WO2017191627A1

WO2017191627A1 - Antibodies to interleukin 13 receptor alpha-1 (il-13r alpha 1) and uses thereof

Info

Publication number: WO2017191627A1
Application number: PCT/IL2017/050479
Authority: WO
Inventors: Itai Benhar; Ariel Munitz; Danielle KARO-ATAR; Almog BITTON
Original assignee: Ramot At Tel-Aviv University Ltd.
Priority date: 2016-05-02
Filing date: 2017-04-30
Publication date: 2017-11-09

Abstract

Disclosed are immunoglobulin molecules that bind interleukin 13 receptor alpha-1(IL-13R alpha 1). Polynucleotide sequences encoding these immunoglobulins and therapeutic and diagnostic compositions comprising them are also disclosed. Further disclosed are methods of treating and diagnosing conditions, such as asthma and atopic dermatitis that involved binding of IL-13R alpha 1 to at least one ligand.

Description

ANTIBODIES TO INTERLEUKIN 13 RECEPTOR ALPHA- 1 (IL-13R ALPHA 1)

AND USES THEREOF

FIELD OF THE INVENTION

The invention relates to antibodies and antibody fragments which bind to Interleukin 13 (IL-13) receptor alpha- 1, to polynucleotide sequences encoding these antibodies and to cells producing these antibodies. The invention further relates to therapeutic and diagnostic compositions comprising these antibodies and to methods of treating and diagnosing diseases, using these antibodies.

BACKGROUND OF THE INVENTION

Asthma and atopic dermatitis (AD) are chronic and complex inflammatory diseases. The incidence of these two diseases is steadily increasing, mostly affecting children but also adults.

Asthma is a chronic and complex inflammatory disease of the airways characterized by airflow obstruction, airway hyperresponsiveness and airway inflammation. It is the commonest chronic illness of childhood, affecting up to 20% of children and 7% of adults in western countries, resulting in approximately 320 million asthmatics worldwide (Fanta CH et al., The New England journal of medicine, 2009;360: 1002-14). The incidence as well as the number of hospital admissions attributable to asthma are continuously rising and are now considered an epidemic (Barnes PJ, Nature reviews Drug discovery, 2004;3:831-44; Wills- Karp M, The Journal of allergy and clinical immunology, 2001 ;107:9-18). Although asthma can be controlled by using inhaled corticosteroids and bronchodilator combination therapy, 10-25% of asthmatics suffer a persistent form of the disease and develop progressive deterioration, fixed airway obstruction and irreversible fibrosis.

Atopic dermatitis is a chronic and complex inflammatory disorder of the skin. AD is an eczema characterized by cutaneous hyperreactivity to environmental triggers leading to a pruritic, swollen skin (Howell MD et al., Allergy, 2015;70:887-96; Jin H et al., The Journal of investigative dermatology, 2009;129:31-40). AD's prevalence is steadily increasing and affects roughly 20% of the population: up to 25% children and 10% adults. Moreover, after childhood AD is often followed by a subsequent development of food allergy, allergic rhinitis, and asthma, a phenomenon called "the atopic march" (Howell MD et al. ibid; Silverberg JI et al., The Journal of allergy and clinical immunology, 2013;132:1132-8). In a study conducted with AD patients up to the age of 7, 43% developed asthma and 45% developed allergic rhinitis (Gustafsson D et al., Allergy, 2000;55:240-5).

Asthmatic responses are associated with increased levels of inflammatory cells, in particular activated CD4+ T helper type 2 (Th2) lymphocytes and eosinophils. The elevated levels of T-lymphocytes and eosinophils correlate with disease severity (Miloux B et al., FEBS letters. 1997;401 :163-6; Wynn TA et al., Annual review of immunology. 2003;21 :425- 56). Th2 cells are thought to induce asthma through the secretion of an array of cytokines (e.g. IL-4, IL-5, and IL-13) that activate inflammatory and residential effector pathways both directly and indirectly. In particular, IL-4 and IL-13 are produced at elevated levels in allergic tissues and are central regulators of many of the hallmark features of the disease. Traditionally, IL-4 has been considered an immunoregulatory cytokine since it regulates Th2 cell differentiation and IgE production (Wills-Karp M., ibid). In contrast, IL-13 is viewed as an effector cytokine since it induces mucus hypersecretion, airway hyper reactivity (AHR), over expression of adhesion molecules and chemokines (such as the eosinophil-specific eotaxins) and has key roles in fibrosis (Wills-Karp M., ibid). Chronic overexpression of IL-13 can induce emphysematous-like and fibrotic changes in the murine lung (Zheng T et al., The Journal of clinical investigation, 2000;106:1081-93). Furthermore, IL-13 expression has been shown to regulate various profibrogenic mediators including TGF-β, matrix metalloproteinases (MMPs), tenascin, periostin, and fibronectin (Jinnin M et al., The Journal of investigative dermatology, 2006;126:551-60), and is considered to be a critical factor in airway remodeling (Wills-Karp M et al., Science, 1998;282:2258-61). Similar to IL-13, and aside of its immunoregulatory role, IL-4 is also capable of inducing "effector" functions even in the absence of IL-13. Studies using 1113 ^", 1147^" and 1113/1147^" mice have clearly demonstrated the crucial function of these cytokines in asthma. Based on such studies, antibody-based strategies targeting these cytokines are under development and in clinical trials as potential therapeutic modalities (Munitz A et al., Proceedings of the National Academy of Sciences of the United States of America, 2008;105:7240-5 ; Ramalingam TR et al., Nature immunology, 2008;9:25-33; Rothenberg ME et al., Journal of immunology, 2011 ;187:4873-80).

Inflammation triggered by IL-4/IL-13 is amplified by the expression of IL-4 and IL-13 receptors in multiple cell types in the tissue (including in epithelial cells, smooth muscle and endothelial cells), as well as in infiltrative cells such as macrophages, dendritic cells and eosinophils. IL-4 exerts its activities by interacting with a specific cell surface receptor comprised of IL-4 receptor a and the common γ (yc) chain (designated the type 1 IL-4R). IL-4 can also utilize the type 2 IL-4R, comprised of IL-4Ra and the IL-13Rotl chain (Jinnin M et al., ibid), a receptor complex that is also the cognate functional receptor for IL-13 (McCormick SM et al., Cytokine, 2015 ;75:38-50; Wills-Karp M, ibid). IL-13 mediates its effects via a complex receptor system that includes IL-4Ra and at least two other cell surface proteins, IL-13Ral and IL-13Ra2 (Wills-Karp M, ibid). In order to initiate cellular signaling via type 1 or type 2 IL-4R, a critical and sequential receptor dimerization step occurs between IL-4R and one of the IL-13 receptors. Similar to studies with 1147^", 1113 ^" and 114/11137^" mice, mice lacking IL-4/IL-13 signaling components such as I14ra^~/^~, I113ral7^~ and Stat67^~ have demonstrated critical roles for these signaling complexes in asthma. Recent analyses of I113ral7^~ mice have demonstrated the essential role for this receptor in mediating the effects of IL-13 and allergen in the lungs, including AHR, mucus production and fibrosis (Munitz A et al., ibid). Notably, these studies have also shown that the key features of asthma induced by IL-4 are mediated by IL-13Rotl while the type 1 IL-4 receptor appears more important for lung eosinophilia (Munitz A. et al., ibid). Consistently, it has recently been shown that IL- 13Rotl regulates the key clinical features of asthma (namely, AHR and mucus production) in response to various aeroallergens (i.e. Aspergillus fumigatus and house dust mite) (Rothenberg ME. et al., ibid).

Several monoclonal antibodies have entered the pipeline for the evaluation of their potential to treat allergies such as asthma and AD. Omalizumb (antagonizing IgE-receptor crosslinking on the surface of mast cells) and Mepolizumab (IL-5 inhibitor) are approved monoclonal antibodies for treating asthma, and are currently under evaluation for the treatment of AD. Many efforts to inhibit the IL-4/IL-13 axis have been reported; for instance, antibodies targeting IL-13, IL-4, and IL-4Rot are in clinical evaluation. Dupilumab (Dupixen), previously proposed for treatment of asthma (Vatrella at al., J Asthma Allergy, 2014; 7: 123- 130), was recently approved by the FDA for treatment of Atopic Dermatitis.

Nevertheless, there is a need in the art for new therapeutic strategies and agents, such as efficient and specific antibodies against Interleukin-13 Receptor alpha 1 (IL-13Rotl), that are more effective and robust and that allow efficient treatment of various related conditions, such as asthma and acute dermatitis. Preferred antibodies will target only type 2 IL-4 receptor activation and thus will have fewer side effects as therapeutics. SUMMARY OF THE INVENTION

The present invention provides human immunoglobulin molecules, including antibodies and antibody fragments, that specifically recognize Interleukin-13 Receptor alpha 1 (IL-13Rotl) and inhibit its activity. The disclosed immunoglobulin molecules can further inhibit activation of the receptor with high affinity and specificity. In some embodiments, the antibodies or compositions comprising the same are used as therapeutics for prevention, treatment and diagnosis of conditions involving high expression of IL-13 or IL-13Rotl, such as asthma, atopic dermatitis and other allergic and fibrotic diseases and several malignancies.

Binding molecules, e.g., monoclonal antibodies (mAbs) according to the present invention being specific to human IL-13Rotl , inhibit activation of IL-13 and type 2 IL-4 receptors, without affecting type 1 IL-4R activation. This eliminates dangerous adverse effects that may arise from IL-4 signaling via the type 1 IL-4 receptor which is important for homeostasis of the immune system.

In some exemplary embodiments, several monospecific single-chain human antibodies (scFvs) that bind IL-13Rotl were identified and isolated by antibody phage-display and yeast- display technologies. An exemplary clone (referred to herein as 1A6), was shown to bind recombinant human IL-13Rotl with an affinity constant (KD) in the nanomolar range. The 1A6 clone was used to produce mAb that was also shown to bind whole human blood granulocytes, and neutralizes IL-13- signaling in human lung carcinoma and erythro leukemia cells. Furthermore, 1A6 mAb binds the Cynomolgus monkey IL-13Rotl homolog in vitro.

At least some of the immunoglobulin molecules of the present invention are capable of binding and at least partially inhibiting or neutralizing the activity or activation of IL- 13Rotl receptor in various assays and cellular systems. In some exemplary embodiments, a full size human IgG mAb that targets IL-13Rotl is provided for the neutralization of IL-4- and IL-13-mediated pathways, and uses thereof in treating asthma, atopic dermatitis and other allergic and fibrotic conditions.

Some of the single chain antibodies and the mAbs of the present invention exhibit increased specificity toward human IL-13Rotl , in particular, the extracellular domain (ECD) thereof, and do not bind with high affinity to the mouse receptor. Furthermore, some of the antibodies, single chain antibodies and fragments of the present invention cross-react with the Cynomolgus receptor, a property that will enable testing them in vivo in primates. Other monoclonal antibodies, including the clone denoted Bl, bind human IL-13Rotl as well as its rodent counterpart, making it a valuable tool for in vivo studies in rodents.

The present invention provides, according to some embodiments, an immunoglobulin molecule, comprising at least the antigen binding portion, that recognizes human IL-13Rotl with high affinity and specificity and can inhibit its interaction with its ligand, IL-13. According to some embodiments, the immunoglobulin molecule of the present invention is selected from a single chain antibody (scFv), a monoclonal antibody (mAb), and fragments thereof comprising at least the antigen binding site.

Some of the immunoglobulins of the present invention are shuffled IgG antibodies each comprising several domains of different human constant region isotypes.

The present invention provides, according to one aspect, an immunoglobulin comprising a set of at least three complementarity determining region (CDR) amino acid sequences, said set is selected from the group consisting of: i. three CDRs from a heavy chain molecule selected from the group consisting of: SEQ ID Nos: 35, 49, 57, 59, 61 , and 63; and ii. three CDRs from a light chain molecule selected from the group consisting of: SEQ ID Nos: 36, 50, 58, 60, 62, and 64, or an analog or derivative thereof having at least 90% sequence identity with said heavy or light chain sequence.

According to some embodiments, the present invention provides an isolated immunoglobulin which binds to human IL-13Rotl, wherein the isolated immunoglobulin comprises a set of six complementarity determining region (CDR) amino acid sequences selected from the group consisting of: i. three CDRs of a heavy-chain (HC) variable region comprising SEQ ID NO: 35 and three CDRs of a light-chain (LC) variable comprising SEQ ID NO: 36, or an analog or derivative thereof having at least 90% sequence identity with said immunoglobulin sequence;

ii. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 49 and three CDRs of a light-chain variable region comprising SEQ ID NO: 50, or an analog or derivative thereof having at least 90% sequence identity with said immunoglobulin sequence; iii. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 57 and three CDRs of a light-chain variable region comprising SEQ ID NO: 58, or an analog or derivative thereof having at least 90% sequence identity with said immunoglobulin sequence;

iv. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 59 and three CDRs of a light-chain variable region comprising SEQ ID NO: 60, or an analog or derivative thereof having at least 90% sequence identity with said immunoglobulin sequence;

v. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 61 and three CDRs of a light-chain variable region comprising SEQ ID NO: 62, or an analog or derivative thereof having at least 90% sequence identity with said immunoglobulin sequence; and

vi. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 63 and three CDRs of a light-chain variable region comprising SEQ ID NO: 64, or an analog or derivative thereof having at least 90% sequence identity with said immunoglobulin sequence.

According to some embodiments, the immunoglobulin is selected from the group consisting of: a monoclonal antibody (mAb), an antibody fragment comprising at least the antigen binding portion, and a single chain antibody.

The present invention provides, according to some embodiments, a mAb, a single chain antibody, or a fragment thereof, comprising a set of six CDR amino acid sequences wherein three CDRs are from a heavy chain molecule selected from the group consisting of: SEQ ID Nos: 35, 49, 57, 59, 61 , and 63; and three CDRs are from a light chain molecule selected from the group consisting of: SEQ ID Nos: 36, 50, 58, 60, 62, and 64, or an analog or derivative thereof having at least 90% sequence identity with said heavy or light chain sequence.

The present invention provides, according to some embodiments, a mAb, a single chain antibody or a fragment thereof, comprising a set of six CDR amino acid sequences selected from the groups consisting of:

1. SEQ ID Nos: 167, 168, 169, 170, 171 , and 172

11. SEQ ID Nos: 209, 210, 211, 212, 213, and 214

in. SEQ ID Nos: 233, 234, 235, 236, 237, and 238 iv. SEQ ID Nos: 239, 240, 241, 242, 243, and 244;

v. SEQ ID Nos: 245, 246, 247, 248, 249, and 250; and

vi. SEQ ID Nos: 251, 252, 253, 254, 255, and, 256.

There are several methods known in the art for determining the CDR sequences of a given antibody molecule, but there is no standard unequivocal method. Determination of CDR sequences from antibody heavy and light chain variable regions can be made according to any method known in the art, including but not limited to the methods known as KABAT, Chothia and IMGT. A selected set of CDRs may include sequences identified by more than one method, namely, some CDR sequences may be determined using KABAT and some using IMGT, for example.

The present invention provides, according to some embodiments, a mAb, a single chain antibody or a fragment thereof, comprising a heavy chain variable region set forth in a sequence selected from the group consisting of: 35, 49, 57, 59, 61, and 63.

