Bispecific antibody

Abstract

To provide a therapeutic antibody useful for therapy of various diseases and disorders and methods for producing and using such antibody.SOLUTION: A bispecific antibody is produced by identifying a monospecific antibody which has an electrostatic or hydrophobic light chain variable region VL residue, and performing modification of a nucleic acid sequence for encoding one or more residues which can approach a VH solvent of the antibody alone, or performing the modification in combination with modification of a nucleic acid sequence for encoding a VL of the antibody. The modified VH and VL are expressed, and the bispecific antibody or an antigen-coupling fragment thereof is selected. An exemplary bispecific antibody and a use method of the antibody are provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to bispecific antibodies and methods of making and using such antibodies. [Background technology]

[0002] Antibodies are specific immunoglobulin polypeptides produced by the immune system of vertebrates in response to challenge with foreign proteins, glycoproteins, cells, or other antigenic foreign substances. A key part of this process is the production of antibodies that specifically bind to a particular foreign substance. The binding specificity of such polypeptides for particular antigens is highly refined, and the large number of specificities that can be produced in individual vertebrates is remarkable for their complexity and variability. Thousands of antigens can elicit a response, with each response being directed almost exclusively to the particular antigen that elicited it.

[0003] Specific recognition of antigens is essential for antibodies to function in adaptive immune responses. The combination of heavy chain (HC) and light chain (LC) associations is conserved in all vertebrates, creating antibody repertoires. However, the diversity of these heavy and light chains is asymmetric. HC(V) H ) variable domain contains significantly more sequence diversity, and LC(V L However, because such variability allows antibodies to recognize and bind to specific foreign substances, some antibodies may have a higher antigen-binding energy than the V L is heavily dependent on

[0004] The specificity of antibodies and antibody fragments for a particular antigen or antigens makes them desirable therapeutic agents. Antibodies and antibody fragments can be used to target specific antigens (e.g., cytokines) that play diverse biological roles. Thus, there is an ongoing need to identify and characterize therapeutic antibodies, particularly antibodies, fragments, and derivatives thereof, that are useful in the treatment of various diseases and disorders, including allergic, inflammatory, autoimmune, and proliferative diseases. Summary of the Invention

[0005] The present invention provides methods for making bispecific antibodies and antibody fragments. The present invention also provides specific antibodies identified using these methods and uses thereof.

[0006] Generally, in the methods of the invention, the V H are diversified to generate bispecific antibody variants that can be stably expressed in a library. In one embodiment, the antibody before diversification contains H and V L are paired together to form an antigen-binding site that specifically binds to a first epitope but not a second epitope. L Such antibodies having hydrophobic or electrostatic residues at one or more of these positions are then characterized as having electrostatic or hydrophobic residues at any one, two, or three amino acids found at positions 32, 50, or 91 (Kabat numbering system) of V. H (e.g., solvent-exposed amino acid residues). H and V L are expressed (e.g., as a library), and the expressed V H and V L From the above, diversified bispecific antibodies, or antigen-binding fragments thereof, capable of specifically binding to the first and second epitopes are selected.

[0007] In one embodiment, the variable heavy domain (V H ) and variable light chain domain (V L ), wherein the V of such bispecific antibody H and V L and V are paired together to form an antigen-binding site that specifically binds to a first epitope and a second epitope, the method comprising: (a) forming a bispecific antibody or antigen-binding fragment thereof; H and V L wherein said V H and V L pair together to form an antigen-binding site that binds to the first epitope but not the second epitope, and such an antibody has V L (b) at least one electrostatic or hydrophobic amino acid at position 32, 50, or 91 of the antibody of step (a); H (c) modifying a nucleic acid sequence encoding V L and V modified in step (b). H and (d) V L and V modified in step (c). H and selecting a bispecific antibody, or antigen-binding fragment thereof, comprising: H and V L The invention features such methods that include the steps of: pairing together to form an antigen binding site that specifically binds to the first epitope and the second epitope.

[0008] In some embodiments, at least two of the amino acids at positions 32, 50, or 91 are electrostatic or hydrophobic. In some embodiments, all three amino acids at positions 32, 50, and 91 are electrostatic or hydrophobic. In some embodiments, the electrostatic residue is tyrosine. In some embodiments, the hydrophobic residue is tryptophan. In some embodiments, V H The nucleic acid sequence encoding the heavy chain amino acid sequence is modified based on the diversity of multiple naturally occurring heavy chain amino acid sequences. In some embodiments, the solvent-accessible residue positions are VH In some embodiments, the V of the antibody of step (a) is at an amino acid residue position selected from the group consisting of positions 33, 34, 50-58, and 95-97. L In some embodiments, the nucleic acid sequence encoding the V is further modified, wherein one or more solvent-accessible amino acid residues are modified. L In some embodiments, during the selection step (d), the modified V H V Land displayed on the phage. In some embodiments, the antibody of step (a) comprises light chain variable region complementarity determining regions: CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO: 9), CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO: 10), and CDRL3 comprising the amino acid sequence QQDYTSPWTF (SEQ ID NO: 11). In some embodiments, the antibody of step (a) comprises heavy chain variable region complementarity determining regions: CDRH1 comprising the amino acid sequence DYSMH (SEQ ID NO: 13), CDRH2 comprising the amino acid sequence VWINTETGEPTYADDFK (SEQ ID NO: 17), and CDRH3 comprising the amino acid sequence GGIFYGM DY (SEQ ID NO: 20). In some embodiments, the antigen-binding site of the bispecific antibody of step (d) exclusively binds both the first epitope and the second epitope. In other embodiments, the antigen-binding site of the bispecific antibody of step (d) simultaneously binds the first epitope and the second epitope. In some embodiments, the first epitope is derived from one biological molecule and the second epitope is derived from the same biological molecule. In other embodiments, the first epitope is derived from the first biological molecule and the second epitope is derived from the second biological molecule. In some embodiments, the first and second biological molecules are selected from the group consisting of IL4 / IL5 and IL4 / IL13. In some embodiments, the first and second biological molecules are cytokines. In some embodiments, the first or second biological molecule is a molecule capable of extending the half-life of a bispecific antibody when bound to an antibody in vivo. In some embodiments, the first or second biological molecule is serum albumin or fetal Fc receptor (FcRn). In some embodiments, the first or second biological molecule is a molecule capable of enhancing the effector function of a bispecific antibody when bound to an antibody in vivo. In some embodiments, the first or second biological molecule binds to a cell surface protein on natural killer cells or macrophages. In some embodiments, the cell surface protein is an Fc receptor or C1q. In some embodiments, the V of the bispecific antibody H and VL pair together to form a first epitope or a second epitope. -6 The following K D In some embodiments, the V of the bispecific antibody forms an antigen-binding site that specifically binds to the V H and V L pair together to form a first epitope or a second epitope. -9 The following K D In some embodiments, the V of the bispecific antibody forms an antigen-binding site that specifically binds to the V H and V L pair together to form a first epitope or a second epitope. -12 The following K D In some embodiments, the V of the bispecific antibody forms an antigen-binding site that specifically binds to the V H and V L pair together to form a first epitope and a second epitope. -6 The following K D In some embodiments, the V of the bispecific antibody forms an antigen-binding site that specifically binds to the V H and V L pair together to form a first epitope and a second epitope. -9 The following K D In some embodiments, the V of the bispecific antibody forms an antigen-binding site that specifically binds to the V H and V L pair together to form a first epitope and a second epitope. -12 The following K D In some embodiments, the first biological molecule and the second biological molecule are structurally dissimilar. In some embodiments, the selecting in step (d) comprises deep sequencing, ultra-deep sequencing, and / or next-generation sequencing.

[0009] Exemplary antibodies generated using the methods of the invention include antibodies that bind both interleukin 4 (IL4) and interleukin 5 (IL5), and antibodies that bind both IL4 and interleukin 13 (IL13), as described below. The successful generation of these antibodies demonstrates that modifying the heavy chain variable domain sequence of an antibody can be a general engineering avenue toward generating antibodies with dual specificity and function. Bispecific antibodies, including but not limited to the IL4 / IL5 and IL4 / IL13 antibodies described herein, have the potential to simultaneously target two pathways (redundant or non-redundant) and are useful for treating a variety of diseases and disorders, including but not limited to immune, inflammatory, and proliferative disorders.

[0010] Thus, in another aspect, the invention features an isolated bispecific antibody, or antigen-binding fragment thereof, produced by the above-described method of the invention. In some embodiments, the bispecific antibody is a monoclonal antibody. In some embodiments, the fragment is a Fab or scFv. In some embodiments, the bispecific antibody is an IgG.

[0011] In another aspect, the invention features an isolated bispecific antibody, or an antigen-binding fragment thereof, comprising the amino acid sequence of any one of the antibodies of Figures 4A, 4B, 4C, 7A, 7C, or 7D.

[0012] In another aspect, the invention features an isolated bispecific antibody, or an antigen-binding fragment thereof, comprising the following six CDRs: (i) a CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO:9); (ii) a CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO:10); (iii) a CDRL3 comprising the amino acid sequence QQDYTSPWTF (SEQ ID NO:11); (iv) a CDRH1 comprising the amino acid sequence DYDIH (SEQ ID NO:14); (v) a CDRH2 comprising the amino acid sequence VWINTETGEPTYADDFK (SEQ ID NO:17); and (vi) a CDRH3 comprising the amino acid sequence EILFYGMDY (SEQ ID NO:21).

[0013] In another aspect, the invention features an isolated bispecific antibody, or an antigen-binding fragment thereof, comprising the following six CDRs: (i) a CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO:9); (ii) a CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO:10); (iii) a CDRL3 comprising the amino acid sequence QQDYTSPWTF (SEQ ID NO:11); (iv) a CDRH1 comprising the amino acid sequence DYFIH (SEQ ID NO:15); (v) a CDRH2 comprising the amino acid sequence AGIVYDATGFTTYADDFK (SEQ ID NO:18); and (vi) a CDRH3 comprising the amino acid sequence GGIFYGMDY (SEQ ID NO:20).

[0014] In another aspect, the invention features an isolated bispecific antibody, or an antigen-binding fragment thereof, comprising the following six CDRs: (i) a CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO: 9); (ii) a CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO: 10); (iii) a CDRL3 comprising the amino acid sequence QQDYTPFPLTF (SEQ ID NO: 12); (iv) a CDRH1 comprising the amino acid sequence DYLMH (SEQ ID NO: 16); (v) a CDRH2 comprising the amino acid sequence AVIVSITGRTYYADDFK (SEQ ID NO: 19); and (vi) a CDRH3 comprising the amino acid sequence GGIFYGMDY (SEQ ID NO: 20).

[0015] In another aspect, the invention features an isolated bispecific antibody, or an antigen-binding fragment thereof, comprising the following six CDRs: (i) a CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO: 9); (ii) a CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO: 10); (iii) a CDRL3 comprising the amino acid sequence QQDYTSPWTF (SEQ ID NO: 11); (iv) a CDRH1 comprising the amino acid sequence DYSMH (SEQ ID NO: 13); (v) a CDRH2 comprising the amino acid sequence GVIFQSGATYYADDFK (SEQ ID NO: 22); and (vi) a CDRH3 comprising the amino acid sequence GGIFYGMDY (SEQ ID NO: 20).

[0016] In another aspect, the invention features an isolated bispecific antibody, or an antigen-binding fragment thereof, comprising the following six CDRs: (i) a CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO:9); (ii) a CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO:10); (iii) a CDRL3 comprising the amino acid sequence QQDYTSPWTF (SEQ ID NO:11); (iv) a CDRH1 comprising the amino acid sequence DYSMH (SEQ ID NO:13); (v) a CDRH2 comprising the amino acid sequence GIIFYTGHTYYADDFK (SEQ ID NO:23); and (vi) a CDRH3 comprising the amino acid sequence GGIFYGMDY (SEQ ID NO:20).

[0017] In another aspect, the present invention provides a CDRH1 comprising the following six CDRs: (i) a CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO:9); (ii) a CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO:10); (iii) a CDRL3 comprising the amino acid sequence QQDYX1X2PWTF (SEQ ID NO:24), wherein X1 is Thr, Ie, Leu, or Lys, and X2 is Ser or His; (iv) a CDRH1 comprising the amino acid sequence DYFIH (SEQ ID NO:15); (v) a CDRH2 comprising the amino acid sequence X1GIVYDATGFTX2YAX3X4FK (SEQ ID NO:25), wherein X3 is Ala or Gly, X2 is Thr, Ie, Val, or Ala, X3 is Asp, Val, or Glu, and X4 is Asp, Glu, Asn, Ser, Ie, Leu, Thr, Ala, or Phe; and (vi) an amino acid sequence GGIFYGM The present invention features an isolated bispecific antibody, or antigen-binding fragment thereof, comprising: a CDRH3 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO: 9); (ii) a CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO: 10); (iii) a CDRL3 comprising the amino acid sequence QQDYTHPWTF (SEQ ID NO: 27); (iv) a CDRH1 comprising the amino acid sequence DYFIH (SEQ ID NO: 15); (v) a CDRH2 comprising the amino acid sequence GGIVYDATGFTTYAEEFK (SEQ ID NO: 28); and (vi) a CDRH3 comprising the amino acid sequence GGIFYGMDY (SEQ ID NO: 20). In some embodiments, the bispecific antibody, or antigen-binding fragment thereof, comprises the following six CDRs: (i) a CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO: 9); (ii) a CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO: 10); (iii) a CDRL3 comprising the amino acid sequence QQDYTHPWTF (SEQ ID NO: 27); (iv) a CDRH1 comprising the amino acid sequence DYFIH (SEQ ID NO: 15); (v) a CDRH2 comprising the amino acid sequence GGIVYDATGFTTYAEEFK (SEQ ID NO: 28); and (vi) a CDRH3 comprising the amino acid sequence GGIFYGMDY (SEQ ID NO: 20). In some embodiments, the bispecific antibody, or antigen-binding fragment thereof, comprises the following six CDRs: (i) CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO: 9); (ii) CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO: 10); (iii) CDRL3 comprising the amino acid sequence QQDYKHPWTF (SEQ ID NO: 31); (iv) CDRH1 comprising the amino acid sequence DYFIH (SEQ ID NO: 15); (v) CDRH2 comprising the amino acid sequence AGIVYDATGFTVYADDFK (SEQ ID NO: 32); and (vi) CDRH3 comprising the amino acid sequence GGIFYGM DY (SEQ ID NO: 20).In some embodiments, the bispecific antibody, or antigen-binding fragment thereof, further comprises a framework region 3 (FR3) comprising the amino acid sequence GRX1TITX2DX3STSTX4 (SEQ ID NO: 26), where X1 is Val or Phe, X2 is Arg or lie, X3 is Thr, Phe, Met, or Pro, and X4 is Ala or Val.

