isN,P, S,or D;
X13 is Q, E, D, P, or S;
X14 is K, M, S, T, A, or V;
X15 is F,L,V,or M;
X16 is K,R,or Q, and
X17 is D, G, or S; residues 50-65 of SEQ ID NO:44; residues 52-67 of SEQ ID NO:46; residues 52-67 of SEQ ID NO:48; residues 52-67 of SEQ ID NO:50; residues 50-65 of SEQ ID NO:54; residues 50-65 of SEQ ID NO:56; residues 50-65 of SEQ ID NO:58; residues 50-65 of SEQ ID NO:60; and residues 50-65 of SEQ ID NO:62; CDR-H3is selected from the group consisting of:
X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 (SEQ ID NO: 66), wherein;
X1 is W, T, G, Y, D, or I;
X2 is R, A, S, G, or V;
X3 is T, F, Y, or S;
X4 is S, T, or Y;
X5 is Y, F, or G;
X6 is F, or Y;
X7 is S,Y,I, or F;
X8 is D,L,Y, or P;
X9 is Y;
X10 is G;
X11is Y, A,P, or E;
X12 is F,M,S,L,or I;
X13 is D, V, N, or K; and
X14 is Y, or F; residues 99-105 of SEQ ID NO:32; residues 99-105 of SEQ ID NO:34;

residues 99-109 of SEQ ID NO:36;
residues 99-109 of SEQ ID NO:38;
residues 99-100 of SEQ ID NO:42;
residues 98-106 of SEQ ID NO:44;
residues 100-112 of SEQ ID NO:46;
residues 100-112 of SEQ ID NO:48;
residues 100-112 of SEQ ID NO:50;
residues 99-107 of SEQ ID NO:52;
residues 98-107 of SEQ ID NO:54;
residues 98-107 of SEQ ID NO:56;
residues 98-107 of SEQ ID NO:58;
residues 98-107 of SEQ ID NO:60; and
residues 98-107 of SEQ ID NO:62;
CDR-L1 is selected from the group consisting of:
X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17 (SEQ ID
NO: 67), wherein;
X, is K,or R;
X2 is S, or A;
X3 is S or T;
X4 is Q, K, or I;
X5 is N, S, T, G, or E;
X6 is L, T, or S;
X7 is L, Q, or V;
X8 isY,N,H,D, orT;
X9 is S, I, or T;
X10 is S, D, N, H, orY;
X11 is N, or G;
X12 is Q;
X13 is K,F,N,E, or S;
X14 is N, T, or S;
X15 isY,or F;
X16isL, A, or M; and
X17 is A,D,E, H, or N; residues 24-39 of SEQ ED N0.33; residues 24-39 of SEQ ED NO:35; residues 24-39 of SEQ ED NO:37; residues 24-39 of SEQ ED NO:40;

residues 24-39 of SEQ ID NO:43;
residues 24-34 of SEQ ID NO:47;
residues 24-34 of SEQ ID NO:49;
residues 24-34 of SEQ ID NO:51;
residues 23-36 of SEQ ID NO:53;
residues 24-38 of SEQ ID NO:55;
residues 24-38 of SEQ ID NO:57;
residues 24-38 of SEQ ID NO:59;
residues 24-38 of SEQ ID NO:61; and
residues 24-38 of SEQ ID NO:63;
CDR-L2 is X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 68), wherein;
X1is L, S,K,T, W,or Y;
X2 is V, T, or A;
X3 is S, or N;
X4 is N,K, T,M, or R;
X5 is R, K, or L;
X6 is F, D, E, H, P, or A; and
X7 is S, R,or P; and
CDR-L3 is selected from the group consisting of: X1-X2-X3-X4-X5-Xs-X7-X8-X9 (SEQ ID NO: 69), wherein;
X, is F, W, Q or A;
X2 is Q or L;
X3 is H, G,Y,W,or N;
X4 is N, S, T, L, or Y;
X5 is Y, T, S, E, or H;
X6 isL, V,F,Y,N,G,P,or D;
X7 is P, or, H;
X8 is L, F, Y, W, or R; and
X9 is T, or V; and residues 94-102 of SEQ ID NO:40.
2. The binding protein according to claim 1, wherein at least one of the CDR comprises an amino acid sequence selected from the group consisting of:
residues 31-35 of SEQ ID NO.:32 residues 24-34 of SEQ ID NO.:47
(CDR-H1); (CDR-L1);

residues 50-66 of SEQ ID NO.:32 residues 50-56 of SEQ ID NO.:47
(CDR-H2); (CDR-L2);
residues 99-105 of SEQ ID NO.:32 residues 89-97 of SEQ ID NO.:47
(CDR-H3); (CDR-L3);
residues 24-39 of SEQ ID NO.:33 residues 31-37 of SEQ ID NO.:48
(CDR-L1); (CDR-H1);
residues 55-61 of SEQ ID NO.:33 residues 52-67 of SEQ ID NO.:48
(CDR-L2); (CDR-H2);
residues 94-102 of SEQ ID NO.:33 residues 100-112 of SEQ ID NO.:48
(CDR-L3); (CDR-H3);
residues 31-35 of SEQ ID NO:34 residues 24-34 of SEQ ID NO.:49
(CDR-H1); (CDR-L1);
residues 50-66 of SEQ ID NO. :34 residues 50-56 of SEQ ID NO. :49
(CDR-H2); (CDR-L2);
residues 99-105 of SEQ ID NO.:34 residues 89-97 of SEQ ID NO.:49
(CDR-H3); (CDR-L3);
residues 24-39 of SEQ ID NO.:35 residues 31-37 of SEQ ID NO.:50
(CDR-L1); (CDR-H1);
residues 55-61 of SEQ ID NO.:35 residues 52-67 of SEQ ID NO:50
(CDR-L2); (CDR-H2);
residues 94-102 of SEQ ID NO.:35 residues 100-112 of SEQ ID NO.:50
(CDR-L3); (CDR-H3);
residues 31-35 of SEQ ID NO.:36 residues 24-34 of SEQ ID NO.:51
(CDR-H1); (CDR-L1);
residues 50-66 of SEQ ID NO.: 36 residues 60-66 of SEQ ID NO.: 51
(CDR-H2); (CDR-L2);
residues 99-109 of SEQ ID NO.:36 residues 89-97 of SEQ ID NO.:51
(CDR-H3); (CDR-L3);
residues 24-39 of SEQ ID NO.,37 residues 31-35 of SEQ ID NO.:52
(CDR-L1); (CDR-H1);
residues 55-61 of SEQ ID N0..37 residues 50-66 of SEQ ID N0..52
(CDR-L2); (CDR-H2);
residues 94-102 of SEQ ID NO.:37 residues 99-107 of SEQ ID NO.:52
(CDR-L3); (CDR-H3);
residues 31-35 of SEQ ID NO.:38 residues 23-36 of SEQ ID NO.:53
(CDR-H1); (CDR-L1);