According to some embodiments, the present invention provides a mAb, a single chain antibody or a fragment thereof, comprising a light chain variable region set forth in a sequence selected from the group consisting of: 36, 50, 58, 60, 62, and 64.

Selected mAbs, single chain antibodies or fragments thereof recognize human IL- 13Rotl with an affinity of at least 10^"8M. Other mAbs, single chain antibodies or fragments thereof recognize human IL-13Rotl with an affinity of at least 10^"7M. According to other

-8 -9 embodiments, an antibody or antibody fragment binds with an affinity of 10^" M, 5x10^" M, 10^" ⁹M, 5xl0^"10M, 10^"10M, 5xl0^"nM or even higher to human IL-13Rotl . Each possibility represents a separate embodiment of the invention.

According to some embodiments, the immunoglobulin of the invention specifically binds human IL-13Rotl.

According to other embodiments, selected immunoglobulins of the invention bind human IL-13Rotl as well as mouse IL-13Rotl.

According to some embodiments, the mAb or fragment thereof is selected from the group consisting of: (i) a niAb denoted 1A6 or a fragment thereof comprising heavy-chain CDR1 comprising the sequence set forth in SEQ ID NO: 167, heavy-chain CDR2 comprising the sequence set forth in SEQ ID NO: 168, heavy-chain CDR3 comprising the sequence set forth in SEQ ID NO: 169, light-chain CDR1 comprising the sequence set forth in SEQ ID NO: 170, light-chain CDR2 comprising the sequence set forth in SEQ ID NO: 171 , and light-chain CDR3 comprising the sequence set forth in SEQ ID NO: 172;

(ii) a mAb denoted 6A8 or a fragment thereof comprising heavy-chain CDR1 comprising the sequence set forth in SEQ ID NO: 209, heavy-chain CDR2 comprising the sequence set forth in SEQ ID NO: 210, heavy-chain CDR3 comprising the sequence set forth in SEQ ID NO: 211 , light-chain CDR1 comprising the sequence set forth in SEQ ID NO: 212, light-chain CDR2 comprising the sequence set forth in SEQ ID NO: 213, and light-chain CDR3 comprising the sequence set forth in SEQ ID NO: 214;

(iii) a mAb denoted A2 or a fragment thereof comprising heavy-chain CDR1 comprising the sequence set forth in SEQ ID NO: 233, heavy-chain CDR2 comprising the sequence set forth in SEQ ID NO: 234, heavy-chain CDR3 comprising the sequence set forth in SEQ ID NO: 235, light-chain CDR1 comprising the sequence set forth in SEQ ID NO: 236, light-chain CDR2 comprising the sequence set forth in SEQ ID NO: 237, and light-chain CDR3 comprising the sequence set forth in SEQ ID NO: 238;

(iv) a mAb denoted Bl or a fragment thereof comprising heavy-chain CDR1 comprising the sequence set forth in SEQ ID NO: 239, heavy-chain CDR2 comprising the sequence set forth in SEQ ID NO: 240, heavy-chain CDR3 comprising the sequence set forth in SEQ ID NO: 241 , light-chain CDR1 comprising the sequence set forth in SEQ ID NO: 242, light-chain CDR2 comprising the sequence set forth in SEQ ID NO: 243, and light-chain CDR3 comprising the sequence set forth in SEQ ID NO: 244;

(v) a mAb denoted H4 or a fragment thereof comprising heavy-chain CDR1 comprising the sequence set forth in SEQ ID NO: 245, heavy-chain CDR2 comprising the sequence set forth in SEQ ID NO: 246, heavy-chain CDR3 comprising the sequence set forth in SEQ ID NO: 247, light-chain CDRl comprising the sequence set forth in SEQ ID NO: 248, light-chain CDR2 comprising the sequence set forth in SEQ ID NO: 249, and light-chain CDR3 comprising the sequence set forth in SEQ ID NO: 250; and

(vi) a mAb denoted L2 or a fragment thereof comprising heavy- chain CDRl comprising the sequence set forth in SEQ ID NO: 251, heavy-chain CDR2 comprising the sequence set forth in SEQ ID NO: 252, heavy-chain CDR3 comprising the sequence set forth in SEQ ID NO: 253, light-chain CDRl comprising the sequence set forth in SEQ ID NO: 254, light-chain CDR2 comprising the sequence set forth in SEQ ID NO: 255, and light-chain CDR3 comprising the sequence set forth in SEQ ID NO: 256.

The present invention provides, according to some embodiments, a mAb that specifically binds the human protein IL-13Rotl , a single chain antibody, or a binding fragment thereof, comprising a set of six CDR sequences wherein the set is selected from the group consisting of: i. SEQ ID Nos: 167, 168, 169, 170, 171 , and 172;

ii. SEQ ID Nos: 209, 210, 211, 212, 213, and 214;

iii. SEQ ID Nos: 233, 234, 235, 236, 237, and 238;

iv. SEQ ID Nos: 239, 240, 241, 242, 243, and 244;

v. SEQ ID Nos: 245, 246, 247, 248, 249, and 250; and

vi. SEQ ID Nos: 251, 252, 253, 254, 255, and, 256.

The present invention provides, according to other embodiments, a mAb that specifically binds the human protein IL-13Rotl , a single chain antibody, or a binding fragment thereof, comprising a set of six CDR sequences wherein the set is selected from the group consisting of: i. SEQ ID Nos: 167, 168, 169, 170, 258, and 172;

ii. SEQ ID Nos: 209, 210, 211, 212, 265, and 214;

iii. SEQ ID Nos: 233, 234, 235, 236, 269, and 238 ;

iv. SEQ ID Nos: 239, 240, 241, 242, 270, and 244;

v. SEQ ID Nos: 245, 246, 247, 248, 271 , and 250; and

vi. SEQ ID Nos: 251, 252, 253, 254, 272, and, 256. The present invention also provides immunoglobulins , comprising a heavy chain and a light chain, wherein said chains comprises a set of heavy chain variable region sequence and light chain variable region sequence, said set is selected from the group consisting of: i. SEQ ID NOs: 35, 36;

ii. SEQ ID NOs: 49, 50;

iii. SEQ ID NOs: 57, 58;

iv. SEQ ID NOs: 59, 60;

v. SEQ ID NOs: 61 , 62;

vi. SEQ ID NOs: 63, 64.

According to some embodiments, the immunoglobulin is selected from the group consisting of: a mAb, an antibody fragment comprising at least the antigen binding portion, and a single chain antibody.

According to some embodiments, the immunoglobulin comprises the CDR sequences of a scFv antibody clone selected from the group consisting of: 1A6, 6A8, A2, Bl , H4 and L2. Each possibility is a separate embodiment.

According to some embodiments, the immunoglobulin comprises the heavy chain variable region and light chain variable region of a scFv antibody clone selected from the group consisting of: 1A6, 6A8, A2, Bl , H4 and L2. Each possibility is a separate embodiment.

According to some embodiments, the scFv antibody comprises a light chain variable region and a heavy chain variable region, covalently connected through an amino acid linker.

According to some molecules, the amino acid linker comprises the sequence GS AGGGGSGGGGS GGGGS (SEQ ID NO: 296).

According to some specific embodiments, the amino acid linker consists of the sequence GSAGGGGSGGGGSGGGGS (SEQ ID NO: 296).

According to some embodiments, the scFv antibody comprises a heavy chain variable region having a sequence selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 49, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63; and a light chain variable region having a sequence selected from the group consisting of: SEQ ID NO: 36, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64, covalently connected through an amino acid linker.

According to yet other embodiments, the immunoglobulin molecule is a full size human mAb comprising a heavy chain variable region selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 49, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63; a light chain variable region having a sequence selected from the group consisting of: SEQ ID NO: 36, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64, and at least one constant region sequence of a human IgG.

According to some embodiments, for the antibody heavy chain, the constant human IgG sequence comprises gamma 1 heavy chain sequence (IgGl).

According to some embodiments, for the antibody heavy chain, the constant human IgG sequence comprises gamma 4 heavy chain sequence (IgG4).

According to some embodiments, for the antibody light chain, the constant human IgG sequence comprises C-kappa or C-lambda light chain sequence.

According to other embodiments, the antibody comprises a framework sequence that combines CH domains originating from more than a single isotype, namely, the antibody is a shuffled antibody, comprising constant domains of various IgG isotypes.

According to some embodiments, the IgG isotypes are selected from the group consisting of: human IgGl , human IgG2, human IgG3, and human IgG4. Each possibility represents a separate embodiment of the present invention.

According to some embodiments, the constant human IgG comprises at least one portion of an IgGl sequence and at least one portion of an IgG4 sequence.

According to some specific embodiments, the constant domains of the heavy chain of the mAb comprise: CHI domain of the human gamma-1 isotype, CH2 of the human gamma-4 isotype and CH3 domain of the human gamma-1 isotype.

According to some embodiments, the shuffled mAb, comprises VH domain having a sequence selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 49, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63, a VL domain having a sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64, a CH2 domain of IgG4 isotype, and CHI and CH3 domains of either IgGl or IgG4 isotypes.

According to some exemplary embodiments, the shuffled mAb comprises VH and VL domains having amino acid sequence SEQ ID NO: 35 and SEQ ID NO: 36, respectively, a human IgG4 CHI domain, a human IgG4 CH2 domain, and a human IgGl CH3 domain.

In other exemplary embodiments, the CHI domain is of human IgGl , the CH2 domain is of human IgG4 and the CH3 domain is of human IgG4.

In other exemplary embodiments, the CHI domain is of human IgGl , the CH2 domain is of human IgG4 and the CH3 domain is of human IgGl .

Non-limiting examples of amino acid sequences of constant domains (human IgG constant regions) that may be used according to the present invention, together with the variable regions described above are: i. Human C-Lambda: (SEQ ID NO: 274);

ii. Human C-Kappa: (SEQ ID NO: 276);

iii. Human CHI (including hinge) (γΐ): (encoded by DNA SEQ ID NO: 277);

iv. Human CH2 (γΐ): (encoded by DNA SEQ ID NO: 278);

v. Human CH3 (γΐ): (encoded by DNA SEQ ID NO: 279);

vi. Entire human gammal constant region amino acid sequence: SEQ ID NO: 280; vii. Human CHI (including hinge) (γ4): (encoded by DNA SEQ ID NO: 281);

viii. Human CH2 (γ4): (encoded by DNA SEQ ID NO: 282);

ix. Human CH3 (γ4): (encoded by DNA SEQ ID NO: 283); and/or

x. Entire human gamma4 constant region amino acid sequence: SEQ ID NO: 297.

The full size heavy chain amino acid sequences of the 1A6 clones are set forth in SEQ ID NO: 290 (IgGl); SEQ ID NO: 291 (IgG4); SEQ ID NO: 292 (Shuffled Ά': CHl(y4) - CH2(y4) - CH3(yl)); SEQ ID NO: 293 (Shuffled 'Β': CHl(yl) - CH2(y4) - CH3(y4)); and SEQ ID NO: 294 (Shuffled 'C: CHl(yl) - CH2(y4) - CH3(yl).

The full size light chain amino-acid sequence of clone 1A6 is set forth in SEQ ID NO:

295. Polynucleotide sequences encoding monoclonal antibodies or fragments thereof, having high affinity and specificity for IL-13Rotl protein, as well as vectors and host cells carrying these polynucleotide sequences, are provided according to another aspect of the present invention.

According to some embodiments, a polynucleotide sequences defined above encodes a molecule selected from the group consisting of: an antibody an antibody fragment comprising at least an antigen-binding portion, or an antibody conjugate comprising said antibody or antibody fragment. Each possibility represents a separate embodiment of the present invention.

According to some embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising the amino acid sequence set forth in SEQ ID NO: 35.

According to some embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising the amino acid sequence set forth in SEQ ID NO: 36.

According to some embodiments, a polynucleotide sequence is provided comprising SEQ ID NO: 3 that encodes the heavy chain sequence of a monoclonal antibody, or single chain antibody, or a variant thereof having at least 90% sequence identity.

According to some embodiments, a polynucleotide sequence is provided comprising SEQ ID NO: 4 that encodes the light chain sequence of a monoclonal antibody, or single chain antibody, or a variant thereof having at least 90% sequence identity.

According to yet some embodiments, a polynucleotide sequence according to the invention encodes an antibody or antibody fragment or chain comprising the six CDR nucleic acid sequences: heavy chain CDR1 sequence: GGATTCACCTTCAGTAATCATGAA (SEQ ID NO: 71), heavy chain CDR2 having the sequence: ATTAGTTGGAATGGAAGTAGCCTA (SEQ ID NO: 72), heavy chain CDR3 having the sequence: GCGAGAGATGGGGCAGCAGTTGGCTGGTTCGACCCC (SEQ ID NO: 73), light chain CDR1 having the sequence: AGCTCCAACATTGGGAGTAATTAT (SEQ ID NO: 74), light chain CDR2 having the sequence: GGTAATGAT (SEQ ID NO: 75), and light chain CDR3 having the sequence: GGAACATGGGATAGCAGCCTGAGTGCTGGGGTA (SEQ ID NO: 76).

According to some embodiments, polynucleotide sequences encoding the amino acid sequences of HC variable region and light LC variable region described above are provided. In some embodiments, the present invention provides a polynucleotide sequence selected from the group consisting of: SEQ ID NOs: 3, 17, 25, 27, 29, and 31, encoding an antibody heavy chain variable domain.

In some embodiments, the present invention provides a polynucleotide sequence selected from the group consisting of: SEQ ID NOs: 4, 18, 26, 28, 30, and 32, encoding an antibody light chain variable domain. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the antibodies of the present invention comprise at least one CDR sequence encoded by a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 71-76, SEQ ID NOs: 113-118, SEQ ID NOs: 137-142, SEQ ID NOs: 143-148, SEQ ID NOs: 149-154, and SEQ ID NOs: 155-160. Each possibility represents a separate embodiment of the invention.

A mAb, single chain antibody or an antibody fragment according to some embodiments of the present invention comprises a set of six CDR sequences from heavy- and light- chain variable regions, the variable regions are encoded by a set of two polynucleotide sequences, wherein the set of polynucleotide sequences is selected from the group consisting of: i. SEQ ID NOs. 3 and 4;

ii. SEQ ID NOs. 17 and 18;

iii. SEQ ID NOs. 25 and 26;

iv. SEQ ID NOs. 27 and 28;

v. SEQ ID NOs. 29 and 30;

vi. SEQ ID NOs. 31 and 32.

Non-limiting examples of nucleic acid sequences encoding constant domains (human IgG constant regions) that may be used according to the present invention, together with the variable regions described above are: i. Human C-Lambda: (SEQ ID NO: 273);

ii. Human C-Kappa: (SEQ ID NO: 275);

iii. Human CHI (including hinge) (γΐ): (SEQ ID NO: 277);

iv. Human CH2 (γΐ): (SEQ ID NO: 278); v. Human CH3 (γΐ): (SEQ ID NO: 279);

vi. Human CHI (including hinge) (γ4): (SEQ ID NO: 281);

vii. Human CH2 (γ4): (SEQ ID NO: 282); and/or

viii. Human CH3 (γ4): (SEQ ID NO: 283).