[0018] In another aspect, the invention features an isolated bispecific antibody, or antigen-binding fragment thereof, comprising a light chain variable region selected from the amino acid sequence of SEQ ID NO: 1, 5, 29, or 33, and a heavy chain variable region selected from SEQ ID NO: 2, 3, 4, 6, 7, 8, 30, or 34. In some embodiments, the antibody or antigen-binding fragment thereof binds IL4 with a Kd of 500 nM or less and IL5 with a Kd of about 900 nM or less. In some embodiments, the antibody or antigen-binding fragment thereof binds IL4 with a Kd of 100 nM or less and IL5 with a Kd of about 100 nM or less. In some embodiments, the antibody or antigen-binding fragment thereof binds IL4 with a Kd of 10 nM or less and IL5 with a Kd of about 50 nM or less. In other embodiments, the isolated bispecific antibody or antigen-binding fragment thereof binds IL4 with a Kd of 500 nM or less and IL13 with a Kd of about 900 nM or less. In some embodiments, the antibody, or antigen-binding fragment thereof, binds IL4 with a Kd of 100 nM or less and IL13 with a Kd of about 100 nM or less. In some embodiments, the antibody, or antigen-binding fragment thereof, inhibits or blocks IL4, IL5, or IL13 from binding to their receptors. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antigen-binding fragment is a Fab fragment or a single-chain variable fragment (scFv). In some embodiments, at least a portion of the framework sequences are human consensus framework sequences. In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0019] Pharmaceutical compositions comprising any one of the above-described bispecific antibodies, or antigen-binding fragments thereof, are also provided. In another aspect, the invention features isolated nucleic acids encoding any of the bispecific antibodies disclosed herein, including vectors (e.g., expression vectors) for expressing such antibodies.

[0020] In another aspect, the invention features a host cell containing the above-described nucleic acid and / or vector. In some embodiments, the host cell is a mammalian cell (e.g., a Chinese hamster ovary (CHO) cell). In other embodiments, the host cell is a prokaryotic cell (e.g., an E. coli cell). Methods of making any one of the above-described bispecific antibodies are also provided, comprising culturing a host cell that produces the bispecific antibody and recovering the bispecific antibody from the host cell or the culture medium.

[0021] In another aspect, the invention features a method of treating asthma in a subject, the method including administering to the subject any of the bispecific antibodies disclosed herein for a duration and in an amount sufficient to treat or prevent asthma in the subject. In some embodiments, the method further includes administering at least one additional asthma therapy selected from the group consisting of an IgE antagonist, an antihistamine, theophylline, salbutamol, beclomethasone dipropionate, sodium cromoglycate, a steroid, and an anti-inflammatory agent. In some embodiments, the asthma is allergic asthma.

[0022] In yet another aspect, the invention features a method of treating a proliferative disorder in a subject, the method comprising administering to the subject any of the bispecific antibodies disclosed herein for a duration and in an amount sufficient to treat the proliferative disorder in the subject. In some embodiments, the proliferative disorder is cancer. In some embodiments, the method further comprises administering to the subject an additional antiproliferative agent selected from the group consisting of a chemotherapeutic agent, a cytotoxic agent, and an anti-angiogenic agent. [Brief explanation of the drawings]

[0023] [Figure 1A-1B] Figures 1A and 1B are graphs showing mutagenesis mapping of the CDRs of the hu19C11 antibody. To measure the relative antigen-binding affinity of the hu19C11 Fab variants, binding of serially diluted phage displaying anti-IL4 hu19C11 wild-type (wt) or LC CDR alanine mutants to anti-gD antibody (A) or IL4 (B) coated on ELISA wells was detected by Fab expression and anti-M13 phage-horseradish peroxidase (HRP) conjugate, respectively, to quantify antigen binding. gD is an expressed peptide tag fused to the C-terminus of the light chain. Anti-gD antibody was coated directly on the ELISA wells, while IL4 was captured with a non-blocking anti-IL4 antibody coated on the ELISA wells. [Figure 1C] Figure 1C graphically depicts the effect of alanine mutations at individual CDR sites, performed in the assays described in Figures 1A and 1B, by comparing the relative IL4-binding affinities of phage displaying Fab variants. Single-letter amino acid codes are used. Data were fitted to a linear regression model, and the relative IL4-binding strength was determined by dividing the slope of IL4 binding (vs. phage concentration) by the slope of Fab expression. Low values ​​(below the dotted line) were considered to indicate disruptive mutations with IL4-binding affinities lower than hu1911 wt. [Figure 2A]Figure 2A shows the structure of trastuzumab Fab (PDB: 1FDV) with the key IL4-binding residues of hu19C11 mapped onto a top-down view. A structural model of hu19C11 was generated using MOE, using POB entries 3SQO and 3BEI as templates for the heavy and light chains, respectively. The residues important for IL4 binding are residues 31, 32, 50, 53, 91, and 92 in the LC and residues 31, 32, 96, 98, and 99 in the HC, according to Kabat numbering. Residues are colored red in the model structure in the top-down view of the antigen-binding site. A library of phage-displayed hu19C11 Fab variants was generated by site-directed mutagenesis. The HC residues, for which all 20 amino acids show preference over the wild-type residue, are shown in bold. The other HC and LC residues shown were restricted for mutation to mimic natural diversity. [Figure 2B] Figure 2B is a table showing the combinatorial library design for mobilizing a second antigen specificity into hu19C11. The CDR sequences of selected clones are shown, including anti-IL5 (5A), anti-IL4 / 5 (E7, B1), and anti-IL4 / 13 (F1, F2) antibody clones with mutations from the parental hu19C11 wild-type. The residues randomized in the library and mutated in the isolated clones are shaded according to their properties: Y, W, and F have aromatic side chains; L, I, V, and M are hydrophobic; K, R, and H are basic; D and E are acidic; S, T, N, and Q are polar; and P and G are acidic. Relative antigen-binding affinities, measured by IC50 in phage competition assays, are shown. Fab-displaying phages were first incubated with serial dilutions of the respective antigens in solution for 2 hours. Unbound phages were then briefly captured in antigen-coated ELISA wells and detected with anti-M13-HRP conjugate. The IC50 was calculated as the antigen concentration that inhibited 50% of phage binding to the antigen-coated wells. NB means that there was no detectable direct binding of the phage clone to the antigen-coated wells. [Figure 3]FIG. 3 is a graph showing the titration of the 10 phage display libraries (2144-1 to 2144-10) generated for IL4 binding ability. [Figure 4A] FIG. 4A shows the amino acid sequences of the light chain variable domains of bispecific anti-IL4 / IL5 variants of hu19C11 (E7 (SEQ ID NO: 1) and B1 (SEQ ID NO: 5)) and anti-IL5 specific 5A (SEQ ID NO: 1) aligned with anti-IL4 specific hu19C11 (SEQ ID NO: 1). [Figure 4B] Figure 4B shows the amino acid sequences of the heavy chain variable domains of bispecific anti-IL4 / IL5 variants of hu19C11 (E7 (SEQ ID NO: 4) and B1 (SEQ ID NO: 6)) and anti-IL5 specific 5A (SEQ ID NO: 3) aligned with anti-IL4 specific hu19C11 (SEQ ID NO: 2). [Figure 4C] Figure 4C shows the amino acid sequences of the heavy chain variable domains of bispecific anti-IL4 / IL13 variants of hu19C11 (F1 (SEQ ID NO: 7) and F2 (SEQ ID NO: 8)) aligned with the anti-IL4 specific hu19C11 (SEQ ID NO: 2). [Figure 5] Figure 5 is a graph showing the bispecificity of selected variants of hu19C11 as IgG. The antigen-binding specificity of selected variants of hu19C11 was assessed by binding of these variants in a human IgG1 format to the target antigen(s) or several unrelated proteins coated on ELISA wells at 250 nM, detected with an anti-Fc antibody-HRP conjugate. [Figure 6] Figure 6 is a graph characterizing the binding specificity of bispecific variants of hu19C11 (anti-IL4-specific wild-type: 19C11; anti-IL5-specific: 5A; and anti-IL4 / IL5-specific: E7 and B1) when blocking IL5 interaction with its receptor at the indicated antibody concentrations. The level of biotinylated IL5 receptor alpha bound to IL5 immobilized on ELISA wells in the presence of increasing concentrations of humanized 19C11 or variants as IgG was detected using streptavidin-HRP conjugate. [Figure 7A] Figure 7A is a table showing the amino acid sequences of affinity-matured variants of IL4 / IL5-specific E7 and their relative affinities for their target antigens, as measured in phage competition assays. To improve affinity, phage libraries displaying E7 variants were constructed using "homologous" (bold), "limited" (italic), and "soft" (gray) randomization methods, i.e., mutating selected residues to wild-type homologous amino acids, limited diversity based on natural antibodies, or randomizing approximately 50% of wild-type and approximately 50% of all other amino acids, respectively. CDR H2 was primarily subjected to homologous mutations to subtly optimize binding function to the newly recruited IL5. Sites unimportant for IL4 binding were targeted for the other CDRs. The sequences of selected clones were aligned with E7, and mutations are shown. The relative affinity of each clone was evaluated by phage IC50 as described above. [Figure 7B] Figure 7B is a table showing the affinities of E7 and its affinity-matured variants 1C36 and 1C60 purified as Fab, measured by Biacore at 25°C using a CM5 sensor chip immobilized with human IL5 (R&D Systems) or IL4. [Figure 7C] Figure 7C shows the alignment of the amino acid sequences of the light chain variable domains of affinity-improved bispecific anti-IL4 / IL5 variants of E7, 1C36 (SEQ ID NO: 29) and 1C60 (SEQ ID NO: 33), with the amino acid sequence of the light chain variable domain of E7 (SEQ ID NO: 1). [Figure 7D] Figure 7D shows the amino acid sequences of the heavy chain variable domains of affinity-improved bispecific anti-IL4 / IL5 variants of E7, 1C36 (SEQ ID NO: 30) and 1C60 (SEQ ID NO: 34), aligned with the amino acid sequence of the heavy chain variable domain of E7 (SEQ ID NO: 4). [Figure 8A-8B]Figures 8A and 8B are graphs showing the characterization of the binding specificity of E7 and affinity-matured variants of E7 (1C36 and 1C60). Direct binding of E7 and affinity-improved variants of E7 as IgG (100 nM) to immobilized antigen and to an unrelated protein on an ELISA plate was detected with anti-IgG-HRP (A). Binding of biotinylated IL5 to the IL5 receptor coated on ELISA wells in the presence of buffer (PBS) or 50 nM E7, 1C36, or 1C60 was detected with streptavidin-HRP complex (B). DETAILED DESCRIPTION OF THE INVENTION

[0024] Many disease pathways are driven by the action of more than one protein or a single protein with more than one function. For example, multiple cytokines are often involved in allergic, inflammatory, or autoimmune disorders (e.g., asthma). Bispecific antibodies are useful in both therapeutic and diagnostic applications where targeting more than one antigen is desired. We have discovered a novel method for generating bispecific antibodies. The V of antibodies L contains residues important for antibody-antigen interaction, such antibodies may be H The residue can be placed alone or in a separate V L and framework residues can be combined to create diversity.

[0025] Generally, the methods of the invention involve the V H The method involves diversifying the V gene to generate variants that can be stably expressed in a library. Generally, the V gene specifically binds to a first epitope but not a second epitope, and L and an antibody characterized as having electrostatic or hydrophobic residues at any one, two, or three amino acids found at positions 32, 50, or 91 (according to Kabat numbering) of V. H Then, modifications are made to one or more amino acid residues (e.g., solvent-exposed amino acid residues) in V H and VL and generating a diversified bispecific antibody, or antigen-binding fragment thereof, capable of specifically binding to the first and second epitopes from the expressed V. H and V L Choose from:

[0026] Exemplary antibodies generated using the methods of the invention include antibodies that bind both interleukin 4 (IL4) and interleukin 5 (IL5), and antibodies that bind both IL4 and interleukin 13 (IL13), as described below. These antibodies demonstrate that mutations within the heavy chain variable domain (e.g., CDRs) of the IL4 antibody confer dual binding capability for IL4 and another unrelated protein, and also demonstrate the use of V to confer bispecificity. H This provides proof of concept for a general method of modifying residues within the IL4 / IL5 and IL4 / IL13 domains. The bispecific antibodies described herein, including but not limited to IL4 / IL5 and IL4 / IL13 antibodies, have the potential to simultaneously target multiple antigens, e.g., redundant and non-redundant cytokine pathways, making them useful for treating a variety of diseases and disorders, including cytokine-mediated diseases (e.g., asthma).

