Abstract:
The invention relates to a human Interleukin-2 (hIL-2) specific monoclonal antibody (mAb), or antigen binding fragment thereof, the binding of which to hIL-2 inhibits binding of hIL-2 to CD25 and the antibody is characterized by any of the parameters: the variable chain of the mAb comprises the amino acid sequence of SEQ ID NO 005 or SEQ ID NO 006; the binding to hIL-2 is characterized by a dissociation constant (K D )≦7.5 nmol/L; the binding to hIL-2 is characterized by an off-rate (K off )≦1×10 −4  s −1  and/or the antibody displays no measurable cross-reactivity to murine IL-2.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This is application is a 35 USC §371 National Stage Application of International Application No. PCT/IB2015/055226, filed Jul. 10, 2015, which claims benefit of European Application No. 14176619.6, filed Jul. 10, 2014, each of which are herein incorporated by reference in their entireties. 
     
    
     SEQUENCE LISTING 
       [0002]    The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 4, 2017, is named PAT056093_ST25.txt and is 39,591 bytes in size. 
       FIELD OF THE INVENTION 
       [0003]    The present invention relates to antibodies binding to human interleukin-2 (hIL-2). The invention more specifically relates to antibodies specifically binding a particular epitope of hIL-2 and when bound to this epitope are capable of inhibit binding of hIL-2 to CD25. Furthermore, the invention relates to in vitro and in vivo therapeutic applications of the antibodies. 
       BACKGROUND OF THE INVENTION 
       [0004]    Malignant melanoma is a frequent cancer type in men and women. Once melanoma becomes metastatic and spreads to distant sites, the 5-year survival rate is quite poor, calculated at about 15%. Currently available treatment strategies for metastatic melanoma barely improve this survival rate. 
         [0005]    Interleukin-2 (IL-2) is a cytokine able to potently stimulate cytotoxic lymphocytes against metastatic tumours. However, IL-2 is also able to stimulate so-called CD25 +  CD4 +  regulatory T cells (Treg cells) that are crucial for prevention of autoimmune disease. Importantly, Treg cells can significantly dampen anti-tumour responses by cytotoxic lymphocytes, thus somewhat antagonizing the beneficial anti-tumour effects of IL-2. Moreover, at doses required to achieve a clinical anti-tumour response, IL-2 can exert toxic adverse effects. 
         [0006]    Standard IL-2 immunotherapy has been used since the early 1980&#39;s for the immunotherapy of metastatic melanoma and metastatic renal cell carcinoma, leading to the approval by the FDA for these indications in 1996 and 1992, respectively. While IL-2 given at high doses has shown objective response rates in about 17% and complete regression in about 6-9% of patients suffering from these deadly metastatic cancers, IL-2 given at these doses frequently led to toxic adverse effects, such as hypotension, pulmonary edema, liver cell damage, gastrointestinal toxicity, and general edema. Moreover, as mentioned above, IL-2 is able to stimulate Treg cells, which in turn are able to dampen the activity of anti-tumour CD8 +  T cells and NK cells. 
         [0007]    The combination of IL-2 with a particular anti-IL-2 monoclonal antibody (mAb) has been shown to improve IL-2 therapy in experimental murine models of cancer immunotherapy by 
         [0000]    (1) directing IL-2 preferentially to cytotoxic lymphocytes, but not Treg cells, and by
 
(2) rendering IL-2 more potent but less toxic (Boyman O, Kovar M, Rubinstein M P, Surh C D, and Sprent J. Selective stimulation of T cell subsets with antibody-cytokine immune complexes.  Science  (2006) 311:1924-1927; Krieg C, Letourneau S, Pantaleo G, and Boyman O. Improved IL-2 immunotherapy by selective stimulation of IL-2 receptors on lymphocytes and endothelial cells.  Proceedings of the National Academy of Sciences USA  (2010) 107:11906-11911).
 
         [0008]    This approach has the advantage that unmutated, natural IL-2 is delivered via anti-IL-2 mAb to CD8 +  T cells and NK cells, which subsequently exert potent anti-tumour properties, while IL-2 complexed to this kind of anti-IL-2 mAb barely activates Treg cells. Moreover, IL-2 complexed to this kind of anti-IL-2 mAb is much less toxic than standard IL-2 immuno therapy in mice. However, this therapy has up to date not been available for use in patients due to the lack of appropriate anti-human IL-2 mAbs. 
       SUMMARY OF THE INVENTION 
       [0009]    The problem addressed by the present invention is to provide an anti-human IL-2 monoclonal antibody able to recognize and bind a specific epitope of human IL-2, thereby favoring the stimulation of cytotoxic T cells and NK cells compared to Treg cells, for use in in vitro and in vivo therapeutic applications. This problem is solved by the subject-matter of the independent claims. 
         [0010]    According to a first aspect of the invention a human interleukin-2 (hIL-2) specific monoclonal antibody (mAb), or antigen binding fragment thereof, is provided, wherein the antibody is able to bind to a particular epitope in hIL-2 thereby inhibiting the binding to CD25, thus modulating the immunological effects of hIL-2/IL-2R interaction. The antibody of the invention is further characterized by at least one of the parameters:
       a) the variable chain of the mAb comprises an amino acid sequence having an identity of ≧85%, ≧90%, ≧95%, or ≧99% compared to SEQ ID NO 005 or SEQ ID NO 006;   b) the antibody binding to hIL-2 is, i.e. the reaction mAb+hIL-2         mAb*hIL-2, wherein mAb*hIL-2 symbolizes the bound complex of antibody and interleukin, is characterized by a dissociation constant (K D )≦7.5 nmol/L, ≦5 nmol/L, ≦3 nmol/L, ≦2 nmol/L or ≦1.5 nmol/L;   c) the antibody binding to hIL-2 is characterized by an off-rate (K off )≦1×10 −4  s −1 , ≦8×10 −5  s −1 , ≦6×10 −5  s −1 , ≦4×10 −5  s −1 , ≦3×10 −5  s −1  or ≦2.1×10 −5  s −1 ;   d) upon mAb binding to hIL-2, the resulting mAb*hIL-2 complex cannot efficiently bind human IL-2 receptor α (also known as CD25) anymore, effectively rendering the binding of human CD25 to mAb*hIL-2 to background levels as compared to the binding of human CD25 to free (non-complexed) hIL-2 when measured by surface plasmon resonance; and/or   e) the antibody displays no measurable cross-reactivity to murine IL-2.       
 
         [0016]    A lack of cross-reactivity with murine IL-2 is advantageous for preclinical studies, which usually involve mouse models, such as the use of mAb*hIL-2 complexes for the treatment of murine tumour models where a cross-reactive anti-IL-2 mAb might bind and seclude endogenous murine IL-2 from endogenous murine Treg cells, thus enhancing the anti-tumour response. 
         [0017]    A lack of cross-reactivity with murine IL-2 is also advantageous for preclinical safety and efficacy studies conducted prior to development of a candidate mAb in human patients. 
         [0018]    In certain embodiments the hIL-2 mAb comprises at least one V H  and/or V L  sequence having an identity of ≧80%, ≧85%, ≧90%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97% or ≧98% compared to SEQ ID NOs 019 or SEQ ID NO 020. 
         [0019]    In certain embodiments the variable chain of the hIL-2 mAb comprises an amino acid sequence having an identity of ≧85%, ≧90%, ≧95%, or ≧99% compared to SEQ ID NOs 003, 004, 005 or 006 and the hIL-2 mAb is characterized by a dissociation constant ≦7.5 nmol/L, ≦5 nmol/L, ≦3 nmol/L, ≦2 nmol/L or ≦1.5 nmol/L. 
         [0020]    In certain embodiments the variable chain of the hIL-2 mAb comprises an amino acid sequence having an identity of ≧85%, ≧90%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98% or ≧99% compared to SEQ ID NO 005 or 006 and the hIL-2 mAb is characterized by an off-rate ≦1×10 −4  s −1 , ≦8×10 −5  s −1 , ≦6×10 −5  s −1 , ≦4×10 −5  s −1 , ≦3×10 −5  s −1  or ≦2.1×10 −5  s −1 . 
         [0021]    In certain embodiments the variable chain of the hIL-2 mAb comprises an amino acid sequence having an identity of ≧85%, ≧90%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98% or ≧99% compared to SEQ ID NO 005 or 006 and the hIL-2 mAb displays no measurable cross-reactivity to murine IL-2. 
         [0022]    In certain embodiments the sequence of the hIL-2 mAb is humanized for administration to human patients to prevent adverse reactions. 
         [0023]    In certain embodiments the hIL-2 mAb is provided as fragment antigen-binding (Fab) or single-chain variable fragment (scFv). 
         [0024]    In certain embodiments the hIL-2 mAb comprises at least one complementarity determining (CDR) sequence having an identity of ≧80%, ≧85%, ≧90%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97% or ≧98% compared to SEQ ID NOs 007, 008, 009, 010, 011 or 012. 
         [0025]    According to a second aspect of the invention, a nucleic acid molecule encoding the monoclonal antibody, or antigen binding fragment thereof, able to bind to human interleukin-2 according to the first aspect of the invention is provided. 
         [0026]    In certain embodiments the nucleic acid molecule according to the second aspect of the invention has ≧60%, ≧70%, ≧80%, ≧90%, ≧95%, or ≧99% sequence identity compared to SEQ ID NOs 003 to 004. 
         [0027]    According to a third aspect of the invention a vector comprising the nucleic acid molecule according to the invention is provided. 
         [0028]    According to a fourth aspect of the invention, a cell is provided, comprising or expressing the nucleic acid molecule according to the invention. 
         [0029]    According to a fifth aspect of the invention a cell able to produce the antibodies according to the first aspect of the invention is provided. 
         [0030]    According to a sixth aspect of the invention a monoclonal antibody-producing hybridoma cell line is provided, characterized in that the antibodies produced are those of the first aspect of the invention. 
         [0031]    According to a seventh aspect of the invention a therapeutic formulation for use in the treatment of cancer or other diseases benefiting from immune stimulatory therapy, such as viral infections, comprising
       i. the monoclonal antibody (mAb) according to the first aspect of the invention, and/or   ii. human interleukin-2 or human IL-2 mutants, administered to the subject either contemporaneously or at different time points.       
 