According to some embodiments, the antibodies of the present invention comprise at least one human IgG constant region encoded by nucleic acid sequences selected from the group consisting of SEQ ID Nos. 273, 275, 278, 279, 282, and 283.

Full size heavy chain nucleic acid sequences of the various 1A6 clones (including hinge regions) are set forth in SEQ ID NO: 284 (IgGl); SEQ ID NO: 285 (IgG4); SEQ ID NO: 286 (Shuffled Ά': CHl(y4) - CH2(y4) - CH3(yl); SEQ ID NO: 287 (Shuffled 'Β': CHl(yl) - CH2(y4) - CH3(y4); and SEQ ID NO: 288 (Shuffled 'C: CHl(yl) - CH2(y4) - CH3(yl);

The full size light chain nucleic acid sequence of clone 1A6 is set forth in SEQ ID

NO: 289.

The present invention provides, according to some embodiments, a polypeptide comprising at least one sequence encoded by at least one of the polynucleotide sequences disclosed above.

In a further aspect the present invention provides a nucleic acid construct comprising a nucleic acid molecule encoding at least one antibody chain or fragment thereof of a monoclonal antibody or antibody fragment according to the present invention. According to some embodiments the nucleic acid construct is a plasmid.

In still another aspect the present invention provides a cell (hybridoma or tissue culture cell) capable of producing an antibody or an antibody fragment comprising the specific CDR sequences and/or specific heavy and light chain variable regions defined above.

According to some embodiments, a tissue culture cell is provided comprising at least one polynucleotide sequence disclosed herein.

According to some embodiments, the monoclonal antibody, or scFv, or fragments thereof denoted as "1A6" comprises heavy chain variable region having the amino acid sequence denoted by SEQ ID NO: 35, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 1 A6 comprises light chain variable region having the amino acid sequence as denoted by SEQ ID NO:36, or an analog or derivative thereof having at least 90% sequence identity with the light chain sequence. In some embodiments, 1A6 comprises heavy chain variable region having the nucleic acid sequence denoted by SEQ ID NO: 3, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 1A6 comprises light chain variable region having the nucleic acid sequence as denoted by SEQ ID NO:4, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 1A6 comprises heavy chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 71-73, respectively. In some embodiments, 1A6 comprises heavy chain CDRl , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 167-169, respectively. In some embodiments, 1A6 comprises light chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 74-76, respectively. In some embodiments, 1A6 comprises light chain CDRl , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 170-172, respectively; as well as analogs and derivative thereof.

According to some embodiments, the monoclonal antibody, or scFv, or fragments thereof denoted as "6A8" comprises heavy chain variable region having the amino acid sequence denoted by SEQ ID NO: 49, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 6A8 comprises light chain variable region having the amino acid sequence as denoted by SEQ ID NO: 50, or an analog or derivative thereof having at least 90% sequence identity with the light chain sequence. In some embodiments, 6A8 comprises heavy chain variable region having the nucleic acid sequence denoted by SEQ ID NO: 17, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 6A8 comprises light chain variable region having the nucleic acid sequence as denoted by SEQ ID NO: 18, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 6A8 comprises heavy chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 113-115, respectively. In some embodiments, 6A8 comprises heavy chain CDRl , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 209-211, respectively. In some embodiments, 6A8 comprises light chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 116-118, respectively. In some embodiments, 6A8 comprises light chain CDRl , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 212-214, respectively; as well as analogs and derivative thereof.

According to some embodiments, the monoclonal antibody, or scFv, or fragments thereof denoted as "A2" comprises heavy chain variable region having the amino acid sequence denoted by SEQ ID NO: 57, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, A2 comprises light chain variable region having the amino acid sequence as denoted by SEQ ID NO: 58, or an analog or derivative thereof having at least 90% sequence identity with the light chain sequence. In some embodiments, A2 comprises heavy chain variable region having the nucleic acid sequence denoted by SEQ ID NO: 25, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, A2 comprises light chain variable region having the nucleic acid sequence as denoted by SEQ ID NO: 26, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, A2 comprises heavy chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 137-139, respectively. In some embodiments, A2 comprises heavy chain CDRl , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 233-235, respectively. In some embodiments, A2 comprises light chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 140-142, respectively. In some embodiments, A2 comprises light chain CDRl , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 236-238, respectively; as well as analogs and derivative thereof.

According to some embodiments, the monoclonal antibody, or scFv, or fragments thereof denoted as "Bl" comprises heavy chain variable region having the amino acid sequence denoted by SEQ ID NO: 59, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, Bl comprises light chain variable region having the amino acid sequence as denoted by SEQ ID NO: 60, or an analog or derivative thereof having at least 90% sequence identity with the light chain sequence. In some embodiments, Bl comprises heavy chain variable region having the nucleic acid sequence denoted by SEQ ID NO: 27, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, B l comprises light chain variable region having the nucleic acid sequence as denoted by SEQ ID NO: 28, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, Bl comprises heavy chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 143-145, respectively. In some embodiments, B l comprises heavy chain CDRl , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 239-241, respectively. In some embodiments, Bl comprises light chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 146-148, respectively. In some embodiments, Bl comprises light chain CDRl, CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 242-244, respectively; as well as analogs and derivative thereof.

According to some embodiments, the monoclonal antibody, or scFv, or fragments thereof denoted as "H4" comprises heavy chain variable region having the amino acid sequence denoted by SEQ ID NO: 61, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, H4 comprises light chain variable region having the amino acid sequence as denoted by SEQ ID NO: 62, or an analog or derivative thereof having at least 90% sequence identity with the light chain sequence. In some embodiments, H4 comprises heavy chain variable region having the nucleic acid sequence denoted by SEQ ID NO: 29, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, H4 comprises light chain variable region having the nucleic acid sequence as denoted by SEQ ID NO: 30, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, H4 comprises heavy chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 149-151 , respectively. In some embodiments, H4 comprises heavy chain CDRl , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 245-247, respectively. In some embodiments, H4 comprises light chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 152-154, respectively. In some embodiments, H4 comprises light chain CDRl , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 248-260, respectively; as well as analogs and derivative thereof.

According to some embodiments, the monoclonal antibody, or scFv, or fragments thereof denoted as "L2" comprises heavy chain variable region having the amino acid sequence denoted by SEQ ID NO: 63, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, L2 comprises light chain variable region having the amino acid sequence as denoted by SEQ ID NO: 64, or an analog or derivative thereof having at least 90% sequence identity with the light chain sequence. In some embodiments, L2 comprises heavy chain variable region having the nucleic acid sequence denoted by SEQ ID NO: 31, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, L2 comprises light chain variable region having the nucleic acid sequence as denoted by SEQ ID NO: 32, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, L2 comprises heavy chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 155-157, respectively. In some embodiments, L2 comprises heavy chain CDRl , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 251-253, respectively. In some embodiments, L2 comprises light chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 158-160, respectively. In some embodiments, L2 comprises light chain CDRl, CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 254-256, respectively; as well as analogs and derivative thereof.

According to some embodiments, a monoclonal antibody, or scFv, or fragments thereof comprises the complementarity determining region (CDR) sequences of a monoclonal antibody denoted herein 1A6, namely, the three CDR amino acid sequences contained in heavy chain variable region set forth in SEQ ID NO: 35 and the three CDR sequences contained in light chain variable region set forth in SEQ ID NO: 36. Determination of CDR sequences can be made according to any method known in the art, including but not limited to the methods known as KABAT, Chothia and IMGT. A selected set of CDRs may include sequences identified by more than one method, namely, some CDR sequences may be determined using KABAT and some using IMGT, for example.

According to some specific embodiments the monoclonal antibody comprises heavy chain CDRl (^HCCDR1) comprising the amino acid sequence: GFTFSNHE (SEQ NO: 167), heavy chain CDR2 (^HCCDR2) having the sequence: ISWNGSSL (SEQ NO: 168), heavy chain CDR3 (^HCCDR3) having the sequence: ARDGAAVGWFDP (SEQ NO: 169), light chain CDRl (^LCCDR1) having the sequence: SSNIGSNY (SEQ NO: 170), light chain CDR2 (^LCCDR2) having the sequence: GND, and light chain CDR3 (^LCCDR3) having the sequence: GTWDSSLSAGV (SEQ NO: 172), and analogs and derivative thereof.

According to some embodiments, the monoclonal antibody, or scFv, or fragment thereof comprises heavy chain variable region having the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSNHEMNWVRQAPGKGLEWVSGISWNGS SLGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGAAVGWFDPWGQ GTLVTVSS (SEQ ID NO: 35), or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence.

According to some embodiments, the monoclonal antibody, or scFv, or fragment thereof comprises light chain variable region having the amino acid sequence: DIVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVSWYQQLPGKAPKLLIYGNDQRPSGV SDRFSGSKSGTSASLAISGLRSEDEADYYCGTWDSSLSAGVFGGGTKVTVL (SEQ ID NO: 36), or an analog or derivative thereof having at least 90% sequence identity with the light chain sequence.

According to a specific embodiment, the monoclonal antibody, or scFv, or fragment thereof comprises a heavy chain variable region having the sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSNHEMNWVRQAPGKGLEWVSGISWNGS SLGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGAAVGWFDPWGQ GTLVTVSS (SEQ ID NO: 35), and a light chain variable region having the sequence: DIVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVSWYQQLPGKAPKLLIYGNDQRPSGV SDRFSGSKSGTSASLAISGLRSEDEADYYCGTWDSSLSAGVFGGGTKVTVL (SEQ ID NO: 36), or an analog or derivative thereof having at least 90% sequence identity with the light and/or heavy chain sequence.

Analogs and derivatives of the isolated mAb antibodies, the single chain antibodies and the antibody fragments described above, are also within the scope of the invention. In some embodiments, particular analogs of mAbs, single chain antibodies or fragment thereof comprising at least one variable region set forth in a sequence selected from the group consisting of: SEQ ID NOs: 33-64 are provided. Each possibility represents a separate embodiment of the present invention.

According to some embodiments, an antibody, or antibody fragment analog has at least 95% sequence identity with the hypervariable region of the reference antibody or fragment sequence, or at least 90% sequence identity with the heavy or light chain variable regions of the reference antibody or fragment sequence.

According to certain embodiments, the analog or derivative of the isolated antibody or fragment thereof has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with a variable region of the reference antibody sequence. Each possibility represents a separate embodiment of the invention. According to some embodiments, the antibody or antibody fragment comprises a hypervariable region having light and heavy chain regions defined above, in which 1, 2, 3, 4, or 5 amino acids were substituted, deleted and/or added. According to specific embodiments, the antibody or antibody fragment comprises a hypervariable region having a set of CDR sequences as defined above, in which no more than one amino acid is substituted, deleted or added to each CDR sequence. Each possibility represents a separate embodiment of the present invention.

According to some embodiments, the analog comprises no more than one amino acid substitution, deletion or addition to one or more CDR sequences of the hypervariable region, namely, any one of the CDR sequences set forth in SEQ ID NOs: 167-172, 209-214, 233-238, 239-244, 245-250, or 251-256. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the amino acid substitution is a conservative substitution.

According to another aspect the present invention provides a monoclonal antibody or an antibody fragment comprising an antigen binding domain (ABD) comprising three CDRs of a light chain and three CDRs of a heavy chain, wherein said CDRs have at least 90% sequence identity or similarity with the CDRs of the ABD of a an antibody (herein identified as 1 A6 or 1A6 clone) comprising a heavy chain variable region of SEQ ID NO: 35 and a light chain variable region of SEQ ID NO: 36. According to some embodiments, the CDRs have at least 91%, at least 92%, at least 93% or at least 94% sequence identity or similarity with those of 1A6. According to other embodiments, the ABD has at least 95%, at least 96%, or at least 97%, at least 98% or at least 99% sequence identity or similarity with 1A6.

According to some embodiments, an antibody or antibody fragment according to the invention comprises a heavy chain of SEQ ID NO: 35, or an analog having at least 95% sequence similarity with said sequence.

According to other embodiments, an antibody or antibody fragment comprises a light chain of SEQ ID NO: 36, or an analog having at least 95% sequence similarity with said sequence.

According to some embodiments, an antibody or antibody fragment according to the invention comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 35 and the light chain comprises SEQ ID NO: 36. Analogs of the antibodies or fragments, having at least 95% sequence similarity with said heavy or light chains are also included.

According to some embodiments, the antibodies or antibody fragments of the present invention are capable of recognizing IL-13Rotl protein expressed by various cells, such as, human carcinoma and leukemia cells. In some embodiments, the antibodies or antibody fragments of the present invention are capable of recognizing membrane embedded IL-13Rotl protein.

According to a specific embodiment, the antibody fragment is selected from the group consisting of: Fab, Fab', F(ab')2, Fd, Fd', Fv, dAb, isolated CDR region, single chain antibody (ScFv), "diabodies", and "linear antibodies". Each possibility represents a separate embodiment of the present invention.

The present invention provides, according to other embodiments, a pharmaceutical composition comprising as an active ingredient, at least one mAb, single chain antibody, antibody fragment or conjugates thereof, that recognizes IL-13Rotl with high affinity and specificity and inhibits its interaction with one of its ligands and/or activity and optionally a pharmaceutical acceptable excipient, diluent, salt or carrier.

According to some embodiments, the antibody, single chain antibody, antibody fragment or conjugates thereof are as described hereinabove.

According to some embodiments, the pharmaceutical composition comprises a monoclonal antibody, single chain antibody, or a fragment thereof which is capable of binding to IL-13Rotl and inhibit activation or activity thereof. According to some embodiments, the antibody comprises CDRs as described hereinabove. According to some exemplary embodiments, the antibody is 1A6, comprising a heavy chain variable region having the amino acid sequence as denoted by SEQ ID NO: 35 and/or a light chain variable region having the amino acid sequence as denoted by SEQ ID NO: 36.

According to some embodiments, the pharmaceutical composition according to the present invention is for treating various conditions associated with expression or activation or signaling of IL-13Rotl. According to some embodiments the condition is a disease or disorder characterized by increased IL-13 and/or IL-4 levels as well as increased activation of the adaptor molecule STAT6.

According to some embodiments, the condition is selected from the group consisting of: allergic disease or disorder, fibrotic disease or disorder and proliferative disease or disorder.

In some embodiments, the condition is selected from the group consisting of: asthma, Chronic obstructive pulmonary disorder (COPD), atopic dermatitis, eosinophilic esophagitis, allergic conditions and fibrotic diseases (including heart, kidney and liver fibrotic diseases), as well as various solid tumors characterized with elevated levels of IL-13, IL-13Rotl or IL-4.

According to some embodiments, the condition is an allergic disease or disorder.

According to some embodiments, the condition is selected from the group consisting of: asthma, atopic dermatitis and eosinophilic esophagitis.

According to some embodiments, the solid tumor is breast or lung cancer.

According to some embodiments, the condition is a fibrotic disease or disorder.

According to some embodiments, the fibrotic disease or disorder is selected from the group consisting of: COPD, ulcerative colitis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, liver fibrosis, cardiac fibrosis, endomyocardial fibrosis, atrial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, chronic kidney disease, nephrogenic systemic fibrosis, Crohn's disease, hypertrophic scarring, keloid, scleroderma, organ transplant-associated fibrosis, or ischemia- associated fibrosis.