[0027] I. Definition The term "multispecific antibody" is used in the broadest sense and specifically refers to antibodies that contain heavy chain variable domains (V H ) and the light chain variable domain (V L ), including V H V L These include antibodies in which the V units have polyepitopic specificity (i.e., can bind to two different epitopes on one biological molecule or to epitopes on different biological molecules). Such multispecific antibodies include full-length antibodies, antibodies containing two or more V units, and antibodies containing two or more V units. L Domain and V HThese include, but are not limited to, antibody fragments such as domain-containing antibodies, Fabs, Fvs, dsFvs, scFvs, diabodies, bispecific diabodies, and triabodies, as well as covalently or non-covalently linked antibody fragments. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). "Dual specificity" or "bispecificity" refers to the ability to specifically bind to two different epitopes on the same or different target(s). However, in contrast to bispecific antibodies, dual-specific antibodies are based on the natural IgG antibody model, in which the two antigen-binding arms are identical in amino acid sequence, and each Fab arm can recognize two different antigens. Dual specificity allows an antibody to interact with two different antigens with high affinity as a single Fab or IgG molecule. According to one embodiment, the IgG1-type multispecific antibody binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM or 0.1 μM to 0.001 pM. "Monospecific" refers to the ability to bind to only one epitope.

[0028] Generally, antibodies comprise a basic four-chain antibody unit, a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (IgM antibodies are composed of five basic heterotetrameric units with an additional polypeptide called the J chain, thus containing 10 antigen-binding sites, and secretory IgA antibodies can polymerize to form multivalent assemblies containing two to five basic four-chain units with the J chain). In the case of IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain contains a variable domain (V) at its N-terminus. H ), followed by three constant domains in the α and γ chains (C H ) and four CH domains in the μ and ε isotypes. Each L chain has a variable domain (V L ) at the other end of which is a constant domain (C L ) followed by V L is V H It is lined up with C L is the heavy chain (C H 1) is aligned with the first constant domain. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain. H and V L pair with each other to form an antigen-binding site. For the structure and properties of the various classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and chapter 6. Light chains from any vertebrate can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins are composed of their heavy chains (C HDepending on the amino acid sequence of the constant domain of the immunoglobulin, they can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains designated α, δ, γ, ε, and μ, respectively. Both the γ and α classes are further divided into C H IgA1 and IgA2 are expressed in humans.

[0029] The term "variable" refers to the fact that certain segments of the variable domains vary significantly in sequence among antibodies. The variable or "V" domain mediates antigen binding and determines the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed across the 110 amino acid length of the variable domain. Instead, the V domains consist of relatively invariant stretches of 15-30 amino acids, called framework regions (FRs), separated by short, highly variable regions of 9-12 amino acids in length, called "hypervariable regions." Native heavy and light chain variable domains each contain four FRs, which are primarily arranged in a beta-sheet configuration, and three connecting hypervariable regions form loops that span, and in some cases, form part of, the beta-sheet structure. The hypervariable regions of each chain are held in close proximity by the FR regions and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not directly involved in binding the antibody to an antigen but exhibit various effector functions, such as the participation of antibodies in antibody-dependent cellular cytotoxicity (ADCC).

[0030] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen binding. Hypervariable regions generally consist of amino acid residues of the "complementarity determining regions" or "CDRs" (e.g., V L Around residues 24-34 (L1), 50-56 (L2), and 89-97 (L3), and V H around residues 26-35 (H1), 49-65 (H2), and 95-102 (H3) of (in one embodiment, H1 is around residues 31-35); Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), and / or residues in the "hypervariable loops" (e.g., V L Residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) of V H 26-32 (H1), 53-55 (H2), and 96-101 (H3; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)).

[0031] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., an antibody in which the individual antibodies comprising the population are substantially similar and bind to the same epitope(s), except for variations that may arise during monoclonal antibody production, which are generally minor, if any. Such monoclonal antibodies typically include antibodies comprising a variable region that binds a target, wherein the antibody has been obtained by a process that includes selecting the antibody from a plurality of antibodies. For example, such a selection process can involve selecting a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that the selected antibody can be further modified, for example, to improve affinity for the target, humanize the antibody, improve its production in cell culture, reduce its immunogenicity in vivo, create multispecific antibodies, etc., and that antibodies comprising modified variable region sequences are also monoclonal antibodies of the present invention. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically free of contaminating immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be made by a variety of techniques, including hybridoma methods (e.g., Kohler et al., Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681, (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display methods (e.g., Clackson et al.,Nature,352:624-628(1991);Marks et al.,J.Mol.Biol.,222:581-597(1991);Sidhu et al.,J.Mol.Biol.338(2):299-310(2004);Lee et al. al., J.Mol.Biol.340(5):1073-1093(2004); Fellouse, Proc.Nat.Acad.Sci.USA101(34):12467-12472(2004); and Lee et al. J.Immunol.Methods 284(1-2):119-132 (2004)), as well as techniques for producing human or human-like antibodies from animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (e.g., WO98 / 24893, WO / 9634096, WO / 9633735, and WO / 9110741; Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669 (all GenPharm); 5,545,807; WO 97 / 17852, U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, as well as Marks et al., Bio / Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnology, 14:845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13:65-93 (1995).

[0032] The monoclonal antibodies of the present invention specifically include chimeric antibodies, humanized antibodies, fully human antibodies, and affinity-matured antibodies. Chimeric antibodies are antibodies in which portions of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) are identical to or homologous to corresponding sequences in antibodies from another species or belonging to a different antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein contain variable domain antigen-binding sequences derived from constant region sequences of a non-human primate (e.g., Old World monkey, ape, etc.) and a human.

[0033] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient antibody are substituted by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired antibody specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, such humanized antibodies will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of each FR is that of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0034] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human and / or an antibody made using any technique for producing human antibodies. This definition of human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues.

[0035] An "affinity matured" antibody is an antibody with one or more modifications in one or more CDRs of the antibody that improve its affinity for an antigen over that of a parent antibody that does not have these modifications. Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al. Bio / Technology 10:779-83 (1992) describes affinity matured antibodies as: H and V L Affinity maturation by domain shuffling has been described. Random mutagenesis of CDR and / or framework residues has been described by Barbas et al., Proc. Nat. Acad. Sci. USA 91:3809-13 (1994); Schier et al., Gene 169:147-55 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-19 (1995); and Hawkins et al., J. Mol. Biol. 226:889-96 (1992).

[0036] An "intact" antibody is one that contains both the antigen-binding site and the C L and at least heavy chain constant domain C H 1. C H 2, and C H 3. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof. Preferably, the intact antibody has one or more effector functions.

[0037] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments; bispecific antibodies; linear antibodies (see Example 2 of US Patent No. 5,641,870; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0038] The expression "linear antibody" generally refers to antibodies as described in Zapata et al., Protein Eng., 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-C) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions. H 1-VH-C H 1) Linear antibodies can be bispecific or monospecific.

[0039] Digestion of an antibody with papain yields two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment (the name stands for the ease with which it can be crystallized). The Fab fragment contains the entire light chain along with the heavy chain (V H ) variable region domain and one heavy chain (C H 1) and the first constant domain of antibody fragment 1. Treatment of an antibody with pepsin yields a single large F(ab')2 fragment, roughly equivalent to two disulfide-linked Fab fragments, which has bivalent antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments in that they contain a few additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. As used herein, Fab'-SH refers to Fab' in which the cysteine ​​residue(s) of the constant domain bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments, which have hinge cysteine ​​residues between them. Other chemical couplings of antibody fragments are also known.

[0040] The Fc fragment contains the carboxy-terminal portions of both heavy chains held together by disulfides. The effector functions of an antibody are determined by the sequences in the Fc region; this region is also recognized by Fc receptors (FcRs) found on certain types of cells.

[0041] An "Fv" consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. These two domains combine to form six hypervariable loops (three H-chain loops and three L-chain loops) that contribute to antigen-binding and confer antigen-binding specificity to the antibody. However, a single variable domain (or half of an Fv containing only three CDRs specific for one antigen) can still recognize and bind antigen, although often with lower affinity than the complete binding site.

[0042] "Single-chain Fv" is abbreviated as "sFv" or "scFv", and refers to VFvs linked in a single polypeptide chain. H and V L Preferably, the sFv polypeptide contains a polypeptide linker between the V and V domains that enables the sFv to form the desired structure for antigen binding. H Domains and V L Further interdomain fragments. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995.

[0043] The term "diabody" refers to a diabody that combines V domains in a single V-domain, such that pairing of the V domains occurs between chains but not within chains, resulting in bivalent fragments, i.e., fragments with two antigen-binding sites. H Domains and V LA bispecific diabody is a heterodimer of two "crossover" sFv fragments, in which the V domains of the two antibodies are linked together. H Domain and V L The domains are present on different polypeptide chains. Diabodies are described in further detail in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0044] "Electrostatic" means having a charge. Generally, electrostatic amino acids have polar or charged side chains. Examples of amino acids with polar side chains include serine, threonine, tyrosine, cysteine, asparagine, and glutamine. Examples of amino acids with negatively charged side chains include aspartic acid and glutamic acid. Examples of amino acids with positively charged side chains include lysine, arginine, and histidine.

[0045] "Hydrophobic" means incompatible with water, i.e., not readily dissolved in, absorbed into, or mixed with water. Generally, hydrophobic amino acids have nonpolar side chains and examples include alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine.

[0046] The term "cytokine" is a general term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of cytokines include lymphokines, monokines, and general polypeptide hormones. These cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin and proinsulin; relaxin and prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factors α and β; Müllerian inhibitory factor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet growth factor (PGF); factors); transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factors I and II; erythropoietin (EPO); bone morphogenetic factors; interferons such as interferon-α, -β, and -γ; colony-stimulating factors (CSFs) such as macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF); interleukins (ILs) such as IL1, IL1α, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL11, IL12, and IL13; tumor necrosis factors such as TNF-α or TNF-β; and other polypeptide factors, including LIF and Kit ligand (KL). As used herein, the term cytokine includes biologically active equivalents of wild-type sequence cytokines, whether derived from natural sources or from recombinant cell culture.

[0047] As used herein, a "codon set" refers to a set of different nucleotide triplets used to encode desired modified amino acids. A set of oligonucleotides, representing all possible combinations of nucleotide triplets provided by the codon set, contains sequences encoding the desired amino acids and can be synthesized, for example, by solid-phase synthesis. A standard format for codon designation is the IUB code, which is known in the art and described herein. Codon sets are typically represented by three capital letters in italics, e.g., NNK, NNS, XYZ, DVK, etc. (e.g., the NNK codon encodes all 20 naturally occurring amino acids, with N=A / T / G / C at positions 1 and 2 of the codon and K=G / T in equimolar ratios at position 3). Thus, a "non-random codon set" as used herein refers to a codon set that encodes selected amino acids that partially, or preferably completely, satisfy the amino acid selection criteria described herein. Synthesis of oligonucleotides with "degeneracy" of selected nucleotides at specific positions is known in the art, for example, the TRIM method (Knappek et al., J. Mol. Biol. 296:57-86, 1999; Garrard and Henner, Gene 128:103, 1993). Such sets of oligonucleotides with specific codon sets can be synthesized using commercially available nucleic acid synthesizers (e.g., available from Applied Biosystems, Foster City, CA), although commercially available alternatives are also available (e.g., Life Technologies, Rockville, MD). Thus, a set of synthetic oligonucleotides with a specific codon set will typically include multiple oligonucleotides that differ in sequence, with the differences established by the codon set present within the overall sequence. Oligonucleotides used in accordance with the invention have sequences that allow hybridization to a variable domain nucleic acid template and may, but need not necessarily, contain useful restriction enzyme sites (e.g., for cloning).

[0048] An antibody of the present invention that "binds" an antigen of interest is one that binds the antigen with sufficient affinity so that it is useful as a diagnostic and / or therapeutic agent in targeting proteins, cells, or tissues expressing the antigen and does not significantly cross-react with other proteins. In such embodiments, the extent to which the antibody binds to "non-target" proteins will be less than about 10% of the extent to which the antibody binds to its specific target protein, as measured by fluorescence activated cell sorting (FACS) analysis, radioimmunoprecipitation (RIA), or ELISA. With respect to antibody binding to a target molecule, the terms "specific binding" or "specifically binds to" or "specific for" a particular polypeptide target or epitope on a particular polypeptide refer to binding that is measurable as distinct from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be determined by competing with a control molecule that resembles the target molecule, e.g., an excess of unlabeled target molecule. In this case, specific binding occurs if the binding of the labeled target molecule to the probe is competitively inhibited by an excess of unlabeled target molecules. In the present invention, the terms "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or epitope on a particular polypeptide target refer to, for example, the K D is 10 -4 M or less, alternatively 10 -5 M or less, alternatively 10 -6 M or less, alternatively 10 -7 M or less, alternatively 10 -8 M or less, alternatively 10 -9 M or less, alternatively 10 -10 M or less, alternatively 10 -11 M or less, alternatively 10 -12 M or less or K D is 10 -4 M~10 -12 M or 10 -6 M~10 -10 M or 10 -7 M~10 -9As will be appreciated by those skilled in the art, affinity and K D The values ​​are inversely correlated. The higher the affinity of the antigen, the higher the measured K D In one embodiment, the term "specific binding" refers to binding when a molecule binds to a particular polypeptide or an epitope on a particular polypeptide and does not substantially bind to any other polypeptides or epitopes of polypeptides.

[0049] "Biologically active" and "biological activity" and "biological characteristics" with respect to the polypeptides of the present invention mean, unless otherwise specified, having the ability to bind to biological molecules.

[0050] "Biological molecule" refers to nucleic acids, proteins, carbohydrates, lipids, and combinations thereof. In one embodiment, the biological molecule is naturally occurring.

[0051] "Isolated," as used in describing various antibodies disclosed herein, refers to an antibody that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Contaminant components of its natural environment are substances that would typically interfere with diagnostic or therapeutic uses of the polypeptide and can include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antibody is purified (1) using a spinning cup sequenator to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, or (2) to homogeneity by SDS-PAGE under reducing or nonreducing conditions with Coomassie blue or, preferably, silver staining. Since at least one component of the polypeptide's natural environment will not be present, isolated antibody includes the antibody in situ within recombinant cells. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

[0052] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

[0053] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous with each other; and, in the case of a secretory leader, the sequences are contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0054] With respect to the polypeptide sequences identified herein, "percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a comparison polypeptide, after sequence alignment and, if necessary, after inserting gaps to maximize the percent sequence identity, excluding any conservative substitutions that qualify as sequence identity. Alignment for determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximal alignment across the full length of the comparison sequences. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and its source code, along with user documentation, has been submitted to the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is published by Genentech, Inc., South San Francisco, California. The ALIGN-2 program must be compiled for use on a UNIX operating system, preferably Digital UNIX V4.0D. All sequence comparison parameters are preset in the ALIGN-2 program and should not be altered.