         [0034]    According to an eighth aspect of the invention a fusion protein is provided. The fusion protein comprises:
       a. an hIL-2 binding polypeptide fragment, wherein said polypeptide is characterized by any one of the parameters:
           i. the hIL-2 binding polypeptide fragment comprises an amino acid sequence having an identity of ≧85%, ≧90%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97% or ≧98% compared to SEQ ID NO 021 or SEQ ID NO 022;   ii. the hIL-2 binding of said polypeptide fragment to hIL-2 is characterized by a dissociation constant (K D )≦7.5 nmol/L, ≦5 nmol/L, ≦3 nmol/L, ≦2 nmol/L or ≦1.5 nmol/L;   iii. the binding of said hIL-2 binding polypeptide fragment to hIL-2 is characterized by an off-rate (K off )≦1×10 −4  s −1 , ≦8×10 −5  s −1 , ≦6×10 −5  s −1 , ≦4×10 −5  s −1 , ≦3×10 −5  s −1  or 2.1×10 −5  s −1 ;   and/or   iv. the hIL-2 binding polypeptide fragment displays no measurable crossreactivity to murine IL-2.   
           b. a human IL-2 polypeptide fragment having an identity of ≧85%, ≧90%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97% or ≧98% compared to SEQ ID NO 001, and, optionally,   c. an amino acid linker of 1 to 50, particularly of 5 to 40, more particularly of 10 to 30, even more particularly of approx. 15 to 25 amino acids, linking the hIL-2 binding polypeptide fragment to the human IL-2 polypeptide fragment as one single polypeptide chain.       
 
         [0043]    In other words the fusion protein retains the ability of the antibody to bind and direct human interleukin-2 to stimulate selected immune cells, such as CD8 +  T cells and NK cells. 
         [0044]    The advantage of using such fusion protein is that human IL-2 will not be able to dissociate from the antibody and that the therapy will be composed of one single product instead of two, facilitating various aspects of manufacture, dosing and regulatory compliance. 
         [0045]    According to a ninth aspect of the invention, an isolated antibody or antigen binding fragment thereof binding a specific epitope is provided. Said epitope can be the epitope to which an isolated antibody or antigen binding fragment thereof according to other aspects of the invention binds. In an embodiment, the isolated antibody or molecule binds to a human interleukin-2 (hIL-2) epitope which comprises the amino acids K52, P54, K55, T57, R58, T61, F62, K63, Q94, and K96. In another embodiment, the isolated antibody or molecule binds to an epitope further comprising any one or more of the amino acids N50, N53, N91, L92, A93, and N97. An isolated antibody or molecule, which comprises an antigen recognition surface having epitope recognition characteristics equivalent to an antibody or antigen binding fragment thereof according to other aspects is also provided. 
         [0046]    Wherever alternatives for single separable features such as, for example, a coding sequence or binding epitope are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein. 
         [0047]    The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope. 
       Definitions 
       [0048]    By “human interleukin-2” or “hIL-2” is meant the protein designated UniProt ID P60568 and is reproduced as SEQ ID NO: 1. 
         [0049]    Identity in the context of the present specification is a single quantitative parameter representing the result of a sequence comparison position by position. Methods of sequence comparison are known in the art; the BLAST algorithm available publicly is an example. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1.-2; Gap costs: Linear. In the absence of other measurement variables, identity shall be measured according to the specification above. 
         [0050]    In the context of the present specification, the term antibody is used in its meaning known in the art of cell biology and immunology; it refers to whole antibodies, any antigen binding fragment or single chains thereof and related or derived constructs. A whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (V H ) and a heavy chain constant region (C H ). The heavy chain constant region is comprised of three domains, C H 1, C H 2 and C H 3. Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and a light chain constant region (C L ). The light chain constant region is comprised of one domain, C L . The V H  and V L  regions can be further subdivided into regions of hypervariability, termed complementarity determining regions 20 (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V H  and V L  is composed of three CDRs and four FRs arranged from amino-terminus to carboxyterminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. 
         [0051]    In the context of the present specification, the term antigen binding portion or antigen binding fragment is used in its meaning known in the art of cell biology and immunology; it refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., interleukin-2). Antigen binding functions of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term antigen binding portion or antigen binding fragment of an antibody include a Fab fragment, a monovalent fragment consisting of the V L , V H , C L  and C H  domains; a F(ab) 2  fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of the V H  and C H  domains; an Fv fragment consisting of the V L  and V H  domains of a single arm of an antibody; a single domain antibody (dAb) fragment, which consists of a V H  domain or a V L  domain; and an isolated complementarity determining region (CDR). HCDR means a CDR of the heavy chain and LCDR means a CDR of the light chain. 
         [0052]    In the context of the present specification, the term chimeric antibody is used in its meaning known in the art of cell biology and immunology; it refers to an antibody molecule in which the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, cytokine, toxin, hormone, growth factor, drug, etc. For example, an antibody can be modified by replacing its constant region with a cytokine. Due to the replacement with a cytokine, the chimeric antibody can retain its specificity in recognizing the antigen while having also the function, or part thereof, of the original cytokine molecule. 
         [0053]    In the context of the present specification, the term hybridoma is used in its meaning known in the art of cell biology and biochemistry; it refers to a hybrid cell created by fusion of a specific antibody-producing B-cell with a myeloma (B-cell cancer) cell. Hybridoma cells can be grown in tissue culture and produce antibodies of a single specificity (monoclonal antibodies). 
         [0054]    In the context of the present specification, the term single-chain variable fragment (scFv) is used in its meaning known in the art of cell biology and biochemistry; it refers to a fusion protein of the variable regions of the heavy (V H ) and light chains (V L ) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. The scFv retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. 
         [0055]    In the context of the present specification, the term fragment antigen-binding (Fab) is used in its meaning known in the art of cell biology and immunology; it refers to a region on an antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy (V H ) and light chains (V L ) of immunoglobulins. These domains shape the antigen-binding site at the amino terminal end of the monomer. 
         [0056]    In the context of the present specification, the term dissociation constant (K D ) is used in its meaning known in the art of chemistry and physics; it refers to an equilibrium constant that measures the propensity of a larger object to dissociate reversibly into smaller components, as when a complex falls apart into its component molecules. K D  is expressed in molar units [M] and corresponds to the concentration of [Ab] at which the binding sites of [Ag] are half occupied. In other words the concentration of unbound [Ab] equals the concentration of the [AbAg] complex. The dissociation constant can be calculated according to the following formula: 
         [0000]    
       
         
           
             
               K 
               D 
             
             = 
             
               
                 
                   [ 
                   Ab 
                   ] 
                 
                 * 
                 
                   [ 
                   Ag 
                   ] 
                 
               
               
                 [ 
                 AbAg 
                 ] 
               
             
           
         
       
     
         [0000]    [Ab]: concentration of antibody; [Ag]: concentration of antigen; [AbAg]: concentration of antibodyantigen complex 
         [0057]    In the context of the present specification, the terms off-rate (Koff; [1/sec]) and on-rate (Kon; [1/sec*M]) are used in their meaning known in the art of chemistry and physics; they refer to a rate constant that measures the dissociation (Koff) or association (Kon) of 5 an antibody with its target antigen. Koff and Kon can be experimentally determined using methods well established in the art. A method for determining the Koff and Kon of an antibody employs surface plasmon resonance. This is the principle behind biosensor systems such as the Biacore® or the ProteOn® system. They can also be used to determine the dissociation constant KD by using the following formula: 
         [0000]    
       
         
           
             
               K 
               D 
             
             = 
             
               
                 [ 
                 
                   K 
                   off 
                 
                 ] 
               
               
                 [ 
                 
                   K 
                   on 
                 
                 ] 
               
             
           
         
       
