Method for identifying bacteria comprising binding polypeptides

Abstract

To provide a method for identifying bacteria comprising binding polypeptides.SOLUTION: There is provided a method for identifying bacteria comprising binding polypeptides that specifically bind to a target molecule, the method including the steps of: (a) providing bacteria having an outer membrane transmitting molecules having molecular weight exceeding 10 kDa, wherein the bacteria develop a nucleic acid coding candidate binding polypeptides, and the candidate binding polypeptides are present in the periplasm of the bacteria; (b) bringing the bacteria into contact with a target molecule labeled to enable detection of the bacteria; and (c) identifying the bacteria as including the binding polypeptides with the presence of the labeled target molecule in the periplasm, wherein the bacteria remain viable after the step (c).SELECTED DRAWING: None

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on April 20, 2016, is entitled 50474-102WO2_Sequence_Listing_4.20.16_ST25 and is 26,384 bytes in size.

[0002] FIELD OF THEINVENTION The present invention is directed to methods for identifying bacteria comprising binding polypeptides, methods for identifying binding polypeptides with improved expression, methods for identifying bacteria with improved expression of binding polypeptides, engineered bacteria suitable for use in the methods, compositions obtained using the methods, such as antibodies with improved expression and / or stability (e.g., anti-interleukin-13 (IL-13) antibodies and anti-vascular endothelial growth factor (VEGF) antibodies with improved expression and / or stability), and libraries comprising binding polypeptide (e.g., antibody) variants. [Background technology]

[0003] Display of binding polypeptides (e.g., antibodies or fragments thereof) on the surface of bacteriophages or cells (e.g., bacterial, yeast, or mammalian cells) is a widely used approach for protein engineering. While the rapid replication time of bacteria provides an accelerated round-by-round progression for bacterial display compared to other cell display techniques, these advantages are generally offset by the compromises required to retain candidate binding polypeptides within the cell and maintain antigen accessibility. It can be difficult to present large molecules as fusions to bacterial outer membrane proteins, limiting the use of these systems to peptides or small proteins. Another approach is the display of larger proteins in the periplasm. However, under most conditions, the outer membrane is impermeable to the diffusion of hydrophilic molecules with molecular weights above about 600 daltons into the periplasm (see, e.g., Decad et al. J. Bacteriology 128(1):325-336, 1976). Current approaches to periplasmic display of binding polypeptides to larger ligands involve removal of the outer membrane, which requires tethering the binding polypeptide to the outside of the inner membrane (e.g., by using a fusion protein or other tether) to maintain the association of the binding polypeptide with the bacterial inner membrane. Furthermore, disruption of the outer membrane results in cell death, which requires time-consuming molecular manipulation between rounds of bacterial display.

[0004] Thus, there remains a need for improved methods for live cell bacterial display that are compatible with full-length binding polypeptides (e.g., antibodies) and target molecules (e.g., antigens) that can be used, for example, to identify protein variants with desired characteristics (e.g., improved expression, stability, and / or affinity), as well as to identify bacteria with improved expression of the binding polypeptides. Summary of the Invention

[0005] The present invention is directed to methods for identifying bacteria harboring desired binding polypeptides, methods for identifying binding polypeptides with improved expression, methods for identifying bacteria with improved expression of binding polypeptides, engineered bacteria suitable for use in the methods, compositions obtained using the methods, such as antibodies (e.g., anti-IL-13 and anti-VEGF antibodies) with improved expression and / or stability, and libraries comprising binding polypeptide (e.g., antibody) variants.

[0006] In one aspect, the invention features a method for identifying bacteria that contain a binding polypeptide that specifically binds to a target molecule, the method comprising the steps of: (a) providing a bacterium with an outer membrane that is permeable to molecules having a molecular weight of more than 10 kDa, where the bacterium expresses a nucleic acid encoding a candidate binding polypeptide, and the candidate binding polypeptide is present in the periplasm of the bacterium; (b) contacting the bacterium with a detectably labeled target molecule; and (c) identifying the bacterium as containing the binding polypeptide by the presence of the labeled target molecule in the periplasm, where the bacterium remains viable after step (c). In some embodiments, the target molecule has a molecular weight of less than 250 kDa. In some embodiments, the target molecule has a molecular weight of less than 150 kDa.

[0007] In another aspect, the invention features a method for identifying bacteria having improved expression of a binding polypeptide that specifically binds to a target molecule, the method comprising the steps of: (a) providing a bacterium having an outer membrane that is permeable to molecules having a molecular weight of more than 10 kDa, where the bacterium expresses a nucleic acid encoding a binding polypeptide, and the binding polypeptide is present in the periplasm of the bacterium; (b) contacting the bacterium with a detectably labeled target molecule; and (c) identifying the bacterium as having improved expression of the binding polypeptide by the presence of the labeled target molecule in the periplasm, where the bacterium remains viable after step (c). In some embodiments, the target molecule has a molecular weight of less than 250 kDa. In some embodiments, the target molecule has a molecular weight of less than 150 kDa.

[0008] In another aspect, the invention features a method for identifying bacteria containing a binding polypeptide that specifically binds to a target molecule, the method including the steps of: (a) providing a bacterium containing a mutation that allows diffusion of a membrane impermeable molecule across the outer membrane into the periplasm, where the bacterium expresses a nucleic acid encoding a candidate binding polypeptide, and the candidate binding polypeptide is present in the periplasm of the bacterium; (b) contacting the bacterium with a detectably labeled target molecule; and (c) identifying the bacterium as containing the binding polypeptide by the presence of the labeled target molecule in the periplasm, where the bacterium remains viable after step (c).

[0009] In another aspect, the invention features a method for identifying bacteria having improved expression of a binding polypeptide that specifically binds to a target molecule, the method including the steps of: (a) providing a bacterium that includes a mutation that allows diffusion of a membrane impermeable molecule across an outer membrane into the periplasm, where the bacterium expresses a nucleic acid encoding the binding polypeptide, and the binding polypeptide is present in the periplasm of the bacterium; (b) contacting the bacterium with a detectably labeled target molecule; and (c) identifying the bacterium as having improved expression of the binding polypeptide by the presence of the labeled target molecule in the periplasm, where the bacterium remains viable after step (c).

[0010] In some embodiments of any of the aforementioned aspects, the method further comprises, prior to step (b), subjecting the bacterium to conditions that permeabilize the outer membrane of the bacterium. In some embodiments, the method further comprises resealing the outer membrane of the bacterium after contacting the bacterium with the detectably labeled target molecule.

[0011] In another aspect, the invention features a method for identifying a bacterium containing a binding polypeptide that specifically binds to a target molecule, the method including the steps of: (a) providing a bacterium expressing a nucleic acid encoding a candidate binding polypeptide, where the candidate binding polypeptide is present within the periplasm of the bacterium; (b) subjecting the bacterium to conditions that permeabilize the bacterial outer membrane; (c) contacting the bacterium with a detectably labeled target molecule; (d) after step (c), resealing the bacterial outer membrane; and (e) identifying the bacterium as containing the binding polypeptide due to the presence of the labeled target molecule in the periplasm. In another aspect, the invention features a method for identifying bacteria having improved expression of a binding polypeptide that specifically binds to a target molecule, the method including the steps of: (a) providing a bacterium expressing a nucleic acid encoding a binding polypeptide, wherein the binding polypeptide is present within the periplasm of the bacterium; (b) subjecting the bacterium to conditions that permeabilize the outer membrane of the bacterium; (c) contacting the bacterium with a detectably labeled target molecule; (d) after step (c), resealing the outer membrane of the bacterium; and (e) identifying the bacterium as having improved expression of the binding polypeptide due to the presence of the labeled target molecule in the periplasm.

[0012] In some embodiments of any of the aforementioned aspects, the method further comprises repeating the steps of the method at least once without an intervening step of isolating the nucleic acid, hi some embodiments, the method further comprises incubating the bacteria in a growth medium prior to repeating the method.

[0013] In some embodiments of any of the foregoing aspects, the bacterium comprises a mutation that allows diffusion of membrane impermeable molecules across the outer membrane into the periplasm. In some embodiments, the mutation is in a gene that encodes an outer membrane protein or a protein that affects lipopolysaccharide (LPS) composition. In some embodiments, the outer membrane protein is major outer membrane lipoprotein Lpp (Lpp), a porin, or TolC. In some embodiments, the protein that affects LPS composition is selected from RfaD, RfaE, RfaH, RfaRd, TolA, TolB, and TolD.

[0014] In some embodiments of any of the aforementioned aspects, subjecting the bacteria to conditions that permeabilize the outer membrane of the bacteria comprises treating the bacteria with a permeabilizing agent. In some embodiments, the permeabilizing agent is a divalent cation chelator, NaCl, sucrose, antibiotics, detergents, lysozyme, Tris, Tris-EDTA, ascorbate, polylysine, benzalkonium chloride, protamine, bactericidal / permeability enhancing protein (BPI), serum, complement, and Ca. 2+ In some embodiments, the divalent cation chelator is EDTA. In some embodiments, the antibiotic is selected from the group consisting of an aminoglycoside, polymyxin B, deacylated polymyxin, octapeptin, and benzylpenicillin.

[0015] In some embodiments of any of the aforementioned aspects, resealing the outer membrane of the bacterium comprises treating the bacterium with Mg 2+ , Mn 2+ , Ca 2+ , Zn 2+ , Fe 2+ , Na + , or K + In some embodiments, the cation is selected from the group consisting of Mg 2+ In some embodiments, Mg 2+ The salt is MgCl 2 It is.

[0016] In some embodiments of any of the aforementioned aspects, the contacting step further comprises contacting the bacteria with a nucleic acid dye. In some embodiments, the nucleic acid dye is selected from the group consisting of SYTO® 9, SYTO® 41, SYTO® 43, SYTO® 42, SYTO® 44, SYTO® 40, and SYTO® 45. In some embodiments, the nucleic acid dye is SYTO® 9 or SYTO® 41.

[0017] In some embodiments of any of the foregoing aspects, the detectably labeled target molecule comprises a fluorescent label. In some embodiments, the fluorescent label comprises a fluorescent dye or a fluorescent polypeptide. In some embodiments, the fluorescent dye is selected from the group consisting of an ALEXA® dye or a DYLIGHT® dye. In some embodiments, the ALEXA® dye is ALEXA FLUOR® 488 or ALEXA FLUOR® 647. In some embodiments, the DYLIGHT® dye is DYLIGHT® 649.

[0018] In some embodiments of any of the aforementioned aspects, the method further comprises at least one wash step comprising resuspending the bacteria in a wash buffer after contacting the bacteria with the detectably labeled target molecule.

[0019] In some embodiments of any of the foregoing aspects, the identifying step comprises flow cytometry. In some embodiments, the flow cytometry comprises sorting single cells and sorting based on a signal of a detectably labeled target molecule. In some embodiments, the method further comprises sorting based on a signal of a nucleic acid dye. In some embodiments, the flow cytometry sequentially comprises (i) sorting based on a signal of a nucleic acid dye, (ii) sorting single cells, and (iii) sorting based on a signal of a detectably labeled target molecule.

[0020] In some embodiments of any of the aforementioned aspects, the bacterium is a gram-negative bacterium. In some embodiments, the gram-negative bacterium is an E. coli bacterium.

[0021] In some embodiments of any of the aforementioned aspects, the binding polypeptide is expressed in a soluble form within the periplasm of the bacteria.

[0022] In some embodiments of any of the foregoing aspects, the binding polypeptide is an antibody. In some embodiments, the antibody is a full length antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is a half antibody. In some embodiments, the antibody is an antibody fragment. In some embodiments, antibody fragments include Fab, Fab'-SH, Fv, scFv, and (Fab') 2 The nucleic acid is selected from the group consisting of:

[0023] In some embodiments of any of the foregoing aspects, step (a) comprises providing a plurality of bacteria, each of the plurality of bacteria comprising a library of nucleic acids encoding a candidate binding polypeptide. In some embodiments, the library comprises a plurality of nucleic acids encoding candidate antibody variants, each of the candidate antibody variants comprising an amino acid residue change in a FR of a VH or VL compared to a reference antibody, the amino acid residue change being in an amino acid residue that is (a) identified in a naturally occurring antibody having the same subtype as the reference antibody, and (b) predicted to be buried. In some embodiments, the amino acid residue change in the naturally occurring antibody is due to somatic hypermutation. In some embodiments, identifying the candidate binding polypeptide as having an increased expression level based on the amount of labeled target molecule in the periplasm. In some embodiments, the method further comprises identifying the candidate binding polypeptide as having an increased stability based on the amount of labeled target molecule in the periplasm.

[0024] In some embodiments of any of the aforementioned aspects, the method further includes isolating the nucleic acid after the particular step.