residues 50-66 of SEQ ID NO.:38 residues 52-58 of SEQ ID NO.:53
(CDR-H2); (CDR-L2);
residues 99-109 of SEQ ID NO.:38 residues 91-99 of SEQ ID NO.:53
(CDR-H3); (CDR-L3);
residues 31-35 of SEQ ID NO.:39 residues 31-35 of SEQ ID NO.:54
(CDR-H1); (CDR-H1);
residues 50-66 of SEQ ID NO.:39 residues 50-65 of SEQ ID NO.:54
(CDR-H2); (CDR-H2);
residues 99-112 of SEQ ID NO.:39 residues 98-107 of SEQ ID NO.:54
(CDR-H3); (CDR-H3);
residues 24-39 of SEQ ID NO.:40 residues 24-38 of SEQ ID NO.:55
(CDR-L1); (CDR-L1);
residues 55-61 of SEQ ID NO.:40 residues 54-60 of SEQ ID NO.:55;
(CDR-L2); (CDR-L2);
residues 94-102 of SEQ ID NO.:40 residues 93-101 of SEQ ID NO.:55
(CDR-L3); (CDR-L3);
residues 31-35 of SEQ ID NO.:41 residues 31-35 of SEQ ID NO.:56
(CDR-H1); (CDR-H1);
residues 50-66 of SEQ ID NO. :41 residues 50-65 of SEQ ID NO.: 56
(CDR-H2); (CDR-H2);
residues 99-112 of SEQ ID N0..41 residues 98-107 of SEQ ID N0..56
(CDR-H3); (CDR-H3);
residues 31-35 of SEQ ID NO.:42 residues 24-38 of SEQ ID NO.:57
(CDR-H1); (CDR-L1);
residues 50-66 of SEQ ID NO.:42 residues 54-60 of SEQ ID NO.:57
(CDR-H2); (CDR-L2);
residues 99-100 of SEQ ID NO.:42 residues 93-101 of SEQ ID NO.:57
(CDR-H3); (CDR-L3);
residues 24-39 of SEQ ID NO.:43 residues 31-35 of SEQ ID NO.:58
(CDR-L1); (CDR-H1);
residues 55-61 of SEQ ID NO.:43 residues 50-65 of SEQ ID NO.:58
(CDR-L2); (CDR-H2);
residues 94-102 of SEQ ID NO.:43 residues 98-107 of SEQ ID NO.:58
(CDR-L3); (CDR-H3);
residues 31-35 of SEQ ID NO.:44 residues 24-38 of SEQ ID NO.:59
(CDR-H1); (CDR-L1);

residues 50-65 of SEQ ID NO.:44 residues 54-60 of SEQ ID NO.:59
(CDR-H2); (CDR-L2);
residues 98-106 of SEQ ID NO.:44 residues 93-101 of SEQ ID NO.:59
(CDR-H3); (CDR-L3);
residues 24-40 of SEQ ID NO.:45 residues 31-35 of SEQ ID NO.:60
(CDR-L1); (CDR-H1);
residues 56-62 of SEQ ID NO.:45 residues 50-65 of SEQ ID NO. :60
(CDR-L2); (CDR-H2);
residues 95-103 of SEQ ID NO.:45 residues 98-107 of SEQ ID NO.:60
(CDR-L3); (CDR-H3);
residues 31-37 of SEQ ID NO.:46 residues 24-38 of SEQ ID NO.:61
(CDR-H1); (CDR-L1);
residues 52-67 of SEQ ID NO.:46; residues 54-60 of SEQ ID NO.:61
(CDR-H2); (CDR-L2);
residues 100-112 of SEQ ID NO.:46 residues 93-101 of SEQ ID NO.:61
(CDR-H3); (CDR-L3);
residues 31-35 of SEQ ID NO.:62
(CDR-H1);
residues 50-65 of SEQ ID NO.:62
(CDR-H2);
residues 98-107 of SEQ ID NO.:62
(CDR-H3);
residues 24-38 of SEQ ID NO.:63
(CDR-L1);
residues 54-60 of SEQ ID NO.:63
(CDR-L2); and
residues 93-101 of SEQ ID NO.:63
(CDR-L3).
3. The binding protein according to claim 1, wherein at least 3 CDRs are selected from a variable domain CDR set selected from the group consisting of:

(Table Removed)
4. The binding protein according to claim 3, comprising at least two variable domain CDR sets.
5. The binding protein according to claim 4, wherein said at least two variable domain
CDR sets are selected from a group consisting of:
VH 25C8 CDR Set & VL 25C8 CDR Set; VH 9C11 CDR Set & VL 9C11 CDR Set; VH 21D9 CDR Set & VL 21D9 CDR Set; VH 22D10 CDR Set & VL 22D10 CDR Set; VH 5F1 CDR Set & VL 5F1 CDR Set; VH 5G1 CDR Set & VL 5G1 CDR Set; VH 3H7 CDR Set & VL 3H7 CDR Set; VH 14B2 CDR Set & VL 14B2 CDR Set; VH 13C5 CDR Set & VL 13C5 CDR Set; VH 29G5 CDR Set & VL 29G5 CDR Set; VH 33C3 CDR Set & VL 33C3 CDR Set; VH 4A8 CDR Set & VL 4A8 CDR Set; VH 1B6 CDR Set & VL 1B6 CDR Set; VH 3E5CDR Set & VL 3E5 CDR Set; VH 6C8 CDR Set & VL 6C8 CDR Set; VH 5D3 CDR Set & VL 5D3 CDR Set; and VH 8B6 CDR Set & VL 8B6 CDR Set.
6. The binding protein according to claim 5, further comprising a human acceptor framework.
7. The binding protein according to claim 6, wherein said human acceptor framework comprises an amino acid
sequence selected from the group consisting of:
SEQ ID NO.:6 SEQ ID NO.:16 SEQ ID NO. :26
SEQ ID NO.:7 SEQ ID NO.:17 SEQ ID NO.:27
SEQ ID NO.:8 SEQ ID NO.:18 SEQ ID NO.:28
SEQ ID NO..9 SEQ ID NO.:19 SEQ ID NO..29
SEQ ID NO.:10 SEQ ID NO.:20 SEQ ID NO.:30
SEQ ID NO.:ll SEQ ID NO.:21 AND
SEQ ID NO.:12 SEQ ID NO.:22 SEQ ID NO.:31.
SEQ ID NO.:13 SEQ ID NO.:23
SEQ ID NO.:14 SEQ ID NO.:24
SEQ ID NO.:15 SEQ ID NO.:25
8. The binding protein according to claim 6 wherein said human acceptor framework comprises at least one
Framework Region amino acid substitution, wherein the amino acid sequence of the framework is at least 65% identical to the sequence of said human acceptor framework and comprises at least 70 amino acid residues identical to said human acceptor framework.
9. The binding protein according to claim 7, wherein said human acceptor framework comprises at least one
Framework Region amino acid substitution at a key residue, said key residue selected from the group
consisting of:
a residue adjacent to a CDR;
a glycosylation site residue;
a rare residue;
a residue capable of interacting with human IL-13;
a residue capable of interacting with a CDR;
a canonical residue;
a contact residue between heavy chain variable region and light chain variable region,
a residue within a Vernier zone ;and
a residue in a region that overlaps between a Chothia-defined variable heavy chain CDR1
and a Kabat-defined first heavy chain framework.
10. The binding protein according to claim 9, wherein the key residue
is selected from the group consisting of 2L, 15L, 22L, 41L, 42L, 44L, 49L, 50L, 51L, 62L, 71L, 73L, 10H, 44H, 46H, 48H, 67H, 68H, 70H, 72H, 74H, 76H, 83H, 84H, 86H, 87H, and 97H.
11. The binding protein according to claim 10, wherein the binding protein is a consensus human variable domain.
12. The binding protein according to claim 1, wherein said binding protein comprises at least one variable domain
having an amino acid sequence selected from the group consisting of:
SEQ ID NO.:70 SEQ ID NO.:81
SEQ ID NO.:71 SEQ ID NO.:82
SEQ ID NO.:72 SEQ ID NO.:83
SEQ ID NO.:73 SEQ ID NO.:84
SEQ ID NO.:74 SEQ ID NO.:85
SEQ ID NO.:75 SEQ ID NO.:92
SEQ ID NO.:76 SEQ ID NO.:93
SEQ ID NO.:77 and
SEQ ID NO. :78 SEQ ID NO. :94.
SEQ ID NO.:79
SEQ ID NO.:80
13. The binding protein according to claim 12 wherein said binding protein comprises two variable domains,
wherein said two variable domains have amino acid sequences selected from the group consisting of:
SEQ ID NO.:70 & SEQ ID NO.:71,
SEQ ID NO.:72 & SEQ ID NO: 73, SEQ ID NO.:74 & SEQ ID NO.:75, SEQ ID NO.:76 & SEQ ID NO.:77, SEQ ID NO.:78 & SEQ ID NO.:79, SEQ ID NO.:80 & SEQ ID NO.:81, SEQ ID NO.:82 & SEQ ID NO: 83, SEQ ID NO.:84 & SEQ ID NO: 85 SEQ ID NO.:80 & SEQ ID NO.:92, SEQ ID NO..80 & SEQ ID N0..93, AND SEQ ID NO.:80 & SEQ ID NO.:94.
14 The binding protein according to claim 10, wherein said binding protein comprises at least one variable
domain having an amino acid sequence selected from the group consisting of:
SEQ ID NO. :70 SEQ ID NO.:78 SEQ ID NO.:92
SEQ ID NO.:71 SEQ ID NO.:79 SEQ ID NO.:93
SEQ ID NO. :72 SEQ ID NO. :80 and
SEQ ID NO.:73 SEQ ID NO.:81 SEQ ID NO.:94.
SEQ ID NO.:74 SEQ ID NO.:82
SEQ ID NO. :75 SEQ ID NO. :83
SEQ ID NO.:76 SEQ ID NO.:84
SEQ ID NO.:77 SEQ ID NO.:85
15. The binding protein according to claim 1 or 13, wherein the binding protein is capable of modulating a
biological function of IL-13.
16. The binding protein according to claim 1 or 13, wherein the binding protein is capable of neutralizing IL-13.
17. The binding protein according to claim 1 or 13, wherein said binding protein has an on rate constant (Kon) to
said target selected from the group consisting of: at least about 102M-1s-1; at least about 103M1-s-1; at least about 104M-1s-1; at least about 105M-1s-1; and at least about 106M-1s-1; as measured by surface plasmon resonance.
18. The binding protein according to claim 1 or 13, wherein said binding protein has an off rate constant (Koff) to
said target selected from the group consisting of: at most about 10-3s-1; at most about 10-4s-1; at most about 10-5s-1; and at most about 10-6s-1, as measured by surface plasmon resonance.
19. The binding protein according to claim 1 or 13 wherein said binding protein has a dissociation constant (KD) to
said target selected from the group consisting of: at most about 10-7 M; at most about 10-8 M; at most about 10-9 M; at most about 10-10 M; at most about 10-11 M; at most about 10-12 M; and at most 10-13 M.
20. An antibody construct comprising a binding protein described in claim 1, said antibody construct further
comprising a linker polypeptide or an immunoglobulin constant domain.
21. The antibody construct according to claim 20, wherein the antibody construct is selected from the group
consisting of:
an immunoglobulin molecule, a disulfide linked Fv,
a monoclonal antibody, a scFv,
a chimeric antibody, a single domain antibody,
a CDR-grafted antibody, a diabody,
a humanized antibody, a multispecific antibody,
a Fab, a dual specific antibody, and
a Fab', a bispecific antibody.
a F(ab')2,
aFv,
22. The antibody construct according to claim 20, wherein the antibody construct comprises a heavy chain
immunoglobulin constant domain selected from the group consisting of:
a human IgM constant domain, a human IgG4 constant domain,
a human IgGl constant domain, a human IgE constant domain,
a human IgG2 constant domain, and
a human IgG3 constant domain, a human IgA constant domain.
23. The antibody construct according to claim 20, wherein the antibody construct comprises an immunoglobulin
constant domain having an amino acid sequence selected from the group consisting of.
SEQ ID NO.:2 SEQ ID NO.:3
SEQ ID NO.:4
and
SEQ ID NO.:5.
24. An antibody conjugate comprising an antibody construct of claim 21, said antibody conjugate further
comprising an agent selected from the group consisting of:
an immunoadhesion molecule, an imaging agent, a therapeutic agent, and a cytotoxic agent.
25. The antibody conjugate according to claim 24, wherein said agent is an imaging agent selected from the group
consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic
label, and biotin.
26. The antibody conjugate according to claim 24, wherein said imaging agent is a radiolabel selected from the
group consisting of: 3H, 14C, 35S,90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm
27. The antibody conjugate according to claim 24, wherein said agent is a therapeutic or cytotoxic agent selected
from the group consisting of; an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an
anti-angiogenic agent, an anti-mitotic agent, an anthracycline, toxin, and an apoptotic agent.
28. The antibody construct according to claim 21, wherein the antibody construct possesses a human glycosylation
pattern.
29. The binding protein according to claim 1, wherein said binding protein is a crystallized binding protein.
30. The antibody construct according to claim 20, wherein said antibody construct is a crystallized antibody construct.
31. The antibody construct according to claim 30, wherein said crystallized antibody construct is a carrier-free pharmaceutical controlled release crystallized antibody construct.
32. The antibody construct according to claim 31, wherein said antibody construct has a greater half life in vivo than the soluble counterpart of said antibody construct.
33. The antibody construct according to claim 31, wherein said antibody construct retains biological activity.
34. An isolated nucleic acid encoding a binding protein amino acid sequence of claim 1.
35. An isolated nucleic acid encoding an antibody construct amino acid sequence of claim 20.
36. A vector comprising an isolated nucleic acid according to claim 35.
37. The vector of claim 36 wherein said vector is selected from the group consisting of pcDNA, pTT, pTT3. pEFBOS, pBV, pJV, and pBJ.
38. A host cell comprising a vector according to claim 36.
39. The host cell according to claim 38, wherein said host cell is a prokaryotic cell.
40. The host cell according to claim 39, wherein said host cell is E.Coli.
41. The host cell according to claim 38, wherein said host cell is a eukaryotic cell.
42. The host cell according to claim 41, wherein said eukaryotic cell is selected from the group consisting of protist cell, animal cell, plant cell and fungal cell.
43. The host cell according to claim 41, wherein said eukaryotic cell is an animal cell selected from the group consisting of; a mammalian cell, an avian cell, and an insect cell.
44. The host cell according to claim 41, wherein said host cell is a CHO cell.
45. The host cell according to claim 41, wherein said host cell is COS.
46. The host cell according to claim 41, wherein said host cell is a yeast cell.