     
         [0058]    In the context of the present specification, the term humanized antibodies is used in its meaning known in the art of cell biology and biochemistry; it refers to antibodies originally produced by immune cells of a non-human species, whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. 
         [0059]    In the context of the present specification, the term no measurable cross-reactivity refers to the lacking capability of an antibody to recognize and bind to orthologous proteins from other species. For example, an antibody directed against human interleukin-2 would have no measurable cross-reactivity to murine interleukin-2 if, under suitable conditions, binding of the antibody to murine interleukin-2 could not be detected with sufficiently sensitive methods such as surface plasmon resonance. One such example of no measurable cross-reactivity is shown in  FIG. 9  for the antibody in the lower panel (NARA1). 
         [0060]    As used herein, an antibody or a protein that “specifically binds to hIL-2” is intended to refer to an antibody or protein that binds to human IL-2 polypeptide with a K D  of 100 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, or 10 pM or less. An antibody that “cross-reacts with an antigen other than human IL-2” is intended to refer to an antibody that binds that antigen with a K D  of 10 nM or less, 1 nM or less, or 100 pM or less. An antibody that “does not cross-react with a particular antigen” is intended to refer to an antibody that binds to that antigen, with a K D  of 100 nM or greater, or a K D  of 1 μM or grater, or a K D  of 10 μM or greater. In certain embodiments, such antibodies that do not cross-react with the antigen exhibit essentially undetectable binding against these proteins in standard binding assays. 
         [0061]    The term “epitope” means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. 
         [0062]    The term “epitope binding domain” or “EBD” refers to portions of a binding molecule (e.g., an antibody or epitope-binding fragment or derivative thereof), that specifically interacts with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) a binding site on a target epitope. EBD also refers to one or more fragments of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) a IL-2 epitope and inhibit signal transduction. Examples of antibody fragments include, but are not limited to, an scFv, a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab) 2  fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR). 
         [0063]    EBDs also include single domain antibodies, maxibodies, unibodies, minibodies, triabodies, tetrabodies, v-NAR and bis-scFv, as is known in the art (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23: 1126-1136), bispecific single chain diabodies, or single chain diabodies designed to bind two distinct epitopes. EBDs also include antibody-like molecules or antibody mimetics, which include, but not limited to minibodies, maxybodies, Fn3 based protein scaffolds, Ankrin repeats (also known as DARpins), VASP polypeptides, Avian pancreatic polypeptide (aPP), Tetranectin, Affililin, Knottins, SH3 domains, PDZ domains, Tendamistat, Neocarzinostatin, Protein A domains, Lipocalins, Transferrin, and Kunitz domains that specifically bind epitopes, which are within the scope of the invention. Antibody fragments can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). 
         [0064]    The present invention also encompasses an antibody to human IL-2, which is an isolated antibody. 
         [0065]    The phrase “isolated antibody”, as used herein, refers to antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds hIL-2 is substantially free of antibodies that specifically bind antigens other than hIL-2). An isolated antibody that specifically binds hIL-2 may, however, have cross-reactivity to other antigens, such as IL-2 molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. 
         [0066]    The terms “nucleic acid” and “polynucleotide” or “nucleotide coding sequences” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, siRNAs, shRNAs, RNAi agents, and primers. A polynucleotide can be modified or substituted at one or more base, sugar and/or phosphate, with any of various modifications or substitutions described herein or known in the art. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. 
         [0067]    The term “polypeptide” is used interchangeably with the term “protein” and in its broadest sense refers to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits can be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc. 
         [0068]    As used herein, the term “treating” or “treatment” of any disease or disorder (e.g. cancer) refers in one embodiment, to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and/or prevention of disease are generally known in the art, unless specifically described hereinbelow. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0069]      FIG. 1  shows anti-human IL-2 binders. Supernatants of B cell clones obtained after B cell hybridoma fusion were added to a plate previously coated with human IL-2. The anti-human IL-2 mAbs were detected using a biotinylated anti-mouse IgG antibody. 
           [0070]      FIG. 2  shows screening of anti-human IL-2 mAbs for binding to presumed specific human IL-2 epitope. Plates were coated with 5344 (a hIL-2 mAb without the herein targeted superagonistic behaviour) and blocked, followed by addition of human IL-2 in order to allow the cytokine to bind to 5344, thus covering a specific epitope of the IL-2. Then the supernatants giving a positive signal in the first screening (see  FIG. 1 ) were added. After allowing the mAbs in the supernatants to bind to the IL-2-5344 complex, a biotinylated MAB602 antibody was added to the plate in order to assess whether the tested mAbs of the supernatants bound to the same (so-called “competitors”) or to a different region than MAB602. The competitor mAbs led to an absorbance (OD450) that is two-fold lower than the absorbance obtained with MAB602 alone (in this case OD=1.1, as shown in H11). 
           [0071]      FIG. 3  shows concentration-dependent competition of B cell hybridomas. The supernatants of 8 competitor B cell hybridoma clones of the first screening (see  FIG. 2 ) were expanded and concentrated before use in this assay. The supernatants of these 8 competitor B cell hybridoma clones (labeled 1 to 8) were added in increasing quantities. Competent competitor B cell hybridoma clones reduced the OD450 as much as MAB602 or even more, which is evident for clones 1 and 2. MAB602 at different concentrations (green open circles) served as a control. 
           [0072]      FIG. 4  shows in vivo proliferation of CD8+ T cells. Carboxyfluorescein succinimidyl ester (CFSE)-labeled CD8+ T cells of CD45.1-congenic IL-7 transgenic mice were transferred to CD45.2-congenic WT recipient mice, followed by daily injections of phosphate-buffered saline (PBS), IL-2, IL-2 plus MAB602 (IL-2/MAB602), IL-2 plus 5344 (IL-2/5344), IL-2 plus hybridoma 1 (IL-2/Hyb#1), or IL-2 plus hybridoma 2 (IL-2/Hyb#2) for 4 days. On day 5, lymph nodes and spleens were analyzed for CFSE profiles of donor CD45.1+CD8+ T cells. Shown are the results obtained with the lymph nodes, similar results were obtained in the spleens. 
           [0073]      FIG. 5  shows phenotypic characterisation of endogenous CD8+ T cells and NK cells following in vivo treatment using IL-2 plus hybridoma 1 and 2. Mice were treated as in  FIG. 4 , followed by assessment by flow cytometry of endogenous CD8+ T cell subsets and NK cells in the lymph nodes and spleen. Shown are (A) CD8 vs. CD3 profiles of total lymph node cells (left graphs) and CD44 (activated or memory T cells) vs. CD122 (IL-2 receptor P3-subunit, present on activated or memory T cells) profiles of CD3+CD8+ lymph node cells, or (B) NK1.1 vs. CD3 profiles of mice receiving the indicated treatment. Activated/memory CD8+ T cells are high for CD44 and intermediate to high for CD122. NK cells are CD3 negative and NK1.1 positive. Similar results were obtained using spleen cells. 
           [0074]      FIG. 6  shows total cell counts of activated/memory CD8+ T cells and NK cells in lymph nodes and spleens. Animals were treated and analyzed as in  FIG. 5 . Shown are absolute cell counts of CD44high CD8+ T cells (so-called memory phenotype, MP CD8+) and of CD3 negative NK1.1+NK cells in lymph nodes (top panel) and spleen (lower panel). 
           [0075]      FIG. 7  shows surface plasmon resonance binding curves of the commercially available monoclonal antibody MAB602 (left graph) and the monoclonal antibody NARA1 (right graph), which is the subject of this invention, to human IL-2. For this experiment an amine coupling GLM chip was used. The activation of the carboxylic acid groups in the chip was done using a mix of 1-ethyl-3-3-dimethylaminopropyl carbodiimide hydrochlorid (EDC at 0.2 M) and sulfo N-hydroxysulfosuccinimids (s_NHS at 0.05M) at 30 ml/min for 420 seconds (s). The antibodies NARA1 and MAB602 were coated in the chip at 100 mg/ml in a sodium acetate buffer (10 mM pH 4.5). Deactivation was followed adding ethanolamine HCl at 30 ml/min for 300 s. Finally human IL-2 was added at different concentrations (starting from 100 nM and followed by three-fold dilutions) at 100 ml/min, 600 s association, and 240 s dissociation. 
           [0076]      FIG. 8  shows surface plasmon resonance binding curves of human IL-2 bound to the monoclonal antibody NARA1 with the IL-2 receptors subunits CD25 (used here as an Fc fusion of CD25-Fc), CD122, the monoclonal antibody MAB602 or an anti-hIL-2 antibody binding to a different human IL-2 epitope than NARA1 and MAB602. The chip described in  FIG. 7  coated with NARA1 and MAB602 was re-used. Regeneration of the chip was done using 10 mM glycine, pH 2.5, 30 ml/min, 60 s. Human IL-2 was added at saturating concentration (1 mM), at 100 ml/min, 120 s association, and 0 s dissociation. Immediately after IL-2 association to the antibodies, the second analytes were added at 100 ml/min, 120 s association, and 240 s dissociation. The concentration used for the cross-binding were: MAB602: 50 nM; NARA1: 50 nM; positive control: 50 nM; CD25-Fc: 500 nM; CD122: 138 nM. When hIL-2 is bound to NARA1, an anti-hIL-2 antibody that recognizes a different hIL-2 epitope (here termed ‘positive control’) binds strongly to the hIL-2/NARA1 complex as expected (green line in  FIG. 8 ). Alternatively, IL-2Ra (in the form of CD25-Fc) cannot bind to hIL-2 when hIL-2 is already bound to NARA1 (pink line,  FIG. 8 ), however, IL-2Rβ (CD122) still binds to hIL-2 when hIL-2 is already bound to NARA1 (orange line,  FIG. 8 ). 
           [0077]      FIG. 9  shows surface plasmon resonance binding curves of the monoclonal antibodies MAB602 (top graph) and NARA1 (lower graph) to murine IL-2. The same chip used for the generation of the data in  FIGS. 7 and 8  was re-used. Regeneration of the chip was done with 10 mM glycine, pH 2.5, 30 ml/min, 60 s. Mouse IL-2 (mIL-2) or human IL-2 (hIL-2) starting at 10 nM and then doing a three-fold dilution was injected at 100 ml/min, 120 s association, 5 and 240 s dissociation. In the top graph MAB602 shows cross-reactivity by binding to mouse IL-2. Especially, with higher concentrations of murine interleukin-2 (≧1 nM) the binding curves differ significantly from background levels with response units 10 (RU) well above 10. Whereas NARA1 (lower graph) displays no measurable cross-reactivity to murine IL-2 at all concentrations tested. 
           [0078]      FIG. 10  provides the overview of the three-dimension structure of Proleukin/Fab-NARA1 complex as obtained in Example 1. 
           [0079]      FIG. 11  provides further analysis of epitope residues. The X-axis lists the amino acid sequence and numbering according to SEQ ID No 1. The upper side of Y-axis shows the total number of atoms of NARA1-Fab that are within 4 Å from corresponding residue from Proleukin and the lower side of Y-axis shows the reduced solvent-accessible area (Å 2 ) of corresponding residue from Proleukin as a consequence of binding to NARA1-Fab. 
           [0080]      FIG. 12  illustrates the most critical epitope residue recognized by the NARA1-Fab. 
           [0081]      FIG. 13  shows the overlay of Proleukin/NARA1-Fab complex with IL-2/CD25/CD122/CD132 quaternary complex. 
           [0082]      FIG. 14  displays the overlay of C helices from IL-2_C145A (PDB: 3INK), Superkine (PDB: 3QB1), IL-2/CD25/CD122/CD132 (PDB: 2B5I), and Proleukin/NARA1-Fab. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0083]    Until now, no monoclonal antibodies suitable for the disclosed invention have been available. The inventors disclose their anti-human IL-2 mAbs that allow the following crucial steps towards the use and commercialization of this technology in clinical applications:
       Further sequencing and fine characterization of the anti-human IL-2 mAbs.   Humanization of the anti-human IL-2 mAbs, which is essential to avoid (or minimize) immunogenicity in patients.   Generation of different formats of anti-human IL-2 mAbs, such as IgG, IgG1, IgG4, Fab, and single-chain Fv (scFv).   Generation of a fusion protein consisting of human IL-2 and an anti-human IL-2 mAb (or a fragment of the anti-human IL-2 mAb): such a construct has the advantage of consisting of one component only, instead of two as in IL-2 bound to an anti-human IL-2 mAb.       
 