[0055] The amino acid sequences described herein are contiguous amino acid sequences unless otherwise specified.

[0056] "Structurally dissimilar" biological molecules according to the present invention refer to biological molecules that are not in the same class (proteins, nucleic acids, lipids, carbohydrates, etc.), or, for example, when referring to proteins, biological molecules that have less than 60% amino acid identity, less than 50% amino acid identity, less than 40% amino acid identity, less than 30% amino acid identity, less than 20% amino acid identity, or less than 10% amino acid identity with each other.

[0057] The "stringency" of a hybridization reaction can be readily determined by one of ordinary skill in the art and is generally calculated empirically depending on probe length, washing temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are in an environment below their melting temperature. The higher the desired degree of homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. Consequently, higher relative temperatures tend to make the reaction conditions more stringent, while lower temperatures tend to make them less so. For further details and explanations regarding stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

[0058] "Stringent conditions" or "high stringency conditions," as defined herein, include: (1) low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (3) low ionic strength and high temperature for hybridization, e.g., 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM pH 6.5 sodium phosphate buffer, 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (4) low ionic strength and high temperature for hybridization, e.g., 50% formamide, 5x SSC (0.75 M Hybridization can be performed overnight at 42°C in a solution containing 50 mM NaCl, 0.075 M sodium citrate, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 pg / ml), 0.1% SDS, and 10% dextran sulfate, followed by a 10-minute high-stringency wash at 55°C in 0.2x SSC (sodium chloride / sodium citrate).

[0059] "Moderately stringent conditions" can be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of less stringent wash solutions and hybridization conditions (e.g., temperature, ionic strength, and % SDS). An example of moderately stringent conditions is overnight incubation at 37°C in a solution containing 20% ​​formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 mg / ml fragmented, denatured salmon sperm DNA, followed by washing the filter in 1x SSC at approximately 37-50°C. Those skilled in the art will recognize how to adjust temperature, ionic strength, and other parameters as needed to accommodate factors such as probe length.

[0060] Antibody "effector functions" refer to biological activities attributable to the Fc region of an antibody (whether a wild-type sequence Fc region or an amino acid sequence variant Fc region) and which vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0061] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "arm" the cytotoxic cells, and this arming is absolutely necessary for such killing. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcRs expressed on hematopoietic cells are summarized in Table 3, page 464, of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or 5,821,337, can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of a molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. (Proc. Natl. Acad. Sci. USA) 95:652-656 (1998).

[0062] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. A preferred FcR is a wild-type sequence human FcR. Further, a preferred FcR is one that binds IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see Daeron, Annu. Rev. Immunol. 15:203-234; for review, see M. (1997)). FcRs are reviewed in Ravetch and Kinet, (Annu. Rev. Immunol. 9:457-492 (1991)); Capel et al., (Immunomethods 4:25-34 (1994)); and de Haas et al., (J. Lab. Clin. Med. 126:330-41 (1995)). Other FcRs yet to be identified are encompassed by the term "FcR" herein. The term also includes the fetal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0063] "Human effector cells" are leukocytes that express one or more FcRs and are responsible for effector function. Preferably, such cells express at least FcγRIII and are responsible for ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being preferred. Effector cells can be isolated from natural sources, such as blood.

[0064] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that are bound to their cognate self antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed.

[0065] The term "therapeutically effective amount" refers to an amount of an antibody or antibody fragment to treat a disease or disorder in a subject. In the case of allergic, inflammatory, or autoimmune diseases (e.g., asthma, arthritis, etc.), a therapeutically effective amount of an antibody or antibody fragment (e.g., a bispecific or multispecific antibody or antibody fragment against IL4 and IL5 or IL4 and IL13) may ameliorate or treat the disease, or prevent, reduce, ameliorate, or treat symptoms associated with the disease. In the case of proliferative diseases (e.g., tumors), a therapeutically effective amount of an antibody or antibody fragment may reduce the number of cancer cells; reduce the size of the primary tumor; inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion into surrounding organs; inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with the disorder. To the extent that the antibody or antibody fragment can inhibit the growth and / or kill existing cancer cells, the drug may be cytostatic and / or cytotoxic. For cancer therapy, in vivo efficacy can be measured, for example, by assessing survival, time to disease progression (TTP), disease-free survival (DFS), progression-free survival (PFS), response rate (RR), duration of response, and / or quality of life.

[0066] By "reduce or inhibit" is meant the ability to bring about an overall decrease, preferably of 20% or more, more preferably 50% or more, and most preferably 75%, 85%, 90%, 95% or more. Reduce or inhibit may refer to the symptoms of the disorder being treated, the presence or size of metastases, the size of the primary tumor, or the size or number of blood vessels in an angiogenic disorder.

[0067] As used herein, "inflammatory disease" refers to a pathological condition that results in inflammation, typically inflammation due to neutrophil chemotaxis.

[0068] As used herein, an "autoimmune disease" is a disease or disorder that arises from and is directed against an individual's own tissues, or a condition that occurs concurrently with, develops in, or is caused by it.

[0069] Examples of diseases or disorders that are inflammatory or autoimmune, or both, include asthma, such as asthmatic bronchitis (asthmatic bronchitis), bronchial asthma, and autoimmune asthma; arthritis (such as acute arthritis, rheumatoid arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, osteoarthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, progressive chronic arthritis, osteoarthritis, chronic primary polyarthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, progressive chronic arthritis, osteoarthritis, chronic primary polyarthritis, and juvenile-onset rheumatoid arthritis. primaria), reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases; psoriasis, such as plaque psoriasis, guttate psoriasis, pustular psoriasis, and nail psoriasis; dermatitis, including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis; X-linked hyper-IgM syndrome; urticaria, such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria; polymyositis / dermatomyositis; juvenile dermatomyositis; toxic epidermal necrolysis; scleroderma (including systemic sclerosis); systemic sclerosis, spino-optical multiple sclerosis, primary progressive (primary Multiple sclerosis (MS), such as progressive multiple sclerosis (PPMS) and relapsing-remitting multiple sclerosis (RRMS), sclerosis, such as progressive systemic sclerosis, atherosclerosis, arteriosclerosis, generalized sclerosis (sclerosis disseminata), and dysplastic sclerosis; inflammatory bowel disease (IBD), such as Crohn's disease, autoimmune-mediated gastrointestinal diseases, ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, and polypoid colitis colitis such as necrotizing enterocolitis and transmural colitis, and autoimmune inflammatory bowel disease); pyoderma gangrenosum; erythema nodosum; primary sclerosing cholangitis; episcleritis); respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS); meningitis; inflammation of all or part of the uvea;Iritis; choroiditis; autoimmune blood disorders; rheumatoid spondylitis; sudden hearing loss; IgE-mediated disorders such as anaphylaxis and allergic and atopic rhinitis; hyper-IgE syndrome; encephalitis such as Rasmussen's encephalitis and limbic encephalitis and / or brainstem encephalitis; uveitis such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis; primary glomerulonephritis, immune-mediated glomerulonephritis, membranous glomerulonephritis (membranous nephropathy), idiopathic membranous glomerulonephritis or idiopathic membranous nephropathy, membranoproliferative or membranous, including types I and II. Glomerulonephritis (GN), with or without nephrotic syndrome, such as chronic or acute glomerulonephritis, including progressive (proliferative) glomerulonephritis (MPGN), and rapidly progressive glomerulonephritis; allergic conditions; allergic reactions; eczema, including allergic or atopic eczema; scleroderma; Whipple's disease, hypertrophic scars, pre-eclampsia, abdominal adhesions, conditions with T-cell infiltration and chronic inflammatory responses; chronic inflammatory lung diseases; autoimmune myocarditis; leukocyte adhesion deficiency; systemic lupus erythematosus (SLE), i.e., cutaneous lupus erythematosus, subacute cutaneous lupus erythematosus, Systemic lupus erythematosus, such as neonatal lupus syndrome (NLE), disseminated lupus erythematosus, and lupus (nephritis, encephalitis, pediatric, non-renal, extrarenal, discoid, alopecia); juvenile-onset (Type 1) diabetes, including insulin-dependent diabetes mellitus (IDDM); adult-onset diabetes mellitus (Type 2); autoimmune diabetes; idiopathic diabetes insipidus; immune responses related to cytokine- and T-lymphocyte-mediated acute and delayed hypersensitivity; tuberculosis; sarcoidosis; granulomatous diseases, including lymphomatoid granulomatosis and Wegener's granulomatosis; agranulocytosis;Vasculitis (including large-vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu's) arteritis), medium-vessel vasculitis (including Kawasaki disease and polyarteritis nodosa), microscopic polyarteritis, central nervous system (CNS) vasculitis, necrotizing, cutaneous, or hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis such as Churg-Strauss vasculitis or Churg-Strauss syndrome (CSS)); temporal arteritis; aplastic anemia; autoimmune aplastic anemia; Coombs-positive anemia; Diamond-Blackfan anemia; hemolytic anemia or immune-mediated hemolytic anemia, including autoimmune hemolytic anemia (AIHA); pernicious anemia (anemia perniciosa); Addison's disease; erythroid anemia or true red cell aplasia (PRCA); Factor VIII Factor deficiency; hemophilia A; autoimmune neutropenia; pancytopenia; leukopenia; diseases associated with leukocyte emigration; central nervous system (CNS) inflammatory disorders; multiorgan injury syndromes such as those secondary to sepsis, trauma, or hemorrhage; antigen-antibody complex-mediated diseases; antiglomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Behçet's disease; Castleman syndrome; Goodpasture's syndrome; Raynaud's syndrome; Sjögren's syndrome; Stevens-Johnson syndrome; pemphigoid, such as bullous pemphigus and cutaneous pemphigus; pemphigus (including pemphigus vulgaris, pemphigus foliaceus, mucous membrane pemphigoid, and pemphigus erythematosus); autoimmune polyendocrinopathy; Reiter's disease or syndrome; immune complex nephritis; antibody-mediated nephritis neuromyelitis optica; polyneuropathy; chronic neuropathies such as IgM polyneuropathy or IgM-mediated neuropathy; thrombocytopenia (e.g., occurring in patients with myocardial infarction), including autoimmune or immune-mediated thrombocytopenia such as thrombotic thrombocytopenic purpura (TTP) and chronic or acute idiopathic thrombocytopenic purpura (ITP); autoimmune diseases of the testes and ovaries, including autoimmune orchitis and oophoritis; primary hypothyroidism; hypoparathyroidism; autoimmune endocrine disorders, including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis; autoimmune thyroid disease; idiopathic hypothyroidism; Graves' disease;polyglandular syndromes, such as autoimmune polyglandular syndrome (or polyendocrinopathy syndrome); paraneoplastic syndromes, including neurological paraneoplastic syndromes, such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man syndrome or stiff-person syndrome; encephalomyelitis, such as allergic encephalomyelitis (also known as encephalomyelitis allergica) and experimental allergic encephalomyelitis (EAE); myasthenia gravis, such as thymoma-associated myasthenia gravis; cerebellar degeneration; neuromyotonia; opsoclonus or opsoclonus-myoclonus syndrome (OMS), and sensory neuropathies; multifocal motor neuropathy; Sheehan's syndrome; autoimmune hepatitis; chronic hepatitis; lupoid hepatitis; giant cell hepatitis; chronic active hepatitis or autoimmune chronic active hepatitis; Lymphocytic interstitial pneumonia; bronchiolitis obliterans (non-transplant) and nonspecific interstitial pneumonia (NSIP); Guillain-Barré syndrome; Berger's disease (IgA nephropathy); idiopathic IgA nephropathy; linear IgA bullous dermatosis; primary biliary cirrhosis; pulmonary fibrosis; autoimmune enteropathy syndrome; celiac disease; celiac sprue (gluten enteropathy); refractory sprue; idiopathic sprue; cryoglobulinemia; cryoglobulinemia; amyotrophic lateral sclerosis (ALS); Lou Gehry syndrome autoimmune hearing disorders, such as autoimmune inner ear disease (AIED); autoimmune hearing disorders, such as autoimmune deafness; opsoclonus-myoclonus syndrome (OMS); polychondritides, such as refractory or relapsing polychondritis; pulmonary alveolar proteinosis; amyloidosis; scleritis; non-cancerous lymphocytosis; monoclonal B-cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance (MGA)); Primary lymphocytosis, including GUS; peripheral neuropathy, paraneoplastic syndromes; epilepsy, migraine, cardiac arrhythmias, myopathy, hearing loss, blindness, periodic paralysis, and channelopathies of the central nervous system (CNS); autism, inflammatory myopathies, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveitis, chorioretinitis, autoimmune liver disease, fibromyalgia, multiple endocrine deficiencies, Schmidt's syndrome, adrenal inflammation, gastrotrophy, and presenile dementia;Demyelinating diseases such as autoimmune demyelinating diseases, diabetic nephropathy, Dressler's syndrome, alopecia areata, crest syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia); male and female autoimmune infertility, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent miscarriage, farmer's lung, erythema multiforme, postpericardiectomy syndrome, Cushing's syndrome, bird fancier's lung, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport syndrome; alveolitis such as allergic alveolitis and fibrosing alveolitis; interstitial lung disease, transfusion reactions, leprosy; malaria; Leishmaniasis; Kypanosomiasis; Schistosomiasis; Ascariasis; Aspergillosis; Sampter's syndrome; Kaplan's syndrome; Dengue; Endocarditis; Endocardial fibrosis; Diffuse interstitial pulmonary fibrosis; Interstitial pulmonary fibrosis; Idiopathic pulmonary fibrosis; Cystic fibrosis; Endophthalmitis; Persistent elevatum et diutinum erythema; erythroblastosis fetalis; eosinophilic fasciitis; Charmant's syndrome; Felty's syndrome; filariasis; cyclitis such as chronic cyclitis, heterochromic iritis, iridocyclitis, or Fuchs' cyclitis; Henoch-Schönlein purpura; human immunodeficiency virus (HIV) infection; echovirus infection; cardiomyopathy; Alzheimer's disease; parvovirus infection; rubella virus infection; post-vaccination syndrome; congenital rubella infection; Epstein-Barr virus infection; mumps; Evans syndrome; autoimmune dysgonadism; Sydenham's chorea; poststreptococcal nephritis; thromboangitis ubiterans); thyrotoxicosis; tabes dorsalis; choroiditis; giant cell polymyalgia; endocrine ophthalmopathy; chronic hypersensitivity pneumonitis; conjunctivitis sicca; epidemic keratoconjunctivitis; idiopathic nephritic syndrome; minimal change nephropathy; benign familial and ischemia-reperfusion injury; retinal autoimmunity; arthritis; bronchitis; chronic obstructive airway disease; silicosis; aphthous stomatitis; arteriosclerotic disorders, aspermiogenesis; autoimmune hemolysis; Beck's disease; cryoglobulinemia; Dupuytren's contracture; lenticular hypersensitivity endophthalmitis; allergic enteritis (enteritis allergica); erythema nodosum leprosum; idiopathic facial nerve palsy; chronic fatigue syndrome; rheumatic fever (febris rheumatica); Hamann-Rich syndrome; sensorineural hearing loss; paroxysmal hemoglobinuria paroxysmatica); hypogonadism; regional ileitis (ileitis regionalis); leukopenia; infectious mononucleosis (mononucleosis infectiosa); transverse myelitis; primary idiopathic myxedema; nephrosis; sympathetic ophthalmia; granulomatous orchitis (orchitis granulomatosa); pancreatitis; acute polyradiculitis (polyradiculitis acuta); pyoderma gangrenosum; Quervain's thyroiditis; acquired splenic atrophy;Infertility due to antispermatozoan antibodies; nonmalignant thymoma; vitiligo; severe combined immunodeficiency (SCID) and Epstein-Barr virus-related diseases; acquired immunodeficiency syndrome (AIDS); parasitic diseases such as leishmaniasis; toxic shock syndrome; food poisoning; conditions involving T cell infiltration; leukocyte adhesion deficiency; immune responses related to cytokines and T lymphocyte-mediated acute and delayed hypersensitivity; diseases involving leukocyte emigration; multiple organ injury syndrome; antigen-antibody complex-mediated diseases; antiglomerular basement membrane diseases; allergic neuritis; autoimmune polyendocrinopathy; oophoritis; primary myxedema; autoimmune atrophic gastritis; sympathetic ophthalmia; rheumatic diseases; mixed connective tissue disease; Nephropathy Rothe's syndrome; insulitis; polyglandular deficiency; peripheral neuropathy; autoimmune polyglandular syndrome type I; adult-onset idiopathic hypoparathyroidism (AOIH); alopecia totalis; dilated cardiomyopathy; epidermolysis bullosa acquisita (EBA); hemochromatosis; myocarditis; nephrotic syndrome; primary sclerosing cholangitis; suppurative or non-suppurative sinusitis; acute or chronic sinusitis; sinusitis (ethmoid, frontal, maxillary, or sphenoid); autoimmune bullous disease; eosinophilia; idiopathic hypereosinophilic syndrome; eosinophilic pulmonary infiltrate; eosinophilia-myalgia syndrome; Löffler's syndrome; chronic eosinophilic pneumonia; tropical pulmonary eosinophilia; bronchopulmonary aspergillosis aspergillosis; aspergilloma; or eosinophil-related disorders such as eosinophil-containing granulomas; anaphylaxis; seronegative spondyloarthritis spondyloarthritides); polyglandular autoimmune disease; sclerosing cholangitis; scleritis; episcleritis; chronic mucocutaneous candidiasis; Bruton's syndrome; transient hypogammaglobulinemia of infancy; Wiskott-Aldrich syndrome; ataxia-telangiectasia; autoimmune diseases associated with collagen diseases, rheumatism, neurological diseases, ischemia-reperfusion injury, hyporesponsiveness of blood pressure, vascular dysfunction, vasoectasia, tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and diseases involving angiogenesis; allergic hypersensitivity disorders; glomerulonephritis (glomerulonephritides); reperfusion injury; reperfusion injury of the myocardium or other tissues; acute inflammatory components; skin diseases accompanied by acute purulent meningitis or other central nervous system inflammatory disorders; inflammatory disorders of the eye and orbit; granulocyte transfusion-associated syndrome;These include, but are not limited to, cytokine-induced toxicity; acute severe inflammation; chronic intractable inflammation; pyelitis; pulmonary fibrosis; diabetic retinopathy; diabetic large arteriopathy; endarterial hyperplasia; peptic ulcer; valvular inflammation; and endometriosis.