[0025] In some embodiments of any of the foregoing aspects, the bacterium comprises a mutation affecting a transcriptional regulator gene. In some embodiments, step (a) comprises providing a plurality of bacteria, the plurality of bacteria comprising a library of nucleic acids each encoding a variant of a transcriptional regulator gene. In some embodiments, the transcriptional regulator gene is selected from a transcription initiation factor, an anti-sigma factor, an RNA polymerase subunit, or a transcription factor. In some embodiments, the transcription initiation factor is a sigma factor. In some embodiments, the sigma factor is RpoD (σ 70 ), RpoE(σ 24 ), RpoH(σ 32 ), RpoS(σ 38 ), RpoN(σ 54 ), RpoF(σ 28 ), and FecI(σ 19In some embodiments, the sigma factor is selected from the group consisting of RpoD (σ 70 ). In some embodiments, the mutation affecting the transcriptional regulator gene is present on a synthetic plasmid. In some embodiments, the mutation affecting the transcriptional regulator gene is present in the endogenous bacterial genome. In some embodiments, the mutation affecting the transcriptional regulator gene is due to error-prone PCR mutagenesis, chemical mutagenesis, transposition mutagenesis, or targeted mutagenesis. In some embodiments, the mutation affecting the transcriptional regulator gene is due to error-prone PCR mutagenesis. In some embodiments, the method identifies bacteria with improved expression of a binding polypeptide.

[0026] In another aspect, the invention features a bacterium that includes (i) a loss-of-function mutation in a gene encoding Lpp, and (ii) an expression construct encoding an antibody. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is a half antibody. In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment is a Fab, Fab'-SH, Fv, scFv, and (Fab') 2In some embodiments, the expression construct comprises a promoter operably linked to a gene encoding the antibody. In some embodiments, the expression construct comprises a synthetic plasmid. In some embodiments, the bacterium further comprises a mutation affecting a transcription control gene. In some embodiments, the transcription control gene is selected from the group consisting of a transcription initiation factor, an anti-sigma factor, an RNA polymerase subunit, or a transcription factor. In some embodiments, the transcription initiation factor is a sigma factor. In some embodiments, the sigma factor is RpoD (σ 70 ), RpoE(σ 24 ), RpoH(σ 32 ), RpoS(σ 38 ), RpoN(σ 54 ), RpoF(σ 28 ), and FecI(σ 19 In some embodiments, the sigma factor is selected from the group consisting of RpoD (σ 70 ). In some embodiments, the mutation affecting the transcriptional regulator gene is present on a synthetic plasmid. In some embodiments, the mutation affecting the transcriptional regulator gene is present in the endogenous bacterial genome. In some embodiments, the mutation affecting the transcriptional regulator gene is due to error-prone PCR mutagenesis, chemical mutagenesis, transposition mutagenesis, or targeted mutagenesis. In some embodiments, the mutation affecting the transcriptional regulator gene is due to error-prone PCR mutagenesis.

[0027] In another aspect, the invention features a bacterium that includes (i) a mutation that allows diffusion of a membrane impermeable molecule across the outer membrane into the periplasm, (ii) an expression construct encoding a binding polypeptide, and (iii) a mutation that affects a transcription control gene. In some embodiments, the transcription control gene is selected from a transcription initiation factor, an anti-sigma factor, an RNA polymerase subunit, or a transcription factor. In some embodiments, the transcription initiation factor is a sigma factor. In some embodiments, the sigma factor is RpoD (σ 70 ), RpoE(σ 24 ), RpoH(σ 32 ), RpoS(σ 38 ), RpoN(σ 54 ), RpoF(σ 28 ), and FecI(σ 19 In some embodiments, the sigma factor is selected from the group consisting of RpoD (σ 70 ). In some embodiments, the mutation affecting the transcriptional regulator gene is present on a synthetic plasmid. In some embodiments, the mutation affecting the transcriptional regulator gene is present in the endogenous bacterial genome. In some embodiments, the mutation affecting the transcriptional regulator gene is due to error-prone PCR mutagenesis, chemical mutagenesis, translocation mutagenesis, or targeted mutagenesis. In some embodiments, the mutation affecting the transcriptional regulator gene is due to error-prone PCR mutagenesis. In some embodiments, the binding polypeptide is an antibody. In some embodiments, the antibody is a full length antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is a half antibody. In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment is a Fab, Fab'-SH, Fv, scFv, and (Fab') 2In some embodiments, the antibody is selected from the group consisting of EnvA, RfaD, RfaE, RfaH, RfaRd, TolA, TolB, and TolD. In some embodiments, the antibody is expressed in a soluble form in the periplasm of the bacteria. In some embodiments, the mutation that allows diffusion of membrane impermeable molecules across the outer membrane into the periplasm is in a gene that encodes an outer membrane protein or a protein that affects LPS composition. In some embodiments, the outer membrane protein is the major outer membrane lipoprotein Lpp (Lpp), a porin, or TolC. In some embodiments, the outer membrane protein is Lpp. In some embodiments, the protein that affects LPS composition is selected from EnvA, RfaD, RfaE, RfaH, RfaRd, TolA, TolB, and TolD.

[0028] In another aspect, the invention features an isolated antibody that specifically binds to interleukin-13 (IL-13), the antibody comprising: (a) a light chain variable region comprising the amino acid sequence of SEQ ID NO:1; and (b) a heavy chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:5.

[0029] In another aspect, the invention features an isolated antibody that specifically binds to IL-13, the antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2; and (b) a light chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:6.

[0030] In another aspect, the invention features an isolated antibody that specifically binds to IL-13, the antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3; and (b) a light chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:6.

[0031] In another aspect, the invention features an isolated antibody that specifically binds to IL-13, the antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4; and (b) a light chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:6.

[0032] In another aspect, the invention features an isolated antibody that specifically binds to IL-13, the antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5.

[0033] In another aspect, the invention features an isolated antibody that specifically binds to IL-13, the antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4.

[0034] In another aspect, the invention features an isolated antibody that specifically binds to IL-13, the antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:6 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2.

[0035] In another aspect, the invention features an isolated antibody that specifically binds to IL-13, the antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:6 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3.

[0036] In another aspect, the invention features an isolated antibody that specifically binds to IL-13, the antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:6 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4.

[0037] In some embodiments of any of the aforementioned aspects, the antibody further comprises six HVRs: (a) HVR-H1 comprising the amino acid sequence of AYSVN (SEQ ID NO: 7); (b) HVR-H2 comprising the amino acid sequence of MIWGDGKIVYNSALKS (SEQ ID NO: 8); (c) HVR-H3 comprising the amino acid sequence of DGYYPYAMDN (SEQ ID NO: 9); (d) HVR-L1 comprising the amino acid sequence of RASKSVDSYGNSFMH (SEQ ID NO: 10); (e) HVR-L2 comprising the amino acid sequence of LASNLES (SEQ ID NO: 11); and (f) HVR-L3 comprising the amino acid sequence of QQNNEDPRT (SEQ ID NO: 12).

[0038] In some embodiments, any one of the aforementioned antibodies has a K D In some embodiments, the antibody specifically binds human IL-13 with a K of about 1 pM to about 50 pM. D In some embodiments, the antibody specifically binds human IL-13 with a K of about 29 pM to about 40 pM. D It specifically binds to human IL-13.

[0039] In some embodiments, any one of the foregoing antibodies is monoclonal, human, humanized, or chimeric. In some embodiments, the antibody is a full length antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is a half antibody. In some embodiments, the antibody is an antibody fragment that binds to IL-13. In some embodiments, the antibody fragment is an antibody fragment that includes Fab, Fab'-SH, Fv, scFv, and (Fab') 2 In some embodiments, the antibody is selected from the group consisting of: a monospecific antibody; a multispecific antibody; and a bispecific antibody.

[0040] In another aspect, the invention features an isolated nucleic acid encoding any of the antibodies described herein. In another aspect, the invention features a vector (e.g., an expression vector) comprising the isolated nucleic acid for expressing the antibody. In another aspect, the invention features a host cell comprising the aforementioned nucleic acid and / or vector. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is an E. coli cell.

[0041] In another aspect, the invention features a method of producing any of the antibodies described herein, the method including culturing a host cell containing any of the foregoing vectors (e.g., expression vectors) in a culture medium. In some embodiments, the method further includes recovering the antibody from the host cell or culture medium.

[0042] In another aspect, the invention features a composition comprising any one of the aforementioned antibodies. In some embodiments, the composition further comprises a pharma- ceutically acceptable carrier, excipient, or diluent. In some embodiments, the composition is a pharmaceutical composition.

[0043] In some embodiments, any one of the foregoing antibodies may be used as a medicament.

[0044] In some embodiments, any one of the aforementioned antibodies may be used in the treatment of an IL-13 mediated disorder.

[0045] In another aspect, the invention features a method of treating a patient suffering from asthma, comprising administering to the patient an effective amount of any one of the preceding antibodies to reduce asthma symptoms.

[0046] In another aspect, the invention features a method for inhibiting IgE antibody production in a patient, comprising administering to the patient an effective amount of any one of the aforementioned antibodies. In some embodiments, the inhibition of IgE antibody production is intended to treat bronchial asthma, allergic rhinitis, allergic dermatitis, bronchial asthma, allergic rhinitis, urticaria, anaphylaxis, or atopic dermatitis.

[0047] In another aspect, the invention features a method of treating an IL-13 mediated disorder in a patient, comprising administering to the patient an effective amount of any one of the aforementioned antibodies. In some embodiments, the disorder is allergic asthma, non-allergic (intrinsic) asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, eczema, urticaria, food allergy, chronic obstructive pulmonary disease, ulcerative colitis, RSV infection, uveitis, scleroderma, or osteoporosis.

[0048] In another aspect, the invention features a method of treating an IgE-mediated disorder in a patient, comprising administering to the patient an effective amount of any one of the aforementioned antibodies. In some embodiments, the IgE-mediated disorder is bronchial asthma, allergic rhinitis, allergic dermatitis, urticaria, anaphylaxis, or atopic dermatitis.

[0049] In some embodiments of the foregoing methods of treatment, the antibody inhibits binding of IL-13 to its receptor and inhibits one or more functions associated with the binding of IL-13 to its receptor.

[0050] In some embodiments of the foregoing methods of treatment, the antibody is administered by one or more of the routes selected from the group consisting of intravenous, intraperitoneal, inhalation, intramuscular, subcutaneous, and oral. In some embodiments, the route is subcutaneous.

[0051] In some embodiments of the foregoing methods of treatment, the patient is a human.

[0052] In another aspect, the invention features a library comprising a plurality of nucleic acids encoding candidate antibody variants, each candidate binding polypeptide variant comprising an amino acid residue change in a FR of a VH or VL compared to a reference antibody, the amino acid residue change being in an amino acid residue that is (a) identified in a naturally occurring antibody having the same subtype as the reference antibody and (b) predicted to be buried. In some embodiments, the amino acid residue change in the naturally occurring antibody is due to somatic hypermutation. [Brief description of the drawings]

[0053] [Figure 1A] Figure 1 is a panel of fluorescent micrographs showing specific staining of antibody-expressing bacterial cells with IL-13 antigen. Cells expressing anti-IL-13 antibody or empty vector control (pBR322) were stained with SYTO® 41 (nucleic acid dye), IL-13 labeled with ALEXA FLUOR® 488, or anti-human Fc F(ab')2 antibody labeled with DYLIGHT® 649. Only cells expressing the antibody stain positively with the IL-13 antigen and anti-human Fc antibody. [Figure 1B] Shown are images of bacterial culture plates spotted with serial dilutions of cells expressing different anti-IL-13 antibody formats or an empty control vector (pBR322) before ("pre-staining") or after ("post-staining") ethylenediaminetetraacetic acid (EDTA) treatment, antigen staining, and MgCl2. No substantial differences in viability were observed between unstained and stained cells. [Figure 1C] Graph showing flow cytometry analysis of cells expressing different formats of anti-IL-13 antibodies (IgG, Half-Ab, and Fab) or control cells transfected with the empty control vector pBR322 (black line, shaded) after staining with fluorescently labeled IL-13. All formats showed a shift in the IL-13 signal over the pBR322 control. [Figure 1D]FIG. 1 is a schematic diagram of an exemplary method of the invention. Cells are permeabilized by treatment with EDTA, incubated with fluorescently labeled antigen, and outer membrane integrity is restored by addition of MgCl2. The top ≦1% antigen positive cells are sorted by flow cytometry and either progressed to a subsequent round after regrowth or collected for analysis. SSC, side scatter. [Figure 2A] Shown are images of bacterial culture plates spotted with serial dilutions of bacteria carrying a plasmid encoding an anti-IL-13 antibody conferring carbenicillin (Carb) and tetracycline (Tet) resistance spiked at 1:105 or 1:106 into bacteria transformed with a plasmid encoding an anti-VEGF antibody conferring carbenicillin resistance. The presence of rare anti-IL-13 expressing bacteria was visualized by plating the serial dilutions onto carbenicillin (total bacteria) and tetracycline (only bacteria carrying the anti-IL-13 plasmid) containing plates and confirming the expected ratios. [Figure 2B] Graph showing percentage of antigen positive cells after one or two rounds (Rd) of bacterial antibody display (BAD). Spiked cultures were BAD selected with antigen labeled with ALEXA FLUOR® 647. Cells presenting anti-IL-13 (circles) or anti-VEGF-antibodies (squares) spiked at a ratio of 1:106 into a pool of cells presenting unbound antibodies are rapidly enriched by two rounds of BAD selection. [Figure 2C] 2C is a series of graphs showing flow cytometry analysis of the anti-VEGF spiking experiment shown in Figure 2B. After two rounds of selection, a significant portion of the population binds to the VEGF antigen. [Figure 3A] Graph showing expression levels of anti-VEGF.2 variants as determined by Western blot. Non-reduced soluble samples were detected with anti-human Fc antibody and quantified using a LI-COR ODYSSEY® instrument. Signals were normalized to the half-antibody band of anti-VEGF.2 wild type (WT). n=3 experiments. All variants have increased expression levels compared to wild type. [Figure 3B] Graph showing flow cytometry analysis of individual anti-VEGF.2 variants. Anti-VEGF.2 wild type has a higher shift than unstained cells (black, shaded). All variants have a higher shift than wild type, correlating with the expression rank determined by Western blot (see FIG. 3A). Anti-VEGF.2 wild type, L4M.E6Q.M34I, F71Y.E6Q.M34I, and S24R.E6Q.M34I are shown. [Figure 3C]