         [0088]    The inventors have generated and characterized specific anti-human IL-2 mAbs that are able to bind human IL-2 and, when tested in mice, are able to exert specific and potent stimulation of cytotoxic lymphocytes, including CD8 +  T cells and natural killer (NK) cells. Towards these ends several difficulties had to be overcome.
       Human IL-2 shows high similarity with mouse and rat IL-2, thus human IL-2 is able to stimulate mouse lymphocytes in vitro and in vivo. Moreover, IL-2 is present at high concentrations in the primary immune organs (such as the bone marrow), which is the reason why IL-2 is somewhat a “forbidden” antigen, meaning it is very difficult to generate B cell responses leading to neutralizing antibodies against IL-2. Nevertheless, the inventors were able to elicit polyclonal anti-human IL-2 antibody responses, following immunization of C57BL/6 mice using purified recombinant human IL-2 plus adjuvant.   Of the generated antibody responses, only some mAbs efficiently bound to IL-2 (socalled “binders”) and of those only about 0.35% interacted with the presumed active site of IL-2.   Finally, of these anti-human IL-2 mAbs some showed the desired specific and potent in vivo activity as assessed by specialized in vivo assays in mice that are not replaceable by in vitro experiments.       
 
         [0092]    The inventors have developed specific screening assays that allow detection of specific antihuman IL-2 antibodies (so-called “binders”) in the serum of immunized animals and in the supernatant of the B cell clones obtained after B cell hybridoma fusion. In a second step it was discriminated between standard binders and those targeting a presumed specific epitope of the human IL-2 molecule. One example of such an in vitro enzyme-linked immunosorbent assay (ELISA) performed with different B cell clones, is shown in  FIGS. 1 to 3 . 
         [0093]    After the in vitro screening of the anti-human IL-2 mAbs, these mAbs were characterised in vivo. To this end and in order to obtain sufficient amounts of mAbs, the mAbs were concentrated from the supernatant of the hybridomas, the amount was estimated using an ELISA and finally the anti-human IL-2 mAbs was tested in mice. The results obtained on proliferation and expansion of CD8 +  T cells and NK cells is shown in  FIGS. 4 to 6 . 
         [0094]    In order to characterize the binding properties of the anti-human IL-2 mAbs the binding to human interleukin-2 was tested with surface plasmon resonance binding assays. The commercially available anti-human IL-2 mAb MAB602 was measured as a comparison. In  FIG. 7  binding curves of MAB602 (left graph) and NARA1 (an antibody according to this invention; right graph) to human interleukin-2 at varying concentrations are shown. The dissociation constant (K D ) as well as the rate constants K on  and K off  measured for MAB602 and NARA1 are shown in Table 1. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Table 1: Binding properties of anti- 
               
               
                 human IL-2 mAbs to human IL-2 
               
             
          
           
               
                   
                 K on  (M*s −1 ) 
                 K off  (s −1 ) 
                 K D  (nM) 
               
               
                   
                   
               
             
          
           
               
                   
                 MAB602 
                  5.8 × 10 4   
                 4.94 × 10 −4   
                 9.7 
               
               
                   
                 NARA1 
                 1.78 × 10 4   
                 2.08 × 10 −5   
                 1.2 
               
               
                   
                   
               
             
          
         
       
     
       Examples 
       [0095]    Antibodies of the invention include the antibody NARA1, which was derived, isolated and structurally characterized by its full length heavy chain according to SEQ ID NO: 5 and its full length light chain amino acid sequences according to SEQ ID NO: 6. 
         [0096]    The corresponding variable regions, V H  and V L  amino acid sequences of NARA1 are. SEQ ID NO: 19 (variable heavy) and SEQ ID NO: 20 (variable light). 
         [0097]    Full length light and heavy chains nucleotide coding sequences of NARA1 are SEQ ID NO: 3 (heavy chain coding sequence, including leader sequence) and SEQ ID NO: 4 (light chain coding sequence, including leader sequence). 
         [0098]    Variable light and heavy chains nucleotide coding sequences of NARA1 are SEQ ID NO: 21 (variable heavy coding sequence) and SEQ ID NO: 22 (variable light coding sequence). 
         [0099]    The CDR regions of NARA1 are delineated using the Kabat system (Kabat, E. A., et al. 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, see also Zhao&amp;Lu 2009, Molecular Immunology 47:694-700). For the ease of reading, when CDR regions are delineated according to Kabat definition, they are called hereafter HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 respectively. The CDR regions of NARA1 are: HCDR1 according to SEQ ID NO: 7, HCDR2 according to SEQ ID NO: 8, HCDR3 according to SEQ ID NO: 9, LCDR1 according to SEQ ID NO: 10, LCDR2 according to SEQ ID NO: 11, LCDR3 according to SEQ ID NO: 12. 
         [0100]    Nucleotide coding sequences for the CDR regions of NARA1 are: HCDR1 coding sequence according to SEQ ID NO: 13, HCDR2 coding sequence according to SEQ ID NO: 14, HCDR3 coding sequence according to SEQ ID NO: 15, LCDR1 coding sequence according to SEQ ID NO: 16, LCDR2 coding sequence according to SEQ ID NO: 17, LCDR3 coding sequence according to SEQ ID NO: 18. 
         [0101]    Fusion proteins are also provided according to SEQ ID NO: 23 and SEQ ID NO: 24. SEQ ID NO: 23 is a fusion protein comprising the variable heavy chain of NARA1 with its N-terminus fused to the C-terminus of hIL-2 via a GxS linker. SEQ ID NO: 24 is a fusion protein comprising the variable light chain of NARA1 with its N-terminus fused to the C-terminus of hIL-2 via a GxS linker. 
       (a) Example 1. Crystal Structure of NARA1 
       [0102]    (i) Material and Methods 
         [0103]    The complex structure of a human Interleukin 2 mutant (SEQ ID NO:2), called “Proleukin”, bound to the Fab fragment of antibody “NARA 1” (SEQ ID NO: 5 and 6) was determined. The resulting numbering of residues on Proleukin is given according to the numbering of wt IL-2. 
         [0104]    As will be discussed in detail below, the differences in sequence between Proleukin and wt hIL-2 are irrelevant and Proleukin is a valid model for structural analysis of hIL-2. 
         [0105]    To define the epitope, X-ray crystallography was used to solve the atomic-resolution structure of the complex mentioned above. X-ray crystallography is a technology that has become routinely and widely used to generate structural data for biomolecules including antibodies and their complexes with antigens (Adms et al, (2013) Annual Review Biophysics 42:265-287; Garman, (2014) Science 343:1102-1108; Joachimiak, (2009) Current Opinio Structural Biology 19:573-584.) 
         [0106]    The antigen, Proleukin, is commercially available as lyophilyzed powder together with excipients (every 1 mg Proleukin is mixed with approximately 50 mg mannitol, 0.18 mg sodium dodecyl sulfate, 0.173 mg sodium dihydrogen phosphate, and 0.89 mg disodium hydrogen phosphate). Before used for complex formation, Proleukin was purified by reverse-phase HPLC to remove the excipients. 
         [0107]    The Fab fragment of NARA1 (NARA1-Fab) was generated by papain cleavage of the full-length antibody followed by Protein A chromatography. Briefly, 6.5 ml full-length NARA1 (9 mg/ml in 50 mM citrate buffer with 90 mM sodium chloride at pH 7.0) was mixed with 5 mM DTT and 590 ug Papain (Roche). The cleavage reaction was kept at room temperature for 16 h and stopped by addition of 15 ul 56 mM E64 solution (Roche). The cleavage solution was then diluted 10 times with 25 mM Tris, 25 mM NaCl, pH 8.0 and loaded onto a 5 ml Protein A column (GE Healthcare) equilibrate with 5 column volume of 25 mM Tris, 25 mM NaCl, pH 8.0 and Fab fragment was in the loading-through fraction and Fc fragment was bound to the Protein A column. 
         [0108]    To form complex, Proleukin powder after HPLC was dissolved in H 2 O at the concentration of 5.5 mg/ml. 6.6 mg Proleukin, in excess, was added to 11.5 mg NARA1 Fab fragment solution drop by drop. Centrifugation was used to remove the excess Proleukin that was precipitated under current condition. The complex was then purified by gel filtration with Superdex 200 10×300 (GE Healthcare) with running buffer of 25 mM Tris, 25 mM NaCl, pH 7.4. 
         [0109]    Proleukin/NARA1-Fab complex after gel filtration was concentrated to 14 mg/ml and was screened by vapour diffusion method as sitting drops. The protein solution was mixed 1:1 with reservoir buffer to a total size of 0.4 ul. The experiments were set up with Phoenix robotic system (Art Robbins Instruments), stored in a RockImager hotel (Formulatrix) at 19° C., and imaged automatically. Crystals were harvested 4 days after screening under condition of 20% w/v polyethylene Glycol 3350 and 0.2M sodium nitrate. Crystals were cryo-protected with reservoir buffer containing 10% glycerol and flashed frozen in liquid nitrogen prior to data collection. Diffraction data were collected at the Swiss Light Source (Villigen, Switzerland) at beam-line PX-II with a Pilatus pixel detector using x-ray radiation wavelength of 0.99998 Å. 
         [0110]    The dataset was processed with XDS and XSCALE (version Dec. 6, 2010) and the structure was resolved with molecular replacement method with the program PHASER by using Protein Data Bank entry “31NK” as search model for IL-2 and Protein Data Bank entry “3TTI” as search model for Fab fragment. Iterative model building and refinement were performed with the programs Coot (Crystallographic Object-Oriented Toolkit) and AUTOBUSTER (Bricogne et al., 2011). All figures were generated with the program PyMOL (Molecular Graphics System; DeLano Scientific: Palo Alto, Calif.; http://www.pymol.org). 
         [0111]    Epitope residues are defined as those residues from Proleukin that are within 4 Å distance from any atom in Fab fragment of NARA1 and are further confirmed by CCP4 program CONTACT and AREAIMOL (Collaborative Computational Project, Number 4, version 6.4.0). Similarly paratope residues are defined as those residues from NARA1-Fab that are within 4 Å distance from any atom in Proleukin. 
         [0112]    (ii) Results 
         [0113]    The Proleukin/NARA1-Fab complex was solved to 1.95 Å in space group C 1 2 1 with unit cell dimension a=201.8 Å, b=36.2 Å, c=88.7 Å, alpha=90°, beta=102.9°, gamma=90°. Please refer to Table 2 for detailed structure statistics. In each asymmetric unit, there is one complex molecule. 
         [0000]    
       