[0070] As used herein, an "allergic disease" refers to a disease or disorder in which an individual is hypersensitized to and mounts an immune response against a normally non-immunogenic substance. Allergic diseases are generally characterized by the activation of mast cells by IgE, which triggers an inflammatory response and can result in a variety of symptoms, ranging from benign symptoms such as a runny nose to life-threatening anaphylactic shock and death. Examples of allergic diseases include, but are not limited to, asthma (e.g., allergic asthma), allergic rhinitis (e.g., hay fever), allergic dermatitis (e.g., eczema), contact dermatitis, food allergies, and urticaria.

[0071] Those skilled in the art will appreciate that the above examples are not all inclusive and that a disease or disorder may fall into various categories, for example, asthma is both an allergic and an inflammatory disease, but is considered by some clinicians to be an autoimmune disease.

[0072] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers.

[0073] The term "precancerous" refers to a condition or growth that typically precedes or develops into cancer.

[0074] As used herein, the term "proliferative disorder" refers to a disease or disorder associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, colon cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), B-cell lymphoma, B-cell leukemia, multiple myeloma, renal cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and glioblastoma.

[0075] The term "tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0076] "Non-metastatic" means a cancer that is benign or confined to the primary site and has not spread into the lymphatic or vascular system or to tissues other than the primary site. Generally, a non-metastatic cancer is any cancer that is stage 0, stage I, or stage II, and sometimes stage III.

[0077] A "subject" is a vertebrate, preferably a mammal, and more preferably a human. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice, and rats.

[0078] II. Generation of bispecific antibodies Heavy chain variable domain (V H ) contains significantly higher sequence diversity, and the light chain variable domain (V L ) contribute more to the determinants of antigen recognition than V. Our previous results demonstrate for the first time that it is possible to engineer light chain variable domains to create a single antibody with bispecificity (see US Patent Application Publication No. 20080069820 and Bostrom et al., Science 232:1610-1614 (2009), which are incorporated herein by reference in their entireties. We believe that V H By modifying the amino acid residues of V LV, which is important for antigen recognition, including the absence of mutations in L We have discovered that bispecific antibodies can be generated from antibodies containing these residues. These novel and unexpected methods are detailed below.

[0079] We are V L It was found that certain residues of V can be identified as being electrostatic or hydrophobic, which is L In such cases, the V of the antibody can be used to generate a bispecific antibody that binds both the first and second epitopes. H Specifically, if any one, two, or three of the amino acid residues at positions 32, 50, or 91 (according to Kabat numbering) of the antibody are electrostatic (e.g., tyrosine) or hydrophobic (e.g., tryptophan), the V H a nucleic acid sequence encoding one or more solvent accessible amino acid residues, H The codons are modified in one or more of the following ways:

[0080] In various embodiments, V H Residues that may be targeted for modification include any one or more of amino acids 33, 34, 50-58, or 95-97. Optionally, amino acid residues 93-96 of the light chain may be modified in addition to the heavy chain residues. For the heavy chain, solvent accessibility or importance for antigen recognition may be determined using standard techniques known in the art, including, but not limited to, structural mapping and alanine scanning mutagenesis.

[0081] Then V L and modified V H is expressed (e.g., as a library) and V L and modified V H A bispecific antibody, or antigen-binding fragment thereof, is selected that specifically binds to the first epitope and the second epitope. L is V HThe amino acid residues in the framework region of the first antibody may or may not be modified in addition to the modifications in V. H Modifications other than those mentioned above may or may not be made.

[0082] The bispecific antibody or antigen-binding fragment thereof identified by the above selection method may be further modified, for example, by affinity maturation or other methods known in the art, to enhance its affinity for one or both target antigens. The affinity maturation selection process may include applying massively parallel sequencing methods (e.g., deep sequencing, ultra-deep sequencing, or next-generation sequencing) to identify residue(s) (e.g., solvent-exposed or non-solvent-exposed residues) that contribute to binding to one or both target antigens (e.g., contributing to high affinity for the target antigen). See, for example, Fowler et al. Nat. Methods. 7(9):741-746, 2010. Bispecific antibodies may also be modified to improve stability or half-life, or to reduce immunogenicity. Such modifications are known to those skilled in the art.

[0083] III. Therapeutic Uses The bispecific antibodies, or antigen-binding fragments thereof, described herein bind to both IL4 and IL5 (e.g., B1, E7, and E7 affinity matured variants) or both IL4 and IL13 (e.g., F1 and F2) and can be used to treat, inhibit, or prevent diseases such as allergic, inflammatory, and autoimmune diseases (e.g., asthma); IL4-mediated diseases; IL5-mediated diseases; IL13-mediated diseases; IL4 / IL5-mediated diseases; IL4 / IL13-mediated diseases; and / or proliferative disorders (e.g., cancer).

[0084] Examples of inflammatory and autoimmune diseases or disorders that can be treated with bispecific antibodies, or antigen-binding fragments thereof, are described above. In some embodiments, the disease or disorder includes, but is not limited to, asthma, such as asthma-bronchitis, bronchial asthma, and autoimmune asthma.

[0085] Asthma is described as a chronic lung disease characterized by airway inflammation, hyperresponsiveness, and obstruction. Physiologically, airway hyperresponsiveness is evidenced by reduced bronchial airflow following bronchial provocation with methacholine or histamine. Other triggers of airway obstruction include cold air, exercise, viral upper respiratory tract infections, smoking, and respiratory allergens. Bronchial provocation with allergens immediately induces an immunoglobulin E (IgE)-mediated early-phase airflow reduction, and in most patients, an IgE-mediated delayed-phase response persists, resulting in reduced bronchial airflow for 4 to 8 hours. The immediate response is caused by the rapid release of inflammatory substances such as histamine, PGD2, leukotrienes, tryptase, and platelet-activating factor (PAF), whereas the delayed response is caused by the de novo synthesis of proinflammatory cytokines (e.g., TNFα, IL4, IL13) and chemokines (e.g., MCP-1 and MIP-1α) (Busse et al. In: Allergy: Principles and Practice, Ed. Middleston, 1173 (1998)). In chronic asthmatics, persistent pulmonary symptoms are mediated by an enhanced Th2 cell response. Th2 cytokines, particularly IL-13 and IL-4 produced by Th2 cells with an NK phenotype (NKT) in the airways, are thought to play an important role in the disease, as demonstrated in rodent models of asthma (Akbari et al., Nature Med., 9:582 (2003)). These cytokines are produced by Th2 cells with an NK phenotype (NKT) in the airways (Larche et al., J. Allergy Clin. Immunol., 111:450 (2003)). The overall pathology of the asthmatic airways is characterized by lung hyperinflation, smooth muscle hypertrophy, thickening of the reticular layer, mucosal edema, epithelial cell desquamation, ciliated cell destruction, and mucus gland hypersecretion. Microscopically, asthma is characterized by increased numbers of eosinophils, neutrophils, lymphocytes, and plasma cells in bronchial tissue, bronchial secretions, and mucus. Initially, activated CD4+ T lymphocytes recruit leukocytes from the circulation to the airways. Activated T lymphocytes also induce the release of inflammatory mediators from eosinophils, mast cells, and lymphocytes. Additionally, Th2 cells produce IL4, IL5, IL9, and IL13.IL4, together with IL13, signals the switch from IgM to IgE antibodies.

[0086] When allergens crosslink membrane-bound IgE molecules, mast cells degranulate, releasing histamine, leukotrienes, and other mediators that perpetuate airway inflammation. IL-5 stimulates the recruitment and activation of eosinophils. Activated mast cells and eosinophils also produce their own cytokines, which help sustain inflammation. Repeated cycles of this inflammation in the lungs, involving damage to lung tissue followed by its repair, can lead to long-term structural changes ("remodeling") of the airways.

[0087] Moderate asthma is currently treated with daily inhaled anti-inflammatory corticosteroids or mast cell inhibitors, such as cromolyn sodium or nedocromil, plus inhaled beta-2 agonists as needed (3–4 times daily) to relieve breakthrough symptoms, i.e., allergen- or exercise-induced asthma. Cromolyn sodium and nedocromil block bronchospasm and inflammation, but are usually only effective in allergen- or exercise-related asthma, typically in younger asthmatics. Inhaled corticosteroids improve inflammation, airway hyperresponsiveness, and obstruction, reducing the number of acute exacerbations. However, it takes at least one month for a clear effect to appear, and up to one year for significant improvement. The most common side effects are hoarseness and oral fungal infections, i.e., candidiasis. More serious side effects, such as partial adrenal suppression, growth inhibition, and decreased bone formation, have been reported, but only at high doses. Beclomethasone, triamcinolone, and flunisolide are likely to be similar in efficacy, whereas budesonide and fluticasone are reported to be more potent and have fewer systemic side effects.