According to some embodiments, the present invention provides a method of inhibiting binding of IL-13Rotl to at least one ligand by using a monoclonal antibody or antibody fragment defined above. In some embodiments, there is provided a method of reducing or inhibiting activation of IL-13Rotl by using a monoclonal antibody or antibody fragment defined above. In some embodiments, there is provided a method of reducing or inhibiting activation of IL-13Rotl signaling pathways by using a monoclonal antibody or antibody fragment defined above. According to some embodiments, there is provided a method of treating a subject having a disease or condition associated with expression or activation of IL-13Rotl , the method comprising administering to the subject a pharmaceutical composition comprising at least one antibody, antibody fragment or conjugate thereof, that recognizes human IL-13Rotl . In some embodiments, the conditions may be selected from: asthma, COPD, atopic dermatitis, eosinophilic esophagitis, allergic conditions, fibrotic diseases and various solid tumors. Each possibility represents a separate embodiment of the invention. In some exemplary embodiments, the condition is asthma.

According to some embodiments, there is provided a method of treating asthma in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising at least one antibody, antibody fragment or conjugate thereof, that recognizes human IL-13Rotl with high affinity and specificity and capable of inhibiting its binding to its ligand and/or activation thereof.

In some embodiments, the mAb, single chain antibody, antibody fragment or conjugates thereof are as defined above.

In some embodiments, the mAb, single chain antibody, antibody fragment or conjugate thereof is 1A6 antibody clone. In some embodiments, the antibody or antibody fragment comprises a heavy chain variable region having the amino acid sequence as denoted by SEQ ID NO: 35 and/or a light chain variable region having the amino acid sequence as denoted by SEQ ID NO: 36.

The present invention further comprises, according to another aspect, a method of determining or quantifying the expression of 13Rotl , the method comprising contacting a biological sample with an immunoglobulin molecule, and measuring the level of complex formation, wherein the immunoglobulin molecule is selected from the group consisting of monoclonal antibody, single chain antibody or antibody fragment comprising at least the antigen binding domain, and wherein the immunoglobulin molecule comprises a CDR set of a mAb selected from the group consisting of: 1A6, 6A8, A2, Bl, H4 and L2.

According to some embodiments, the method of determining or quantifying the expression of 13Rotl is used in research. Determining and quantifying methods may be performed in-vitro or ex-vivo according to some embodiments or may be used in diagnosing conditions associated with expression of 13Rotl . The antibodies according to the present invention may be also used to configure screening methods. For example, an enzyme-linked immunosorbent assay (ELISA), or a radioimmuno assay (RIA) can be constructed for measuring levels of secreted or cell- associated polypeptide using the antibodies and methods known in the art.

According to one embodiment a method is provided for detecting or quantifying the presence of 13Rotl, comprising the steps of: i. incubating a sample with an antibody specific to 13Rotl or an antibody fragment thereof comprising at least an antigen-binding portion;

ii. detecting the bound 13Rotl using a detectable probe.

According to some embodiments, the method further comprises the steps of: iii. comparing the amount of (ii) to a standard curve obtained from a reference sample containing a known amount of 13Rotl ; and

iv. calculating the amount of the 13Rotl in the sample from the standard curve.

According to some particular embodiments the sample is a body fluid.

According to some embodiments, the method is performed in-vitro or ex-vivo.

A kit for measuring the expression of 13Rotl in biological sample is also provided comprising at least one antibody or antibody fragment comprising the CDRs contained in an antibody selected from the group consisting of: 1A6, 6A8, A2, Bl , H4 and L2.

In some embodiments, the invention provides a method of diagnosing, assessing the severity or staging disease or condition associated with elevated expression of 13Rotl , comprising determining the expression or activity of 13Rotl in a sample from a subject using an antibody according to the present invention or a fragment or conjugate thereof, and comparing the expression or activity of 13Rotl to a reference amount of 13Rotl expression or activity. Said reference amount may be obtained from a sample taken from a normal subject, from the same subject while being in a different stage of the disease or is determined from clinical data of a large population of subjects. Antibodies, antibody fragments or conjugates thereof, according to the present invention may be used in any diagnostic, therapeutic or prophylactic method that utilizes binding to the IL-13Rotl protein, as long as they are capable of specifically binding to said protein and inhibiting it's binding to at least one ligand and/or inhibiting the activity or activation thereof.

Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 - A schematic illustration of IL-4 and IL-13 receptor structure and activity.

Fig. 2 - A representative Phage ELISA. A phage-display library of fully human scFv antibodies was subjected to affinity selection. ELISA plates were coated with 5 μg/ml of rhIL-13Rotl or BSA and blocked with PBS + 3% skim milk. Single phage clones were grown in 96-well plates and phage-containing media were added to the coated ELISA. Secondary antibody horseradish peroxidase (HRP)-conju gated anti-M13 Fd fragment was added before Tetramethylbenzidine (TMB) development.

Fig. 3 - A pictogram of SDS-PAGE analysis of the identified anti-hIL-13Rotl, human IgGl monoclonal antibodies. Eleven hIL-13Rotl antibodies were expressed as human IgGls in Expi293F cell system. The cells were transiently transfected with pcDNA3.4 based IgH & IgL vectors. At days 5-7 post transfection, the mAbs were purified from the conditioned media using MabSelect SuRe columns (GE Healthcare), ^g of purified protein samples were electrophoresed on 10% SDS/polyacrylamide gel and were stained with GelCode Blue™ Stain reagent. Erbitux, commercial anti epidermal growth factor receptor (EGFR) antibody served as a reference IgGl for size and purity.

Figs. 4A-B - Line graphs showing binding assays of various hIL-13Rotl mAbs. The binding abilities of the anti-hIL-13Rotl human IgGl mAbs were assessed by ELISA. Plates were coated with ^g/ml of rhIL-13Rotl or b-gal as control antigen and blocked with 3% skim milk. IgGl dilutions were added as indicated, before secondary HRP-conjugated human IgG H+L polyclonal Ab was reacted with TMB. Fig. 4A - dose dependent binding curve of IL- 13Ral mAb (clone 1A6). 1A6 binds rhIL-13Rotl with an EC₅₀ of less than InM. Fig. 4B - dose dependent binding curves of IL-13Ral mAbs (clones 1C6, 6A8 and 2H12). Clones 1C6, 6A8, and 2H12 bind rhIL-13Rotl with EC50s of -ΙΟΟηΜ or higher.

Fig. 5 - Line graphs showing BIAcore assay to determine the binding affinity of 1A6 human IgGl to rhIL-13Rotl. The interaction of 1A6 IgGl with a recombinant extra cellular domain (ECD) of the IL-13Ral human receptor was monitored by surface plasmon resonance (SPR) detection using a BIAcore 3000 instrument (Biacore Inc.). IL-13Rotl ECD was immobilized onto a flow cell of a CM5 sensor chip using amine- coupling chemistry at a surface density of about 300 response units (RU). The first flow cell was used as a blank control surface. All binding experiments were performed in standard BIAcore buffer [10 mM Hepes (pH 7.4)/150 mM NaCl/0.005 (vol/vol) surfactant P20] at 25°C using a concentration range of 3-100 nM. The IgG flow rate was 20μνπιίη. The binding data was obtained by subtracting the signal of flow cell 1 from the sample flow cell. Multi-concentration data were globally fit using BIAevaluation 3.2 software. The binding data was fitted to a 1 :1 binding model to determine the dissociation constant (KD) value for the 1A6 - IgG/IL-13Rotl ECD interaction. 1A6 was determined to bind rhIL-13Rotl with a K_D of 1.57nM.

Figs. 6A-B - line graphs of ELISA binding assays of 1A6 antibody with Extracellular domains (ECD) of IL-13Rotl receptors of other species. The ability of 1A6 mAb to bind IL- 13Rotl ECDs from other species than human was assessed by ELISA. Plates were coated with ^g/ml antigen and blocked with 3% skim milk. IgGl dilutions were added as shown before secondary HRP-conjugated human Lambda chain polyclonal Ab was reacted with TMB. Fig. 6A - 1A6 mAb binds mouse IL-13 Rod receptor ECD (mIL-13Rotl) with an EC₅₀ of more than ΙΟΟΟηΜ. Fig. 6B - 1A6 mAb binds Expi293F-expressed cynomulus IL-13Rotl (cyIL-13Rotl) with an EC50 of about 20nM.

Figs. 7A-7B - Line graphs showing binding assays of shuffled various IL-13Rotl mAbs (clone 1A6). Fig. 7A - The ability of the various anti-hIL-13Ral shuffled IgGl/IgG4 mAbs to bind to rhIL-13Ral was assessed by ELISA. Plates were coated with ^g/ml of rhlL- 13Ral and blocked with PBS + 5% skim milk. Antibody dilutions were added as indicated, before the secondary HRP-conjugated human IgG H+L polyclonal Antibody was reacted with TMB. Fig. 7B - The ability of the various anti-hIL-13Ral shuffled human IgGl/IgG4 mAbs to bind to rhCD16a was assessed by ELISA. Plates were coated with 15μg/ml of the various mAb formats (IgGl, IgG4, Shuffled Ά, Shuffled 'Β', or Shuffled 'Q and blocked with PBS + 5% skim milk. Dilutions of a His-tagged CD16a protein were added as indicated, before a secondary mix (mouse anti-His-tag + HRP-conjugated goat anti mouse IgG) was reacted with TMB.

Figs. 8A-8D - Representative graphs of binding affinities of scFv antibodies A2, Bl, H4, and L2. ELISA plates were coated with ^g/ml of rhIL-13Ral or control antigen and blocked with 3% skim milk. IgGl dilutions were added as indicated (in triplicates), before secondary HRP-conjugated human IgG H+L polyclonal Ab was reacted with TMB. The reactions were stopped by 1M H₂SO₄. Plates were read at 450nm.

Figs. 9A-9B - binding of mAb IgG Bl clone to human and murine cells. A549 (Fig. 9A) and MC38 (Fig. 9B) cells were stained with biotinylated human IgGl mAbs (avastin or Bl) + StreptAvidin-APC, and DAPI, and then analyzed by flow cytometry. DAPI-negative cells were defined as living cells. Data were analyzed by unpaired t-test using GraphPad Prism 5 (San Diego,CA, USA). Data are presented as mean ± SD, and values of P < 0.05 were considered statistically significant.

DETAILED DESCRIPTION OF THE INVENTION

The present invention utilized a library of human single chain antibodies in order to identify variable and hypervariable human sequences of IL-13Rotl binding immunoglobulins. The use of libraries of human immunoglobulin repertoire enables selection of human hypervariable sequences and eliminates the need of immunizing mice (or other animals), identifying murine binding molecules and humanizing them by grafting their non-human CDRs into human immunoglobulin molecules.

Phage and yeast display technologies were utilized to select scFvs targeting human IL- 13Rotl from a human synthetic combinatorial library. The specificities of the selected antibodies were confirmed by flow cytometry and/or by ELISA as human IgGl antibodies. The pathological effects of IL-13 and IL-4 are mediated via an overlapping set of receptors that display differential distribution among structural and hematopoietic cells. For example, the type 1 IL-4 receptor is predominantly expressed by hematopoietic cells indicating its importance in homeostasis of immune cells. The type 2 IL-4 receptor is expressed mainly by cells that are not from the hematopoietic origin (e.g. fibroblasts, epithelial cells, smooth muscle cells). Thus, the available therapeutic approaches that target the IL-13/IL-4 signaling pathway (e.g. Anti-IL-4Ra, Anti-Ill ^', Anti-114 or bispecific anti- Ill 3/114 antibodies) may display unique and non-overlapping phenotypes. Neutralizing IL-4Ra will block signals from both the type 1 and type 2 IL-4 receptors. Neutralizing IL-13 alone will still leave free IL-4 that can bind its receptors. Conversely, neutralizing IL-4 will leave free IL-13 that will still mediate signals through its receptors. Moreover, bispecific targeting of both IL-4 and IL-3 will block all signals, a fact that might have serious side effects since cognate IL-4 signaling via the type 1 IL-4 receptor may be important for homeostasis of the immune system. Thus, the only way to exclusively block the pathological effects driven by the type 2 IL-4R and leaving the type 1 IL-4R intact is via targeting IL-13Rotl.

Definitions

To facilitate an understanding of the present invention, a number of terms and phrases are defined below. It is to be understood that these terms and phrases are for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

The term "antigen" as used herein refers to a molecule or a portion of a molecule capable of eliciting antibody formation and being bound by an antibody. An antigen may have one or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens. An antigen according to some embodiments of the present invention is an Interleukin 13 receptor alpha 1.

The terms "Interleukin 13 receptor alpha 1", "IL13Rotl" and "IL13Ral" may interchangeably be used. The term is directed to receptor type alpha (a) 1 of interleukin 13 (IL-13). In some embodiments, the receptor is membrane embedded. In some embodiments, the IL13Rotl may be of various sources, such as, for example, human, mouse, monkey, and the like. In some embodiments, the receptor is a recombinant protein. In some embodiments, the receptor is isolated.

The term "antigenic determinant" or "epitope" as used herein refers to the region of an antigen molecule that specifically reacts with a particular antibody. Peptide sequences derived from an epitope can be used, alone or in conjunction with a carrier moiety, applying methods known in the art, to immunize animals and to produce additional polyclonal or monoclonal antibodies. Isolated peptides derived from an epitope may be used in diagnostic methods to detect antibodies and as therapeutic agents when inhibition of said antibodies is required.

Full size antibodies comprise two heavy chains linked together by disulfide bonds and two light chains, each light chain being linked to a respective heavy chain by disulfide bonds in a "Y" shaped configuration. Proteolytic digestion of an antibody yields Fv (Fragment variable) and Fc (Fragment crystalline) domains. The antigen binding domains, Fab, include regions where the polypeptide sequence varies. The term F(ab')₂ represents two Fab' arms linked together by disulfide bonds. The central axis of the antibody is termed the Fc fragment. Each heavy chain has at one end a variable domain (V_H) followed by a number of constant domains (CH). Each light chain has a variable domain (VL) at one end and a constant domain (CL) at its other end, the light chain variable domain being aligned with the variable domain of the heavy chain and the light chain constant domain being aligned with the first constant domain of the heavy chain (CHI). The variable domains of each pair of light and heavy chains form the antigen-binding site. The domains on the light and heavy chains have the same general structure and each domain comprises four framework regions, whose sequences are relatively conserved, joined by three hyper- variable domains known as complementarity determining regions (CDRs 1-3). These domains contribute specificity and affinity of the antigen-binding site. The isotype of the heavy chain (gamma, alpha, delta, epsilon or mu) determines immunoglobulin class (IgG, IgA, IgD, IgE or IgM, respectively). The light chain is either of two isotypes (kappa, κ or lambda, λ) found in all antibody classes.