47. The host cell according to claim 46, wherein said yeast cell is Saccharomyces cerevisiae.
48. The host cell according to claim 41, wherein said host cell is an insect Sf9 cell.
49. A method of producing a protein capable of binding IL-13, comprising culturing a host cell of claim 38 in culture medium under conditions sufficient to produce a binding protein capable of binding IL-13.
50. A protein produced according to the method of claim 49.
51. A composition for the release of an antibody construct said composition comprising:

(a) a formulation, wherein said formulation comprises a crystallized antibody construct according to claim 30, and an ingredient; and
(b) at least one polymeric carrier.
52. The composition according to claim 51, wherein said polymeric carrier is a polymer selected from one or more
of the group consisting of: poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly
(depsipeptide), poly (esters), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate),
poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide, poly
[(organo) phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride- alkyl
vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin,
gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends and copolymers
thereof.
53. The composition according to claim 51, wherein said ingredient is selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-ß- cyclodextrin, methoxypolyethylene glycol and polyethylene glycol.
54. The composition according to claim 51 for use in treating a mammal.
55. A pharmaceutical composition comprising the binding protein of claim 1, and a pharmaceutically acceptable carrier.
56. The pharmaceutical composition of claim 55, wherein said pharmaceutically acceptable carrier functions as adjuvant useful to increase the absorption, or dispersion of said binding protein.
57. The pharmaceutical composition of claim 56, wherein said adjuvant is hyaluronidase.
58. The pharmaceutical composition of claim 55, further comprising at least one additional therapeutic agent for
treating a disorder in which IL-13 activity is detrimental.
59. The pharmaceutical composition of claim 55 comprising an additional agent, wherein said additional agent is selected from the group consisting of: a therapeutic agent, an imaging agent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, a co-stimulation molecule blocker, an adhesion molecule blocker, an anti-cytokine antibody or functional fragment thereof; methotrexate, a cyclosporin, a rapamycin, an FK506, a detectable label or reporter, a TNF antagonist, an anti-rheumatic, a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteroid, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressive, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an oral steroid, an epinephrine or analog, a cytokine, and a cytokine antagonist.
60. The pharmaceutical composition of claim 55 for reducing human IL-13 activity.