[0088] Even patients with mild disease exhibit airway inflammation, including infiltration of mucosal membranes and epithelium by activated T cells, mast cells, and eosinophils. T cells and mast cells release cytokines that promote eosinophil proliferation and maturation and IgE antibody production. These cytokines, in turn, increase microvascular permeability, disrupt epithelium, and stimulate neural reflexes and mucus-secreting glands. The result is airway hyperresponsiveness, bronchoconstriction, and hypersecretion, manifested as wheezing, coughing, and dyspnea.

[0089] Traditionally, asthma has been treated with oral and inhaled bronchodilators. While these medications can help with asthma symptoms, they have no effect on the underlying inflammation. Over the past decade, recognition of the importance of inflammation in the pathogenesis of asthma has led to increased use of corticosteroids, but many patients still suffer from uncontrolled asthma.

[0090] Bispecific antibodies, or antigen-binding fragments thereof, having specificity for IL4, IL5, and / or IL13 appear to target multiple pathogenic pathways and can be used as therapeutic agents for the treatment of asthma, either alone or in combination with other therapies (e.g., known in the art or described above).

[0091] In another embodiment, bispecific antibodies, or antigen-binding fragments thereof, may be used to treat cancer. The term cancer collectively encompasses proliferative disorders, including, but not limited to, precancerous growths, benign tumors, and malignant tumors. Benign tumors remain localized to the site of their origin and lack the ability to infiltrate, invade, or metastasize to distant sites. Malignant tumors invade and damage other tissues in their vicinity. Malignant tumors can also acquire the ability to break away from their site of origin and spread (metastasize) to other parts of the body, usually via the blood circulation or the lymphatic system where lymph nodes are located. Primary tumors are classified by the type of tissue from which they originate, and metastatic tumors are classified by the type of tissue from which the cancer cells originate. Over time, cells in malignant tumors become more abnormal and less like normal cells. This change in appearance of cancer cells is called tumor grade, and cancer cells are described as well-differentiated, moderately differentiated, poorly differentiated, or undifferentiated. A fully differentiated cell is completely normal in appearance and resembles the normal cell from which it originates. An undifferentiated cell is one that has become so abnormal that it is no longer possible to determine its origin.

[0092] IV. Dosage and Formulation Antibody or antibody fragment compositions are formulated, dosed, and administered in accordance with good medical practice. Factors to be considered in this context include the particular disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of drug delivery, the method of administration, the administration schedule, and other factors known to physicians. The "therapeutically effective amount" of the antibody or antibody fragment to be administered will be determined taking such factors into consideration and will be the minimum amount necessary to prevent, ameliorate, or treat an allergic, inflammatory, autoimmune, or proliferative disease or disorder, or a symptom thereof. The dosage and timing of administration of the antibody or antibody fragment of the present invention will depend on various clinical factors, such as the subject's overall health and condition, e.g., the severity of symptoms of an allergic disorder. The present invention includes the use of antibodies or antibody fragments to treat, prevent, or reduce an allergic disorder or its symptoms, or the risk of developing an allergic disorder, in a subject. The antibody or antibody fragment can be administered at any time, for example, after an allergic disorder or a condition associated with an allergic disorder has been diagnosed or detected, or after a subject has been determined to be at risk for developing an allergic disorder to prevent an allergic disorder in a subject who has not yet been diagnosed with an allergic disorder but is at risk for developing such a disorder (e.g., a subject suffering from or being treated for an immunodeficiency).

[0093] The bispecific antibodies of the present invention, or antigen-binding fragments thereof, can be formulated and administered in a variety of ways, e.g., by routes known for the particular indication, including, but not limited to, inhalation, topical, oral, subcutaneous, bronchial injection, intravenous, intracerebral, nasal, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, intra-arterial, intracerebrospinal, intra-articular, intrasynovial, intralesional, parenteral, intracerebroventricular, or intraocular. For example, the antibody or antibody fragment can be in the form of a pill, tablet, capsule, liquid, or sustained-release tablet for oral administration; or a liquid for intravenous, subcutaneous, or other administration; a polymeric or other sustained-release vehicle for topical administration; or an ointment, cream, gel, liquid, or patch for topical administration.

[0094] Local administration may be particularly desirable when extensive side effects or toxicity are associated with IL4, IL5, or IL13 antagonism. Ex vivo approaches can also be used for therapeutic applications. In ex vivo approaches, cells obtained from a subject are transfected or transduced with a polynucleotide encoding the bispecific antibody or its antigen-binding fragment. The transfected or transduced cells are then returned to the subject. The cells can be of any of a wide variety of types, including, but not limited to, hematopoietic cells (e.g., bone marrow cells, macrophages, monocytes, dendritic cells, T cells, or B cells), fibroblasts, epithelial cells, endothelial cells, keratinocytes, or muscle cells.

[0095] For example, continuous systemic infusion or periodic injection / infusion of bispecific antibodies or antigen-binding fragments thereof can be used to treat or prevent disorders. Treatment can be continued for periods ranging from one day to the lifespan of the subject, more preferably 1-100 days, most preferably 1-20 days, and most preferably until the allergic, inflammatory, autoimmune, or proliferative disease or disorder, or its symptoms, is alleviated or eliminated. This varies depending on the dose, compound, and severity of the condition. Bispecific antibodies or antigen-binding fragments thereof can be administered continuously by infusion using an implantable pump with a constant or programmable flow rate, or by periodic injection. Sustained-release systems can also be used. In certain circumstances, semipermeable, implantable membrane devices are also useful as a means of delivering bispecific antibodies or antigen-binding fragments thereof. In another embodiment, bispecific antibodies or antigen-binding fragments thereof can be administered locally, e.g., by inhalation, and periodically repeated. Pulmonary administration is also possible, e.g., using an inhaler or nebulizer, or a formulation containing an aerosolizing agent. The antibody may also be administered to the patient's lungs in the form of a dry powder composition (see, eg, US Pat. No. 6,514,496).

[0096] The dosage of the bispecific antibody, or antigen-binding fragment thereof, will depend on other clinical factors, such as the subject's weight and condition, as well as the route of administration of the compound. For treatment of a subject, approximately 0.1 mg / kg to 500 mg / kg of body weight of the bispecific antibody, or antigen-binding fragment thereof, may be administered. A more preferred range is 1 mg / kg to 50 mg / kg of body weight, with the most preferred range being 1 mg / kg to 25 mg / kg of body weight. Depending on the half-life of the antibody or antibody fragment in a particular subject, the antibody or antibody fragment may be administered several times daily to once weekly. The methods of the present invention provide for single and multiple administrations, given either simultaneously or over an extended period of time.

[0097] Preferably, the bispecific antibody or its antigen-binding fragment is administered parenterally or intravenously by continuous infusion, or locally by inhalation. The dosage and administration regimen depend on the severity of the disease and the overall health of the subject. For parenteral administration, the bispecific antibody or its antigen-binding fragment is formulated with a pharmacologically acceptable parenteral vehicle in an injectable unit dosage form (solution, suspension, emulsion). Such vehicles are essentially non-toxic and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as fixed oils and ethyl oleate may also be used. Liposomes may be used as carriers. The vehicle may contain small amounts of additives, such as substances that enhance isotonicity and chemical stability, such as buffers and preservatives. The bispecific antibody or its antigen-binding fragment is typically formulated in such a vehicle at a concentration of about 1 mg / ml to 10 mg / ml. For inhalation administration, the bispecific antibody or antigen-binding fragment thereof may be formulated in any suitable manner to form the aerosol form of the present invention. Compositions containing the bispecific antibody or antigen-binding fragment thereof may be vaporized or nebulized, for example, using an inhaler, with or without a pharmacologically acceptable excipient, to generate a vapor that condenses and can be inhaled directly into the lungs. The pharmacologically acceptable excipient may be volatile; such excipient classes are known in the art and include, but are not limited to, gaseous, supercritical fluid, liquid, and solid solvents. Exemplary carriers within these classes include, but are not limited to, water, sterile saline, physiological buffers such as phosphate-buffered saline, terpenes, alcohols, propylene glycol, glycerin, and other similar alcohols, dry ice, dimethylformamide, dimethylacetamide, supercritical carbon dioxide, and mixtures thereof.

[0098] The required dosage will depend on the choice of route of administration, the nature of the formulation, the nature of the subject's illness, the subject's size, weight, body surface area, age, and sex, other administered medications, and the judgment of the attending physician. It is expected that the required dosage will vary widely, given the types of polypeptides and fragments available and the varying efficiencies of various administration routes. For example, oral administration may require higher dosages than administration by intravenous injection. These variations in dosage levels can be adjusted and optimized using standard empirical routines and are conventionally recognized. Administration may be single or multiple (e.g., 2, 3, 6, 8, 10, 20, 50, 100, 150, or more) doses. Encapsulating the polypeptide in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may enhance the effectiveness of delivery, particularly oral delivery.

[0099] In one embodiment for the treatment of asthma, the bispecific antibody, or antigen-binding fragment thereof, is optionally, but not necessarily, formulated or administered in combination (simultaneously or sequentially) with one or more drugs currently used to prevent or treat asthma or the risk of developing asthma. The antibody, or antigen-binding fragment thereof, can be formulated with, for example, an IgE antagonist, a bronchodilator (e.g., a beta-2-adrenergic receptor agonist, a xanthine, a cholinergic receptor antagonist), an anti-inflammatory agent (e.g., an antihistamine such as dicromoglycate sodium (DSCG), nedocromil sodium, or ketotifen, or a corticosteroid such as prednisolone), theophylline, salbutamol, beclomethasone dipropionate, or another asthma medication known in the art. The effective amount of such other drug will depend on the amount of bispecific antibody, or antigen-binding fragment thereof, in the formulation, the type of disorder or treatment, and other factors discussed above.

[0100] Therapeutic formulations are prepared by mixing the active ingredient having the desired purity with optional physiologically acceptable carriers, excipients, or stabilizers using standard methods known in the art (Remington's Pharmaceutical Sciences (2010)). th(Edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA). Acceptable carriers include saline or buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or PEG.

[0101] Optionally, but preferably, the formulation contains a pharmacologically acceptable salt, preferably sodium chloride, preferably at near-physiological concentrations. Optionally, the formulation of the present invention may contain a pharmacologically acceptable preservative. In some embodiments, the preservative concentration is 0.1-2.0%, typically in the range of 0.1-2.0 v / v. Suitable preservatives include those known in the pharmaceutical arts. Benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben are exemplary preservatives. Optionally, the formulation of the present invention may contain a pharmacologically acceptable surfactant at a concentration of 0.005-0.02%.

[0102] The formulations herein may also contain more than one active compound as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other, and such molecules are suitably present in combination in amounts that are effective for the purpose intended.

[0103] The active ingredient may also be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

[0104] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide (US Patent No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. If encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and altered immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is found to be the formation of intermolecular S—S bonds via thio-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solution, controlling the water content, using appropriate additives, and developing specific polymer matrix compositions.

[0105] In one example, the bispecific antibody, or antigen-binding fragment thereof, is administered locally, for example, by direct injection, which may be repeated periodically, or by inhalation, as the condition permits. The bispecific antibody, or antigen-binding fragment thereof, may be delivered to the subject systemically or directly to the affected area.

[0106] The present invention also provides a composition comprising a bispecific antibody, or an antigen-binding fragment thereof, and a pharmacologically acceptable carrier or diluent. This therapeutic composition is sterile and may be lyophilized. It is also contemplated that the bispecific antibody, or antigen-binding fragment thereof, of the present invention can be used in the manufacture of a medicament for treating the indications described herein. The composition can further comprise a second therapeutic agent, such as an anti-asthmatic agent, an anti-inflammatory agent, or an anti-proliferative agent (e.g., a chemotherapeutic agent, a cytotoxic agent, or an anti-angiogenic agent).

[0107] V. Manufactured Articles and Kits Another embodiment of the present invention is an article of manufacture comprising materials useful for treating a disease or disorder (e.g., an allergic disease or disorder or asthma). Yet another embodiment of the present invention is an article of manufacture comprising materials useful for treating inflammatory, autoimmune, and proliferative diseases or disorders. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition effective for treating a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a bispecific antibody or antigen-binding fragment of the present invention. The label or package insert indicates that the composition is used to treat a particular disease. The label or package insert will further include instructions for administering the antibody composition to a patient. Articles of manufacture and kits containing the combination therapies described herein are also contemplated.

[0108] Package insert refers to instructions customarily included in commercial packaging of a therapeutic product, which contain information about the indications, uses, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic product. In one embodiment, the package insert indicates that the composition is used to treat asthma.

[0109] Additionally, the article of manufacture may include a second container containing a pharmacologically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0110] Kits useful for various purposes, such as purifying or immunoprecipitating IL4, IL5, or IL13 from cells, are also provided. For isolation or purification of IL4, IL5, or IL13, the kits may contain IL4 / IL5 or IL4 / IL13 antibodies coupled to beads (e.g., Sepharose beads). Kits may be provided that contain antibodies for in vitro detection and quantitation of IL4, IL5, or IL13, for example, in ELISA or Western blot. Similar to an article of manufacture, the kit comprises a container and a label or package insert on or associated with the container. The container holds a composition comprising at least one bispecific or multispecific antibody or antibody fragment of the invention. Separate containers containing, for example, diluents and buffers or control antibodies may be included. The label or package insert may provide a description of the composition and instructions for its intended in vitro or diagnostic use.

[0111] The following examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various other modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[0112] Example Commercially available reagents referred to in the examples below were used according to manufacturer's instructions unless otherwise specified. The source of cells identified by ATCC accession numbers in the following examples and throughout the specification is the American Type Culture Collection, Manassas, VA. Unless otherwise noted, the present invention employs standard procedures of recombinant DNA technology as described herein and in the following text. Sambrook et al., supra; Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience,NY,1989);Innis et al., PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc.:NY,1990);Harlow et al.. Antibodies: A Laboratory Manual (Cold Spring Harbor Press:Cold Spring Harbor,1988);Gait, Oligonucleotide Synthesis (IRL Press:Oxford,1984);Freshney, Animal Cell Culture ,1987;Coligan et al. Current Protocols In Immunology, 1991.