         
               
             
               
               
               
             
               
             
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Structure statistics for Proleukin/NARA1-Fab complex 
               
               
                   
               
             
             
               
                 Data collection 
               
             
          
           
               
                   
                 Space group 
                 C1 2 1 
               
               
                   
                 Cell dimensions 
               
               
                   
                 a, b, c (Å) 
                 201.757, 36.233, 88.707 
               
               
                   
                 a, b, g (°) 
                 90, 102.93, 90 
               
               
                   
                 Resolution (Å) 
                 58.74-1.95 
               
               
                   
                 R merge   
                 0.066 (0.472) 
               
               
                   
                 I |σ| 
                 14.18 (2.59)  
               
               
                   
                 Completeness (%) 
                 84.8 (96)   
               
               
                   
                 Redundancy 
                 3.19 
               
             
          
           
               
                 Refinement 
               
             
          
           
               
                   
                 Resolution (Å) 
                 58.74-1.95 
               
               
                   
                 No. reflections 
                 34750 
               
               
                   
                 R work /R free   
                 0.2052/0.2872 
               
               
                   
                 Ramachandran plot 
               
               
                   
                 Outliners 
                 0.0162 
               
               
                   
                 Allowed 
                 0.0378 
               
               
                   
                 Favored 
                 0.9459 
               
               
                   
                 R.m.s. deviations 
               
               
                   
                 Bond lengths (Å) 
                 0.01 
               
               
                   
                 Bond angles (°) 
                 1.7 
               
               
                   
                   
               
             
          
         
       
     
         [0114]    1) Epitope and Paratope Analysis 
         [0115]      FIG. 10  provides the overview of the three-dimension structure of Proleukin/Fab-NARA1 complex as obtained in Example 1. Light chain of Fab fragment of NARA1 is designated A, heavy chain of Fab fragment of NARA1 is shown as B, epitope residues recognized by NARA1-Fab are designated D, and Proleukin is designated C and the mutation, C145S, is highlighted. 
         [0116]      FIG. 11  provides further analysis of epitope residues. The X-axis lists the amino acid sequence and numbering according to SEQ ID No 1. The upper side of Y-axis demonstrates the total number of atoms of NARA1-Fab that are within 4 Å from corresponding residue from Proleukin and the lower side of Y-axis demonstrates the reduced solvent-accessible area (Å 2 ) after binding to NARA1-Fab. 
         [0117]    Proleukin used in Example 1 contains mutation of C145S. As shown in  FIG. 10 , C145S is far away from the epitope region. In addition the superposition of Ca atoms between Proleukin in Example 1 with Ca atoms from wt hIL-2 in complex with CD25, CD122, and CD132 (PDB: 2B5I) shows r.m.s.d of 0.447 Å, which indicates that the mutation does not disturb the over-all structure. Hence Proleukin with C145S mutation is a valid model for structural analysis for wt hIL-2. 
         [0118]    hIL-2 is 4-helix bundle protein and the 4 helices are named from N-terminus to C-terminus as A, B, C, and D, respectively. The epitope recognized by NARA1-Fab as shown in  FIG. 10  is a conformational epitope and spans two regions as shown in  FIG. 11 : one region (N50-K63) comprises a loop and a short helix and connects helix A and B, and the other region (N91-N97) comprises a loop and connects helix B and C. 
         [0119]    The epitope residues together with interacting paratope residues from NARA1-Fab are summarized in Table 3. Among all the epitope residues, Arg58 as shown in  FIG. 11  is the most critical epitope residue for binding with NARA1-Fab, as this residue alone has 42 interacting atoms from NARA1-Fab and accounts for 17.7% of total reduced solvent-accessible surface area as a consequence of binding to NARA1-Fab. Furthermore Arg58, as shown in  FIG. 12 , forms two strong salt-bridges with Glu35 in HCDR1 and with Asp100 from LCDR3, respectively. Arg58 also makes π-action interaction with the aromatic ring of Try100 from LCDR3. Residues K52, P54, K55, T57, T61, F62, K63, Q94, and K96 are also considered important for the binding to NARA1-Fab, since they all show equal to/more than 5 interacting atoms from NARA1-Fab and larger than 30 Å 2  reduced solvent-accessible area as shown in  FIG. 11 . 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Epitope and paratope summary 
               
             
          
           
               
                 Light chain residue 
                 Epitope residue 
                 Heavy chain residue 
               
               
                   
               
               
                 Y31 
                 N50 
                   
               
               
                 Y31 
                 K52 
               
               
                 Y31 
                 N53 
               
               
                 Y31, Y36, S95, N96 
                 P54 
               
               
                   
                 K55 
                 W99, G101, G103, Y105 
               
               
                 D98 
                 T57 
               
               
                 D98, Y100 
                 R58 
                 L33, E35, W47, W99 
               
               
                   
                 T61 
                 N52, S55, N59 
               
               
                   
                 F62 
                 L33, N52 
               
               
                   
                 K63 
                 S55 
               
               
                   
                 N91 
                 G101, D102, G103 
               
               
                   
                 L92 
                 W99, G101 
               
               
                   
                 A93 
                 G101 
               
               
                   
                 Q94 
                 D102, G103, Y104 
               
               
                 D32, D34 
                 K96 
                 Y104 
               
               
                 D32 
                 N97 
               
               
                   
               
             
          
         
       
     