The term "immunoglobulin" as used herein refers to a polypeptide comprising an antigen binding domain which binds to an antigen. The term "immunoglobulin" is used in the broadest sense and includes monoclonal antibodies (including full length or intact monoclonal antibodies and single chain antibodies), polyclonal antibodies, multivalent antibodies, multi- specific antibodies (e.g., bi-specific antibodies), and antibody fragments long enough to exhibit the desired biological activity. The antibody according to the present invention is a molecule comprising at least the antigen-binding portion of an antibody. Antibody or antibodies according to the invention include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs), single chain antibodies, such as, scFv, as well as proteolytic fragments thereof, such as the Fab or F(ab')2 fragments. In some embodiments, the antibody or antibody fragment may contain a set of CDR sequences contained in a scFv or mAb selected from any of the antibody clones 1A6, 6A8, H4, L2, A2 and Bl .

"Antibody fragments" comprise only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen. Examples of antibody fragments encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CHI domains; (ii) the Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain; (iii) the Fd fragment having VH and CHI domains; (iv) the Fd' fragment having VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain; (v) the Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., Nature 1989, 341 , 544-546) which consists of a VH domain; (vii) isolated CDR regions; (viii) F(ab')2 fragments, a bivalent fragment including two Fab' fragments linked by a disulphide bridge at the hinge region; (ix) single chain antibody molecules (e.g. single chain Fv; scFv) (Bird et al., Science 1988, 242, 423-426; and Huston et al, PNAS (USA) 1988, 85,5879-5883); (x) "diabodies" with two antigen binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93/11161 ; and Hollinger et al, Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448); (xi) "linear antibodies" comprising a pair of tandem Fd segments (VH-CHl-VH-CHl) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng., 1995, 8, 1057-1062; and U.S. Pat. No. 5,641,870).

CDR determination - CDR identification from a given heavy or light chain variable sequence, is typically made using one of few methods known in the art. For example, such determination can be made according to IMGT (Lefranc M-P, et al., Dev Comp Immunol, 2003, 27:55-77) and/or the Kabat (Wu T.T and Kabat E.A., J Exp Med, 1970; 132:211-50). Single chain antibodies can be single chain composite polypeptides having antigen binding capabilities and comprising amino acid sequences homologous or analogous to the variable regions of an immunoglobulin light and heavy chain, i.e. linked VH-VL or single chain Fv (scFv).

The term "neutralizing antibody" as used herein refers to a molecule having an antigen-binding site to a specific receptor or ligand target capable of reducing or inhibiting (blocking) activity and/or signaling through a receptor, as determined by in- vivo or in-vitro assays, as per the specification.

The term "monoclonal antibody" ("mAbs"), as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. mAbs may be obtained by methods known to those skilled in the art. For example, the monoclonal antibodies to be used in accordance with the present invention may be produced by hybridoma method or by recombinant DNA methods. The "monoclonal antibodies" may also be isolated from phage antibody libraries.

Besides the conventional method of raising antibodies in vivo, antibodies can be generated in vitro using phage display technology. Such a production of recombinant antibodies is much faster compared to conventional antibody production and they can be generated against an enormous number of antigens. Furthermore, when using the conventional method, many antigens prove to be non-immunogenic or extremely toxic, and therefore cannot be used to generate antibodies in animals. Moreover, affinity maturation (i.e., increasing the affinity and specificity) of recombinant antibodies is very simple and relatively fast. Finally, large numbers of different antibodies against a specific antigen can be generated in one selection procedure. To generate recombinant mAbs various methods based on display libraries may be used to generate a large pool of antibodies with different antigen recognition sites. Such a library can be made, for example, in several ways: a synthetic repertoire may be generated by cloning synthetic CDR regions in a pool of H chain germline genes and thus generating a large antibody repertoire, from which recombinant antibody fragments with various specificities can be selected. Lymphocyte pool of humans as starting material for the construction of an antibody library may be used. It is possible to construct naive repertoires of human IgM antibodies and thus create a human library of large diversity. This method has been widely used successfully to select a large number of antibodies against different antigens. Protocols for bacteriophage library construction and selection of recombinant antibodies are provided in various reference text, such as, Current Protocols in Immunology, Colligan et al (Eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1.

Techniques for the production of single-chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single-chain antibodies to polypeptides or polynucleotides of this invention. Also, transgenic mice, or other organisms such as other mammals, can be used to express humanized antibodies specific to the polypeptides or polynucleotides of the invention.

The invention also provides conservative amino acid variants of the peptides and antibody molecules according to the invention. Variants according to the invention also may be made that conserve the overall molecular structure of the encoded proteins or peptides. Given the properties of the individual amino acids comprising the disclosed protein products, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e. "conservative substitutions," may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.

In some embodiments, the invention provides a monoclonal antibody or an antibody fragment comprising an antigen binding domain (ABD) which comprises three CDRs of a light chain and three CDRs of a heavy chain, wherein said ABD has at least 90% sequence identity or similarity with an ABD of a monoclonal antibody or scFv, having the CDR sequences disclosed in the present application.

According to some embodiments, the antibodies of the present invention may be "shuffled antibodies", which include constant antibodies domains from various IgG isotypes. For example, in some embodiments, the shuffled antibodies may include the VH and VL domains having the sequences described in present application, together with CH2 domain of IgG4 isotype (for example, human IgG4), CHI and CH3 domains of either IgGl or IgG4 isotypes. In some embodiments, the construction of shuffled antibodies may result in improved antibodies which retain the high antigen recognition specificity and retain the binding and neutralization properties for the IL-13Rotl receptor, but which lack the ability to mediate antibody dependent cell death.

Sequence identity is the amount of amino acids or nucleotides which match exactly between two different sequences. Sequence similarity permits conservative substitution of amino acids to be determined as identical amino acids.

The invention also provides conservative amino acid variants of the antibody molecules according to the invention. Variants according to the invention also may be made that conserve the overall molecular structure of the encoded proteins. Given the properties of the individual amino acids comprising the disclosed protein products, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e. "conservative substitutions," may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. The term "antibody analog" as used herein refers to an antibody derived from another antibody by one or more conservative amino acid substitutions.

The term "antibody variant" as used herein refers to any molecule comprising the antibody of the present invention. For example, fusion proteins in which the antibody or an antigen-binding-fragment thereof is linked to another chemical entity is considered an antibody variant.

Analogs and variants of the antibody sequences are also within the scope of the present application. These include but are not limited to conservative and non-conservative substitution, insertion and deletion of amino acids within the sequence. Such modification and the resultant antibody analog or variant are within the scope of the present invention as long as they confer, or even improve the binding of the antibody to the human IL-13Rotl .

The term "amino acid" as used herein refers to an organic compound comprising both amine and carboxylic acid functional groups, which may be either a natural or non-natural amino acid.

Conservative substitutions of amino acids as known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions include replacement of one amino acid with another having the same type of functional group or side chain, e.g., aliphatic, aromatic, positively charged, negatively charged. These substitutions may enhance oral bioavailability, penetration into the islets, targeting to specific beta cell populations, immunogenicity, and the like. One of skill will recognize that individual substitutions, deletions or additions to a peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, according to one table known in the art, the following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Antibody Fragments

Various techniques have been developed for the production of antibody fragments. Such fragments can be derived via proteolytic digestion of intact antibodies, or produced directly by recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments. According to another approach, F(ab')₂ fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In some exemplary embodiments, the antibody of choice is a single chain Fv fragment (scFv).

The term "human antibody" as used herein refers to an antibody which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art.

The terms "molecule having the antigen-binding portion of an antibody" and "antigen- binding-fragments" as used herein is intended to include not only intact immunoglobulin molecules of any isotype and generated by any animal cell line or microorganism, but also the antigen-binding reactive fraction thereof, including, but not limited to, the Fab fragment, the Fab' fragment, the F(ab')2 fragment, the variable portion of the heavy and/or light chains thereof, Fab mini-antibodies, dimeric bispecific mini-antibodies and single-chain antibodies incorporating such reactive fraction, as well as any other type of molecule in which such antibody reactive fraction has been physically inserted. Such molecules may be provided by any known technique, including, but not limited to, enzymatic cleavage, peptide synthesis or recombinant techniques.

In some embodiments, the invention provides a monoclonal antibody or an antibody fragment comprising an antigen binding domain (ABD) which comprises three CDRs of a light chain and three CDRs of a heavy chain, wherein said ABD has at least 90% sequence identity or similarity with an ABD of a monoclonal antibody or scFv, having the CDR sequences as listed in the present invention. Further provided are pharmaceutical compositions comprising the same, and uses thereof for treating related conditions.

According to an aspect, the present invention provides an isolated immunoglobulin molecule which binds to human IL-13Rotl, said antibody molecule is selected from a monoclonal antibody, an antibody fragment comprising at least the antigen binding portion, and a single chain antibody, wherein the isolated antibody molecule comprises a set of six complementarity determining region (CDR) amino acid sequences selected from the group consisting of: i. three CDRs of a heavy-chain (HC) variable region comprising SEQ ID NO: 35 and three CDRs of a light-chain (LC) variable comprising SEQ ID NO: 36, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence;

ii. three complementarity determining regions (CDRs) of a heavy-chain variable region comprising SEQ ID NO: 49 and three CDRs of a light-chain variable region comprising SEQ ID NO: 50, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence;

iii. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 57 and three CDRs of a light-chain variable region comprising SEQ ID NO: 58, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence;

iv. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 59 and three CDRs of a light-chain variable region comprising SEQ ID NO: 60, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence;

v. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 61 and three CDRs of a light-chain variable region comprising SEQ ID NO: 62, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence; vi. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 63 and three CDRs of a light-chain variable region comprising SEQ ID NO: 64, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence; vii. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 33 and three CDRs of a light-chain variable region comprising SEQ ID NO: 34, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence;

viii. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 37 and three CDRs of a light-chain variable region comprising SEQ ID NO: 38, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence;

ix. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 39 and three CDRs of a light-chain variable region comprising SEQ ID NO: 40, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence;

x. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 41 and three CDRs of a light-chain variable region comprising SEQ ID NO: 42, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence;

xi. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 43 and three CDRs of a light-chain variable region comprising SEQ ID NO: 44, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence;

xii. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 45 and three CDRs of a light-chain variable region comprising SEQ ID NO: 46, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence;

xiii. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 47 and three CDRs of a light-chain variable region comprising SEQ ID NO: 48, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence;

xiv. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 51 and three CDRs of a light-chain variable region comprising SEQ ID NO: 52, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence;

xv. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 53 and three CDRs of a light-chain variable region comprising SEQ ID NO: 54, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence; and

xvi. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 55 and three CDRs of a light-chain variable region comprising SEQ ID NO: 56, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence.

According to some embodiments, the monoclonal antibody, single chain antibody or fragment thereof, comprises a set of six complementarity determining region (CDR) amino acid sequences selected from the groups consisting of:

1. SEQ ID Nos: 167, 168, 169, 170, 171 , and 172;

11. SEQ ID Nos: 209, 210, 211, 212, 213, and 214;

111. SEQ ID Nos: 233, 234, 235, 236, 237, and 238; iv. SEQ ID Nos: 239, 240, 241, 242, 243, and 244;

v. SEQ ID Nos: 245, 246, 247, 248, 249, and 250;

vi. SEQ ID Nos: 251, 252, 253, 254, 255, and, 256;

vii. SEQ ID Nos: 161, 162, 163, 164, 165, and 166;

viii. SEQ ID Nos: 173, 174, 175, 176, 177, and 178;

ix. SEQ ID Nos: 179, 180, 181, 182, 183, and 184;

x. SEQ ID Nos: 185, 186, 187, 188, 189, and 190;

xi. SEQ ID Nos: 191, 192, 193, 194, 195, and 196;

xii. SEQ ID Nos: 197, 198, 199, 200, 201 , and 202;

xiii. SEQ ID Nos: 203, 204, 205, 206, 207, and 208;

xiv. SEQ ID Nos: 215, 216, 217, 218, 219, and 220;

xv. SEQ ID Nos: 221, 222, 223, 224, 225, and 226; and

xvi. SEQ ID Nos: 227, 228, 229, 230, 231 , and 232.

Each possibility represents a separate embodiment.

According to some embodiments, the monoclonal antibody, single chain antibody or antibody fragment, comprises a set of six CDR sequences from a heavy-chain and light-chain variable regions, said variable regions are encoded by a set of two polynucleotide sequences, wherein the set of polynucleotide sequences is selected from the group consisting of: i. SEQ ID NOs. 3 and 4;

ii. SEQ ID NOs. 17 and 18;

iii. SEQ ID NOs. 25 and 26;

iv. SEQ ID NOs. 27 and 28;

v. SEQ ID NOs. 29 and 30;

vi. SEQ ID NOs. 31 and 32;

vii. SEQ ID Nos. 1 and 2;

viii. SEQ ID Nos. 5 and 6;

ix. SEQ ID Nos. 7 and 8;

X. SEQ ID Nos. 9 and 10;

xi. SEQ ID Nos. 11 and 12; xii. SEQ ID Nos. 13 and 14;

xiii. SEQ ID Nos. 15 and 16;

xiv. SEQ ID Nos. 19 and 20;

XV. SEQ ID Nos. 21 and 22;

xvi. SEQ ID Nos. 23 and 24.

Each possibility represents a separate embodiment of the invention.

In some embodiments, 16 single-chain antibodies (scFvs) that bind IL-13Rotl were identified and isolated by antibody phage-display and yeast display technologies. An exemplary clone (referred to herein as 1A6), was shown to bind recombinant human IL- 13Rotl with an affinity constant (KD) of 1.57nM. The 1A6 antibody was also shown to bind whole human blood granulocytes, as observed by flow cytometry, and neutralizes IL-13- signaling in human A549 and TF-1 cells. Furthermore, 1A6 binds the Cynomolgus monkey IL-13Rotl homolog in vitro. In some exemplary embodiments, there is provided a human monoclonal antibody (referred to herein as 1A6), that targets IL-13Rotl for the neutralization of IL-4- and IL-13-mediated pathways, and uses thereof in treating asthma and other allergic conditions, as well as fibrotic disorders and certain malignancies.

According to some embodiments, the monoclonal antibody, or scFv, or fragments thereof denoted as "1A1" comprises heavy chain variable region having the amino acid sequence denoted by SEQ ID NO: 33, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 1 Al comprises light chain variable region having the amino acid sequence as denoted by SEQ ID NO: 34, or an analog or derivative thereof having at least 90% sequence identity with the light chain sequence. In some embodiments, 1A1 comprises heavy chain variable region having the nucleic acid sequence denoted by SEQ ID NO: 1, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 1A1 comprises light chain variable region having the nucleic acid sequence as denoted by SEQ ID NO: 2, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 1A1 comprises heavy chain CDR1 , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 65-67, respectively. In some embodiments, 1A1 comprises heavy chain CDR1, CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 161-163, respectively. In some embodiments, 1A1 comprises light chain CDR1 , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 68-70, respectively. In some embodiments, 1A1 comprises light chain CDRl, CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 164-166, respectively; as well as analogs and derivative thereof.