[0113] Example 1. Library design and construction Mutations in the light chain (LC) complementarity-determining regions (CDRs) allow monospecific antibodies to recruit a second binding specificity to a new antigen while retaining their primary antigen specificity. Furthermore, dual binding can be achieved with low nanomolar K D The affinity can be matured to a maximum of 1000 kDa. This process of engineering a bispecific antibody is suitable for antibodies that primarily utilize the heavy chain (HC) CDRs to bind their primary antigen. Here, we describe the murine hybridoma-derived anti-interleukin-4 antibody, which relies heavily on the LC CDR for binding energy, and the evolution of this antibody into a bispecific antibody with mutations in the HC CDR alone or additional mutations in the LC CDR. Such bispecific variants for interleukin-4 (IL4) and interleukin-5 (IL5) are described in K. DThe results further highlight the ability of antibodies to evolve into bispecifics and demonstrate a generally applicable engineering approach to generate bispecificity in any antibody, using mutational scanning to identify regions of the antigen-binding site that may tolerate mutations and allow evolution to a second binding specificity.

[0114] We set out to mobilize a second antigen specificity into the humanized antibody 19C11 (hu19C11, humanized in the germline VH1 and kappa I framework). This antibody binds IL4 and blocks its binding to the IL4 receptor α. We first cloned the Fab into a phagemid construct (pV0115) and coexpressed the LC and, bicistronically, the variable domains and constant domain 1 (CH1) of the HC fused C-terminally to the M13 minor coat protein p3 as described (Lee et al., J. Mol. Biol. 340:1073-93, 2004). A portion of the hinge region of IgG, with the amino acid sequence KTHTC, was included between CH1 and M13 p3 to display bivalent Fab (Lee et al., J. Mol. Biol. 340:1073-93, 2004), enhancing binding efficiency to antigens immobilized on solid surface supports. We first demonstrated that Fab-displaying phage bound antibodies bearing an expression tag (gD) fused to LC at the C-terminus and IL4 with high affinity (phage EC) similar to that measured for the antibody as IgG. 50 = 1 nM) and verified successful binding.

[0115] To determine how to engineer a second binding specificity into hu19C11, we first investigated the importance of its three LC CDRs for IL4 binding by mutating two or three residues in each LC CDR to alanine. These LC CDR positions were selected because they were previously identified as key positions for combinatorial mutagenesis of LC libraries to select bispecific clones from antibodies that rely heavily on the HC CDRs for primary antigen binding (Bostrom et al. PLoS One. 6:e17887, 2011). Phage-displayed hu19C11 wild-type and alanine mutants L1 (I30A / N31A / D32A), L2 (Y50A / H53A / R54A), or L3 (D91A / Y92A) were immobilized on anti-gD antibodies (Figure 1A) or IL4 (Figure 1B) and assayed for binding using ELISA. We found that the LC CDRs appear to be energetically important. The phage-displayed Fabs containing alanine mutations in all three LC CDRs showed no detectable IL4 binding, but they bound to anti-gD antibodies, indicating that these mutants were indeed displayed on phage and that their IL4 binding was severely disrupted, albeit at a lower level than the wild-type Fab (Figures 1A and 1B). Therefore, the generation of randomized LC CDR libraries is unlikely to yield bispecific antibodies due to the extremely limited number of amino acid residues available for generating secondary antigen binding sites. Next, we mutated individual key residues in the LC CDRs and surface-accessible HC CDRs as previously described (Sidhu et al. J Mol Biol. 338:299-310, 2004; Lee et al. J Mol Biol. 340:1073-1093, 2004) and assessed their role in IL4 binding by measuring the relative binding affinities of these alanine mutants compared to wild-type hu19C11. Results confirmed the importance of these key LC CDR residues for IL4 binding, although CDR H2 and half of the tested positions in CDRs H1 and H3 all appeared to tolerate mutations (Figure 1C).

[0116] Mapping residues critical for IL4 binding (LC residues 30, 31, 32, 50, 53, 54, 91, 92; HC residues 31, 32, 98, 99 (Kabat numbering)) onto the model structure of trastuzumab Fab (PDB: 1FDV) identified a region concentrated in CDR H2 that was tolerant of mutations and may be suitable for generating secondary specificities (Figure 2A). Next, to focus randomization on CDR H2 for the mutagenesis scheme, we selected a set of residues in CDRs H1 (33, 34), H2 (50-58), H3 (95-97), and L3 (93-96) that tolerated alanine mutations, and used a set of synthetic oligonucleotides to randomize these selected residues in each of the four CDRs according to the mutagenesis method of Kunkel et al. (Kunkel et al., Methods Enzymol. 154:367-82, 1987). The randomization scheme was guided by the diversity of natural antibodies with respect to the variability in amino acid composition and length of CDR H2 and CDR L3 (Figure 2B). The mutagenesis template contained a stop codon only in CDR H2, thereby ensuring the mutation of this CDR in the Fab display library. Each oligonucleotide had a size of 10 8 ~10 9 Ten phage display libraries ranging in size from 1 to 10 were generated. Phage titration of hu19C11 and 10 HC libraries (2144-1 to 2144-10) bound to immobilized IL4 (PeproTech) captured with a non-blocking anti-IL4 antibody revealed that each library as a pool exhibited low levels of IL4 binding, generally significantly lower than the template antibody hu19C11, and that each library disrupted IL4 binding to different levels on average (Figure 3).

[0117] We selected IL5 and IL13 as the second antigens. These two interleukins, like IL4, belong to the four-helix bundle cytokine family, but they are highly divergent in amino acid sequence and structural organization (LaPorte et al. Cell. 132:259-272, 2008; Patino et al. Structure. 19:1864-1875, 2011; Finkelman et al. J Immunol. 184:1663-1674, 2010). IL4 and IL13 share 12% sequence identity, while IL4 and IL5 share 11%. Structurally, IL5 forms a unique intertwined homodimer in which the α-helix of one chain intertwines with three α-helices of the other chain to form a four-helix bundle (Milburn et al. Nature. 363:172-176, 1993; Patino et al. Structure. 19:1864-1875, 2011). IL4 and IL13 are both monomers. However, the three cytokines are related in their biological functions, and bispecific antibodies that bind and block two of these cytokines are likely to be useful as therapeutic agents for allergic diseases such as asthma (Finkelman et al. J Immunol. 184:1663-1674, 2010; Haldar et al. N Engl J Med. 360:973-984, 2009).

[0118] We performed several rounds of panning and enrichment of IL5- or IL13-binding clones that maintained IL4 binding from a phage display library constructed as described previously (Bostrom et al. Science. 323:1610-1614, 2009). By screening approximately 100 clones each, we found three to eight clones that exhibited dual binding to IL5 / IL4 or IL13 / IL4, respectively. Furthermore, sequencing identified two unique IL4 / IL5-binding clones (B1, E7) and two unique IL4 / IL13-binding clones (F1, F2) (Figure 2B). Furthermore, we isolated a clone that bound only to IL5 (e.g., clone 5A). With the exception of clone B1, all clones contained mutations only in the HC CDRs compared to their monospecific parental templates (Figures 4A-4C). This demonstrates that bispecificity can be conferred by mutations in the HC CDRs of monospecific antibodies. We performed phage binding competition assays and predicted clone affinity as the concentration of interleukin required to inhibit 50% of the binding of Fab-displaying phage to immobilized interleukin (IC50). Binding to the second antigen was weak, with IC50s in the micromolar range, whereas IL4 binding was maintained in the low nanomolar range.

[0119] Example 2. Evaluation of library performance To verify the bispecific binding specificity, clones B1, E7, F2, and 5A were expressed as IgGs, and each IgG was confirmed to bind to the desired antigen but not to several other proteins (Figure 5). Further binding specificity was examined by flow cytometry and ELISA, demonstrating minimal binding of the IgGs to the human epithelial kidney cell line 293, which does not express IL4, IL5, or IL13, and to baculovirus (BV) particles produced from an insect cell line (Hotzel et al. MAbs. 4:753-760, 2012). Furthermore, the IL4 / IL5 bispecific antibodies B1 and E7, along with a monospecific IL5-binding antibody, were shown to block IL5 binding to the IL5 receptor α, suggesting that the binding epitope on IL5 overlaps with that of the IL5 receptor (Figure 6). Surface plasmon resonance (SPR) measurements showed that the IL4 / IL5 bispecific clone E7 had low affinity for IL5 (K D = 905 nM), whereas IL4 binding was observed with the high affinity (K D =3.4 nM) was maintained (Figures 7A and 7B).

[0120] Example 3. Affinity maturation of bispecific antigen-binding fragments To improve the dual affinity of E7, we randomized the E7 CDRs by site-directed mutagenesis and displayed the variants on phage for binding selection. As previously described (Lee et al. J Mol Biol. 340:1073-1093, 2004; Bostrom et al. Methods Mol Biol. 525:1-24, 2009; Lee et al. Blood. 108:3103-3111, 2006), we generated three libraries targeting residues in CDR H2 and CDR L3 (H2 / L3 library), residues in CDR H1, H2, and H3 (H1 / H2 / H3 library), or residues in CDR H2, and selected a site in framework region 3 (FR3) of the HC for randomization (H2 / FR3 library) (Figure 7A). Because clone E7 maintained high affinity for IL4 binding, we focused library selection on improving IL5 binding. We found that many clones from the H1 / H2 / H3 library had improved affinity for IL5 but significantly reduced affinity for IL4, whereas clones from the H2 / L3 and H2 / FR3 libraries showed improved IL5 binding without loss of IL4 binding affinity (Figure 4A). Selected clones were purified as IgG and compared with E7. Many variants from the H2 / L3 library exhibited high dual binding as IgG without enhanced binding to a set of non-target proteins (Figure 8A). We further confirmed that off-target binding was low and that the improved clones still blocked IL5 binding to its receptor using BV binding and 293 cell FACS as described above (Figure 8B). The monovalent binding affinities of two improved variants, 1C36 and 1C60 (Figures 7C and 7D), were determined by SPR measurements as Fabs bound to immobilized IL4 or IL5. Both variants maintained high affinity for IL4 (K D =3-4 nM), and the affinity for IL-5 was improved by 37-fold and 20-fold, respectively (1C36 had a K D = 24.1 nM, and 1C60 = 44.4 nM) (Figure 7B).

[0121] In summary, we have demonstrated that mutations in the HC CDR of a monospecific antibody can mobilize a second binding specificity and also improved one isolated bispecific antibody, lowering its nM affinity for both antigens. Previously, we demonstrated that trastuzumab Fab generates bispecificity through mutations in the LC CDR. Since then, other bispecific antibodies have been created using the same LC approach. Together with the current findings, we highlight the potential for antibody binding specificity evolution. We found that antibodies can acquire additional binding specificities by making limited mutations in the CDRs not only on the light chain but also on the heavy chain. In nature, antibodies are constantly undergoing remodeling through gene shuffling and somatic mutation. It has been shown that antibodies can be "reused" by evolving through somatic mutation into antibodies with different binding properties and, therefore, different functions. Antibodies may possess one of the ideal folds and structures for generating binding specificities through limited mutation, which may play a role in the broad ability of the innate immune response to recognize an essentially limitless variety of foreign antigens. Furthermore, this work represents the first general engineering pathway for generating bispecific antibodies from any monospecific antibody, summarized below: Mutagenesis analysis (e.g., alanine scanning) allows for the initial identification of regions of the antigen-binding site that are tolerant to mutations without significantly disrupting binding of its primary antigen.

[0122] Other embodiments All patents, patent applications, patent application publications, and other publications cited or referenced in this specification are hereby incorporated by reference into this specification to the same extent as if each individual patent, patent application, patent application publication, or publication was specifically and individually indicated to be incorporated by reference herein.

Claims

1. Variable heavy chain domain (V Ｈ ) and a variable light chain domain (V Ｌ ), wherein the V of the bispecific antibody or antigen-binding fragment thereof is Ｈ and V Ｌ pair together to form an antigen binding site that specifically binds to the first epitope and the second epitope, and the method comprises (a) V Ｈ and V Ｌ providing an antibody comprising V Ｈ and V Ｌ pair together to form an antigen-binding site that binds to the first epitope but not the second epitope, and the antibody is Ｌ at least one electrostatic or hydrophobic amino acid at position 32, 50, or 91 of (b) the V of the antibody of step (a) Ｈ modifying a nucleic acid sequence encoding the amino acid sequence of (c) V Ｌ and V modified in step (b). Ｈ expressing the (d) V Ｌ and V modified in step (c). Ｈ selecting a bispecific antibody, or antigen-binding fragment thereof, comprising V Ｈ and V Ｌ pair together to form an antigen binding site that specifically binds to the first epitope and the second epitope. A method comprising:

2. 2. The method of claim 1, wherein at least two of the amino acids at positions 32, 50, or 91 are electrostatic or hydrophobic.

3. The method of claim 1, wherein all three amino acids at positions 32, 50 and 91 are electrostatic or hydrophobic.

4. The method of any one of claims 1 to 3, wherein the electrostatic residue is tyrosine.

5. The method according to any one of claims 1 to 3, wherein the hydrophobic residue is tryptophan.

6. V Ｈ The method of any one of claims 1 to 5, wherein the nucleic acid sequence encoding the heavy chain amino acid sequence is modified based on the diversity of multiple naturally occurring heavy chain amino acid sequences.

7. The position of the solvent-exposed residue is V Ｈ The method according to any one of claims 1 to 6, wherein the amino acid residue is selected from the group consisting of positions 33, 34, 50 to 58, and 95 to 97 of

8. V of the antibody of step (a) Ｌ 8. The method of any one of claims 1 to 7, further comprising modifying a nucleic acid sequence encoding: wherein one or more solvent accessible amino acid residues are modified.