         [0120]      FIG. 12  illustrates Arg58 as the most critical epitope residue recognized the NARA1-Fab. A represents Proleukin, B represents heavy chain, and C represents light chain. The involved residues are shown as sticks. 
         [0121]    2) NARA1-Fab Binding Properties 
         [0122]      FIG. 13  shows the overlay of Proleukin/NARA1-Fab complex with IL-2/CD25/CD122/CD132 quaternary complex. The quaternary complex structure comes from PDB entry “2B5I” with cartoon D in pale cyan representing wt hIL-2, cartoon B in red representing CD122, cartoon C in blue representing CD132, and surface A in green representing CD25. In the Proleukin/NARA1-Fab complex structure, cyan cartoon D overlayed with wt hIL-2 represents Proleukin, cartoon E in magenta represents heavy chain, and cartoon F in yellow represents the light chain. 
         [0123]    The structure overlay of the two complexes as shown in  FIG. 13  clearly shows that NARA1-Fab forms direct competition against CD25 but not against CD122/CD132, which is consistent with the observation that IL-2/NARA1 complex demonstrates mainly pro-T effector cell activity rather than pro-Treg activity. 
         [0124]    3) C Helix of Proleukin in Complex with NARA1-Fab Adopts Conformation that is Similar to that in Quaternary Complex 
         [0125]      FIG. 14  displays the overlay of C helices from IL-2_C145A (PDB: 3INK), Superkine (PDB: 3QB1), IL-2/CD25/CD122/CD132 (PDB: 2B5I), and Proleukin/NARA1-Fab. 
         [0126]    The polar interface between helix C in IL-2 and CD122 plays an important role in binding between the two parts (Wang et al (2005) Science 310:1159-1163). In 2012 Levin, et al have demonstrated that superkine, an IL-2 mutant, alone has a Helix C adopting confirmation similar to that in the quaternary complex and superkine showed ˜215 times higher binding affinity towards CD122 than wtIL-2 (Levin et al, (2012) Nature 484:529-533). It was observed that such a conformational change in helix C is associated with conformational stabilization, which then reduces the energetic penalties for binding to CD122. As shown in  FIG. 14 , The conformation of helix C from Proleukin in complex with NARA1-Fab is also similar to that observed in Superkine as well as in IL-2/CD25/CD122/CD132 quaternary complex, therefore it is possible that Proleukin/NARA1-Fab complex may demonstrate higher binding affinity towards CD122 than wt hIL-2 does. 
       (b) Example 2. Linear Peptide Mapping of NARA1 and MAB602 
       [0127]    In order to map the epitope of the NARA1 and MAB602 antibodies, a first library of 15-mer peptides was generated based on the sequence of human IL2. A second library of selected 15-mer peptides was also generated based on the mutation of 3 specific residues F(62), Y(65) and L(92). The latter mutations were done based on the Roche/Glycart IL2 mutein, as disclosed in WO2012/107417A1 which has these 3 mutations. Previous work done in lab Boyman (unpublished) showed that the commercial mouse anti-human IL2 mAb 602 with analogous function as A1 has strongly reduced binding to the F42A mutant of IL2 (one of the IL2 docking sites to CD25). 
         [0128]    (i) Material and Methods 
         [0129]    Accordingly, each peptide in the first library has 15 amino acids and the sequence is derived by scanning the sequence of interest (see Table 4, reference peptides 1 to 41) with a step of 3 residues, starting from the N-terminus. Therefore a ladder is generated and each peptide contains 12 overlapping residues with the previous peptide and 12 overlapping residues with the following peptide in the ladder. In total, 41 peptides were generated from the expressed human IL2 sequence. 
         [0130]    A second library of peptides was generated by mutating F(62), Y(65) and L(92) to alanine in all corresponding peptides in the first library generated as described above (see Table 4, reference peptides no 42 to 60). 
         [0131]    For both libraries, the parental cysteines have been replaced by a serine (underlined residues) to avoid unspecific binding. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Library of reference peptides 
               
             
          
           
               
                   
                 Sequence 
               
               
                   
                 Residue in bold 
               
               
                   
                 are the Alanine 
               
               
                   
                 (A) replacing 
               
               
                   
                 specific residues. 
               
               
                 Reference 
                 Residue are  
               
               
                 Peptide 
                 the serine (S) 
               
               
                 No. 
                 replacing cysteines (C) 
               
               
                   
               
               
                  1 
                 APTSSSTKKTQLQLE 
               
               
                   
               
               
                  2 
                 SSSTKKTQLQLEHLL 
               
               
                   
               
               
                  3 
                 TKKTQLQLEHLLLDL 
               
               
                   
               
               
                  4 
                 TQLQLEHLLLDLQMI 
               
               
                   
               
               
                  5 
                 QLEHLLLDLQMILNG 
               
               
                   
               
               
                  6 
                 HLLLDLQMILNGINN 
               
               
                   
               
               
                  7 
                 LDLQMILNGINNYKN 
               
               
                   
               
               
                  8 
                 QMILNGINNYKNPKL 
               
               
                   
               
               
                  9 
                 LNGINNYKNPKLTRM 
               
               
                   
               
               
                 10 
                 INNYKNPKLTRMLTF 
               
               
                   
               
               
                 11 
                 YKNPKLTRMLTFKFY 
               
               
                   
               
               
                 12 
                 PKLTRMLTFKFYMPK 
               
               
                   
               
               
                 13 
                 TRMLTFKFYMPKKAT 
               
               
                   
               
               
                 14 
                 LTFKFYMPKKATELK 
               
               
                   
               
               
                 15 
                 KFYMPKKATELKHLQ 
               
               
                   
               
               
                 16 
                 MPKKATELKHLQSLE 
               
               
                   
               
               
                 17 
                 KATELKHLQSLEEEL 
               
               
                   
               
               
                 18 
                 ELKHLQSLEEELKPL 
               
               
                   
               
               
                 19 
                 HLQSLEEELKPLEEV 
               
               
                   
               
               
                 20 
                 SLEEELKPLEEVLNL 
               
               
                   
               
               
                 21 
                 EELKPLEEVLNLAQS 
               
               
                   
               
               
                 22 
                 KPLEEVLNLAQSKNF 
               
               
                   
               
               
                 23 
                 EEVLNLAQSKNFHLR 
               
               
                   
               
               
                 24 
                 LNLAQSKNFHLRPRD 
               
               
                   
               
               
                 25 
                 AQSKNFHLRPRDLIS 
               
               
                   
               
               
                 26 
                 KNFHLRPRDLISNIN 
               
               
                   
               
               
                 27 
                 HLRPRDLISNINVIV 
               
               
                   
               
               
                 28 
                 PRDLISNINVIVLEL 
               
               
                   
               
               
                 29 
                 LISNINVIVLELKGS 
               
               
                   
               
               
                 30 
                 NINVIVLELKGSETT 
               
               
                   
               
               
                 31 
                 VIVLELKGSETTFMS 
               
               
                   
               
               
                 32 
                 LELKGSETTFMSEYA 
               
               
                   
               
               
                 33 
                 KGSETTFMSEYADET 
               
               
                   
               
               
                 34 
                 ETTFMSEYADETATI 
               
               
                   
               
               
                 35 
                 FMSEYADETATIVEF 
               
               
                   
               
               
                 36 
                 EYADETATIVEFLNR 
               
               
                   
               
               
                 37 
                 DETATIVEFLNRWIT 
               
               
                   
               
               
                 38 
                 ATIVEFLNRWITFSQ 
               
               
                   
               
               
                 39 
                 VEFLNRWITFSQSII 
               
               
                   
               
               
                 40 
                 LNRWITFSQSIISTL 
               
               
                   
               
               
                 41 
                 NRWITFSQSIISTLT 
               
               
                   
               
               
                 42 
                 INNYKNPKLTRMLTA 
               
               
                   
               
               
                 43 
                 YKNPKLTRMLTAKFY 
               
               
                   
               
               
                 47 
                 YKNPKLTRMLTFKFA 
               
               
                   
               
               
                 52 
                 YKNPKLTRMLTAKFA 
               
               
                   
               
               
                 44 
                 PKLTRMLTAKFYMPK 
               
               
                   
               
               
                 48 
                 PKLTRMLTFKFAMPK 
               
               
                   
               
               
                 53 
                 PKLTRMLTAKFAMPK 
               
               
                   
               
               
                 45 
                 TRMLTAKFYMPKKAT 
               
               
                   
               
               
                 49 
                 TRMLTFKFAMPKKAT 
               
               
                   
               
               
                 54 
                 TRMLTAKFAMPKKAT 
               
               
                   
               
               
                 46 
                 LTAKFYMPKKATELK 
               
               
                   
               
               
                 50 
                 LTFKFAMPKKATELK 
               
               
                   
               
               
                 55 
                 LTAKFAMPKKATELK 
               
               
                   
               
               
                 51 
                 KFAMPKKATELKHLQ 
               
               
                   
               
               
                 56 
                 SLEEELKPLEEVLNA 
               
               
                   
               
               
                 57 
                 EELKPLEEVLNAAQS 
               
               
                   
               
               
                 58 
                 KPLEEVLNAAQSKNF 
               
               
                   
               
               
                 59 
                 EEVLNAAQSKNFHLR 
               
               
                   
               
               
                 60 
                 ANLAQSKNFHLRPRD 
               
               
                   
               
             
          
         
       
     