61. The pharmaceutical composition of claim 55 for reducing human IL-13 activity in a human subject suffering from a disorder in which IL-13 activity is detrimental.
62. The pharmaceutical composition of claim 55 for treating a disease or a disorder in which IL-13 activity is detrimental.
63. The pharmaceutical composition of claim 62, wherein said disorder is selected from the group consisting of respiratory disorders; asthma; allergic and nonallergic asthma; asthma due to infection; asthma due to infection with respiratory syncytial virus (RSV); chronic obstructive pulmonary disease (COPD); other conditions involving airway inflammation; eosinophilia; fibrosis and excess mucus production; cystic fibrosis; pulmonary fibrosis; atopic disorders; atopic dermatitis; urticaria; eczema; allergic rhinitis; and allergic enterogastritis; inflammatory and/or autoimmune conditions of the skin; inflammatory and/or autoimmune conditions of gastrointestinal organs; inflammatory bowel diseases (IBD); ulcerative colitis; Crohn's disease; inflammatory and/or autoimmune conditions of the liver, liver cirrhosis; liver fibrosis; liver fibrosis caused by hepatitis B and/or C virus; scleroderma; tumors or cancers; hepatocellular carcinoma; glioblastoma; lymphoma; Hodgkin's lymphoma; viral infections; HTLV-1 infection (e.g., from HTLV-1); suppression of expression of protective type 1 immune responses, and suppression of expression of protective type 1 immune responses during vaccination.
64. The pharmaceutical composition of claim 61 wherein said disorder is selected from the group consisting of rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, insulin dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitis scleroderma, graft versus host disease, organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignancies, heart failure, myocardial infarction, Addison's disease, sporadic, polyglandular deficiency type I and polyglandular deficiency type II, Schmidt's syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthopathy, arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic synovitis, chlamydia, yersinia and salmonella associated arthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs positive haemolytic anaemia, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic encephalitis/Royal Free Disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired immunodeficiency Disease Syndrome, Acquired Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C, common varied immunodeficiency (common variable hypogammaglobulinaemia), dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian failure, fibrotic lung disease, cryptogenic fibrosing alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonitis, connective tissue disease associated interstitial lung disease, mixed connective tissue disease associated lung disease, systemic sclerosis associated interstitial lung disease, rheumatoid arthritis associated interstitial lung disease, systemic lupus erythematosus associated lung disease,
dermatomyositis/polymyositis associated lung disease, Sjogren's disease associated lung disease, ankylosing spondylitis associated lung disease, vasculitic diffuse lung disease, haemosiderosis associated lung disease, drug-induced interstitial lung disease, fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia, autoimmune neutropaenia, renal disease NOS, glomerulonephritides, microscopic vasulitis of the kidneys, lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjorgren's syndrome, Takayasu's disease/arteritis, autoimmune thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute liver disease, chronic liver diseases, alcoholic cirrhosis, alcohol-induced liver injury, choleosatatis, idiosyncratic liver disease, Drug-Induced hepatitis, Non-alcoholic Steatohepatitis, allergy and asthma, group B streptococci (GBS) infection, mental disorders (e.g., depression and schizophrenia), Th2 Type and Thl Type mediated diseases, acute and chronic pain (different forms of pain), and cancers such as lung, breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectal cancer and hematopoietic malignancies (leukemia and lymphoma), Abetalipoprotemia, Acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, adenocarcinomas, aerial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, alpha-1- antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, angina pectoris, anterior horn cell degeneration, anti cd3 therapy, antiphospholipid syndrome, anti-receptor hypersensitivity reactions, aordic and peripheral aneuryisms, aortic dissection, arterial hypertension, arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrial flutter, atrioventricular block, B cell lymphoma, bone graft rejection, bone marrow transplant (BMT) rejection, bundle branch block, Burkitt's lymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation response, cartilage transplant rejection, cerebellar cortical degenerations, cerebellar disorders, chaotic or multifocal atrial tachycardia, chemotherapy associated disorders, chromic myelocytic leukemia (CML), chronic alcoholism, chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate intoxication, colorectal carcinoma, congestive heart failure, conjunctivitis, contact dermatitis, cor pulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic fibrosis, cytokine therapy associated disorders, Dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever, dermatitis, dermatologic conditions, diabetes, diabetes mellitus, diabetic ateriosclerotic disease, Diffuse Lewy body
disease, dilated congestive cardiomyopathy, disorders of the basal ganglia, Down's Syndrome in middle age, drug-induced movement disorders induced by drugs which block CNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, epstein-barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial hematophagocytic lymphohistiocytosis, fetal thymus implant rejection, Friedreich's ataxia, functional peripheral arterial disorders, fungal sepsis, gas gangrene, gastric ulcer, glomerular nephritis, graft rejection of any organ or tissue, gram negative sepsis, gram positive sepsis, granulomas due to intracellular organisms, hairy cell leukemia, Hallerrorden-Spatz disease, hashimoto's thyroiditis, hay fever, heart transplant rejection, hemachromatosis, hemodialysis, hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, hemorrhage, hepatitis (A), His bundle arrythmias, HIV infection/HIV neuropathy, Hodgkin's disease, hyperkinetic movement disorders, hypersensitity reactions, hypersensitivity pneumonitis, hypertension, hypokinetic movement disorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathic Addison's disease, idiopathic pulmonary fibrosis, antibody mediated cytotoxicity, Asthenia, infantile spinal muscular atrophy, inflammation of the aorta, influenza a, ionizing radiation exposure, iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, kidney transplant rejection, legionella, leishmaniasis, leprosy, lesions of the corticospinal system, lipedema, liver transplant rejection, lymphederma, malaria, malignamt Lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic/idiopathic, migraine headache, mitochondrial multi. system disorder, mixed connective tissue disease, monoclonal gammopathy, multiple myeloma, multiple systems degenerations (Mencel Dejerine-Thomas Shi-Drager and Machado-Joseph), myasthenia gravis, mycobacterium avium intracellulare, mycobacterium tuberculosis, myelodyplastic syndrome, myocardial infarction, myocardial ischemic disorders, nasopharyngeal carcinoma, neonatal chronic lung disease, nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscular atrophies, neutropenic fever, non- hodgkins lymphoma, occlusion of the abdominal aorta and its branches, occulsive arterial disorders, okt3 therapy, orchitis/epidydimitis, orchitis/vasectomy reversal procedures, organomegaly, osteoporosis, pancreas transplant rejection, pancreatic carcinoma, paraneoplastic syndrome/hypercalcemia of malignancy, parathyroid transplant rejection, pelvic inflammatory disease, perennial rhinitis, pericardial disease, peripheral atherlosclerotic disease, peripheral vascular disorders, peritonitis, pernicious anemia, Pneumocystis carinii pneumonia, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome), post perfusion syndrome, post pump syndrome, post-MI cardiotomy syndrome, preeclampsia, Progressive supranucleo Palsy, primary pulmonary hypertension, radiation therapy, Raynaud's phenomenon and disease, Raynoud's disease, Refsum's disease, regular narrow QRS tachycardia, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, Senile Dementia of Lewy body type, seronegative arthropathies, shock, sickle cell anemia, skin allograft rejection, skin changes syndrome, small bowel transplant rejection, solid tumors, specific arrythmias, spinal ataxia, spinocerebellar degenerations, streptococcal myositis, structural lesions of the cerebellum, Subacute sclerosing panencephalitis, Syncope, syphilis of the cardiovascular system, systemic anaphalaxis, systemic inflammatory response syndrome, systemic onset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia, thromboangitis obliterans, thrombocytopenia, toxicity, transplants,