[0023] Figure 1 is a series of graphs showing enrichment of the highest expressing variant from a pool of anti-VEGF.2 wild type and three variants by BAD. The flow cytometry signal of the pool is shifted to a more positive direction over two rounds by sorting the top 1% VEGF binding cells. The lowest expressing anti-VEGF.2 wild type and the highest expressing variant S24R.E6Q.M34I were analyzed in each round and are shown for comparison. [Figure 3D] 1 is a series of pie charts showing enrichment of anti-VEGF.2 variants over two rounds of selection. 96 clones were sequenced and the highest expressing anti-VEGF.2 variants, F71Y.E6Q.M34I and S24R.E6Q.M34I, were enriched after two rounds while the two lowest expressing variants (anti-VEGF.2 wild type and L4M.E6Q.M34I) were depleted. [Figure 4A] 1 is a series of graphs showing round enrichment of anti-IL-13 framework variants. The overlaid FACS™ dot plots show that anti-IL-13 framework (FR) variants shift more positively over each round compared to anti-IL-13 wild type. [Figure 4B] FIG. 1 is a series of pie charts showing round enrichment of an anti-IL-13 library. Data presented from rounds 1 and 2 are from sequencing 186 and 226 clones, respectively. M4L and E6Q are the most enriched variants after two rounds of BAD. [Figure 4C]Graph showing correlation of expression of anti-IL-13 variants and their combinations in mammalian (HEK293T) and E. coli cells. Improved expression is seen in both host systems. For E. coli expression, non-reduced soluble half antibody samples were detected with anti-human Fc antibody and quantified using a LI-COR ODYSSEY® instrument. For HEK293T expression, Protein A purified IgG yield was quantified. Expression was normalized to anti-IL-13 wild type. n=3 experiments. [Figure 4D] Graph showing the thermal stability of anti-IL-13 variants and their combinations measured by Differential Scanning Fluorometry (DSF). All variants containing the M4L mutation show a 5° C. increase in thermal stability, independent of the host expression system. To measure the thermal stability, Fabs and IgGs were purified from E. coli and CHO cells, respectively. The graph shows the results of Fab transitions (the temperature at which half of the Fabs melt), which is equal to the melting temperature Tm. The Tm values ​​of Fab melting for the Fab and IgG antibody formats are comparable. [Figure 5A]

[0023] Figure 1 is a series of graphs showing flow cytometry analysis of the enrichment of the anti-VEGF.3 framework library after each round of FACS™. These data show that after the first round, the anti-VEGF.3 framework (FR) library variants were better biased than anti-VEGF.3 wild-type or unstained cells (black line, shaded), with the bias becoming more pronounced by the second round. [Figure 5B] Graph showing that a subset of anti-VEGF framework variants and their combinations with improved expression in E. coli show a correlated increase in mammalian (HEK293T) expression (outlined box). Expression was normalized to anti-VEGF wild type and determined by anti-Fc Western blot of half antibody (E. coli) or Protein A purified IgG (HEK293T). n=3 experiments. [Figure 5C]1 is a graph showing that anti-VEGF.3 framework variants have increased thermal stability and that combining framework variants had an additive effect. Differential scanning fluorimetry shows that single framework variants E6Q, V37I, V48L or V48I (left boxes) increase thermal stability. The double variant (middle box) increased thermal stability even more, and the triple variant (right box) had the greatest additive increase. IgG Fab transition values ​​are shown, n=3 experiments. [Figure 5D] Rendering of the crystal structure of anti-VEGF G6 in complex with VEGF (RCSB Protein Database (PDB) Accession Number: 2FJG). Identified framework residues that increase expression and thermostability are shown on the X-ray crystal structure of anti-VEGF G6 in complex with VEGF (PDB: 2FJG). V37I and V48I mutations were modeled while Q6 was the native antibody structure. The three residues are located in a plane within the core of the heavy chain variable (VH) domain. E6 and V48I are located on separate chains within the beta barrel core of the VH domain, while V37I is in the buried interface between the VH and light chain variable (VL) domains. Heavy chain constant domain (CH), light chain constant domain (CL). [Figure 6] Figure 1 shows the amino acid sequences of the light and heavy chain anti-IL-13 variable domains from an anti-IL-13 antibody. The antibody variable domain is composed of three hypervariable regions (HVRs, overlined) nestled between four framework regions (FRs). Somatic hypermutation can introduce changes in both FRs and HVRs. Data from the Kabat database was collated to compile all amino acid changes caused by somatic hypermutation in the frameworks of the variable kappa 4 (VK4) and heavy chain 2 (VH2) subtypes, which were then restricted to non-solvent exposed residues (represented by the amino acid residues located under the sequences of SEQ ID NO:5 and SEQ ID NO:6). Single framework variants of the anti-IL-13 half antibody (5 in the light chain and 28 in the heavy chain) were generated, combined and screened by BAD. [Figure 7]Ribbon diagram of the main chain antibody scaffold of the variable domain of the anti-VEGF G6 antibody (Fuh et al. J Biol Chem. 281(10):6625-31, 2006). The light and heavy chain variable regions are labeled. Similar to the approach taken for the anti-IL-13 antibody (see FIG. 6), the Kabat database was scanned to identify residues introduced by somatic hypermutation that were in the FRs of the variable kappa 1 and heavy chain 3 (Vκ1 and VH3, respectively) subtypes. To maintain affinity, HVRs were excluded, while buried residues (spheres) were also selected to reduce the risk of immunogenicity. With these criteria, single framework variants of the anti-VEGF half antibody (47 in the light chain and 36 in the heavy chain) were generated, combined, and screened by BAD. [Figure 8] Images of bacterial media plates spotted with the indicated bacterial strains after expression of anti-VEGF.2 antibodies are shown. Serial dilutions of 1 OD / mL of cells were plated on LB / carbenicillin plates to visualize cell viability. Various wild-type expression strains showed different degrees of viability after expression of anti-VEGF antibodies (left panel). The 62A7 strain showed a 1 log increase in viability compared to strain 64B4. Deletion of lpp in the 64B4 strain background resulted in a 3 log decrease in viability. The 62A7Δlpp strain showed increased viability compared to the 64B4Δlpp strain (right panel) in cells that expressed anti-VEGF or anti-tissue factor (TF) antibodies. [Figure 9]

[0023] Figure 1 is a series of graphs showing flow cytometry analysis of E. coli cells expressing anti-VEGF.V48L stained with the indicated fluorescent nucleic acid dyes. A range of 20-90% of cells positive staining is observed. MFI, mean fluorescent intensity. [Figure 10]1 is a series of graphs showing that cell concentration can affect the presence of cell duplexes during flow cytometry during BAD screening. Two anti-VEGF variants, V48L and A97T, were expressed separately in strain 62A7Δlpp, and cells were labeled with VEGF antigen fluorescently conjugated to ALEXA FLUOR® 488 and ALEXA FLUOR® 647, respectively. These labeled cells were then combined equally and subjected to flow cytometry analysis. When a concentration of 1×108 cells / mL was analyzed, there were cells that were double positive for both ALEXA FLUOR® dyes (top panel). However, when the cell concentration was reduced to 1×106 cells / mL, the duplexes were eliminated and only single positive cells were observed (bottom panel). [Figure 11] Schematic diagram showing an exemplary flow cytometry sorting strategy for bacterial antibody display. Antibodies are expressed in the 62A7Δlpp E. coli expression strain. First, cells are selected based on being nucleic acid dye positive compared to an unstained control. Next, forward and side scatter are used to select cells. Next, two standard gates are implemented using cell complexity (side scatter SSC width and height) and small cell size (forward scatter FSC width and height) to select for doublet exclusion. Finally, antigen positive cells are selected using a gate drawn above the shift seen for cells expressing unbound antibody and unstained cells, and / or empty vector. [Figure 12A]1 is a graph showing that the strategy described in Example 4 can be used to successfully sort differentially labeled antigen positive cells. Two anti-VEGF variants (V48L and A97T) were expressed separately in strain 62A7Δlpp, and cells were labeled with VEGF antigen fluorescently conjugated with ALEXA FLUOR® 488 and ALEXA FLUOR® 647, respectively. These labeled cells were then paired one-to-one and sorted for single antigen positivity (VEGF-ALEXA FLUOR® 488 or VEGF-ALEXA FLUOR® 647), binding to both antigens (double positive), or not binding to either antigen (double negative). Green boxes indicate cells expected to express the V48L variant, while red boxes indicate cells expected to express the A97T variant. Sorting inputs and outputs were plated and 96 colonies were sequenced. The right panel is a table showing the number of events sorted and the percentage of events that were positive after analysis, as determined by running the sorted samples on a flow cytometry instrument to detect the positive percent (positive %) that bind to the antigen. [Figure 12B] 12A is a table showing the results of the sequencing analysis of the experiment presented in FIG. With a 1:1 input of two anti-VEGF variants (V48L and A97T), each could be enriched separately to nearly 95% after one round of sorting. The very few double-positive cells that could be sorted did not yield any colonies for sequencing, while the double-negative cells that did not bind to either labeled antigen were composed of an equal combination of both antibody variants, indicating that there was no selection bias. [Figure 13]Ribbon diagram of main chain antibody scaffold based on anti-IL-13 variable domain subtypes. Somatic hypermutation can introduce changes in both framework and hypervariable regions. Data from the Kabat database was collated to compile all amino acid changes caused by somatic hypermutation in the framework of variable kappa 4 (Vκ4) and heavy chain 2 (VH2) subtypes, and then the selected variants were restricted to non-solvent exposed residues (spheres). Single framework variants of anti-IL-13 half antibodies (5 in the light chain and 28 in the heavy chain) were generated, combined and screened by BAD. [Figure 14] FIG. 1 is a schematic diagram of the domain structure of E. coli sigma factor RpoD (σ70). [Figure 15A] Figures 15A and 15B are schematic diagrams showing an exemplary strategy for constructing a mutant rpoD library by error-prone polymerase chain reaction (PCR). The pACYCrpoD plasmid (6006 base pairs) was mutagenized by error-prone PCR to generate a library size of approximately 10 mutant plasmids (Figure 15A). Upon transformation into E. coli, each mutant expresses a unique phenotype (indicated by the different arrows present in each cell in Figure 15B). [Figure 15B] Figures 15A and 15B are schematic diagrams showing an exemplary strategy for constructing a mutant rpoD library by error-prone polymerase chain reaction (PCR). The pACYCrpoD plasmid (6006 base pairs) was mutagenized by error-prone PCR to generate a library size of approximately 10 mutant plasmids (Figure 15A). Upon transformation into E. coli, each mutant expresses a unique phenotype (indicated by the different arrows present in each cell in Figure 15B). [Figure 16A]16A and 16B are schematic diagrams showing an exemplary strategy for preparing a mutant rpoD plasmid library for BAD screening. Strain 66C4(Δlpp) was transformed with an MD169 plasmid encoding an anti-IL-13 antibody (FIG. 16A). The resulting transformants were further transformed with the mutant rpoD plasmid library to generate a 66C4 bacterial library pool with a diversity of approximately 10 members (FIGS. 16A and 16B). [Figure 16B] 16A and 16B are schematic diagrams showing an exemplary strategy for preparing a mutant rpoD plasmid library for BAD screening. Strain 66C4(Δlpp) was transformed with an MD169 plasmid encoding an anti-IL-13 antibody (FIG. 16A). The resulting transformants were further transformed with the mutant rpoD plasmid library to generate a 66C4 bacterial library pool with a diversity of approximately 10 members (FIGS. 16A and 16B). [Figure 17A] 17A and 17B are schematic diagrams showing an exemplary strategy for BAD screening of bacterial pools transformed with mutant rpoD plasmid libraries. The 66C4 bacterial library pool was grown for 24 hours in CRAP medium at 30° C. with agitation at 225 rpm. One OD unit was harvested, permeabilized by treatment with EDTA, incubated with fluorescently labeled antigen (ALEXA FLUOR® 647-labeled human IL-13 ("ALEXA® 647-huIL-13") and nucleic acid stain (SYTO® 9), and outer membrane integrity was restored by addition of MgCl2. Cells were subjected to flow cytometry using a FACSARIA® device using the gating strategy shown (FIG. 17B). The top 1-3% of cells were sorted based on ALEXA® 647-huIL-13 signal intensity and grown for further rounds of BAD. [Figure 17B]17A and 17B are schematic diagrams showing an exemplary strategy for BAD screening of bacterial pools transformed with mutant rpoD plasmid libraries. The 66C4 bacterial library pool was grown for 24 hours in CRAP medium at 30° C. with agitation at 225 rpm. One OD unit was harvested, permeabilized by treatment with EDTA, incubated with fluorescently labeled antigen (ALEXA FLUOR® 647-labeled human IL-13 ("ALEXA® 647-huIL-13") and nucleic acid stain (SYTO® 9), and outer membrane integrity was restored by addition of MgCl2. Cells were subjected to flow cytometry using a FACSARIA® device using the gating strategy shown (FIG. 17B). The top 1-3% of cells were sorted based on ALEXA® 647-huIL-13 signal intensity and grown for further rounds of BAD. [Figure 18] Graph showing the results of flow cytometry analysis of the indicated samples from a BAD screen of a 66C4 bacterial library pool transformed with a mutant rpoD plasmid library. The inset table shows the samples and the mean fluorescence intensity ("Mean"). [Figure 19] Schematic diagram showing three rpoD mutants identified by BAD screening of 66C4 bacterial library pools transformed with mutant rpoD plasmid libraries. Mutants 1 and 2 are truncation mutants containing the indicated point mutations compared to wild-type RpoD. The pie chart below the figure shows the number of indicated mutants observed among 96 sequenced colonies. [Figure 20] Graph showing the results of flow cytometry analysis of the indicated samples (E. coli expressing the indicated rpoD mutants, wild-type RpoD, or empty vector control). The inset table shows the samples and the mean fluorescence intensity ("Mean"). [Figure 21A] FIG. 1 is an immunoblot showing that expression of the indicated rpoD mutants resulted in increased expression of full-length anti-IL-13 in 66C4 host E. coli cells compared to cells expressing native wild-type RpoD. [Figure 21B] 1 is an immunoblot showing that expression of the indicated rpoD mutants resulted in increased expression of full-length anti-IL-13 in 67A6 host E. coli cells compared to cells expressing native wild-type RpoD. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] I. Definition As used herein, the term "about" refers to a normal range of error for the respective value, which would be readily understood by one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0055] As used herein, "bacteria" refers to a bacterial cell, including an inner membrane (i.e., cell membrane), a periplasm, and an outer membrane. The outer membrane is distinct from the inner membrane and may include, for example, lipopolysaccharide (LPS) and porins. In some embodiments, the bacteria is a gram-negative bacterium. Exemplary bacterial species include Escherichia species (e.g., E. coli), Salmonella species (e.g., S. typhimurium and S. enterica), as well as others known in the art.