         [0132]    Both set of peptides were printed on microarray slides in triplicate, incubated with the antibodies of interest (MAb602 and NARA1) and control antibodies. Additional incubations are with unrelated antibodies from the same isotype (mouse control IgG2a/lambda and mouse control IgG2a/kappa), and secondary antibodies (anti-mouse IgG (Thermo 84545, label DL650) or anti-mouse IgG (JIR 115-175-072, Label Cy5)) to assess unspecific binding due to the detection antibody. The experiments are performed essentially as described in Maksimov P, et al. 2012, PLoS One 7:e34212. doi:10.1371/journal. pone. 0034212. 
         [0133]    The determination of peptide-antibody binding was performed by RepliTope-analysis where the peptide microarray (triplicate) was incubated with the primary antibody followed by a fluorescently labelled secondary antibody directed against the Fc-part of the primary one. All steps were performed on a TECAN microarray processing station enabling highly reliable and reproducible washing and incubation steps. After performing the incubation steps and subsequent to the final washing steps (to remove the unbound secondary antibodies) the microarrays were dried using a nitrogen stream and scanned in a high resolution microarray scanning system with appropriate wavelength settings. Control incubations were performed with an unrelated antibody having the same isotype to exclude false positive signals. 
         [0134]    The resulting images were analyzed und quantified using spot-recognition software GenePix (Molecular Devices). For each spot, the mean signal intensity was extracted (between 0 and 65535 arbitrary units). For further data evaluation, the MMC2 values were determined. The MMC2 equals the mean value of all three instances on the microarray. Except the coefficient of variation (CV)—standard-deviation divided by the mean value—is larger 0.5, in this case the mean of the two closest values (MC2) is assigned to MMC2. 
         [0135]    (ii) Results 
         [0136]    The data are summarized in Table 5. 
         [0137]    The anti-IL2 (NARA1) antibody did not show any significant reactivity towards the immobilized peptides. Only peptide 10 exhibited a weak response, however, this peptide was also weakly recognized by the mouse control antibodies. 
         [0138]    The commercial antibody MAB602 (mIgG2a) provided some weak signals on peptide 22 to 26 and some strong for peptides 10 to 13. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Result of Linear Epitope Mapping 
               
             
          
           
               
                   
                   
                 Signal intensity 
                 Signal intensity 
               
               
                   
                   
                 for MAB602 after 
                 for NARA1 after 
               
               
                 Reference 
                   
                 subtraction of 
                 subtraction of 
               
               
                 peptide 
                   
                 control signal 
                 control signal 
               
               
                 no. 
                 Sequence 
                 (AU) 
                 (AU) 
               
               
                   
               
               
                 10 
                 INNYKNPKLTRMLTF 
                 45954 
                 20883 
               
               
                   
               
               
                 11 
                    YKNPKLTRMLTFKFY 
                 49726 
                  1189 
               
               
                   
               
               
                 12 
                       PKLTRMLTFKFYMPK 
                 28849 
                  1127 
               
               
                   
               
               
                 13 
                          TRMLTFKFYMPKKAT 
                  5250 
                   224 
               
               
                   
               
               
                 22 
                 KPLEEVLNLAQSKNF 
                  4998 
                     0 
               
               
                   
               
               
                 23 
                    EEVLNLAQSKNFHLR 
                 13287 
                    32 
               
               
                   
               
               
                 24 
                       LNLAQSKNFHLRPRD 
                  3289 
                   282 
               
               
                   
               
               
                 25 
                          AQSKNFHLRPRDLIS 
                  5220 
                     0 
               
               
                   
               
               
                 26 
                 KNFHLRPRDLISNIN 
                  7509 
                     0 
               
               
                   
               
             
          
         
       
     
         [0139]    The overlapping sequences within both set of peptides are considered as containing the binding amino acid to the target antibody (Table 5). One stretch is a strong binder to MAB602 whereas the other is rather a weak binder to MAB602: 
       Strong: (57) TRMLTF (62) 
     Weaker: (96) KNF (98) 
       [0140]    Ala mutation on specific residues F42(62), Y45(65), L72(92) showed that residue F42(62) is clearly an important residue for the binding to antibody MAB602 (Table 6). 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Mutagenesis characterization 
               
             
          
           
               
                   
                 Sequence 
                   
                   
               
               
                   
                 Residue in bold 
                 Signal intensity 
                 Signal intensity 
               
               
                   
                 are the Alanine 
                 for MAB602 after 
                 for NARA1 after 
               
               
                 Reference 
                 (A) which are 
                 subtraction of 
                 subtraction of 
               
               
                 peptide 
                 replacing  
                 control signal 
                 control signal 
               
               
                 no. 
                 specific residues 
                 (AU) 
                 (AU) 
               
               
                   
               
               
                 10 
                 INNYKNPKLTRMLTF 
                 45954 
                 20883 
               
               
                   
               
               
                 42 
                 INNYKNPKLTRMLT A   
                   246 
                   162 
               
               
                   
               
               
                 11 
                 YKNPKLTRMLTFKFY 
                 49726 
                  1189 
               
               
                   
               
               
                 43 
                 YKNPKLTRMLT A KFY 
                 42784 
                   507 
               
               
                   
               
               
                 47 
                 YKNPKLTRMLTFKF A   
                 21382 
                   251 
               
               
                   
               
               
                 52 
                 YKNPKLTRMLT A KF A   
                 13089 
                   238 
               
               
                   
               
               
                 12 
                 PKLTRMLTFKFYMPK 
                 28849 
                  1127 
               
               
                   
               
               
                 44 
                 PKLTRMLT A KFYMPK 
                  5027 
                   432 
               
               
                   
               
               
                 48 
                 PKLTRMLTFKF A MPK 
                 13394 
                  6205 
               
               
                   
               
               
                 53 
                 PKLTRMLT A KF A MPK 
                     0 
                    24 
               
               
                   
               
               
                 13 
                 TRMLTFKFYMPKKAT 
                  5250 
                   224 
               
               
                   
               
               
                 45 
                 TRMLT A KFYMPKKAT 
                     0 
                     0 
               
               
                   
               
               
                 49 
                 TRMLTFKF A MPKKAT 
                  3018 
                  1492 
               
               
                   
               
               
                 54 
                 TRMLT A KF A MPKKAT 
                     0 
                     0 
               
               
                   
               
             
          
         
       
     
       Sequence List 
       [0141]    Useful amino acids and nucleotide sequences for practicing the invention are found in Table 7. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Sequence list 
               
             
          
           
               
                 SEQ ID 
                 Ab 
                   
               
               
                 NUMBER 
                 region 
                 Sequence 
               
               
                   
               
               
                 SEQ ID NO: 1 
                 Human 
                 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHL 
               
               
                   
                 IL-2 
                 LLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELK 
               
               
                   
                   
                 HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINV 
               
               
                   
                   
                 IVLELKGSETTFMCEYADETATIVEFLNRWITFCQSI 
               
               
                   
                   
                 ISTLT 
               
               
                   
               
               
                 SEQ ID NO: 2 
                 Proleukin 
                 MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKL 
               
               
                   
                   
                 TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA 
               
               
                   
                   
                 QSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE 
               
               
                   
                   
                 TATIVEFLNRWITFSQSIISILT 
               
               
                   
               
               
                 Antibody 1 
                   
                   
               
               
                 SEQ ID NO: 3 
                 DNA 
                 ATGGAATGGAGCGGAGTCTTTATCTTTCTCCTGTCAG 
               
               
                   
                 Heavy 
                 TAACTGCAGGTGTTCACTCCCAGGTCCAGCTGCAGCA 
               
               
                   
                 Chain 
                 GTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTG 
               
               
                   
                   
                 AAGGTGTCCTGCAAGGCTTCTGGATACGCCTTCACTA 
               
               
                   
                   
                 ATTACTTGATAGAGTGGGTAAAGCAGAGGCCTGGACA 
               
               
                   
                   
                 GGGCCTTGAGTGGATTGGAGTGATTAATCCTGGAAGT 
               
               
                   
                   
                 GGTGGTACTAACTACAATGAGAAGTTCAAGGGCAAGG 
               
               
                   
                   
                 CAACACTGACTGCAGACAAATCCTCCAGCACTGCCTA 
               
               
                   
                   
                 CATGCAGCTCAGCAGCCTGACATCTGATGACTCTGCG 
               
               
                   
                   
                 GTCTATTTCTGTGCAAGATGGAGGGGGGATGGTTACT 
               
               
                   
                   
                 ACGCGTACTTCGATGTCTGGGGCGCAGGGACCACGGT 
               
               
                   
                   
                 CACCGTCTCCTCAGCCAAAACAACAGCCCCATCGGTC 
               
               
                   
                   
                 TATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCT 
               
               
                   
                   
                 CCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTT 
               
               
                   
                   
                 CCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCC 
               
               
                   
                   
                 CTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGC 
               
               
                   
                   
                 AGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGT 
               
               
                   
                   
                 AACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGC 
               
               
                   
                   
                 AATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACA 
               
               
                   
                   
                 AGAAAATTGAGCCCAGAGGGCCCACAATCAAGCCCTG 
               
               
                   
                   
                 TCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGT 
               
               
                   
                   
                 GGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGG 
               
               
                   
                   
                 ATGTACTCATGATCTCCCTGAGCCCCATAGTCACATG 
               
               
                   
                   
                 TGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTC 
               
               
                   
                   
                 CAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACA 
               
               
                   
                   
                 CAGCTCAGACACAAACCCATAGAGAGGATTACAACAG 
               
               
                   
                   
                 TACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCAC 
               
               
                   
                   
                 CAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGG 
               
               
                   
                   
                 TCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAAC 
               
               
                   
                   
                 CATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAG 
               
               
                   
                   
                 GTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTA 
               
               
                   
                   
                 AGAAACAGGTCACTCTGACCTGCATGGTCACAGACTT 
               
               
                   
                   
                 CATGCCTGAAGACATTTACGTGGAGTGGACCAACAAC 
               
               
                   
                   
                 GGGAAAACAGAGCTAAACTACAAGAACACTGAACCAG 
               
               
                   
                   
                 TCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAA 
               
               
                   