trauma/hemorrhage, type III hypersensitivity reactions, type IV hypersensitivity, unstable angina, uremia, urosepsis, urticaria, valvular heart diseases, varicose veins, vasculitis, venous diseases, venous thrombosis, ventricular fibrillation, viral and fungal infections, vital encephalitis/aseptic meningitis, vital-associated hemaphagocytic syndrome, Wernicke- Korsakoff syndrome, Wilson's disease, xenograft rejection of any organ or tissue, Acute coronary syndromes, Acute Idiopathic Polyneuritis, Acute Inflammatory Demyelinating Polyradiculoneuropathy, Acute ischemia, Adult Still's Disease, Alopecia areata, Anaphylaxis, Anti-Phospholipid Antibody Syndrome, Aplastic anemia, Arteriosclerosis, Atopic eczema, Atopic dermatitis, Autoimmune dermatitis, Autoimmune disorder associated with Streptococcus infection, Autoimmune Enteropathy, Autoimmune hearingloss, Autoimmune Lymphoproliferative Syndrome (ALPS), Autoimmune myocarditis, Autoimmune premature ovarian failure. Blepharitis, Bronchiectasis, Bullous pemphigoid, Cardiovascular Disease, Catastrophic Antiphospholipid Syndrome, Celiac Disease, Cervical Spondylosis, Chronic ischemia, Cicatricial pemphigoid, Clinically isolated Syndrome (CIS) with Risk for Multiple Sclerosis, Conjunctivitis, Childhood Onset Psychiatric Disorder, Chronic obstructive pulmonary disease (COPD), Dacryocystitis, dermatomyositis, Diabetic retinopathy, Diabetes mellitus, Disk herniation, Disk prolaps, Drug induced immune hemolytic anemia, Endocarditis, Endometriosis, endophthalmitis, Episcleritis, Erythema multiforme, erythema multiforme major, Gestational pemphigoid, Guillain-Barre Syndrome (GBS), Hay Fever, Hughes Syndrome , Idiopathic Parkinson's Disease, idiopathic interstitial pneumonia, IgE-mediated Allergy, Immune hemolytic anemia, Inclusion Body Myositis, Infectious ocular inflammatory disease , Inflammatory demyelinating disease, Inflammatory heart disease, Inflammatory kidney disease, IPF/UIP, Iritis, Keratitis, Keratojuntivitis sicca, Kussmaul disease or Kussmaul-Meier Disease, Landry's Paralysis, Langerhan's Cell Histiocytosis, Livedo reticularis, Macular Degeneration, Microscopic Polyangiitis, Morbus Bechterev, Motor Neuron Disorders, Mucous membrane pemphigoid, Multiple Organ failure, Myasthenia Gravis, Myelodysplasic Syndrome, Myocarditis, Nerve Root Disorders, Neuropathy, Non-A Non-B Hepatitis, Optic Neuritis, Osteolysis, Pauciarticular JRA , peripheral artery occlusive disease (PAOD), peripheral vascular disease (PVD), peripheral artery disease (PAD), Phlebitis, Polyarteritis nodosa (or periarteritis nodosa), Polychondritis, Polymyalgia Rheumatica, Poliosis, Polyarticular JRA, Polyendocrine Deficiency Syndrome, Polymyositis, polymyalgia rheumatica (PMR), Post-Pump Syndrome, primary parkinsonism, Prostatitis, Pure red cell aplasia, Primary Adrenal Insufficiency, Recurrent Neuromyelitis Optica, Restenosis, Rheumatic heart disease, SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis), Scleroderma, Secondary Amyloidosis, Shock lung, Scleritis, Sciatica, Secondary Adrenal Insufficiency, Silicone associated connective tissue disease, Sneddon-Wilkinson Dermatosis, spondilitis ankylosans, Stevens-Johnson Syndrome (SJS), Systemic inflammatory response syndrome, Temporal arteritis, toxoplasmic retinitis, toxic epidermal necrolysis, Transverse myelitis, TRAPS (Tumor Necrosis Factor Receptor, Type 1 allergic reaction, Type II Diabetes, Urticaria, Usual interstitial pneumonia (UIP), Vasculitis, Vernal conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome), Wet macular degeneration, and Wound healing.
65. The pharmaceutical composition of claim 55 for treating a disorder in which IL-13 is detrimental, further comprising a second agent, which is administered before, concurrent with, or after the administration of the binding
protein, wherein the second agent is selected from the group consisting of inhaled steroids; beta-agonists; short-acting or long- acting beta-agonists; antagonists of leukotrienes or leukotriene receptors; ADVAIR; IgE inhibitors; anti-IgE antibodies; XOLAIR; phosphodiesterase inhibitors; PDE4 inhibitors; xanthines; anticholinergic drugs; mast cell-stabilizing agents; Cromolyn; IL-4 inhibitors; IL-5 inhibitors; eotaxin/CCR3 inhibitors; antagonists of histamine or its receptors including H1, H2, H3, and H4; antagonists of prostaglandin D or its receptors DP1 and CRTH2; TNF antagonists; a soluble fragment of a TNF receptor, ENBREL; TNF enzyme antagonists; TNF converting enzyme (TACE) inhibitors; muscarinic receptor antagonists; TGF-beta antagonists; interferon gamma; perfenidone; chemotherapeutic agents, methotrexate; leflunomide; sirolimus (rapamycin) or an analog thereof, CCI-779; COX2 or cPLA2 inhibitors, NSAIDs; immunomodulators; p38 inhibitors; TPL-2, MK-2 and NFkB inhibitors; budenoside; epidermal growth factor; corticosteroids; cyclosporine; sulfasalazine, aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole; hpoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; anti-IL-1ß antibodies; anti-IL-6 antibodies; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; antibodies or agonists of TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, EMAP-II, GM-CSF, FGF, or PDGF; antibodies of CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands; FK506; rapamycin; mycophenolate mofetil; ibuprofen; prednisolone; phosphodiesterase inhibitors; adensosine agonists; antithrombotic agents; complement inhibitors; adrenergic agents; IRAK, NIK, IKK, p38, or MAP kinase inhibitors; IL-1ß converting enzyme inhibitors; TNFp: converting enzyme inhibitors; T-cell signaling inhibitors; metalloproteinase inhibitors; 6-mercaptopurines; angiotensin converting enzyme inhibitors; soluble cytokine receptors; soluble p55 TNF receptor; soluble p75 TNF receptor; sIL-lRI; sIL-lRII; sIL-6R; anti-inflammatory cytokines; IL-4; IL-10; IL-ll;and T GFß.
66. The pharmaceutical composition according to claim 62, which is administered by at least one mode selected
from parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal,
intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic,
intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal,
intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial,
intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.
67. An isolated antibody, or antigen binding fragment thereof, wherein said antibody, or antigen binding fragment
thereof binds human IL-13 and inhibits the binding of said IL-13 to the IL-13α2 receptor in a cell surface-based
receptor binding assay with an IC50 selected from the group consisting of about 1.5x10-8 to 1x10-8 M, 1x10-8to 1x10-
9 M, 10-9 to 10-10M and 10-10 to 10-11 M, or in an ELISA-based receptor binding assay with an with an IC50 selected
from the group consisting of about 1.8x10-8 to 1x10-8 M, lx10-8 to 1x10-9 M, 10-9 to 10-10M and 10-10 to 10-11 M.
68. An isolated antibody, or antigen binding fragment thereof, wherein said antibody, or antigen binding fragment thereof binds human IL-13 and inhibits AHR by about 50 % in a human IL-13 induced asthma model.
69. The antibody of claim 67, wherein said antibody inhibits AHR by about 80% in a human IL-13 induced asthma model.
70. The antibody of claim 69, wherein said antibody is 13C5.5.
71. An isolated antibody, or antigen binding fragment thereof, wherein said antibody, or antigen binding fragment thereof binds human IL-13 and inhibits AHR by about 50% and inhibits mucus production by about 40% in a human IL-13 induced asthma model.
72. The antibody of claim 71, wherein said antibody is 13C5.5.
73. An isolated antibody, or antigen binding fragment thereof, wherein said antibody, or antigen binding fragment thereof binds human IL-13 and inhibits the binding of said IL-13 to the IL-13α2 receptor in a cell surface-based receptor binding assay or in an ELISA-based receptor binding assay by about 70-100% at a concentration of 100nM.
74. The antibody of claim 67, wherein said antibody is not BAK502G9, mAbl3.2 or MJ2-7.
75. The antibody of claim 67 wherein said antibody is 13C5.5.
76. The antibody of claim 73 wherein said antibody is 13C5.5.
77. An isolated antibody, or antigen binding fragment thereof, wherein said antibody or antigen binding fragment
thereof, binds IL-13 with binding characteristics selected from the group consisting of:
a) an on rate constant (kon) between about 105M-1s-1 to 106M-1s-1 or about 106M-1s-1 to 107M-1s-1;
b) an off rate constant (koff) of about 10-4s-1 to 10-5s-1; or of about 10-5s-1 to 10-6s-1, as measured by surface plasmon resonance; and
c) a dissociation constant (KD) of about 1.5x10-10 to 1x10-10 M or about 10-10 to 10-11 M.
78. The antibody, or antigen binding fragment thereof according to claim 77, wherein said antibody, or antigen
binding fragment thereof, has an on rate constant (kon) to IL-13 selected from the group consisting of: 6.68x105M-1s-1, 7.86xl05M-1s-1, 8.35x105M-1s-1, 8.69X105M-1S-1, 9.15xl05M-1s-1, 1.2x106M-1s-1, 1.7x106M-1s-1 ,and 2.51xl06M-1s-1.
79. The antibody, or antigen binding fragment thereof according to claim 77, wherein said antibody, or antigen binding fragment thereof, has an off rate constant (koff) to IL-13 selected from the group consisting of. 1.23x10-4s-1; 1.76x10-4s-1; 4.74x10-4s-; 1.91xl0-5s-1; 2.14x105s-1, 3.82x105s-1; 8.81x105s-1 and 9.65x105s-1, as measured by surface plasmon resonance.
80 The antibody, or antigen binding fragment thereof according to claim 77, wherein said antibody, or antigen binding fragment thereof, has a dissociation constant (KD) to IL-13 selected from the group consisting of: 1.05xl0-10M; 7.10xl0-10M; 1x10-11 M; 2.20x10-11 M; 2.72x10-11 M; 4.17x10-11 M; 5.68x10-11 M; 7.01x10-11 M; 7.10x10-11 M; and 9.79x10-11 M.
81. The antibody, or antigen binding fragment thereof, according to claim 77, wherein said antibody, or antigen binding fragment thereof, is capable of modulating a biological function of IL-13.
82. The antibody, or antigen binding fragment thereof, according to claim 77, wherein said antibody, or antigen binding fragment thereof, is capable of neutralizing IL-13.
83. The antibody, or antigen binding fragment thereof, according to claim 77, wherein said humanized antibody, or antigen binding fragment thereof, is selected from the group consisting of; an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab', a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody, a diabody, a multispecific antibody, a dual specific antibody, and a bispecific antibody.