[0056] The term "binding polypeptide" refers to a polypeptide that specifically binds to a target molecule. In some embodiments, the binding polypeptide is an antibody. In other embodiments, the binding polypeptide is, for example, an antibody mimic, a cell surface receptor, a cytokine, or a growth factor. The term "target molecule" refers to the specific binding target of a binding polypeptide. A target molecule is typically a small molecule, a polypeptide, or a polypeptide fragment. When a binding polypeptide is an antibody, the target molecule can be, for example, an antigen.

[0057] With respect to the binding of a binding polypeptide (e.g., an antibody) to a target molecule, "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or epitope on a particular target molecule means binding that is measurably different 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 competition with a control molecule similar to the target, for example, an excess of unlabeled target. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the terms "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or epitope on a particular polypeptide target can refer to, for example, binding of a molecule that is more specific than a specific polypeptide or epitope on a particular polypeptide target. -4 M or less, or 10 -5 M or less, or 10 -6 M or less, or 10 -7 M or less, or 10 -8 M or less, or 10 -9 M or less, or 10 -10 M or less, or 10 -11 M or less, or 10 -12 K against targets smaller than M D , or 10 -4 M~10 -6 M or 10 -6 M~10 -10 M or 10 -7 M~10 -9 K within M D As will be appreciated by those of skill in the art, affinity and K D The values ​​are inversely related: a high affinity for an antigen corresponds to a low K D In one embodiment, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptides or epitopes of polypeptides.

[0058] As used herein, a "library" refers to a plurality of binding polypeptide (e.g., antibody or antibody fragment) sequences, or nucleic acids encoding these sequences. Exemplary libraries include libraries containing from about 2 binding polypeptides to about 10 14 2 to about 10, about 2 to about 20, about 2 to about 30, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 70, about 2 to about 70, about 2 to about 80, about 2 to about 100, about 2 to about 10 14 pieces, about 2 to about 10 13 pieces, about 2 to about 10 12 pieces, about 2 to about 10 11 pieces, about 2 to about 10 10 pieces, about 2 to about 10 9 pieces, about 2 to about 10 8 pieces, about 20 to about 10 8 pieces, about 30 to about 10 8 pieces, about 40 to about 10 8 pieces, about 50 to about 10 8 pieces, about 60 to about 10 8 pieces, about 70 to about 10 8 pieces, about 80 to about 10 8 pieces, about 90 to about 10 8 pieces, about 100 to about 10 8 pieces, about 200 to about 10 8 400 to 10 8 pieces, about 600 to about 10 8 pieces, about 800 to about 10 8 pieces, about 10 3 ~about 10 8 pieces, about 10 4 ~about 10 8 pieces, about 10 5 ~about 10 8 pieces, about 10 6 ~about 10 8 Pieces, or about 10 7 ~about 10 8 The size of the binding polypeptide may be 100 or 150 μm.

[0059] A "variant" or "mutant" of a starting or reference binding polypeptide (e.g., a reference antibody) is a mutagenic binding polypeptide that (1) has an amino acid sequence that differs from that of the starting or reference polypeptide, and (2) is derived from the starting or reference polypeptide through either natural or artificial (man-made) mutagenesis. Such variants include, for example, deletions from, insertions into, and / or substitutions of residues in the amino acid sequence of the binding polypeptide of interest, referred to herein as "amino acid residue changes." Thus, a variant HVR refers to an HVR that comprises a variant sequence with respect to a starting or reference polypeptide sequence (such as that of a source antibody or antigen-binding fragment). In this context, an amino acid residue change refers to an amino acid that differs from the amino acid at the corresponding position in the starting or reference polypeptide sequence (such as that of a reference antibody or fragment thereof). Any combination of deletions, insertions, and substitutions can be made to arrive at a final variant or mutant construct, provided that the final construct possesses the desired functional characteristics (e.g., affinity, stability, and / or expression).

[0060] A "wild-type" or "reference" sequence, or a sequence of a "wild-type" or "reference" protein / polypeptide (such as an HVR, FR, or variable domain of a reference antibody), may be a reference sequence from which a variant polypeptide is derived through the introduction of mutations. Generally, the "wild-type" sequence of a given protein is the sequence that is most common in nature. Similarly, a "wild-type" gene sequence is the sequence of that gene that is most commonly found in nature. Mutations can be introduced into a "wild-type" gene (and thus the protein it encodes) through either natural processes or artificial means. The product of such processes is a "variant" or "mutant" form of the original "wild-type" protein or gene.

[0061] A "mutation" is a deletion, insertion, or substitution of a nucleotide(s) compared to a reference nucleotide sequence, such as a wild-type sequence.

[0062] The term "membrane impermeable" refers to the inability of a molecule (e.g., a polypeptide) to diffuse across a biological membrane (e.g., the inner and / or outer membrane of a bacteria) or a channel pore (e.g., a porin). In contrast, a "membrane permeable" molecule is capable of diffusing across a biological membrane. It is understood that under some conditions, e.g., during the methods of the invention, for example, when the integrity of the outer membrane is permanently or transiently compromised, a molecule (e.g., a polypeptide) that is normally membrane impermeable can diffuse into the periplasm of the bacteria.

[0063] As used herein, "permeability" refers to the rate of diffusion of a molecule across a biological membrane. A biological membrane is "permeable" to a given molecule if the molecule can substantially diffuse across the membrane. A biological membrane is "impermeable" to a given molecule if the molecule cannot substantially diffuse across the membrane. The charge, polarity, and size of the molecule can affect whether a biological membrane is permeable or impermeable to the molecule. Typically, hydrophobic molecules and uncharged polar small molecules are permeable across biological membranes, while most water-soluble (hydrophilic) molecules are impermeable. The outer membrane of bacteria is typically impermeable to the diffusion of hydrophilic molecules with a molecular weight greater than about 600 Daltons (see, e.g., Decad et al. J. Bacteriology 128(1):325-336, 1976).

[0064] As used herein, "major outer membrane lipoprotein Lpp" (also referred to in the art as "Lpp", "Braun lipoprotein", and "murein lipoprotein") refers to any native Lpp from any bacterial source, including E. coli or S. typhimurium (also known as lkyD), unless otherwise indicated. The term encompasses "full-length", unprocessed Lpp, and any form of Lpp that results from processing within the cell. Lpp interacts with peptidoglycan and contributes to maintaining the structural and functional integrity of the cell envelope. The amino acid sequence of an exemplary E. coli (strain K12) Lpp can be found, for example, in UniProtKB Accession No. P69776.

[0065] The "periplasm" is the space bounded by two selectively permeable barriers (e.g., biological membranes). In many species of bacteria, including Escherichia coli, the periplasm is the space bounded by the inner and outer membranes.

[0066] As used herein, "permeabilizing agent" refers to a chemical that can increase the permeability of the bacterial outer membrane to molecules (e.g., polypeptides) that are typically membrane impermeable. Exemplary permeabilizing agents include, for example, chelating agents, including divalent cation chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA, also known as edetic acid or 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid), 2-[2-[2-[2-[bis(carboxymethyl)amino]phenoxy]ethoxy]-N-(carboxymethyl)anilino]acetic acid (BAPTA), and 2-[2-[2-[2-[bis(carboxymethyl)amino]ethoxy]ethoxy]ethyl-(carboxymethyl)amino]acetic acid (EGTA)), NaCl (Chen et al., J. Am. Soc. 1999, 143:1311-1322, 1999). al. Nat. Biotech. 19:537-542, 2001), sucrose, antibiotics (e.g., aminoglycosides (e.g., streptomycin and gentamicin), polymyxin B, deacylated polymyxins, octapeptins, and benzylpenicillin), detergents, lysozyme, Tris, Tris-EDTA, ascorbate, polylysine, benzalkonium chloride, protamine, bactericidal / permeability enhancing protein (BPI), serum and / or complement, Ca 2+ or combinations thereof. See, e.g., Hancock, Ann. Rev. Microbiol. 38:237-264, 1984. In some embodiments, the permeabilizing agent is EDTA.

[0067] As used herein, "permeabilize" means to increase the rate of diffusion of a molecule across a biological membrane, e.g., the rate of diffusion of a molecule (e.g., a polypeptide) that is typically membrane impermeable across a bacterial outer membrane.

[0068] "Polypeptide" refers to a peptide or protein containing two or more amino acids linked by peptide bonds, and includes peptides, oligomers, and proteins. Polypeptides may contain natural, modified, or synthetic amino acids. Polypeptides may also be modified naturally, such as by post-translational processing, or chemically, such as by amidation, acylation, and cross-linking.

[0069] As used herein, a "plurality" of a substance, such as a polypeptide or polynucleotide of the present invention, generally refers to a collection of two or more types or varieties of that substance. Two or more types or varieties of a substance will be present when the two or more substances differ from each other with respect to a particular feature, such as a variant amino acid found at a particular amino acid position. For example, a plurality of polypeptides of the present invention will be present when there are two or more substantially identical, preferably identical, polypeptides of the present invention in sequences excluding the sequences of variant framework regions (FRs) or excluding the variant amino acids at particular non-solvent exposed amino acid positions. In another example, a plurality of polynucleotides of the present invention will be present when there are two or more substantially identical, preferably identical, polynucleotides of the present invention in sequences excluding the sequences encoding variant FRs or excluding the sequences encoding variant amino acids at particular non-solvent exposed amino acid positions.

[0070] As used herein, "resealing" refers to substantially restoring the membrane permeability barrier of the bacterial outer membrane. In some embodiments, resealing reverses a previous permeabilization of the bacterial outer membrane.

[0071] As used herein, a "transcriptional regulator gene" refers to any gene that affects the transcription or other aspects of expression (e.g., translation, transport, or stability) of a gene encoding a binding polypeptide (e.g., an antibody), e.g., generally (e.g., affecting transcription of multiple genes) or individually (e.g., affecting transcription of one or several genes). Exemplary, non-limiting transcriptional regulator genes include transcription initiation factors (e.g., sigma factors), anti-sigma factors, RNA polymerase subunits, and transcription factors. In some embodiments, a sigma factor is RpoD (σ 70 ), RpoE(σ 24 ), RpoH(σ 32 ), RpoS(σ 38 ), RpoN(σ 54 ), RpoF(σ 28 ), and FecI(σ 19 In some embodiments, the sigma factor is selected from the group consisting of RpoD (σ 70 ).