According to some embodiments, the monoclonal antibody, or scFv, or fragments thereof denoted as "1C6" comprises heavy chain variable region having the amino acid sequence denoted by SEQ ID NO: 37, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 1C6 comprises light chain variable region having the amino acid sequence as denoted by SEQ ID NO: 38, or an analog or derivative thereof having at least 90% sequence identity with the light chain sequence. In some embodiments, 1C6 comprises heavy chain variable region having the nucleic acid sequence denoted by SEQ ID NO: 5, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 1C6 comprises light chain variable region having the nucleic acid sequence as denoted by SEQ ID NO: 6, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 1C6 comprises heavy chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 77-79, respectively. In some embodiments, 1C6 comprises heavy chain CDRl , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 173-175, respectively. In some embodiments, 1C6 comprises light chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 80-82, respectively. In some embodiments, 1C6 comprises light chain CDRl , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 176-178, respectively; as well as analogs and derivative thereof.

According to some embodiments, the monoclonal antibody, or scFv, or fragments thereof denoted as "2C9" comprises heavy chain variable region having the amino acid sequence denoted by SEQ ID NO: 39, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 2C9 comprises light chain variable region having the amino acid sequence as denoted by SEQ ID NO: 40, or an analog or derivative thereof having at least 90% sequence identity with the light chain sequence. In some embodiments, 2C9 comprises heavy chain variable region having the nucleic acid sequence denoted by SEQ ID NO: 7, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 2C9 comprises light chain variable region having the nucleic acid sequence as denoted by SEQ ID NO: 8, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 2C9 comprises heavy chain CDR1 , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 83-85, respectively. In some embodiments, 2C9 comprises heavy chain CDR1 , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 179-181, respectively. In some embodiments, 2C9 comprises light chain CDR1 , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 86-88, respectively. In some embodiments, 2C9 comprises light chain CDR1, CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 182-184, respectively; as well as analogs and derivative thereof.

According to some embodiments, the monoclonal antibody, or scFv, or fragments thereof denoted as "2H12" comprises heavy chain variable region having the amino acid sequence denoted by SEQ ID NO: 39, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 2H12 comprises light chain variable region having the amino acid sequence as denoted by SEQ ID NO: 40, or an analog or derivative thereof having at least 90% sequence identity with the light chain sequence. In some embodiments, 2H12 comprises heavy chain variable region having the nucleic acid sequence denoted by SEQ ID NO: 9, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 2H12 comprises light chain variable region having the nucleic acid sequence as denoted by SEQ ID NO: 10, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 2H12 comprises heavy chain CDR1 , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 89-91, respectively. In some embodiments, 2H12 comprises heavy chain CDR1, CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 185-187, respectively. In some embodiments, 2H12 comprises light chain CDR1 , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 92- 94, respectively. In some embodiments, 2H12 comprises light chain CDR1 , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 188-190, respectively; as well as analogs and derivative thereof.

According to some embodiments, the monoclonal antibody, or scFv, or fragments thereof denoted as "5D8" comprises heavy chain variable region having the amino acid sequence denoted by SEQ ID NO: 43, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 5D8 comprises light chain variable region having the amino acid sequence as denoted by SEQ ID NO: 44, or an analog or derivative thereof having at least 90% sequence identity with the light chain sequence. In some embodiments, 5D8 comprises heavy chain variable region having the nucleic acid sequence denoted by SEQ ID NO: 11, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 5D8 comprises light chain variable region having the nucleic acid sequence as denoted by SEQ ID NO: 12, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 5D8 comprises heavy chain CDRl, CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 95-97, respectively. In some embodiments, 5D8 comprises heavy chain CDRl, CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 191-193, respectively. In some embodiments, 5D8 comprises light chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 98-100, respectively. In some embodiments, 5D8 comprises light chain CDRl, CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 194-196, respectively; as well as analogs and derivative thereof.

According to some embodiments, the monoclonal antibody, or scFv, or fragments thereof denoted as "5E8" comprises heavy chain variable region having the amino acid sequence denoted by SEQ ID NO: 45, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 5E8 comprises light chain variable region having the amino acid sequence as denoted by SEQ ID NO: 46, or an analog or derivative thereof having at least 90% sequence identity with the light chain sequence. In some embodiments, 5E8 comprises heavy chain variable region having the nucleic acid sequence denoted by SEQ ID NO: 13, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 5E8 comprises light chain variable region having the nucleic acid sequence as denoted by SEQ ID NO: 14, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 5E8 comprises heavy chain CDRl, CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 101-103, respectively. In some embodiments, 5E8 comprises heavy chain CDRl , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 197-199, respectively. In some embodiments, 5E8 comprises light chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 104-106, respectively. In some embodiments, 5E8 comprises light chain CDRl, CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 200-202, respectively; as well as analogs and derivative thereof. According to some embodiments, the monoclonal antibody, or scFv, or fragments thereof denoted as "5G7" comprises heavy chain variable region having the amino acid sequence denoted by SEQ ID NO: 47, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 5G7 comprises light chain variable region having the amino acid sequence as denoted by SEQ ID NO: 48, or an analog or derivative thereof having at least 90% sequence identity with the light chain sequence. In some embodiments, 5G7 comprises heavy chain variable region having the nucleic acid sequence denoted by SEQ ID NO: 15, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 5G7 comprises light chain variable region having the nucleic acid sequence as denoted by SEQ ID NO: 16, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 5G7 comprises heavy chain CDRl, CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 107-109, respectively. In some embodiments, 5G7 comprises heavy chain CDRl, CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 203-205, respectively. In some embodiments, 5G7 comprises light chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 110-112, respectively. In some embodiments, 5G7 comprises light chain CDRl , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 206-208, respectively; as well as analogs and derivative thereof.

According to some embodiments, the monoclonal antibody, or scFv, or fragments thereof denoted as "6C6" comprises heavy chain variable region having the amino acid sequence denoted by SEQ ID NO: 51, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 6C6 comprises light chain variable region having the amino acid sequence as denoted by SEQ ID NO: 52, or an analog or derivative thereof having at least 90% sequence identity with the light chain sequence. In some embodiments, 6C6 comprises heavy chain variable region having the nucleic acid sequence denoted by SEQ ID NO: 19, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 6C6 comprises light chain variable region having the nucleic acid sequence as denoted by SEQ ID NO: 20, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 6C6 comprises heavy chain CDRl, CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 119-121 , respectively. In some embodiments, 6C6 comprises heavy chain CDRl , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 215-217, respectively. In some embodiments, 6C6 comprises light chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 122-124, respectively. In some embodiments, 6C6 comprises light chain CDRl, CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 218-220, respectively; as well as analogs and derivative thereof.

According to some embodiments, the monoclonal antibody, or scFv, or fragments thereof denoted as "6C9" comprises heavy chain variable region having the amino acid sequence denoted by SEQ ID NO: 53, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 6C9 comprises light chain variable region having the amino acid sequence as denoted by SEQ ID NO: 54, or an analog or derivative thereof having at least 90% sequence identity with the light chain sequence. In some embodiments, 6C9 comprises heavy chain variable region having the nucleic acid sequence denoted by SEQ ID NO: 21, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 6C9 comprises light chain variable region having the nucleic acid sequence as denoted by SEQ ID NO: 22, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 6C9 comprises heavy chain CDRl, CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 125-127, respectively. In some embodiments, 6C9 comprises heavy chain CDRl , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 221-223, respectively. In some embodiments, 6C9 comprises light chain CDRl , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 128-130, respectively. In some embodiments, 6C9 comprises light chain CDRl , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 224-226, respectively; as well as analogs and derivative thereof.

According to some embodiments, the monoclonal antibody, or scFv, or fragments thereof denoted as "6D12" comprises heavy chain variable region having the amino acid sequence denoted by SEQ ID NO: 55, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 6D12 comprises light chain variable region having the amino acid sequence as denoted by SEQ ID NO: 56, or an analog or derivative thereof having at least 90% sequence identity with the light chain sequence. In some embodiments, 6D12 comprises heavy chain variable region having the nucleic acid sequence denoted by SEQ ID NO: 23, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 6D12 comprises light chain variable region having the nucleic acid sequence as denoted by SEQ ID NO: 24, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain sequence. In some embodiments, 6D12 comprises heavy chain CDR1 , CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 131-133, respectively. In some embodiments, 6D12 comprises heavy chain CDR1, CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 227-229, respectively. In some embodiments, 6D12 comprises light chain CDR1, CDR2 and CDR3 nucleic acid sequences as denoted by SEQ ID NOs: 134- 136, respectively. In some embodiments, 6D12 comprises light chain CDR1 , CDR2 and CDR3 amino acid sequences as denoted by SEQ ID NOs: 230-232, respectively; as well as analogs and derivative thereof.

According to some embodiments, the VL-CDR2 amino acid sequence of the antibody is selected from the group consisting of: RDNKRPS (SEQ ID NO: 257); GNDQRPS (SEQ ID NO: 258); RDNQRPS (SEQ ID NO: 259); GNNNRPS (SEQ ID NO: 260); GDNNRPS (SEQ ID NO: 261); GNSNRPL (SEQ ID NO: 262); SNHHRPS (SEQ ID NO: 263); GNTLRPS (SEQ ID NO: 264); SNNQRPS (SEQ ID NO: 265); LGSNRAS (SEQ ID NO: 266); GDYHRPS (SEQ ID NO: 267); GNSHRPS (SEQ ID NO: 268); SNHHRPS (SEQ ID NO: 269); DNNKRPS (SEQ ID NO: 270); GNTKRPS (SEQ ID NO: 271); and GNTKRPS (SEQ ID NO: 272). Each possibility represents a separate embodiment of the invention.

In some embodiments, the invention provides a monoclonal antibody or an antibody fragment comprising an antigen binding domain (ABD) which comprises three CDRs of a light chain and three CDRs of a heavy chain, wherein said ABD has at least 90% sequence identity or similarity with an ABD of a monoclonal antibody or scFv (herein identified as 1 A6 or clone 1A6) comprising a heavy variable chain comprising the amino acid sequence SEQ ID NO: 35 and a light variable chain comprising the amino acid sequence SEQ ID NO: 36. Such antibody may have an ABD domain having at least 93%, at least 95%, at least 96, at least 97, at least 98, at least 99% sequence identity or similarity or 100% sequence identity with corresponding ABD of 1 A6.

According to some embodiments, the antibodies of the present invention may be shuffled antibodies, which include domains from various IgG isotypes. In some embodiments, the shuffled antibodies may include the VH and VL domains having the sequences as listed in the present invention, together with CH2 domain of IgG4 isotype, CHI and CH3 domains of either IgGl or IgG4 isotypes. In some exemplary embodiments, the shuffled antibody is of 1A6 antibody, which includes VH and VL having amino acid sequence SEQ ID NO: 35 and SEQ ID NO: 36, respectively, the CHI domain is of IgG4, the CH2 domain is of human IgG4 and the CH3 domain is of IgGl . In other exemplary embodiments, the CHI domain is of IgGl , the CH2 domain is of human IgG4 and the CH3 domain is of IgG4. In other exemplary embodiments, the CHI domain is of IgGl , the CH2 domain is of human IgG4 and the CH3 domain is of IgGl .

Reference is now made to Figure 1, which is a schematic illustration of the structure of IL-4 and IL-13 receptors and the interplay therebetween. As shown in Fig. 1 , IL-4 signals through two possible receptor complexes composed of a heterodimer of the IL-4Rot (140 kDa) and yc chain (60 kDa); type I receptor or the IL-4Rot and IL-13Rotl (65-70 kDa) chain; type II receptor. IL-4 binds IL-4Rot with high affinity, triggering dimerization with the secondary signaling chain. IL-13 binds IL-13Rotl which complexes with the IL-4Rot, forming the type II receptor. Signaling through the type I receptor leads to activation of JAKs and downstream signaling adaptor molecules STAT6 and IRS-2, whereas signaling through the type II receptor predominantly activates STAT6. IL-4 signaling also activates PI3-K and AKT. IL-13 also binds cell-surface and soluble forms of the IL-13Rot2; this so-called inhibitory subunit binds IL-13 with greater affinity than IL-13Rotl and acts as a "cytokine sink". Thus, by inhibiting the activity or activation of the receptor, related conditions affected by the activity of the receptor may be treated.

The pathological effects of IL-13 and IL-4 are thus mediated via an overlapping set of receptors that display differential distribution among structural and hematopoietic cells. The type 1 IL-4 receptor is predominantly expressed by hematopoietic cells indicating its importance in homeostasis of immune cells, while the type 2 IL-4 receptor is expressed mainly by cells that are not from the hematopoietic origin (e.g. fibroblasts, epithelial cells, smooth muscle cells). Neutralizing IL-4Ra will block signals from both the type 1 and type 2 IL-4 receptors. Neutralizing IL-13 alone, as in the present invention, will still leave free IL-4 that can bind its receptors and exert important biological activities, such as immune homeostasis.

Any disease or condition which involves high expression of IL-13 or IL-13Ral is a potential target for treatment or diagnosis with the immunoglobulin molecules of the present invention.

In pharmaceutical and medicament formulations, the active agent (such as antibody or fragment thereof) may be utilized together with one or more pharmaceutically acceptable carrier(s) and optionally any other therapeutic ingredient. The carrier(s) should be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof. The active agent is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in a quantity appropriate to achieve the desired daily dose.

Typically, the molecules of the present invention comprising the antigen binding portion of an antibody or comprising another immunoglobulin including the same CDR set, can be suspended in a sterile saline solution for therapeutic uses. The pharmaceutical compositions may alternatively be formulated to control release of active ingredient (molecule comprising the antigen binding portion of an antibody) or to prolong its presence in a patient's system. Numerous suitable drug delivery systems are known and include, e.g., implantable drug release systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, and the like. Controlled release preparations can be prepared through the use of polymers to complex or adsorb the molecule according to the present invention. For example, biocompatible polymers include matrices of poly(ethylene-co- vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebaric acid. The rate of release of the molecule according to the present invention, i.e., of an antibody or antibody fragment, from such a matrix depends upon the molecular weight of the molecule, the amount of the molecule within the matrix, and the size of dispersed particles.

The pharmaceutical composition of this invention may be administered by any suitable means, such as orally, topically, intranasally, by inhalation, by nebulizer, subcutaneously, intramuscularly, intravenously, intra-arterially, intraarticularly, intralesionally or parenterally. In some embodiments, the pharmaceutical composition of this invention may be administered by inhalation.

The term "administering" or "administration of a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered enterally or parenterally. Enterally refers to administration via the gastrointestinal tract including per os, sublingually or rectally. Parenteral administration includes adminstration intravenously, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, intranasally, by inhalation, intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. In some embodiments, the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self- administer a drug, or to have the drug administered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient.

The molecules of the present invention as active ingredients are dissolved, dispersed or admixed in an excipient that is pharmaceutically acceptable and compatible with the active ingredient as is well known. Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, or the like and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents.

The term "treatment" as used herein refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

It will be apparent to those of ordinary skill in the art that the therapeutically effective amount of the molecule according to the present invention will depend, inter alia upon the administration schedule, the unit dose of molecule administered, whether the molecule is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the molecule administered and the judgment of the treating physician. As used herein, a "therapeutically effective amount" refers to the amount of a molecule required to alleviate one or more symptoms associated with a disorder being treated over a period of time.