9. The position of the solvent-exposed residue is V Ｌ The method according to claim 8, wherein the amino acid residue is selected from amino acids 93 to 96 of

10. During the selection of step (d), the modified V Ｈ V Ｌ The method according to any one of claims 1 to 9, wherein the antigen-binding domain is displayed on a phage together with

11. The method of any one of claims 1 to 10, wherein the antibody in step (a) comprises complementarity-determining regions of the light chain variable region, namely, CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO: 9), CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO: 10), and CDRL3 comprising the amino acid sequence QQDYTSPWTF (SEQ ID NO: 11).

12. The method of any one of claims 1 to 11, wherein the antibody in step (a) comprises complementarity-determining regions of a heavy chain variable region, namely, CDRH1 comprising the amino acid sequence DYSMH (SEQ ID NO: 13), CDRH2 comprising the amino acid sequence VWINTETGEPTYADDFK (SEQ ID NO: 17), and CDRH3 comprising the amino acid sequence GGIFYGMDY (SEQ ID NO: 20).

13. The method of any one of claims 1 to 12, wherein the antigen-binding site of the bispecific antibody in step (d) exclusively binds both the first epitope and the second epitope.

14. The method of any one of claims 1 to 12, wherein the antigen-binding site of the bispecific antibody of step (d) simultaneously binds to the first epitope and the second epitope.

15. The method of any one of claims 1 to 14, wherein the first epitope is derived from one biological molecule and the second epitope is derived from the same biological molecule.

16. The method of any one of claims 1 to 14, wherein the first epitope is derived from a first biological molecule and the second epitope is derived from a second biological molecule.

17. 17. The method of claim 16, wherein the first biological molecule and the second biological molecule are selected from the group consisting of IL4 / IL5, IL4 / IL13.

18. 17. The method of claim 16, wherein the first biological molecule and the second biological molecule are cytokines.

19. 17. The method of claim 16, wherein the first or second biological molecule is a molecule that is capable of extending the half-life of the bispecific antibody when bound to the antibody in vivo.

20. 17. The method of claim 16, wherein the first or second biological molecule is serum albumin or fetal Fc receptor (FcRn).

21. 17. The method of claim 16, wherein the first or second biological molecule is a molecule that can enhance the effector function of the bispecific antibody when bound to the antibody in vivo.

22. 17. The method of claim 16, wherein the first or second biological molecule binds to a cell surface protein on a natural killer cell or a macrophage.

23. 23. The method of claim 22, wherein the cell surface protein is an Fc receptor or C1q.

24. V of bispecific antibodies Ｈ and V Ｌ pair together to bind to the first epitope or the second epitope. －６ The following K Ｄ The method according to any one of claims 1 to 23, wherein an antigen-binding site that specifically binds is formed by

25. V of bispecific antibodies Ｈ and V Ｌ pair together to bind to the first epitope or the second epitope. －９ The following K Ｄ The method of claim 24, wherein the antigen-binding site specifically binds to the

26. V of bispecific antibodies Ｈ and V Ｌ pair together to bind to the first epitope or the second epitope. －１２ The following K Ｄ The method of claim 25, wherein the antigen-binding site specifically binds to the target polypeptide.

27. V of bispecific antibodies Ｈ and V Ｌ pair together to form a first epitope and a second epitope. －６ The following K Ｄ The method according to any one of claims 1 to 23, wherein an antigen-binding site that specifically binds is formed by

28. V of bispecific antibodies Ｈ and V Ｌ pair together to form a first epitope and a second epitope. －９ The following K Ｄ The method of claim 27, wherein the antigen-binding site specifically binds to the

29. V of bispecific antibodies Ｈ and V Ｌ pair together to form a first epitope and a second epitope. －１２ The following K Ｄ The method of claim 28, wherein the antigen-binding site specifically binds to the

30. 30. The method of any one of claims 1 to 29, wherein the first biological molecule and the second biological molecule are structurally dissimilar.

31. The method of any one of claims 1 to 30, wherein the selection step (d) comprises deep sequencing.

32. 10. An isolated bispecific antibody, or antigen-binding fragment thereof, produced by the method of claim 1.

33. 33. The isolated bispecific antibody of claim 32, wherein the bispecific antibody is a monoclonal antibody.

34. 33. The isolated bispecific antibody of claim 32, wherein the fragment is a Fab or an scFv.

35. 33. The isolated bispecific antibody of claim 32, wherein the bispecific antibody is an IgG.

36. An isolated bispecific antibody, or antigen-binding fragment thereof, comprising the amino acid sequence of any one of the antibodies of Figures 4A, 4B, 4C, 7A, 7C, or 7D.

37. The following six CDRs: (i) CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO: 9); (ii) a CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO: 10); (iii) a CDRL3 comprising the amino acid sequence QQDYTSPWTF (SEQ ID NO: 11); (iv) CDRH1 comprising the amino acid sequence DYDIH (SEQ ID NO: 14); (v) a CDRH2 comprising the amino acid sequence VWINTETGEPTYADDFK (SEQ ID NO: 17); and (vi) a CDRH3 comprising the amino acid sequence EILFYGMDY (SEQ ID NO: 21) 1. An isolated bispecific antibody, or an antigen-binding fragment thereof, comprising:

38. The following six CDRs: (i) CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO: 9); (ii) a CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO: 10); (iii) a CDRL3 comprising the amino acid sequence QQDYTSPWTF (SEQ ID NO: 11); (iv) CDRH1 comprising the amino acid sequence DYFIH (SEQ ID NO: 15); (v) a CDRH2 comprising the amino acid sequence AGIVYDATGFTTYADDFK (SEQ ID NO: 18), and (vi) a CDRH3 comprising the amino acid sequence GGIFYGMDY (SEQ ID NO: 20) 1. An isolated bispecific antibody, or an antigen-binding fragment thereof, comprising:

39. The following six CDRs: (i) CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO: 9); (ii) a CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO: 10); (iii) a CDRL3 comprising the amino acid sequence QQDYTPFPLTF (SEQ ID NO: 12); (iv) CDRH1 comprising the amino acid sequence DYLMH (SEQ ID NO: 16); (v) a CDRH2 comprising the amino acid sequence AVIVSITGRTYYADDFK (SEQ ID NO: 19); and (vi) a CDRH3 comprising the amino acid sequence GGIFYGMDY (SEQ ID NO: 20) 1. An isolated bispecific antibody, or an antigen-binding fragment thereof, comprising:

40. The following six CDRs: (i) CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO: 9); (ii) a CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO: 10); (iii) a CDRL3 comprising the amino acid sequence QQDYTSPWTF (SEQ ID NO: 11); (iv) CDRH1 comprising the amino acid sequence DYSMH (SEQ ID NO: 13); (v) a CDRH2 comprising the amino acid sequence GVIFQSGATYYADDFK (SEQ ID NO: 22); and (vi) a CDRH3 comprising the amino acid sequence GGIFYGMDY (SEQ ID NO: 20) 1. An isolated bispecific antibody, or an antigen-binding fragment thereof, comprising:

41. The following six CDRs: (i) CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO: 9); (ii) a CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO: 10); (iii) a CDRL3 comprising the amino acid sequence QQDYTSPWTF (SEQ ID NO: 11); (iv) CDRH1 comprising the amino acid sequence DYSMH (SEQ ID NO: 13); (v) a CDRH2 comprising the amino acid sequence GIIFYTGHTYYADDFK (SEQ ID NO: 23), and (vi) a CDRH3 comprising the amino acid sequence GGIFYGMDY (SEQ ID NO: 20) 1. An isolated bispecific antibody, or an antigen-binding fragment thereof, comprising:

42. The following six CDRs: (i) CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO: 9); (ii) a CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO: 10); (iii) the amino acid sequence QQDYX １ X ２ CDRL3 comprising PWTF (SEQ ID NO: 24), wherein X １ is Thr, Lie, Leu, or Lys; X ２ is Ser or His, (iv) CDRH1 comprising the amino acid sequence DYFIH (SEQ ID NO: 15); (v) amino acid sequence X １ GIVYDATGFTX ２ YAX ３ X ４ a CDRH2 comprising X １ is Ala or Gly, X ２ is Thr, Ie, Val, or Ala, X ３ is Asp, Val, or Glu, and X ４ is Asp, Glu, Asn, Ser, Lie, Leu, Thr, Ala, or Phe, and (vi) a CDRH3 comprising the amino acid sequence GGIFYGMDY (SEQ ID NO: 20) 1. An isolated bispecific antibody, or an antigen-binding fragment thereof, comprising:

43. The following six CDRs: (i) CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO: 9); (ii) a CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO: 10); (iii) a CDRL3 comprising the amino acid sequence QQDYTHPWTF (SEQ ID NO: 27); (iv) CDRH1 comprising the amino acid sequence DYFIH (SEQ ID NO: 15); (v) a CDRH2 comprising the amino acid sequence GGIVYDATGFTTYAEEFK (SEQ ID NO: 28); and (vi) a CDRH3 comprising the amino acid sequence GGIFYGMDY (SEQ ID NO: 20) 42. The isolated bispecific antibody of claim 41 , or an antigen-binding fragment thereof, comprising:

44. The following six CDRs: (i) CDRL1 comprising the amino acid sequence KASQSVINDAA (SEQ ID NO: 9); (ii) a CDRL2 comprising the amino acid sequence YTSHRYT (SEQ ID NO: 10); (iii) a CDRL3 comprising the amino acid sequence QQDYKHPWTF (SEQ ID NO: 31); (iv) CDRH1 comprising the amino acid sequence DYFIH (SEQ ID NO: 15); (v) a CDRH2 comprising the amino acid sequence AGIVYDATGFTVYADDFK (SEQ ID NO: 32); and (vi) a CDRH3 comprising the amino acid sequence GGIFYGMDY (SEQ ID NO: 20) 42. The isolated bispecific antibody of claim 41 , or an antigen-binding fragment thereof, comprising:

45. Amino acid sequence GRX １ TITX ２ DX ３ STSTX ４ (SEQ ID NO: 26), wherein X １ is Val or Phe, X ２ is Arg or lie, X ３ is Thr, Phe, Met, or Pro, and X ４ is Ala or Val.

46. An isolated bispecific antibody, or an antigen-binding fragment thereof, comprising a light chain variable region selected from the amino acid sequence of SEQ ID NO: 1, 5, 29, or 33 and a heavy chain variable region selected from the amino acid sequence of SEQ ID NO: 2, 3, 4, 6, 7, 8, 30, or 34.

47. 47. The isolated bispecific antibody, or antigen-binding fragment thereof, of any one of claims 38, 39, and 42-46, wherein the antibody, or antigen-binding fragment thereof, binds IL4 with a Kd of 500 nM or less and IL5 with a Kd of about 900 nM or less.

48. 47. The isolated bispecific antibody, or antigen-binding fragment thereof, of any one of claims 38, 39, and 42-46, wherein the antibody binds IL4 with a Kd of 100 nM or less and IL5 with a Kd of about 100 nM or less.

49. 47. The isolated bispecific antibody, or antigen-binding fragment thereof, of any one of claims 38, 39, and 42-46, wherein the antibody binds IL4 with a Kd of 10 nM or less and IL5 with a Kd of about 50 nM or less.

50. 47. The isolated bispecific antibody, or antigen-binding fragment thereof, of any one of claims 40, 41, and 46, wherein the antibody binds IL4 with a Kd of 500 nM or less and IL13 with a Kd of about 900 nM or less.

51. 47. The isolated bispecific antibody, or antigen-binding fragment thereof, of any one of claims 40, 41, and 46, wherein the antibody binds IL4 with a Kd of 100 nM or less and IL13 with a Kd of about 100 nM or less.

52. 52. The isolated bispecific antibody, or antigen-binding fragment thereof, of any one of claims 36 to 51, wherein the antibody inhibits or blocks the binding of IL4, IL5, or IL13 to its receptor.

53. 52. The isolated bispecific antibody, or antigen-binding fragment thereof, of any one of claims 36 to 51, wherein the antibody is a monoclonal antibody.

54. 52. The isolated bispecific antibody, or antigen-binding fragment thereof, of any one of claims 36 to 51, wherein the antibody is an IgG antibody.

55. 52. The isolated bispecific antibody, or antigen-binding fragment thereof, of any one of claims 36 to 51, wherein the fragment is a Fab fragment or a single-chain variable fragment (scFv).

56. 52. The isolated bispecific antibody, or antigen-binding fragment thereof, of any one of claims 36 to 51, wherein at least a portion of the framework sequences are human consensus framework sequences.

57. 52. The isolated bispecific antibody, or antigen-binding fragment thereof, of any one of claims 32 to 51, wherein the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.

58. A pharmaceutical composition comprising the antibody of any one of claims 32 to 57.

59. A polynucleotide encoding the isolated bispecific antibody, or antigen-binding fragment thereof, of any one of claims 32 to 57.

60. 60. A vector comprising the polynucleotide of claim 59.

61. 61. A host cell comprising the vector of claim 60.

62. 61. A method for producing the antibody, or antigen-binding fragment thereof, of any one of claims 32 to 57, comprising culturing a host cell comprising the vector of claim 60, and recovering the antibody.

63. 58. A method of treating asthma in a subject, comprising administering to the subject an antibody, or antibody fragment thereof, described in any one of claims 32 to 57, wherein the administration is for a duration and in an amount sufficient to treat or prevent the asthma in the subject.

64. 62. The method of claim 61, wherein the method further comprises administering at least one additional asthma medication selected from the group consisting of an IgE antagonist, an antihistamine, theophylline, salbutamol, beclomethasone dipropionate, sodium cromoglycate, a steroid, and an anti-inflammatory agent.

65. 65. The method of claim 63 or 64, wherein the asthma is allergic asthma.

66. 58. A method of treating a proliferative disorder in a subject, comprising administering to the subject an antibody, or antibody fragment thereof, of any one of claims 32 to 57, wherein said administration is for a duration and in an amount sufficient to treat or prevent said proliferative disorder in said subject.

67. 67. The method of claim 66, wherein the proliferative disorder is cancer.

68. 68. The method of claim 67, further comprising administering to the subject an additional antiproliferative agent selected from the group consisting of a chemotherapeutic agent, a cytotoxic agent, and an anti-angiogenic agent.

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