[0072] "Mutations affecting a transcriptional regulator gene" refers to any mutation that affects the function, expression level, stability, or activity of a transcriptional regulator gene. The mutation is, for example, located in the transcriptional regulator gene itself. In other embodiments, the mutation may be present in a regulatory nucleic acid sequence, for example, in the promoter or enhancer of the transcriptional regulator gene. The mutation may be of any type known in the art, such as point mutations, insertion mutations, deletion mutations, and frameshift mutations.

[0073] "Expression construct" refers to any nucleic acid element capable of conferring expression of a gene in a bacterium, such as a binding polypeptide (e.g., an antibody) or a transcriptional regulator gene. An expression construct may refer to a synthetic plasmid transformed into a bacterium, a synthetic nucleic acid sequence integrated into an endogenous bacterial genome, or any other suitable nucleic acid element. In some embodiments, the expression construct is a synthetic plasmid.

[0074] As used herein, the terms "improved" and "increased" with respect to expression and / or stability of a binding polypeptide refer to an increase or enhancement compared to a baseline expression level of a binding polypeptide and / or a baseline stability of a binding polypeptide. In some embodiments, the baseline expression level may be the expression level of a wild-type binding polypeptide. In other embodiments, the baseline expression level may be the expression level of a variant binding polypeptide. In some embodiments, the baseline expression level may be the expression level of a binding polypeptide in a wild-type bacterium. In some embodiments, the baseline expression level may be the expression level of a binding polypeptide in a mutant bacterium. In some embodiments, the baseline stability may be the stability of a wild-type binding polypeptide. In other embodiments, the baseline stability may be the stability of a variant binding polypeptide.

[0075] "Synthetic plasmid" refers to any non-naturally occurring plasmid (e.g., linear or circular plasmid) that can be used for the purpose of gene expression, such as the expression of a gene encoding a binding polypeptide or a transcriptional regulatory gene. Synthetic plasmids can include, for example, a promoter operably linked to a gene of interest, an antibiotic resistance marker, multiple cloning sites, an origin of replication(s), or any other element known in the art. An "acceptor human framework" for the purposes of this specification is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the sequence of the VL acceptor human framework is identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0076] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative illustrations and exemplary embodiments for measuring binding affinity are described below.

[0077] An "affinity matured" antibody is one that has one or more changes in one or more HVRs and / or framework regions that result in improved affinity of the antibody for antigen compared to a parent antibody that does not have those change(s). 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. For example, Marks et al. Bio / Technology 10:779-783, 1992 describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of HVR and / or framework residues is described by Barbas et al. Proc. Natl. Acad. Sci. USA 91:3809-3813, 1994, Schier et al. Gene 169:147-155, 1995, Yelton et al. J. Immunol. 155:1994-2004, 1995, Jackson et al. J. Immunol. 154(7):3310-3319, 1995, and Hawkins et al. J. Mol. Biol. 226:889-896, 1992.

[0078] As used herein, the term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0079] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the reference antibody from binding to its antigen by 50% or more in a competitive assay, and conversely, a reference antibody that blocks the antibody from binding to its antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein.

[0080] 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') 2and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2, Zapata et al. Protein Eng. 8(10):1057-1062, 1995); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0081] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments and one residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment contains the entire light (L) chain along with the VH domain of the heavy (H) chain, as well as one heavy chain (C H 1) The first constant region domain of the antibody is generated by pepsin treatment to produce a single large F(ab') 2 The resulting fragment generally corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and is still capable of cross-linking antigen. The Fab' fragment is a C fragment containing one or more cysteines from the hinge region of the antibody. H F(ab') differs from Fab fragments by having additional residues at the carboxy terminus of one domain. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab') 2 Antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0082] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0083] An "Fv" consists of a dimer of one heavy and one light chain variable region domain in tight non-covalent association. The folding of these two domains results in six hypervariable loops (three loops each from the H and L chain) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or even half of an Fv containing only three Hs specific for an antigen) has the ability to recognize and bind to an antigen, although the affinity may be lower than that of the entire binding site.

[0084] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising the VH and VL antibody domains joined in a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a general review of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315, 1994.

[0085] The term "diabody" refers to small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (about 5-10 residues) between the VH and VL domains such that inter-chain, but not intra-chain, pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in detail, for example, in EP 404,097, WO 93 / 11161, and Hollinger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993.

[0086] A "blocking" or "antagonist" antibody is one that inhibits or reduces the biological activity of the antigen to which it binds. Certain blocking or antagonist antibodies substantially or completely inhibit the biological activity of an antigen.

[0087] The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these are further subdivided into subclasses (isotypes), e.g., IgG 1 , IgG2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0088] An "effector function" of an antibody refers to a biological activity attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region) and varies with 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 receptor), and B cell activation.

[0089] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) 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 "equip" the cytotoxic cells and are absolutely required for such killing. NK cells, the primary cells mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch et al. Annu. Rev. Immunol. 9:457-492, 1991. To assess the 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 carried out. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of a molecule of interest can be assessed in vivo, for example in an animal model, such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci. USA 95:652-656, 1998.

[0090] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Additionally, a preferred FcR is one that binds IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and / or spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting 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. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see review in M. in Daeron, Annu. Rev. Immunol. 15:203-234, 1997). FcRs are reviewed, for example, in Ravetch et al. 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, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes FcRn, the fetal receptor responsible for the transfer of maternal IgG to the fetus (see, for example, Guyer et al. J. Immunol. 117:587, 1976, and Kim et al. J. Immunol. 24:249, 1994).

[0091] A "human effector cell" is a leukocyte that expresses one or more FcRs and performs effector function. Preferably, the cell expresses at least FcγRIII and performs 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 a natural source, for example from blood.

[0092] "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 bind to their cognate antigen. To assess complement activation, for example, a CDC assay as described in Gazzano-Santoro et al. J. Immunol. Methods 202:163, 1996 may be performed.

[0093] An "epitope" is a portion of an antigen to which an antibody specifically binds. For a polypeptide antigen, an epitope is generally a peptide portion of about 4-15 amino acid residues.

[0094] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a heavy chain having a structure substantially similar to a native antibody structure or containing an Fc region as defined herein.

[0095] As used herein, the term "half antibody" refers to one immunoglobulin heavy chain with one immunoglobulin light chain. One skilled in the art will readily appreciate that a half antibody can encompass fragments thereof and can also have an antigen-binding domain consisting of a single variable domain, for example of camelid origin.

[0096] A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and / or has been produced using any of the techniques for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0097] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup (also referred to herein as a "subtype") of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD, vols. 1-3, 1991. In one embodiment, for VL, the subgroup is subgroup kappa III or kappa IV as in Kabat et al. (supra). In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al. (supra).

[0098] "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 are replaced 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 instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin, and all or substantially all of the FRs being those 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 further 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.

[0099] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), including, but not limited to, a cytotoxic agent.

[0100] The term "isolated" as used to describe various antibodies disclosed herein refers to an antibody that has been identified, separated and / or recovered from the cell or cell culture in which it is expressed. Contaminating components of its natural environment are typically substances that would interfere with diagnostic or therapeutic uses of the polypeptide, and these may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined, for example, by electrophoretic (e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse-phase HPLC) methods. For a review of methods for assessing antibody purity, see, for example, Flatman et al. J. Chromatogr. B 848:79-87, 2007. In a preferred embodiment, the antibody will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. An isolated antibody includes an antibody in situ within a recombinant cell, since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, an isolated polypeptide will be prepared by at least one purification step.

[0101] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, except for possible variant antibodies (such variants are generally present in minor amounts) that contain, for example, naturally occurring mutations or arise during the production of a monoclonal antibody preparation. Typically, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or a portion of the human immunoglobulin loci; such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0102] The term "multispecific antibody" is used in the broadest sense and specifically covers antibodies comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), where the VHVL unit has polyepitopic specificity (i.e., can bind to two different epitopes on one biological molecule or each epitope on a different biological molecule). Such multispecific antibodies include, but are not limited to, full-length antibodies, antibodies with two or more VL and VH domains, antibody fragments (such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and triabodies), antibody fragments that are covalently or non-covalently linked, etc. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). "Bispecific" or "bispecific" refers to the ability to specifically bind to two different epitopes on the same or different target(s). However, in contrast to bispecific antibodies, diabodies have two antigen-binding arms that are identical in amino acid sequence, with each Fab arm being capable of recognizing two antigens. Bispecificity allows an antibody to interact with two different antigens as a single Fab or IgG molecule with high affinity. According to one embodiment, a multispecific antibody in the IgG1 format 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 only to one epitope.

[0103] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0104] The term "variable" refers to the fact that certain segments of the variable domains differ widely in sequence among antibodies. The variable or "V" domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the full length variable domain of 110 amino acids. Instead, the V regions consist of relatively invariable stretches, called framework regions (FR), of 15-30 amino acids separated by shorter regions of extreme variability, called "hypervariable regions", each 9-12 amino acids long. As used herein, the term "hypervariable region" or "HVR" refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable regions generally include amino acid residues from, e.g., about residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the VL, and about residues 26-35 (H1), 49-65 (H2), and 95-102 (H3) in the VH (in one embodiment, H1 is about residues 31-35); Kabat et al., supra), and / or the "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the VL, and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the VH; Chothia et al., supra). et al. J. Mol. Biol. 196:901-917, 1987. Naturally occurring heavy and light chain variable domains each contain four FRs that largely adopt a beta-sheet conformation, which are connected by three hypervariable regions, thereby forming loops that connect, and in some cases form part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs, and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., supra). Thus, the HVR and FR sequences generally occur in the VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as participating in antibody-dependent cellular cytotoxicity (ADCC).

[0105] The terms "Kabat numbering of variable domain residues" or "Kabat numbering of amino acid positions" and variations thereof refer to the numbering system used for the heavy or light chain variable domains of the antibody compilations in Kabat et al. (supra). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FR or HVR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). For a given antibody, the Kabat numbering of residues may be determined by alignment of the antibody sequence with the "standard" Kabat numbered sequence at the regions of homology.

[0106] The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index as reported in Kabat et al., supra). The "EU index as in Kabat" refers to the numbering of residues in the human IgG1 EU antibody. Unless otherwise stated herein, reference to a residue number in a variable domain of an antibody refers to the residue numbering according to the Kabat numbering system. Unless otherwise stated herein, reference to a residue number in a constant domain of an antibody refers to the residue numbering according to the EU numbering system (see, e.g., U.S. Provisional Patent Application No. 60 / 640,323, EU numbering figures).

[0107] As used herein, unless otherwise indicated, the term "interleukin-13 (IL-13)" refers to any native IL-13 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). IL-13 is a cytokine secreted by many cell types, including type 2 T helper (Th2) cells. The term encompasses "full-length," unprocessed IL-13, and any form of IL-13 that results from processing within the cell. An exemplary amino acid sequence of human IL-13 can be found, for example, in UniProtKB Accession No. P35225. IL-13 binds to the IL-13 receptor, which can include IL-4 receptor alpha (IL4Rα) complexed with IL-13 receptor subunit alpha 1 (IL13RA1) or IL-13 receptor subunit alpha 2 (IL13RA2). For example, IL-13 can bind to a complex of IL-4 receptor alpha and IL-13 receptor subunit alpha 1.

[0108] The terms "anti-IL-13 antibody," "antibody that binds IL-13," and "antibody that specifically binds IL-13" refer to an antibody that can bind IL-13 with sufficient affinity such that the antibody is useful in targeting IL-13 as a diagnostic and / or therapeutic agent. In one embodiment, the extent of binding of an anti-IL-13 antibody to an unrelated, non-IL-13 protein is less than about 10% of the antibody's binding to IL-13 as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds IL-13 has an affinity of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13In certain embodiments, the anti-IL-13 antibody binds to an epitope of IL-13 that is conserved among IL-13 from different species. Exemplary anti-IL-13 antibodies include, but are not limited to, lebrikizumab, 228B / C-1, 228A-4, 227-26, and 227-43 (see, e.g., U.S. Patent Nos. 7,674,459, 8,067,199, 8,088,618, 8,318,160, and 8,734,797, which are hereby incorporated by reference in their entireties).