84. The antibody, or antigen binding fragment thereof, according to claim 77, wherein said antibody, or antigen binding fragment thereof, is a humanized antibody.
85. The antibody, or antigen binding fragment thereof, according to claim 77, wherein said antibody, or antigen binding fragment thereof, is 13C5.5 antibody.

86. A pharmaceutical composition comprising the antibody, or antigen binding fragment thereof, according to claim 77 and a pharmaceutically acceptable carrier.
87. The pharmaceutical composition of claim 86 further comprising at least one additional therapeutic agent for treating a disorder in which IL-13 activity is detrimental.
88. An isolated antibody, or antigen binding fragment thereof, wherein said antibody, or antigen binding fragment thereof, binds human IL-13 such that IL-13 with said antibody, or antigen binding fragment thereof, bound to the epitope defined by the topographic regions Ser26-Thr27-Ala28-Leu29-Arg30-Glu31-Leu32-Ile33-Glu34-Glu35-
Leu36-Val37-Asn38 and Lysl23-Lysl24-Leul25-Phel26-Argl27-Glu-128-Glyl29-Argl30 of SEQ ID No. 1 is inhibited from binding to the IL-13 receptor.
89. The isolated antibody, or antigen binding fragment thereof, according to claim 88 wherein said antibody, or antigen bmding fragment thereof binds human IL-13 such that IL-13 with said antibody, or antigen binding fragment thereof, bound to the epitope defined by the topographic regions Arg30-Glu31-Leu32-De33-Glu34-Glu35-Leu36-Val37-Asn38 and Lysl23-Lysl24-Leul25-Phel26-Argl27 of SEQ ID No. 1 is inhibited from binding to the IL-13 receptor.
90. The isolated antibody, or antigen binding fragment thereof, according to claim 88 wherein said antibody, or antigen binding fragment thereof binds human IL-13 such that EL-13 with said antibody, or antigen binding fragment thereof, bound to the epitope defined by the topographic regions Ser26-Thr27-Ala28-Leu29-Arg30-Glu31-Leu32-Ile33-Glu34-Glu35-Leu36-VaI37-Asn38 and Lysl23-Lysl24-Leul25-Phel26-Argl27-Glu-128-Glyl29-Argl30 of SEQ ID No. 1 is inhibited from binding to the IL-13a2 receptor.