The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal, such as allergic condition or other fibrotic conditions. In some embodiments, the conditions may be selected from: asthma, COPD, atopic dermatitis, eosinophilic esophagitis, allergic conditions and fibrotic diseases as well as solid tumors. In an exemplary case of Asthma, the therapeutically effective amount of the drug may reduce the occurrence of Asthma; reduce the severity of asthma; inhibit asthma; and/or relieve to some extent one or more of the symptoms associated with the disorder.

The term "about" means that an acceptable error range, e.g., up to 5% or 10%, for the particular value should be assumed.

As used herein and in the appended claims the singular forms "a", "an", and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to "a trivalent metal cation" includes a plurality of such trivalent metal cations and equivalents thereof known to those skilled in the art, and so forth. It should be noted that the term "and" or the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.

The term "plurality" as used herein, means two or more.

The term "comprising" includes the term consisting of.

While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and subcombinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced be interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

Example 1 - Isolation of human IL-13Ral-specific phage antibodies.

The extracellular domain (ECD) of IL-13Ral , corresponding to amino acid residues Gly22 - Thr343 (Accession no. NP_001551.1), fused with a His tag (denoted "rhIL-13Ral ") was used as bait for affinity selection of the "Ronit 1" human single chain Fv (scFv) antibody phage display library (Azriel-Rosenfeld et al. Journal of Molecular Biology, 2004, 335(1):177-192; Nahary L et al, Methods in molecular biology, 2009;525:61-80, xiv). The rhIL-13Ral protein was purchased from ACROBiosystems Inc., USA. Affinity selection was carried out using alternating complexes with the two carrier proteins (to avoid enrichment for carrier-specific antibodies), namely BSA and skim milk. Dynabeads® His-Tag Isolation and Pulldown (Catalog number: 10103D, ThermoFisher SCIENTIFIC, USA) were used to facilitate complexing with rhIL-13Ral. The first selection cycle was performed with 10¹² phages and 10μg rhIL-13Ral. The second, third, and fourth selection cycles were performed with 10¹¹ phages and 5μg rhIL-13Ral . After four selection cycles were completed, monoclonal phage ELISA was carried out and hits were validated for binding specificity (BSA, streptavidin, and MBP-YFP-6xHis served as negative controls for non-specific binding). Finally, 12 clones were identified to specifically bind rhIL-13Ral and are herein denoted 1A1, 1A6, 1C6, 2C9, 2H12, 5D8, 5E8, 5G7, 6A8, 6C6, 6C9 and 6D12. The results are presented in Fig. 2.

The Ronitl Library was further affinity- selected (by phage display) using recombinant (r) human (h) or mouse (m) IL-13Rotl as bait. After 2 panning cycles the library sizes were reduced to ~ 10⁵ and ~ 10⁶ scFv clones, respectively, and then subcloned into a yeast display vector (Colby DW et al, Methods in enzymology, 2004;388 :348-58; Benatuil L et al, Protein engineering, design & selection: PEDS, 2010;23:155-9).

The two yeast libraries were then assessed for binding biotinylated rIL-13Rotl by flow cytometry. By the end of three sorting cycles, dozens of single clones were randomly picked for further evaluation by flow cytometry and sequencing. After excluding low-affinity clones and/or non-IL-13Rotl -specific clones, sequencing revealed three distinct clones that specifically and selectively bound rhIL-13Rotl (A2, H4 and L2), and only one clone (Bl) that specifically bound recombinant murine IL-13Rotl and cross-react with the human protein. The nucleic acid and amino acid sequences of the variable domains and CDR sequences of the identified anti-IL-13Rotl binding clones are provides in the attached sequence listing and are summarized in Table 1.

Table 1: Sequence identification numbers (SEQ ID NOs) of the amino acid (AA) and nucleic acid (NA) sequences of the variable domains of several anti-IL-13Rotl binding clones.

The CDR sequences of selected anti-IL-13Rot clones identified according to the present invention as determined by the IMGT method, are set forth is SEQ ID NOS. 161-256, while the nucleotide sequences encoding these CDRs are set forth in SEQ ID NOS. 65-160.

For CDR2 of VL of the identified clones, the sequences were also determined by the Kabat method and are as following:

Table 2: VL-CDR2 amino acid sequences

Determined by Determined by

Clone No. SEQ ID NO.

IMGT KABAT

1A1 RDN RDNKRPS 257

1A6 GND GNDQRPS 258

1C6 RDN RDNQRPS 259

2C9 GNN GNNNRPS 260

2H12 GDN GDNNRPS 261 5D8 GNS GNSNRPL 262

5E8 SNH SNHHRPS 263

5G7 GNT GNTLRPS 264

6A8 SNN SNNQRPS 265

6C6 LGS LGSNRAS 266

6C9 GDY GDYHRPS 267

6D12 GNS GNSHRPS 268

A2 SNH SNHHRPS 269

Bl DNN DNNKRPS 270

H4 GNT GNTKRPS 271

L2 GNT GNTKRPS 272

Example 2 - Production of full size human IgGl monoclonal antibodies

The 12 anti- hIL-13Ral scFv sequences isolated from the "Ronit 1 " library were subcloned into pcDNA3.4-based vectors for expression in the mammalian Expi293F cell system. In this system antibodies are secreted to the conditioned medium following transfection. By the end of days 5-7 post transfections, the media were collected after centrifuging the cells, filtered, and adjusted to pH 6.5 - 7.0 for loading onto MabSelect SuRe columns (GE Healthcare). Column-bound proteins were eluted in 1ml fractions of lOOmM citric acid pH 3, neutralized by 250μ1 of 1.5M Tris-HCl pH 8.8, and then dialyzed against PBS overnight at 4°C.

11 out of the 12 sequences were successfully produced as human IgGl and purified (clone 1A1 did not assemble into a full IgGl antibody). Fig. 3 shows the non-reducing SDS- PAGE analysis of the 11 human IgGl mAbs and the commercial therapeutic anti EGFR antibody cetuximab (Erbitux), used as a reference for size and purity.

The nucleic acid sequences of constant domains (IgG) format used (herein and in Example 5, below) are: Human C-Lambda: (SEQ ID NO: 273); Human C-Kappa: (SEQ ID NO: 275); Human CHI (including hinge) (γΐ): (SEQ ID NO: 277); Human CH2 (γΐ): (SEQ ID NO: 278); Human CH3 (γΐ): (SEQ ID NO: 279); Human CHI (including hinge) (γ4): (SEQ ID NO: 281); Human CH2 (γ4): (SEQ ID NO: 282); and Human CH3 (γ4): (SEQ ID NO: 283). Example 3- Binding assays of anti-hIL-13Rotl human IgGl monoclonal antibodies

To assess the binding abilities of the 11 anti- hIL-13Ral mAbs. ELISA assays were utilized. The control antigen for non-specific binding was β-galactosidase ("b-gal"). Four mAbs (out of 11) exhibited binding to rhIL-13Rotl when in the form of IgGl . As shown in Fig. 4A and Fig. 4B, antibody 1A6 binds rhIL-13Rotl in the strongest manner (with an EC₅₀ ~lnM), followed by 1C6, 6A8 (-20 nM), and 2H12 (~500nM). 1A6 was then tested by Surface Plasmon Resonance to accurately determine its affinity. According to BIAcore 3000 measurements, 1A6 antibody, has a calculated KD of 1.57nM (Fig. 5).

To determine their binding affinity, single-chain antibodies A2, Bl , H4, and L2 selected from the yeast display library were cloned from the yeast vectors into IgG expression vectors using human IgGl expression vectors and the "Expi293F" system of Invitrogen. The first two amino acids of the Lambda chains, DI, were substituted with QS when expressed in mammalian Expi293F expression system. ELISA was carried out with Mab-select-purified IgGs to assess their affinities. ELISA plates were coated with ^g/ml of rhIL-13Ral or control antigen and blocked with 3% skim milk. IgGl dilutions were added as indicated (in triplicates), before secondary HRP-conjugated human IgG H+L polyclonal Ab was reacted with TMB. The reactions were stopped by 1M H₂SO₄. Plates were read at 450nm. The data obtained by ELISA are described on Fig. 8 A-D; as shown, the approximate affinities of H4 and L2 to rhIL-13Ral (as estimated by direct ELISA) are 200pM and 400pM, respectively, whilst A2 and Bl have lower affinities (50nM and 200nM respectively).

In addition, an ELISA plate was coated with 5μg/ml of B l mAb (IgGl) and blocked with 3% skim milk. Recombinant, biotinylated antigens (hIL-13Ral, mIL-13Ral , or MBP- LacZ) dilutions were added as indicated, before HRP-Streptavidin was reacted with TMB. The reaction was stopped by 1M H₂SO₄. The Plate was read at 450nm.

To determine binding specificity of Bl IgGl mAb, human A549 cells (expressing hIL-13Rotl) and murine MC38 cells (expressing mIL-13Rotl) were stained with biotinylated human IgG mAbs, Bl or Avastin™ (commercial anti-VEGF IgGl) as isotype control, and analyzed by flow cytometry. The results represented in Fig. 9A and 9B demonstrate that compared with Avastin™, Bl significantly bound human A549 and murine MC38 cells. Example 4- Cross-reactivity of 1A6 antibody with receptor ECD of other species

Antibody 1A6 was then tested for cross-reactivity with the murine (m) and the Cynomolgus (cy) IL-13Rotl homologs. The mouse and human receptors were purchased from R&D systems and ACRObiosystems (respectively), while the Cynomolgus receptor was prepared by cloning into the vectors of the Expi293 system and purified from the conditioned medium by Ni-NTA chromatography. The ELISA results presented in Fig. 6 demonstrate that 1A6 cross-reacts with Cynomolgus receptor, cyIL-13Rotl (estimated EC₅₀ ~20nM), but not with mouse receptor, mIL-13Rotl (EC₅₀ > ΙΟΟΟηΜ). The 20-fold difference between the EC50S of hIL-13Rotl and cyIL-13Rotl may be attributed to impurities in the cyIL-13Rotl sample (commercially available rhIL-13Rotl is >98% pure). All other tested antibodies were also specific to human IL-13Rotl ECD, except for the clone Bl which is cross-reactive with mouse and rat IL-13Ral ECD.

Of note, there is a 96% identity between the human and the Cynomolgus IL-13Rotl ECDs (and 98% similarity). The identity between human and mouse IL-13Rotl ECDs is only 75%.

Example 5 - Construction and evaluation of Shuffled antibodies

In the human body, IgGl antibodies may elicit immune effector functions (ADCC and CDC, mediated by the CH2 domain of the antibody constant region), resulting in death of the targeted cells. To this aim, antibody variants which would retain the binding and neutralizing properties (provided by the variable domains, VH and VL), but lack the ability to mediate antibody-dependent cell death were designed by replacing at least one IgGl constant region with an IgG4 eqvivalent.

To this end, shuffled antibodies which comprise the VH and VL domains of 1A6 mAb, the CH2 domain of human IgG4 (which doesn't elicit ADCC and CDC responses), and CHI & CH3 domains of either isotypes, were prepared. The hinge region (located in the CHI domain) and the CH3 domains of IgGl prevent Fab-arm exchange in vivo.

The following constructs of shuffled constant regions were prepared:

A- CHI (y4) - CH2(y4) - CH3(yl)

B- CHI (γΐ) - CH2(y4) - CH3(y4)

C- CHl (yl) - CH2(y4) - CH3(yl) All five constructs, including above listed A-C, 1A6, IgGl, and 1A6 IgG4, were subcloned into pcDNA3.4 based vectors for expression in the mammalian Expi293F cell system. By the end of day 6 post transfections, the media were collected after centrifuging the cells, filtered, and adjusted to pH 6.5 - 7.0 for loading onto MabSelect SuRe columns (GE Healthcare). Column-bound proteins were eluted in 1ml fractions of lOOmM citric acid pH 3, neutralized by 250μ1 of 1.5M Tris-HCl pH 8.8, and then dialyzed against PBS overnight at 4°C.

In order to confirm functionality of the shuffled antibodies, which include the 1A6 variable domains, ELISA assays were performed. As shown in Fig. 7A, the shuffled antibodies retained the binding properties, with an exceptional slight reduction for variant 'B'. For control, none of the shuffled antibodies bound β-galactosidase.

To verify that the shuffled antibodies do not elicit ADCC, another ELISA was performed with CD 16a (FcyRIIIa) as the antigen. As shown in Fig. 7B, the IgGl antibody could bind CD16a whereas the shuffled antibodies (as well as IgG4) did not.

Altogether, the results suggest that the presented shuffled antibodies can yield improved therapeutic antibodies, as compared to the default IgGl or IgG4 formats.

The full size heavy chain nucleic acid sequences of the various 1A6 clones, that contain the hinge regions in their CHI domains, were as following:

Antibody 1A6 - IgGl : (SEQ ID NO: 284);

Antibody 1A6 - IgG4: (SEQ ID NO: 285);

Shuffled Antibody 1 A6 - Shuffled Ά': CHl(y4) - CH2(y4) - CH3(yl): (SEQ ID NO: 286); Shuffled Antibody 1A6 - Shuffled 'Β': CHl(yl) - CH2(y4) - CH3(y4): (SEQ ID NO: 287); Shuffled Antibody 1A6 - Shuffled 'C: CHl(yl) - CH2(y4) - CH3(yl): (SEQ ID NO: 288); The full size light chain nucleic acid sequence of clone 1A6 is: (SEQ ID NO: 289).

The full size heavy chain amino acid sequences of the 1 A6 clones were as follows: Antibody 1A6 - IgGl : (SEQ ID NO: 290);

Antibody 1A6 - IgG4: (SEQ ID NO: 291);

Shuffled Antibody 1 A6 - Shuffled Ά': CHl(y4) - CH2(y4) - CH3(yl): (SEQ ID NO: 292); Shuffled Antibody 1A6 - Shuffled 'Β': CHl(yl) - CH2(y4) - CH3(y4): (SEQ ID NO: 293); Shuffled Antibody 1A6 - Shuffled 'C: CHl(yl) - CH2(y4) - CH3(yl): (SEQ ID NO: 294); Full size light chain amino- acid sequence of clone 1A6:_(SEQ ID NO: 295);

Example 6- Treatment of Asthma in-vivo

In order to test the in-vivo efficacy of the 1A6 monoclonal antibody, it is tested for its ability to neutralize IL-13Rotl and hence block I1-13/IL-4 dependent responses in monkeys. To this aim, first, IL-13/IL-4-induced effects are neutralized in- vitro using monkey peripheral blood leukocytes. Experimental asthma is then induced in the non-human primates and the animals are treated with the anti-IL-13Rotl antibody (or mistype matched control). Thereafter, asthma-induced airway pathologies are assessed (for example, airway hyper responsiveness mucus production, inflammatory cell recruitment, bronchoalveolar lavage fluid chemokine levels and histopathology). The ability of the anti-IL-13Rotl neutralizing antibody to prevent IL-4 and/or IL-13-induced airway pathology is determined.