[0109] As used herein, the term "vascular endothelial growth factor" or "VEGF" refers to the 165 amino acid human vascular endothelial cell growth factor described by Leung et al. Science 246:1306, 1989, and Houck et al. Mol. Endocrin. 5:1806, 1991, along with their naturally occurring allelic and processed forms, as well as the related 121, 189, and 206 amino acid human vascular endothelial cell growth factors. The term "VEGF" also refers to VEGF derived from non-human species, such as mouse, rat, or primate. VEGF derived from a particular species may be designated by terms such as hVEGF for human VEGF, and mVEGF for mouse VEGF. The term "VEGF" is also used to refer to truncated forms of the polypeptide, including amino acids 8-109 or 1-109 of the 165 amino acid human vascular endothelial cell growth factor. Reference to any such form of VEGF is referred to in this application as, for example, "VEGF 109 ", "VEGF(8-109)", "VEGF(1-109)" or "VEGF 165". The amino acid positions of "truncated" native VEGF are numbered as shown in the native VEGF sequence. For example, amino acid position 17 (methionine) in truncated native VEGF is also position 17 (methionine) in native VEGF. Truncated native VEGF has binding affinity for the KDR and Flt-1 receptors comparable to native VEGF. As used herein, the term "VEGF variant" refers to a VEGF polypeptide that includes one or more amino acid mutations in the native VEGF sequence. Optionally, the one or more amino acid mutations include amino acid substitution(s). For purposes of concisely designating the VEGF variants described herein, it should be noted that the numbers refer to the position of the amino acid residue along the amino acid sequence of the putative native VEGF (provided in Leung et al., supra, and Houck et al., supra). Unless otherwise specified, as used herein, the term "VEGF" refers to VEGF-A.

[0110] The terms "anti-VEGF antibody," "antibody that binds VEGF," and "antibody that specifically binds VEGF" refer to an antibody that can bind VEGF with sufficient affinity such that the antibody is useful in targeting VEGF as a diagnostic and / or therapeutic agent. In one embodiment, the extent of binding of an anti-VEGF antibody to unrelated, non-VEGF proteins is less than about 10% of the binding of the antibody to VEGF, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds VEGF has an affinity of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M) of VEGF. In certain embodiments, the anti-VEGF antibody binds to an epitope of VEGF that is conserved among VEGF from different species.

[0111] Non-limiting examples of anti-VEGF antibodies include, for example, but are not limited to, the anti-VEGF antibody "bevacizumab (BV or Bev)," also known as "rhuMAb VEGF" or "AVASTIN®," which is a recombinant humanized anti-VEGF monoclonal antibody produced according to Presta et al., Cancer Res. 57:4593-4599 (1997). Bevacizumab contains mutated human IgG1 framework regions and antigen-binding complementarity determining regions derived from the murine anti-hVEGF monoclonal antibody A.4.6.1, which blocks binding of human VEGF to its receptor. Approximately 93% of the amino acid sequence of bevacizumab, including most of the framework regions, is derived from human IgG1, and about 7% of the sequence is derived from the murine antibody A4.6.1. Bevacizumab has a molecular mass of about 149,000 daltons and is glycosylated. Other exemplary anti-VEGF antibodies include, for example, those described in U.S. Patent No. 6,884,879 and WO2005 / 044853. A further example includes the anti-VEGF antibody ranibizumab (Lucentis® antibody or rhuFab V2), which is a humanized, affinity-matured anti-human VEGF Fab fragment. Ranibizumab is produced by standard recombinant techniques in E. coli expression vectors and bacterial fermentation. Ranibizumab is not glycosylated and has a molecular mass of about 48,000 daltons. See WO1998 / 45331 and US2003 / 0190317.

[0112] As used herein, "administering" refers to a method of giving a dosage of a compound (e.g., an antibody of the invention or a nucleic acid encoding an antibody of the invention) or composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising an antibody of the invention) to a subject. The compositions utilized in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intravitreally, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, intraorbitally, orally, topically, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by localized perfusion directly bathing target cells, by catheter, by lavage, in a capsule, or in a lipid composition. The compositions utilized in the methods described herein can also be administered systemically or locally. The method of administration can vary depending on a variety of factors, such as the compound or composition being administered and the severity of the condition, disease, or disorder being treated.

[0113] "Angiogenesis" refers to the process by which new blood vessels are formed from pre-existing vessels. Angiogenesis is distinct from vasculogenesis, which is the formation of new endothelial cells from mesodermal cell precursors. Disorders associated with pathological angiogenesis can be treated by the compositions and methods of the invention. These disorders include both non-neoplastic and proliferative disorders. Cellular proliferative disorders include, but are not limited to, those described below.Non-neoplastic disorders include ocular conditions (non-limiting examples of ocular conditions include, for example, retinopathies, including proliferative diabetic retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic and other ischemia-related retinopathies, diabetic macular edema (DME), pathologic myopia, von Hippel-Lindau disease, ocular histoplasmosis, retinal vein occlusion (including central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO) forms), corneal neovascularization, retinal neovascularization, retinopathy of prematurity (ROP), familial exudative vitreoretinopathy (FEVR), Coats' disease, Norrie's osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, and hypertensive retinopathy), unwanted or abnormal chronic hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriatic plaque, sarcoidosis, atherosclerosis, atherosclerotic plaque, vascular restenosis, arteriovenous malformations (AVM), meningiomas, hemangiomas, angiofibromas, thyroid hyperplasia (including Graves' disease), corneal and other tissue transplants, chronic inflammation, pulmonary inflammation, acute lung injury / ARDS, sepsis, primary pulmonary hypertension, malignant pulmonary effusion, cerebral edema (e.g. associated with acute stroke / close head injury / trauma), synovial inflammation, pannus formation in rheumatoid arthritis, myositis ossificans, hypertropic bone formation, osteoarthritis (OA), refractory ascites, polycystic ovarian disease, endometriosis, extracellular fluid disorders (3rd These conditions include, but are not limited to, chronic inflammation such as inflammatory bowel disease (pancreatitis, compartment syndrome, burns, bowel disease), uterine fibroids, preterm labor, chronic inflammation such as IBD (Crohn's disease and ulcerative colitis), renal allograft rejection, inflammatory bowel disease, nephrotic syndrome, unwanted or abnormal tissue mass growth (non-cancerous), hemophilic joints, hypertrophic scars, hair growth inhibition, Osler-Rendu syndrome, pyogenic granulomatous retrolental fibroplasia, scleroderma, trachoma, vascular adhesions, synovitis, dermatitis, preeclampsia, ascites, pericardial effusion (such as that associated with pericarditis), and pleural effusion.

[0114] "Anti-angiogenic agent" or "angiogenesis inhibitor" refers to a small molecular weight substance, polynucleotide, polypeptide, isolated protein, recombinant protein, antibody, or a complex or fusion protein thereof that inhibits angiogenesis, vasculogenesis, or undesired vascular permeability, either directly or indirectly. It should be understood that anti-angiogenic agents include agents that bind to angiogenic factors or their receptors and block their angiogenic activity. For example, anti-angiogenic agents are antibodies or other antagonists against angiogenic agents as defined above, such as antibodies against VEGF-A or antibodies against VEGF-A receptors (e.g., KDR receptor or Flt-1 receptor), anti-PDGFR inhibitors such as GLEEVEC™ (imatinib mesylate). Anti-angiogenic agents also include natural angiogenesis inhibitors, such as angiostatin, endostatin, and the like. See, e.g., Klagsbrun and D'Amore, Annu. Rev. Physiol., 53:217-39 (1991); Streit and Detmar, Oncogene, 22:3172-3179 (2003) (e.g., Table 3, which lists anti-angiogenic therapies in malignant melanoma); Ferrara & Alitalo, Nature Medicine 5(12):1359-1364 (1999); Tonini et al., Oncogene, 22:6549-6556 (2003) (e.g., Table 2, which lists known anti-angiogenic factors); and Sato Int. J. Clin. Oncol., 8:200-206 (2003) (e.g., Table 1, which lists anti-angiogenic agents used in clinical trials).

[0115] "Asthma" refers to a chronic lung disease that may involve airway inflammation, hyperresponsiveness, and obstruction. Physiologically, airway hyperresponsiveness is evidenced by reduced bronchial airflow following bronchial provocation with methacholine or histamine. Bronchial provocation with allergen induces a rapid early-phase immunoglobulin E (IgE)-mediated reduction in bronchial airflow, followed in most patients by a late-phase IgE-mediated response with reduced bronchial airflow for 4-8 hours. The early response is caused by the acute release of inflammatory substances such as histamine, PGD2, leukotrienes, tryptase and platelet activating factor (PAF), while the delayed response is caused by newly synthesized proinflammatory cytokines (e.g., TNFα, IL-4, IL-13) and chemokines (e.g., MCP-1 and MIP-1α) (Busse et al. Allergy: Principles and Practice, Ed. Middleston, 1173, 1998). In chronic asthmatics, persistent pulmonary symptoms are mediated by an increased response of Th2 cells. Th2 cytokines, especially IL-13 and IL-4 produced by Th2 cells with NK phenotype (NKT) in the airways, as shown in rodent asthma models (Akbari et al. Nature Med., 9:582, 2003), are thought to play an important role in the disease (Larche et al. J. Allergy Clin. Immunol. 111:450, 2003). The gross findings of the asthmatic airways present with severe lung inflation, smooth muscle hypertrophy, reticular lamina thickening, mucosal edema, epithelial cell shedding, ciliated cell destruction, and mucus gland hypersecretion. Microscopically, asthma is characterized by the presence of increased numbers of eosinophils, neutrophils, lymphocytes, and plasma cells in bronchial tissue, bronchial secretions, and mucus. Initially, there is recruitment of leukocytes from the bloodstream to the airways by activated CD4+ T lymphocytes. Activated T lymphocytes also direct the release of inflammatory mediators from eosinophils, mast cells, and lymphocytes. In addition, Th2 cells produce IL-4, IL-5, IL-9, and IL-13. IL-4, together with IL-13, signals a switch from IgM to IgE antibodies.

[0116] The terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal tumors), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, and ovarian cancer. cancer), liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer and various types of head and neck cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentigo melanoma, nodular melanoma, and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL, , bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel growth associated with phacomatosis, edema (such as associated with brain tumors), and Meigs' syndrome.

[0117] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0118] The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

[0119] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0120] A "disorder" is any condition that would benefit from treatment, for example, with a therapeutic antibody. In some embodiments, the disorder can be an IL-13-mediated disorder or an IgE-mediated disorder. In other embodiments, the disorder can involve aberrant or pathological angiogenesis (e.g., excessive, inappropriate, or uncontrolled angiogenesis) or vascular permeability. The disorder can be chronic or acute.

[0121] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelanine; Acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecins (including the synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; potassium. Statins; CC-1065 (including its synthetic analogs adrezecin, carzelesin, and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); erytherobin; pancratistatin; sarcodictin; spongiostatins; chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosf nitrogen mustards, such as amide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trophosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; enediyne antibiotics, such as calicheamicin, particularly calicheamicin gamma 1I and calicheamicin omega 1I (see, e.g., Nicolaou et al. Angew. Chem Intl. Ed. Engl., 33:183-186 (1994));CDP323, an oral alpha-4 integrin inhibitor; dynemicin A, including; dynemicin; esperamicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (including MYOCET®), pegylated liposomal doxorubicin (CAELYX®, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, keramycin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilones, and 5-fluorouracil (5-FU );combretastatins;folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate;purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone;antiadrenal agents such as aminoglutethimide, mitotane, trilostane;folic acid replacement fluids such as floric acid;aceglatone;aldophosphamide glycosides;aminolevulinic acid;eniluracil;Amsacrine; Bestravcil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diazicon; Elformithine; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidynin; Maytansinoids such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerin; Pentostatin; Fenameth; Pirarubicin; Rosoxantrone; 2-Ethylhydrazide; Procarbazine; PSK® Polysaccharide Complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triazicon; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veraculin A, roridin A, and anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannommustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ), albumin engineered nanoparticle formulations of paclitaxel (ABRAXANE™), and docetaxel (TAXOTERE®, Rhome-Poulene Rorer, Antony, France; chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vincas that prevent the formation of microtubules from tubulin polymerization, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin;ibandronate; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), bisphosphonates such as tiludronate (SKELID®) or risedronate (ACTONEL®); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways associated with abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R) (e.g., erlotinib (TARCEVA™)); and VEGF-A, which reduces cell proliferation. vaccines such as THERATOOPE® vaccine, and gene therapy vaccines, e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib, Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); Perifosine, COX-2 inhibitors (e.g., e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors; tyrosine kinase inhibitors; serine-tyrosine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®);farnesyltransferase inhibitors such as lonafarnib (SCH6636, SARASAR™); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, which is an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, which is an abbreviation for the treatment regimen of oxaliplatin (ELOXATIN™) in combination with 5-FU and leucovorin, and a pharmaceutically acceptable salt, acid, or derivatives of any of the above; and combinations of two or more of the above;

[0122] Chemotherapeutic agents, as defined herein, include "anti-hormonal agents" or "endocrine therapeutic agents" that act to regulate, reduce, block or inhibit the effects of hormones that may promote the growth of cancer.They may themselves be hormones, including antiestrogens with mixed agonist / antagonist profiles, including tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxyfene, keoxifene, and selective estrogen receptor modulators (SERMs) (such as SERM3); fulvestrant (FASLODEX®); )), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels); steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEX®), letrozole (FEMARA®), and aminoglutethimide. Aromatase inhibitors, including romatase inhibitors, as well as other aromatase inhibitors, include vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and 4(5)-imidazole; luteinizing hormone releasing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; progestins (megestrol acetate and medroxyprogestin acetate); anti-androgens such as flutamide, nilutamide and bicalutamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above; and combinations of two or more of the above.