91. The isolated antibody, or antigen binding fragment thereof, according to claim 88 wherein said antibody, or antigen binding fragment thereof binds human IL-13 such that DL-13 with said antibody, or antigen binding fragment thereof, bound to the epitope defined by the topographic regions Ser26-Thr27-Ala28-Leu29-Arg30-Glu31-Leu32-Ile33-Glu34-Glu35-Leu36-Val37-Asn38 and Lysl23-Lysl24-Leul25-Phel26-Argl27-Glu-128-Glyl29-Argl30 of SEQ ID No. 1 is inhibited from binding to the IL-13a2 receptor, provided said antibody is not BAK502G9 or MJ2-7.
92. The antibody of claim 88 wherein said antibody is 13C5.5.
93. An isolated antibody, or antigen binding fragment thereof, wherein said antibody, or antigen binding fragment thereof, binds amino acid 104-130 of SEQ ID NO. 1.
94. The antibody of claim 93 wherein said antibody is 13C5.5.
95. An isolated antibody, or antigen binding fragment thereof, wherein said antibody or antigen binding fragment
thereof, binds IL-13 and prevents binding of IL-13 to the IL-13a2 receptor with binding characteristics selected from the group consisting of:
a) binding to an epitope on EL-13 including Helix A and D;
b) an on rate constant (kon) between about 105M-1s-1 to 106M-1s-1 or about 106M-1s-1 to 107M-1s-1;
c) an off rate constant (koff) of about 10-4s-1 to 10-5s-1; or of about 10-5s-1 to 10-6s-1, as measured by surface plasmon resonance; and

d) a dissociation constant (KD) of about 1.5xl0-10 to 1x10-10 M or about 10-10 to 10-11 M.
96. An isolated antibody, or antigen binding fragment thereof, wherein said antibody or antigen binding fragment
thereof, binds variant IL-13 with binding characteristics selected from the group consisting of:
a) binding to an epitope on IL-13 including Helix A and D;
b) an on rate constant (kon) between about 105M-1s-1 to 106M-1s-1 or about 106M-1s-1 to 107M-1s-1;

c) an off rate constant (koff) of about 10-4s-1 to 10-5s-1; or of about 10-5s-1 to 10-6s-1, as measured by surface plasmon resonance; and
d) a dissociation constant (KD) of about 1.5xl0-10 to lx10-10 M or about 10-10 to 10-11 M.
97. An isolated antibody, or antigen binding fragment thereof, wherein said antibody or antigen binding fragment
thereof, binds wildtype IL-13 and variant IL-13 with binding characteristics selected from the group consisting of:
a) binding to an epitope on EL-13 including Helix A and D;
b) an on rate constant (kon) between about 105M-1s-1 to 106M-1s-1 or about 106M-1 s-1 to 107M-1s-1;
c) an off rate constant (koff) of about 10-4s-1 to 10-5s-1; or of about 10-5s-1 to 10-6s-1, as measured by surface plasmon resonance; and
d) a dissociation constant (KD) of about 1.5xl10-10 to lx10-10 M or about 10-10 to 10-11 M.

98. The isolated antibody according to claim 91, wherein said antibody inhibits AHR in a human IL-13 induced asthma model by about 50%.
99. The isolated antibody according to claim 91, wherein said antibody inhibits mucus production in a human IL-13 induced asthma model by about 50%.
100. The isolated antibody according to claim 91, wherein said antibody inhibits TARC in a human IL-13 induced
asthma model by about 50%.