                   
                 GCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAAT 
               
               
                   
                   
                 AGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACA 
               
               
                   
                   
                 ATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGG 
               
               
                   
                   
                 TAAATGA 
               
               
                   
               
               
                 SEQ ID NO: 4 
                 DNA 
                 ATGGAGACAGACACAATCCTGCTATGGGTGCTGCTGC 
               
               
                   
                 Light 
                 TCTGGGTTCCAGGCTCCACTGGTGACATTGTGCTGAC 
               
               
                   
                 Chain 
                 CCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAG 
               
               
                   
                   
                 AGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTG 
               
               
                   
                   
                 ATTATGATGGTGATAGTTATATGAACTGGTACCAACA 
               
               
                   
                   
                 GAAACCAGGACAGCCACCCAAACTCCTCATCTATGCT 
               
               
                   
                   
                 GCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTA 
               
               
                   
                   
                 GTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACAT 
               
               
                   
                   
                 CCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTAC 
               
               
                   
                   
                 TGTCAGCAAAGTAATGAGGATCCGTACACGTTCGGAG 
               
               
                   
                   
                 GGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGC 
               
               
                   
                   
                 ACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAG 
               
               
                   
                   
                 TTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGA 
               
               
                   
                   
                 ACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAA 
               
               
                   
                   
                 GATTGATGGCAGTGAACGACAAAATGGCGTCCTGAAC 
               
               
                   
                   
                 AGTTGGACTGATCAGGACAGCAAAGACAGCACCTACA 
               
               
                   
                   
                 GCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTA 
               
               
                   
                   
                 TGAACGACATAACAGCTATACCTGTGAGGCCACTCAC 
               
               
                   
                   
                 AAGACATCAACTTCACCCATTGTCAAGAGCTTCAACA 
               
               
                   
                   
                 GGAATGAGTGTTAG 
               
               
                   
               
               
                 SEQ ID NO: 5 
                 Heavy 
                 MEWSGVFIFLLSVTAGVHSQVQLQQSGAELVRPGTSV 
               
               
                   
                 Chain 
                 KVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGS 
               
               
                   
                   
                 GGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSA 
               
               
                   
                   
                 VYFCARWRGDGYYAYFDVWGAGTIVIVSSAKTTAPSV 
               
               
                   
                   
                 YPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGS 
               
               
                   
                   
                 LSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITC 
               
               
                   
                   
                 NVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLG 
               
               
                   
                   
                 GPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDV 
               
               
                   
                   
                 QISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQH 
               
               
                   
                   
                 QDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQ 
               
               
                   
                   
                 VYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNN 
               
               
                   
                   
                 GKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN 
               
               
                   
                   
                 SYSCSVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
               
               
                 SEQ ID NO: 6 
                 Light 
                 METDTILLWVLLLWVPGSTGDIVLTQSPASLAVSLGQ 
               
               
                   
                 Chain 
                 RATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYA 
               
               
                   
                   
                 ASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYY 
               
               
                   
                   
                 CQQSNEDPYTFGGGTKLEIKRADAAPTVSIFPPSSEQ 
               
               
                   
                   
                 LTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLN 
               
               
                   
                   
                 SWIDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATH 
               
               
                   
                   
                 KTSTSPIVKSFNRNEC 
               
               
                   
               
               
                 SEQ ID NO: 7 
                 HCDR1 
                 NYLIE 
               
               
                 (Kabat) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 8 
                 HCDR2 
                 VINPGSGGTNYNEKFKG 
               
               
                 (Kabat) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 9 
                 HCDR3 
                 WRGDGYYAYFDV 
               
               
                 (Kabat) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 10 
                 LCDR1 
                 KASQSVDYDGDSYMN 
               
               
                 (Kabat) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 11 
                 LCDR2 
                 AASNLES 
               
               
                 (Kabat) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 12 
                 LCDR3 
                 QQSNEDPYT 
               
               
                 (Kabat) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 13 
                 HCDR1 
                 AATTACTTGATAGAG 
               
               
                   
                 DNA 
                   
               
               
                   
               
               
                 SEQ ID NO: 14 
                 HCDR2 
                 GTGATTAATCCTGGAAGTGGTGGTACTAACTACAATG 
               
               
                   
                 DNA 
                 AGAAGTTCAAGGGC 
               
               
                   
               
               
                 SEQ ID NO: 15 
                 HCDR3 
                 TGGAGGGGGGATGGTTACTACGCGTACTTCGATGTC 
               
               
                   
                 DNA 
                   
               
               
                   
               
               
                 SEQ ID NO: 16 
                 LCDR1 
                 AAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTT 
               
               
                   
                 DNA 
                 ATATGAAC 
               
               
                   
               
               
                 SEQ ID NO: 17 
                 LCDR2 
                 GCTGCATCCAATCTAGAATCT 
               
               
                   
                 DNA 
                   
               
               
                   
               
               
                 SEQ ID NO: 18 
                 LCDR3 
                 CAGCAAAGTAATGAGGATCCGTACACG 
               
               
                   
                 DNA 
                   
               
               
                   
               
               
                 SEQ ID NO: 19 
                 VH 
                 QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWV 
               
               
                   
                   
                 KQRPGQGLEWIGVINPGSGGTNYNEKFKGKATLTADK 
               
               
                   
                   
                 SSSTAYMQLSSLTSDDSAVYFCARWRGDGYYAYFDVW 
               
               
                   
                   
                 GAGTTVTVSS 
               
               
                   
               
               
                 SEQ ID NO: 20 
                 VL 
                 DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYM 
               
               
                   
                   
                 NWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTD 
               
               
                   
                   
                 FTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIK 
               
               
                   
               
               
                 SEQ ID NO: 21 
                 DNA VH 
                 CAGGTCCAGCTGCAGCAGTCTGGAGCTGAGCTGGTAA 
               
               
                   
                   
                 GGCCTGGGACTTCAGTGAAGGTGTCCTGCAAGGCTTC 
               
               
                   
                   
                 TGGATACGCCTTCACTAATTACTTGATAGAGTGGGTA 
               
               
                   
                   
                 AAGCAGAGGCCTGGACAGGGCCTTGAGTGGATTGGAG 
               
               
                   
                   
                 TGATTAATCCTGGAAGTGGTGGTACTAACTACAATGA 
               
               
                   
                   
                 GAAGTTCAAGGGCAAGGCAACACTGACTGCAGACAAA 
               
               
                   
                   
                 TCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGA 
               
               
                   
                   
                 CATCTGATGACTCTGCGGTCTATTTCTGTGCAAGATG 
               
               
                   
                   
                 GAGGGGGGATGGTTACTACGCGTACTTCGATGTCTGG 
               
               
                   
                   
                 GGCGCAGGGACCACGGTCACCGTCTCCTCA 
               
               
                   
               
               
                 SEQ ID NO: 22 
                 DNA VL 
                 GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTG 
               
               
                   
                   
                 TGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGC 
               
               
                   
                   
                 CAGCCAAAGTGTTGATTATGATGGTGATAGTTATATG 
               
               
                   
                   
                 AACTGGTACCAACAGAAACCAGGACAGCCACCCAAAC 
               
               
                   
                   
                 TCCTCATCTATGCTGCATCCAATCTAGAATCTGGGAT 
               
               
                   
                   
                 CCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGAC 
               
               
                   
                   
                 TTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATG 
               
               
                   
                   
                 CTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCC 
               
               
                   
                   
                 GTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA 
               
               
                   
               
               
                 SEQ ID NO: 23 
                 Heavy 
                 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHL 
               
               
                   
                 chain 
                 LLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELK 
               
               
                   
                 fusion 
                 HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINV 
               
               
                   
                   
                 IVLELKGSETTFMCEYADETATIVEFLNRWITFCQSI 
               
               
                   
                   
                 ISTLTGGGGSGGGGSGGGGSGGQVQLQQSGAELVRPG 
               
               
                   
                   
                 TSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVIN 
               
               
                   
                   
                 PGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSD 
               
               
                   
                   
                 DSAVYFCARWRGDGYYAYFDVWGAGTIVIVSSAKTTA 
               
               
                   
                   
                 PSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWN 
               
               
                   
                   
                 SGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQS 
               
               
                   
                   
                 ITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPN 
               
               
                   
                   
                 LLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDD 
               
               
                   
                   
                 PDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALP 
               
               
                   
                   
                 IQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVR 
               
               
                   
                   
                 APQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEW 
               
               
                   
                   
                 TNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWV 
               
               
                   
                   
                 ERNSYSCSVVHEGLHNHHTTKSFSRTPGK 
               
               
                   
               
               
                 SEQ ID NO: 24 
                 Light 
                 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHL 
               
               
                   
                 chain 
                 LLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELK 
               
               
                   
                 fusion 
                 HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINV 
               
               
                   
                   
                 IVLELKGSETTFMCEYADETATIVEFLNRWITFCQSI 
               
               
                   
                   
                 ISTLTGGGGSGGGGSGGGGSGGDIVLTQSPASLAVSL 
               
               
                   
                   
                 GQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLI 
               
               
                   
                   
                 YAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAAT 
               
               
                   
                   
                 YYCQQSNEDPYTFGGGTKLEIKRADAAPTVSIFPPSS 
               
               
                   
                   
                 EQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGV 
               
               
                   
                   
                 LNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEA 
               
               
                   
                   
                 THKTSTSPIVKSFNRNEC