[0123] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins (including fragments and / or variants thereof) of bacterial, fungal, plant or animal origin; and various anti-tumor or anti-cancer agents disclosed herein.

[0124] An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0125] As used herein, a "growth inhibitor" refers to a compound or composition that inhibits cell growth either in vitro or in vivo. Thus, a growth inhibitor may significantly reduce the percentage of cells in S phase. Examples of growth inhibitors include agents that block cell cycle progression (outside of S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors (such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin). Agents that arrest G1, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and Ara-C, also extend to S-phase arrest. Further information can be found in Mendelsohn et al. eds., The Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antitineoplastic drugs" by Murakami et al. (WB Saunders, Philadelphia, 1995), e.g., p. 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs that are both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules derived from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.

[0126] As used herein, the term "IL-13-mediated disorder" refers to any disorder or condition mediated by or associated with IL-13. In some embodiments, an IL-13-mediated disorder is associated with excessive IL-13 levels or activity, which may present with atypical symptoms due to local and / or systemic IL-13 levels or activity in the body. IL-13 mediated disorders include, for example, allergies, asthma (including allergic asthma, atopic asthma, severe asthma, and non-allergic (intrinsic) asthma), autoimmune diseases (e.g., psoriasis, rheumatoid arthritis, juvenile rheumatoid arthritis, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), or other inflammatory diseases. For example, IL-13 mediated disorders include allergic rhinitis (e.g., seasonal allergic rhinitis), atopic dermatitis, allergic conjunctivitis, eczema, urticaria, food allergies, food hypersensitivity, abdominal disease, immune-mediated skin diseases (including, for example, bullous dermatitis, erythema multiforme, contact dermatitis), fibrosis, liver fibrosis, endomyocardial fibrosis, chronic bronchitis, bronchiectasis, idiopathic interstitial pneumonia, goblet cell metaplasia, pulmonary inflammatory disorders, inflammatory and fibrotic lung diseases (e.g., sarcoidosis, bronchitis, and pulmonary inflammatory disorders). and chronic obstructive pulmonary disease (COPD)), gluten-sensitive enteropathy, Whipple's disease, pulmonary immune disorders (including, for example, eosinophilic pneumonia (e.g., chronic eosinophilic pneumonia), idiopathic pulmonary fibrosis, allergic bronchopulmonary aspergillosis, and hypersensitivity pneumonitis), Churg-Strauss syndrome (atopy in addition to periarteritis nodosa), eosinophilic myalgia syndrome, hypereosinophilic syndrome, edematous reactions (including, for example, episodic angioedema, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic enteritis, and eosinophilic colitis), nasal micropolyposis and polyposis, RSV infection, uveitis, scleroderma, osteoporosis, and malignancies (including cancers and tumors associated with aberrant expression of IL-13) (including, for example, Hodgkin's lymphoma).

[0127] As used herein, the term "IgE-mediated disorder" refers to any disorder or condition mediated by or associated with IgE. Exemplary IgE-mediated disorders include bronchial asthma, allergic rhinitis, allergic dermatitis, urticaria, anaphylaxis, or atopic dermatitis.

[0128] "Isolated nucleic acid" refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained within a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0129] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0130] The terms "level of expression" or "expression level" are used interchangeably and generally refer to the amount of a polynucleotide or an amino acid product or protein in a biological sample. "Expression" generally refers to the process by which the information encoded by a gene is converted into a structure present and operating in a cell. Thus, according to the present invention, "expression" of a gene may refer to transcription into a polynucleotide, translation into a protein, or even post-translational modification of a protein. Fragments of a transcribed polynucleotide, translated proteins, or post-translationally modified proteins should also be considered as expressed, regardless of whether they originate from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of proteins, for example, by proteolysis. "Expressed genes" include those that are transcribed into polynucleotides as mRNA and then translated into proteins, as well as those that are transcribed into RNA but not translated into proteins (e.g., transfer RNA and ribosomal RNA).

[0131] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0132] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA of a presequence or secretory leader is operably linked to DNA of 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 that 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, and, in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0133] "Percentage (%) amino acid sequence identity" with respect to a polypeptide sequence specified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide being compared, after aligning the sequences and introducing gaps as necessary to obtain the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways that are 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 the appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. 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 created by Genentech, Inc., and its source code has been filed with the U.S. Copyright Office, Washington DC, 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0134] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, it may be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as perfect matches by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program, as described in the immediately preceding paragraph.

[0135] The amino acid sequences described herein are contiguous amino acid sequences unless otherwise specified.

[0136] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications, and / or warnings concerning the use of such therapeutic products.

[0137] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredients contained in the preparation to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation will be administered.

[0138] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0139] The term "prodrug" as used in this application refers to a precursor or derivative form of a pharma- ceutically active substance that has a lower cytotoxicity to tumor cells compared to the parent drug and can be enzymatically activated or converted to a more active parent form.See, for example, Wilman, "Prodrugs in Cancer Chemotherapy," Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al. "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al. (ed.), pp. 247-267, Humana Press (1985). Prodrugs of the present invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs, which may be converted to more active cytotoxicity-free drugs. Examples of cytotoxic drugs that can be derivatized into prodrug forms for use in the present invention include, but are not limited to, the chemotherapeutic agents mentioned above.

[0140] By "reducing or inhibiting" is meant the ability to cause an overall decrease, preferably by 20% or more, more preferably by 50% or more, and most preferably by 75%, 85%, 90%, 95% or more. Reducing or inhibiting can refer to the symptoms of the disorder being treated, the presence or size of metastases, the size of the primary tumor.

[0141] A "patient," "subject," or "individual" is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals (such as cows and sheep), sport animals, pets (such as cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), and rodents (e.g., mice and rats).

[0142] 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 an IL-13-mediated disorder, a therapeutically effective amount of an antibody or antibody fragment (e.g., an anti-IL-13 antibody) can ameliorate or treat the disease, or prevent, reduce, ameliorate, or treat symptoms associated with the disease. In the case of a disorder associated with pathological angiogenesis, a therapeutically effective amount of an antibody or antibody fragment (e.g., an anti-VEGF antibody) can ameliorate or treat the disease, or prevent, reduce, ameliorate, or treat symptoms associated with the disease. In the case of a proliferative disease (e.g., solid tumor), a therapeutically effective amount of an antibody or antibody fragment can reduce the number of cancer cells; reduce the size of the primary tumor; inhibit (i.e., slow down to some extent and preferably stop) the invasion of cancer cells into peripheral organs; inhibit (i.e., slow down to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more of symptoms associated with the disorder. To the extent the antibody or antibody fragment is capable of preventing the growth of and / or killing existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer therapy, efficacy in vivo may 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.

[0143] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequence of the disease, prevention of metastasis, slowing the rate of disease progression, remission or mitigation of disease symptoms, and remission or improvement of prognosis. In some embodiments, the antibodies of the invention are used to delay the development of disease or slow down the progression of disease.

[0144] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication, and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors" or "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" may be used interchangeably.

[0145] II. METHODS AND COMPOSITIONS In one aspect, the invention is based, in part, on novel methods of identifying bacteria that contain binding polypeptides, e.g., screening for bacteria that express antibodies that bind to a labeled antigen. The methods of the invention are useful, e.g., for screening for antibodies with improved expression, stability, and / or affinity. In another aspect, the invention is based, in part, on novel antibodies that bind to IL-13 or VEGF. The anti-IL-13 antibodies of the invention are useful for diagnosing and / or treating IL-13-mediated disorders. The anti-VEGF antibodies described herein are useful, for example, for diagnosing and / or treating disorders associated with pathological angiogenesis (e.g., cancer) and / or inhibiting vascular permeability.

[0146] A. Exemplary Methods of the Invention The invention provides methods for identifying bacteria that comprise a binding polypeptide that specifically binds to a target molecule. The invention also provides methods for identifying bacteria with improved expression of a binding polypeptide. The invention also provides methods for identifying binding polypeptides with improved expression and / or stability.

[0147] For example, in some instances, the present invention provides a method for identifying bacteria comprising a binding polypeptide that specifically binds to a target molecule, the method comprising the steps of: (a) identifying a binding polypeptide that is greater than about 1 kDa (e.g., about 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 25 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 250 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 1100 kDa, 1200 kDa, 1300 kDa, 1400 kDa, 1500 kDa, 1600 kDa, 1700 kDa, 1800 kDa, 1900 kDa, 2000 kDa, 2500 kDa, 300 The present invention provides a method comprising the steps of: (a) providing a bacterium having an outer membrane that is permeable to molecules having a molecular weight of at least about 100 kDa (a, 50 kDa, or greater), wherein the bacterium expresses a nucleic acid encoding a candidate binding polypeptide, and the candidate binding polypeptide is present in the periplasm of the bacterium; (b) contacting the bacterium with a detectably labeled target molecule; and (c) identifying the bacterium as containing the binding polypeptide by the presence of the labeled target molecule in the periplasm. In some embodiments, the bacterium remains viable after step (c). In some embodiments, the target molecule is labeled using a covalently attached label. In other embodiments, the target molecule is labeled using a non-covalently attached label.

[0148] In another example, in some examples, the present invention provides a method for identifying bacteria having improved expression of a binding polypeptide that specifically binds to a target molecule, the method comprising the steps of: (a) identifying a binding polypeptide greater than about 1 kDa (e.g., about 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 25 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 250 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 1100 kDa, 1200 kDa, 1300 kDa, 1400 kDa, 1500 kDa, 1600 kDa, 1700 kDa, 1800 kDa, 1900 kDa, 2000 kDa, 2500 kDa, 3 The present invention provides a method comprising the steps of: (a) providing a bacterium having an outer membrane that is permeable to molecules having a molecular weight of 0 kDa, 50 kDa, or more, wherein the bacterium expresses a nucleic acid encoding a binding polypeptide, and the binding polypeptide is present in the periplasm of the bacterium; (b) contacting the bacterium with a detectably labeled target molecule; and (c) identifying the bacterium as having improved expression of the binding polypeptide by the presence of the labeled target molecule in the periplasm. In some embodiments, the bacterium remains viable after step (c). In some embodiments, the target molecule is labeled using a covalently attached label. In other embodiments, the target molecule is labeled using a non-covalently attached label.

[0149] In some examples of any of the aforementioned methods, the method further comprises repeating the steps of the method at least once. For example, the method may further comprise repeating the steps of the method at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times. In some embodiments, at least one step of the method is repeated. In some examples, all of the steps of the method may be repeated at least once (e.g., repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times). In some examples, all of the steps of the method may be repeated sequentially at least once (e.g., repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times). In some examples, the steps of the method are repeated twice. In some examples, the steps of the method are repeated three times. In some examples, the following steps are repeated: (a) providing a bacterium having an outer membrane that is permeable to molecules having a molecular weight of more than about 1 kDa (e.g., about 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 25 kDa, 30 kDa, 40 kDa, 50 kDa, or more), where the bacterium expresses a nucleic acid encoding a candidate binding polypeptide, and the candidate binding polypeptide is present within the periplasm of the bacterium; (b) contacting the bacterium with a detectably labeled target molecule; and (c) identifying the bacterium as containing the binding polypeptide by the presence of the labeled target molecule in the periplasm. In some examples, the method further comprises incubating the bacteria in a growth medium prior to repeating the steps of the method. Examples of bacterial growth media include CRAP, SOC, Luria Broth (LB), or any other bacterial culture medium known in the art.

[0150] In some examples of any of the aforementioned methods, the target molecule is less than about 500 kDa (e.g., about 500 kDa, about 480 kDa, about 460 kDa, about 440 kDa, about 420 kDa, about 440 kDa, about 420 kDa, about 400 kDa, about 380 kDa, about 360 kDa, about 340 kDa, about 320 kDa, about 30 In some embodiments, the antibody has a molecular weight of less than about 0 kDa, about 280 kDa, about 260 kDa, about 250 kDa, about 220 kDa, about 200 kDa, about 180 kDa, about 160 kDa, about 150 kDa, about 130 kDa, about 120 kDa, about 110 kDa, about 100 kDa, about 80 kDa, about 60 kDa, about 40 kDa, or less than about 20 kDa. Exemplary target molecules include those having a molecular weight of about 1 kDa to about 500 kDa, about 1 kDa to about 400 kDa, about 1 kDa to about 300 kDa, about 1 kDa to about 250 kDa, about 1 kDa to about 230 kDa, about 1 kDa to about 220 kDa, about 1 kDa to about 200 kDa, about 1 kDa to about 180 kDa, about 1 kDa to about 160 kDa, about 1 kDa to about 140 kDa, about 1 kDa to about 120 kDa, about 1 kDa to about 100 kDa, about 1 kDa to about 80 kDa, about 1 kDa to about 60 kDa, about 1 kDa to about 40 kDa, about 1 kDa to about 20 kDa, about The compound has a molecular weight within the range of about 8 kDa to about 10 kDa, about 8 kDa to about 500 kDa, about 8 kDa to about 400 kDa, about 8 kDa to about 300 kDa, about 8 kDa to about 250 kDa, about 8 kDa to about 220 kDa, about 8 kDa to about 200 kDa, about 8 kDa to about 180 kDa, about 8 kDa to about 160 kDa, about 8 kDa to about 140 kDa, about 8 kDa to about 120 kDa, about 8 kDa to about 100 kDa, about 8 kDa to about 80 kDa, about 8 kDa to about 60 kDa, about 8 kDa to about 40 kDa, or about 8 kDa to about 20 kDa.