Claims

CLAIMS What we claim is:

1. An isolated immunoglobulin which binds to human IL-13Rotl, wherein the immunoglobulin comprises complementarity determining region (CDR) amino acid sequences selected from the group consisting of: i. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 35 and three CDRs of a light-chain variable comprising SEQ ID NO: 36, or an analog or derivative thereof having at least 90% sequence identity with said immunoglobulin sequence;

ii. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 49 and three CDRs of a light-chain variable region comprising SEQ ID NO: 50, or an analog or derivative thereof having at least 90% sequence identity with said immunoglobulin sequence;

iii. three CDRs of a heavy-chain variable region comprising SEQ ID NO: 57 and three CDRs of a light-chain variable region comprising SEQ ID NO: 58, or an analog or derivative thereof having at least 90% sequence identity with said immunoglobulin sequence;

2. The isolated immunoglobulin of claim 1 , wherein the immunoglobulin is selected from the group consisting of: a monoclonal antibody (mAb), an antibody fragment comprising at least the antigen binding portion, and a single chain antibody.

3. The isolated immunoglobulin of any one of claims 1 or 2, comprising a set of six CDR amino acid sequences wherein three CDRs are from a heavy chain molecule selected from the group consisting of: SEQ ID Nos: 35, 49, 57, 59, 61, and 63; and three CDRs are from a light chain molecule selected from the group consisting of: SEQ ID Nos: 36, 50, 58, 60, 62, and 64.

4. The isolated immunoglobulin of any one of claims 1 to 3, comprising a set of six CDR amino acid sequences selected from the groups consisting of: i. SEQ ID Nos: 167, 168, 169, 170, 171 , and 172;

ii. SEQ ID Nos: 209, 210, 211 , 212, 213, and 214;

iii. SEQ ID Nos: 233, 234, 235, 236, 237, and 238;

iv. SEQ ID Nos: 239, 240, 241 , 242, 243, and 244;

v. SEQ ID Nos: 245, 246, 247, 248, 249, and 250; and

vi. SEQ ID Nos: 251 , 252, 253, 254, 255, and, 256.

5. The isolated immunoglobulin of any one of claims 1 to 4, comprising a heavy chain variable region set forth in a sequence selected from the group consisting of: 35, 49, 57,

59, 61, and 63.

6. The isolated immunoglobulin of any one of claims 1 to 5, comprising a light chain variable region set forth in a sequence selected from the group consisting of: 36, 50, 58,

60, 62, and 64.

7. The isolated immunoglobulin of any one of claims 1 to 6, said isolated immunoglobulin is selected from the group consisting of: i. a mAb denoted 1A6 or a fragment thereof comprising heavy-chain CDR1 comprising the sequence set forth in SEQ ID NO: 167, heavy-chain CDR2 comprising the sequence set forth in SEQ ID NO: 168, heavy-chain CDR3 comprising the sequence set forth in SEQ ID NO: 169, light-chain CDR1 comprising the sequence set forth in SEQ ID NO: 170, light-chain CDR2 comprising the sequence set forth in SEQ ID NO: 171, and light-chain CDR3 comprising the sequence set forth in SEQ ID NO: 172; ii. a mAb denoted 6A8 or a fragment thereof comprising heavy-chain CDR1 comprising the sequence set forth in SEQ ID NO: 209, heavy-chain CDR2 comprising the sequence set forth in SEQ ID NO: 210, heavy-chain CDR3 comprising the sequence set forth in SEQ ID NO: 211 , light-chain CDR1 comprising the sequence set forth in SEQ ID NO: 212, light-chain CDR2 comprising the sequence set forth in SEQ ID NO: 213, and light-chain CDR3 comprising the sequence set forth in SEQ ID NO: 214; iii. a mAb denoted A2 or a fragment thereof comprising heavy-chain CDR1 comprising the sequence set forth in SEQ ID NO: 233, heavy-chain CDR2 comprising the sequence set forth in SEQ ID NO: 234, heavy-chain CDR3 comprising the sequence set forth in SEQ ID NO: 235, light-chain CDR1 comprising the sequence set forth in SEQ ID NO: 236, light-chain CDR2 comprising the sequence set forth in SEQ ID NO: 237, and light-chain CDR3 comprising the sequence set forth in SEQ ID NO: 238 ; iv. a mAb denoted B l or a fragment thereof comprising heavy-chain CDR1 comprising the sequence set forth in SEQ ID NO: 239, heavy-chain CDR2 comprising the sequence set forth in SEQ ID NO: 240, heavy-chain CDR3 comprising the sequence set forth in SEQ ID NO: 241 , light-chain CDR1 comprising the sequence set forth in SEQ ID NO: 242, light-chain CDR2 comprising the sequence set forth in SEQ ID NO: 243, and light-chain CDR3 comprising the sequence set forth in SEQ ID NO: 244; v. a mAb denoted H4 or a fragment thereof comprising heavy-chain CDR1 comprising the sequence set forth in SEQ ID NO: 245, heavy-chain CDR2 comprising the sequence set forth in SEQ ID NO: 246, heavy-chain CDR3 comprising the sequence set forth in SEQ ID NO: 247, light-chain CDR1 comprising the sequence set forth in SEQ ID NO: 248, light-chain CDR2 comprising the sequence set forth in SEQ ID NO: 249, and light-chain CDR3 comprising the sequence set forth in SEQ ID NO: 250; and vi. a mAb denoted L2 or a fragment thereof comprising heavy-chain CDR1 comprising the sequence set forth in SEQ ID NO: 251 , heavy-chain CDR2 comprising the sequence set forth in SEQ ID NO: 252, heavy-chain CDR3 comprising the sequence set forth in SEQ ID NO: 253, light-chain CDR1 comprising the sequence set forth in SEQ ID NO: 254, light-chain CDR2 comprising the sequence set forth in SEQ ID NO: 255, and light-chain CDR3 comprising the sequence set forth in SEQ ID NO: 256.

8. The isolated immunoglobulin of any one of claims 1 to 7, said isolated immunoglobulin, single chain antibody, or fragment thereof, comprising a set of six CDR sequences wherein the set is selected from the group consisting of: i. SEQ ID Nos: 167, 168, 169, 170, 171 , and 172;

ii. SEQ ID Nos: 209, 210, 211 , 212, 213, and 214;

iii. SEQ ID Nos: 233, 234, 235, 236, 237, and 238;

iv. SEQ ID Nos: 239, 240, 241 , 242, 243, and 244;

v. SEQ ID Nos: 245, 246, 247, 248, 249, and 250;

vi. SEQ ID Nos: 251, 252, 253, 254, 255, and, 256;

vii. SEQ ID Nos: 167, 168, 169, 170, 258, and 172;

viii. SEQ ID Nos: 209, 210, 211 , 212, 265, and 214;

ix. SEQ ID Nos: 233, 234, 235, 236, 269, and 238;

x. SEQ ID Nos: 239, 240, 241 , 242, 270, and 244;

xi. SEQ ID Nos: 245, 246, 247, 248, 271 , and 250; and

xii. SEQ ID Nos: 251, 252, 253, 254, 272, and, 256.

9. The isolated immunoglobulin of any one of claims 1 to 8, said isolated immunoglobulin comprising a heavy chain and a light chain, wherein said chains comprises a set of heavy chain variable region sequence and light chain variable region sequence, said set is selected from the group consisting of: i. SEQ ID NOs: 35, 36;

ii. SEQ ID NOs: 49, 50;

iii. SEQ ID NOs: 57, 58;

iv. SEQ ID NOs: 59, 60;

v. SEQ ID NOs: 61 , 62; and vi. SEQ ID NOs: 63, 64.

10. The isolated immunoglobulin of any one of claims 1 to 9, wherein the isolated immunoglobulin comprises the CDR sequences of a (scFv) antibody clone selected from the group consisting of: 1A6, 6A8, A2, Bl , H4 and L2.

11. The isolated immunoglobulin of any one of claims 1 to 10, wherein the immunoglobulin is a single chain Fv (scFv) antibody comprising a light chain variable region and a heavy chain variable region, covalently connected through an amino acid linker.

12. The isolated immunoglobulin of claim 11, wherein the amino acid linker comprises the sequence GSAGGGGSGGGGSGGGGS (SEQ ID NO: 296).

13. The isolated immunoglobulin of any one of claims 11 or 12, wherein the scFv antibody comprises a heavy chain variable region having a sequence selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 49, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , and SEQ ID NO: 63; and a light chain variable region having a sequence selected from the group consisting of: SEQ ID NO: 36, SEQ ID NO: 50, SEQ ID NO:

58, SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64.

14. The isolated immunoglobulin of any one of claims 1 to 10, wherein the immunoglobulin is a full size human mAb comprising a heavy chain variable region selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 49, SEQ ID NO: 57, SEQ ID NO:

59, SEQ ID NO: 61, and SEQ ID NO: 63; a light chain variable region having a sequence selected from the group consisting of: SEQ ID NO: 36, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64, and at least one sequence of a constant region of a human IgG.

15. The isolated immunoglobulin of claim 14, wherein the at least one sequence of the heavy chain constant region of human IgG is a gamma 1 heavy chain region (IgGl) sequence or gamma 4 heavy chain region (IgG4) sequence.

16. The isolated immunoglobulin of any one of claims 14 or 15, wherein the constant human IgG sequence of the light chain comprises C-kappa or C-lambda light chain sequence.

17. The isolated immunoglobulin of any one of claims 1 to 10, and 16, wherein the antibody is a shuffled antibody, comprising heavy chain constant domains of various IgG iso types.

18. The isolated immunoglobulin of claim 17, wherein the constant human IgG comprises at least one portion of an IgGl sequence and at least one portion of an IgG4 sequence.

19. The isolated immunoglobulin of any one of claims 17 or 18, wherein the shuffled mAb comprises a CH2 domain of IgG4 isotype, and CHI and CH3 domains of either IgGl or IgG4 isotypes.

20. The isolated immunoglobulin of claim 19, wherein the constant domains of the heavy chain of the mAb comprise: CHI domain of the human gamma- 1 isotype, CH2 of the human gamma-4 isotype and CH3 domain of the human gamma- 1 isotype.

21. The isolated immunoglobulin of any one of claims 14 to 20, comprising at least one sequence of a constant domain selected from the group consisting of: i. human C-Lambda: (SEQ ID NO: 274);

ii. human C-Kappa: (SEQ ID NO: 276);

iii. human CHI (including hinge) (γΐ): (encoded by DNA SEQ ID NO: 277);

iv. human CH2 (γΐ): (encoded by DNA SEQ ID NO: 278);

v. human CH3 (γΐ): (encoded by DNA SEQ ID NO: 279);

viii. human CH2 (γ4): (encoded by DNA SEQ ID NO: 282);

ix. human CH3 (γ4): (encoded by DNA SEQ ID NO: 283); and/or

x. entire human gamma4 constant region amino acid sequence: SEQ ID NO: 297.

22. The isolated immunoglobulin of any one of claims 1 to 21 , said isolated immunoglobulin recognizes human IL-13Rotl with an affinity of at least 10^"7M.

23. The isolated immunoglobulin of any one of claims 1 to 22, said isolated immunoglobulin binds human IL-13Rotl as well as mouse IL-13Rotl.

24. The isolated immunoglobulin of any one of claims 1 to 23, wherein the immunoglobulin is a monoclonal antibody comprises heavy chain CDRl comprising the amino acid sequence: GFTFSNHE (SEQ NO: 167), heavy chain CDR2 having the sequence: ISWNGSSL (SEQ NO: 168), heavy chain CDR3 having the sequence: ARDGAAVGWFDP (SEQ NO: 169), light chain CDR1 having the sequence: SSNIGSNY (SEQ NO: 170), light chain CDR2 having the sequence: GND (SEQ NO: 171), and light chain CDR3 having the sequence: GTWDSSLSAGV (SEQ NO: 172), and analogs and derivative thereof.

25. A polynucleotide sequence encoding an isolated immunoglobulin according to any one of claims 1 to 24.

26. A polynucleotide sequence encoding an isolated immunoglobulin which binds to human IL-13Rotl, or a fragment thereof, wherein said isolated immunoglobulin comprises the CDR sequences of a (scFv) antibody clone selected from the group consisting of: 1A6, 6A8, A2, Bl , H4 and L2.

27. The polynucleotide sequence of any one of claims 25 or 26, encoding an antibody heavy chain variable domain, said polynucleotide sequence is selected from the group consisting of: SEQ ID NOs: 3, 17, 25, 27, 29, and 31.

28. The polynucleotide sequence of any one of claims 25 to 27, encoding an antibody light chain variable domain, said polynucleotide sequence is selected from the group consisting of: SEQ ID NOs: 4, 18, 26, 28, 30, and 32.

29. A mAb, single chain antibody or an antibody fragment comprising a set of six CDR sequences from heavy- and light- chain variable regions, the variable regions are encoded by a set of two polynucleotide sequences, wherein the set of polynucleotide sequences is selected from the group consisting of: i. SEQ ID NOs. 3 and 4;

ii. SEQ ID NOs. 17 and 18;

iii. SEQ ID NOs. 25 and 26;

iv. SEQ ID NOs. 27 and 28;

v. SEQ ID NOs. 29 and 30; and

vi. SEQ ID NOs. 31 and 32.

30. A nucleic acid construct comprising a polynucleotide sequence according to any one of claims 25 to 28.

31. A tissue culture cell comprising a polynucleotide sequence according to any one of claims 25 to 28.

32. A pharmaceutical composition comprising as an active ingredient, at least one isolated immunoglobulin according to any one of claims 1 to 24, and a pharmaceutical acceptable excipient, diluent, salt or carrier.

33. The pharmaceutical composition of claim 32 for use in treating conditions associated with expression or activation or signaling of IL-13Rotl .

34. The pharmaceutical composition of claim 33, wherein the condition is a disease or disorder characterized by increased IL-13 and/or IL-4 levels as well as increased activation of the adaptor molecule STAT6.

35. The pharmaceutical composition of any one of claims 33 or 34, wherein the condition is selected from the group consisting of: allergic disease or disorder, fibrotic disease or disorder and proliferative disease or disorder.

36. The pharmaceutical composition of claim 35, wherein the condition is selected from the group consisting of: asthma, atopic dermatitis, eosinophilic esophagitis, and cancer.

37. A method of treating a subject having a disease or condition associated with expression or activation of IL-13Rotl, the method comprising administering to the subject a pharmaceutical composition according to claim 32.

38. A method of determining or quantifying the expression of IL-13Rotl, the method comprising contacting a biological sample with an immunoglobulin molecule, and measuring the level of complex formation, wherein the immunoglobulin molecule is selected from the group consisting of monoclonal antibody, single chain antibody or antibody fragment comprising at least the antigen binding domain, and wherein the immunoglobulin molecule comprises a CDR set of a mAb selected from the group consisting of: 1 A6, 6A8, A2, Bl, H4 and L2.

39. A kit for measuring the expression of IL-13Rotl in biological sample at least one antibody or antibody fragment comprising the CDRs contained in an antibody selected from the group consisting of: 1A6, 6A8, A2, Bl, H4 and L2.

40. An immunoglobulin comprising a set of at least three complementarity determining region (CDR) amino acid sequences, said set is selected from the group consisting of: i. three CDRs from a heavy chain molecule selected from the group consisting of: SEQ ID Nos: 35, 49, 57, 59, 61 , and 63; and ii. three CDRs from a light chain molecule selected from the group consisting of: SEQ ID Nos: 36, 50, 58, 60, 62, and 64, or an analog or derivative thereof having at least 90% sequence identity with said heavy or light chain sequence.