[0151] In some examples of any of the aforementioned methods, the target molecule has a molecular weight of about 10 kDa or more. For example, in some embodiments, the target molecule has a molecular weight of about 10 kDa to about 700 kDa, about 10 kDa to about 600 kDa, about 10 kDa to about 500 kDa, about 10 kDa to about 480 kDa, about 10 kDa to about 470 kDa, about 10 kDa to about 460 kDa, about 10 kDa to about 450 kDa, about 10 kDa to about 440 kDa, about 10 kDa to about 430 kDa, about 10 kDa to about 420 kDa, about 10 kDa to about 410 kDa, about 10 kDa to about 440 kDa, about 10 kDa to about 450 kDa, about 10 kDa to about 460 kDa, about 10 kDa to about 470 kDa, about 10 kDa to about 480 kDa, about 10 kDa to about 490 kDa, about 10 kDa to about 500 kDa, about 10 kDa to about 510 kDa, about 10 kDa to about 520 kDa, about 10 kDa to about 530 kDa, about 10 kDa to about 540 kDa, about 10 kDa to about 550 kDa, about 10 kDa to about 560 kDa, about 10 kDa to about 570 kDa, about 10 kDa to about 580 kDa, about 10 kDa to about 590 kDa, about 10 kDa to about 00kDa, about 10kDa to about 390kDa, about 10kDa to about 380kDa, about 10kDa to about 370kDa, about 10kDa to about 360kDa, about 10kDa to about 350kDa, about 10kDa to about 340kDa, about 10 kDa ~ about 330kDa, about 10kDa - about 320kDa, about 10kDa - about 310kDa, about 10kDa - about 300kDa, about 10kDa - about 290kDa, about 10kDa - about 280kDa, about 10kDa - about 270kD a, about 10kDa - about 260kDa, about 10kDa - about 250kDa, about 10kDa - about 240kDa, about 10kDa - about 230kDa, about 10kDa - about 220kDa, about 10kDa - about 210kDa, about 10kDa - Approximately 200kDa, approximately 10kDa to approximately 190kDa, approximately 10kDa to approximately 180kDa, approximately 10kDa to approximately 170kDa, approximately 10kDa to approximately 160kDa, approximately 10kDa to approximately 150kDa, approximately 10kDa to approximately 140kDa, approximately 1 The molecular weight is within the range of about 0 kDa to about 130 kDa, about 10 kDa to about 120 kDa, about 10 kDa to about 110 kDa, about 10 kDa to about 100 kDa, about 10 kDa to about 90 kDa, about 10 kDa to about 80 kDa, about 10 kDa to about 70 kDa, about 10 kDa to about 60 kDa, about 10 kDa to about 50 kDa, about 10 kDa to about 40 kDa, about 10 kDa to about 30 kDa, about 10 kDa to about 20 kDa, or about 10 kDa to about 15 kDa.

[0152] In some examples, the invention provides a method for identifying bacteria that contain a binding polypeptide that specifically binds to a target molecule, the method comprising the steps of: (a) providing a bacterium that contains a mutation that allows diffusion of a membrane-impermeable molecule across the outer membrane into the periplasm, where the bacterium expresses a nucleic acid encoding a candidate binding polypeptide, and the candidate binding polypeptide is present in the periplasm of the bacterium; (b) contacting the bacterium with a detectably labeled target molecule; and (c) identifying the bacterium as containing the binding polypeptide by the presence of the labeled target molecule in the periplasm. In some embodiments, the bacterium remains viable after step (c). In some examples, the method further comprises subjecting the bacterium to conditions that permeabilize the bacterial outer membrane prior to step (b). In some examples, the method further comprises resealing the bacterial outer membrane after contacting the bacterium with the detectably labeled target molecule.

[0153] In another example, the invention provides a method for identifying bacteria having improved expression of a binding polypeptide that specifically binds to a target molecule, the method comprising the steps of: (a) providing a bacterium that includes a mutation that allows diffusion of a membrane-impermeable molecule across an outer membrane into the periplasm, where the bacterium expresses a nucleic acid encoding the binding polypeptide, and the binding polypeptide is present in the periplasm of the bacterium; (b) contacting the bacterium with a detectably labeled target molecule; and (c) identifying the bacterium as having improved expression of the binding polypeptide by the presence of the labeled target molecule in the periplasm. In some embodiments, the bacterium remains viable after step (c). In some examples, the method further comprises subjecting the bacterium to conditions that permeabilize the outer membrane of the bacterium prior to step (b). In some examples, the method further comprises resealing the outer membrane of the bacterium after contacting the bacterium with the detectably labeled target molecule.

[0154] In some examples of any of the aforementioned methods, the method further comprises repeating the steps of the method at least once. For example, the method may further comprise repeating the steps of the method at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times. In some embodiments, at least one step of the method is repeated. In some examples, all of the steps of the method may be repeated at least once (e.g., repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times). In some examples, all of the steps of the method may be repeated sequentially at least once (e.g., repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times). In some examples, the steps of the method are repeated twice. In some examples, the steps of the method are repeated three times. In some examples, the following steps are repeated: (a) providing a bacterium containing a mutation that allows diffusion of a membrane impermeable molecule across the outer membrane into the periplasm, where the bacterium expresses a nucleic acid encoding a candidate binding polypeptide, and the candidate binding polypeptide is present in the periplasm of the bacterium, (b) contacting the bacterium with a detectably labeled target molecule, and (c) identifying the bacterium as containing the binding polypeptide by the presence of the labeled target molecule in the periplasm. In some examples, the method further comprises incubating the bacterium in a growth medium before repeating the steps of the method.

[0155] In other examples, the invention provides methods for identifying bacteria that contain a binding polypeptide that specifically binds to a target molecule, the methods comprising: (a) providing a bacterium expressing a nucleic acid encoding a candidate binding polypeptide, where the candidate binding polypeptide is present within the periplasm of the bacterium; (b) subjecting the bacterium to conditions that permeabilize the bacterial outer membrane; (c) contacting the bacterium with a detectably labeled target molecule; (d) after step (c), resealing the bacterial outer membrane; and (e) identifying the bacterium as containing the binding polypeptide by the presence of the labeled target molecule in the periplasm. In some examples, the bacterium remains viable after step (e) of the method.

[0156] In yet other examples, the invention provides methods for identifying bacteria having improved expression of a binding polypeptide that specifically binds to a target molecule, the methods comprising: (a) providing a bacterium expressing a nucleic acid encoding a binding polypeptide, wherein the binding polypeptide is present within the periplasm of the bacterium; (b) subjecting the bacterium to conditions that permeabilize the bacterial outer membrane; (c) contacting the bacterium with a detectably labeled target molecule; (d) after step (c), resealing the bacterial outer membrane; and (e) identifying the bacterium as having improved expression of the binding polypeptide by the presence of the labeled target molecule in the periplasm. In some examples, the bacterium remains viable after step (e) of the method.

[0157] In some examples, the method further includes repeating the steps of the method at least once. For example, the method may further include repeating the steps of the method at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times. In some embodiments, at least one step of the method is repeated. In some examples, all of the steps of the method may be repeated at least once (e.g., repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times). In some examples, all of the steps of the method may be repeated sequentially at least once (e.g., repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times). In some examples, the steps of the method are repeated twice. In some examples, the steps of the method are repeated three times. In some examples, the following steps are repeated: (a) providing a bacterium expressing a nucleic acid encoding a candidate binding polypeptide, wherein the candidate binding polypeptide is present within the periplasm of the bacterium; (b) subjecting the bacterium to conditions that permeabilize the bacterial outer membrane; (c) contacting the bacterium with a detectably labeled target molecule; (d) after step (c), resealing the bacterial outer membrane; and (e) identifying the bacterium as containing the binding polypeptide by the presence of the labeled target molecule in the periplasm.

[0158] In any of the foregoing methods, the bacteria may have improved expression of the binding polypeptide by at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, or more. In some examples, the bacteria may have 1-10-fold improved expression, 1-7-fold improved expression, 1-6-fold improved expression, 1-5-fold improved expression, 1-4-fold improved expression, 1-3-fold improved expression, 2-10-fold improved expression, 2-8-fold improved expression, or 2-6-fold improved expression. In some embodiments, the improvement is compared to the expression level of the binding polypeptide in a wild-type bacterium, while in other embodiments, the improvement is compared to the expression level of the binding polypeptide in a mutant bacterium.

[0159] In any of the above methods, the bacteria remain viable after the steps of the method are performed. In some examples, the bacteria may undergo at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more cell divisions after the steps of the method are performed. In some examples, viability is measured using a viability stain, such as SYTOX® Green, propidium iodide, or other viability stains known in the art. In some examples, viability is measured using a live / dead staining kit, such as Life Technologies' LIVE / DEAD® cell viability assay. In some examples, viability is measured by plating the bacteria on a bacterial medium plate. In some examples, after plating on a bacterial medium plate, the viable cells will give rise to colonies.

[0160] In any of the aforementioned methods, the bacteria may contain a mutation that allows diffusion of membrane-impermeable molecules across the outer membrane into the periplasm. Mutations that can allow diffusion of membrane-impermeable molecules across the outer membrane into the periplasm (sometimes also referred to as "periplasmic leaky" or "lky" mutants) are described, for example, in Hancock, Ann. Rev. Microbiol. 38:237-264, 1984, and include, but are not limited to, deep rough (heptose-deficient) mutants of E. coli and S. typhimurium (e.g., rfaD, rfaE, rfaH, and rfaRd), env mutants (e.g., envA, envB, envM-T), and colicin-resistant mutants in E. coli (e.g., tolC and tolA,B), which can affect lipopolysaccharide (LPS) composition in the outer membrane. In some examples, the mutation is in a gene that encodes an outer membrane protein and / or a protein that affects lipopolysaccharide (LPS) composition. In some examples, the mutation is in a gene encoding an outer membrane protein, such as the major outer membrane lipoprotein Lpp (Lpp), porin, or TolC. In some examples, the outer membrane protein is Lpp. Any suitable mutation in a gene encoding Lpp can be used. Exemplary lpp mutations include, but are not limited to, lpp-1; lpp-21; lpp-3; lpp5508; lpp-6; lpp::Tn5KAN-2; lppD70E, G, or S; lppK75S or T; lppK78R; lppR77D or L; lppS22D; lppY76C, D, E, G, N, P, or S; lppY76F, H, I, or L; Δlpp, Δlpp-254, and Δlpp-752::kan, or those described herein. Such lpp mutations can be obtained, for example, from repositories such as The Coli Genetic Stock Center (CGSC). In other examples, mutations in the gene encoding Lpp can be made using standard genetic approaches.In some embodiments, the mutation is in a gene encoding a protein that affects LPS composition, such as an Env protein (e.g., EnvA, EnvB, and EnvM), RfaD, RfaE, RfaH, RfaRd, TolA, TolB, and TolD.

[0161] In any of the foregoing methods, the method may include subjecting the bacteria to conditions that permeabilize the outer membrane. In some examples, the conditions that permeabilize the outer membrane include treating the bacteria with a permeabilizing agent. In some examples, the permeabilizing agent is a chelating agent (including divalent cation chelating agents (e.g., EDTA, BAPTA, and EGTA)), NaCl, sucrose, antibiotics (e.g., aminoglycosides (e.g., streptomycin and gentamicin), polymyxin B, deacylated polymyxin, octapeptin, and benzylpenicillin), detergent, lysozyme, Tris, Tris-EDTA, ascorbate, polylysine, benzalkonium chloride, protamine, bactericidal / permeability enhancing protein (BPI), serum and / or complement, Ca 2+ , or a combination thereof. In some embodiments, the permeabilization agent comprises a chelating agent. In some examples, the chelating agent is a divalent cation chelating agent (including, for example, EDTA, BAPTA, and EGTA). In some embodiments, the divalent cation chelating agent is EDTA. In some examples, the concentration of the chelating agent, e.g., EDTA, is between about 1 μM and about 15 mM, e.g., about 1 μM, 10 μM, 100 μM, 500 μM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, or 15 mM. In some examples, the concentration of EDTA is 5 mM. In some examples, the permeabilization agent further comprises a buffer, e.g., phosphate buffered saline (PBS). In some examples, the permeabilization agent further comprises bovine serum albumin. In some examples, BSA is present at between 1% and about 10% by weight volume, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0162] In any of the aforementioned methods, the method may include resealing the outer membrane of the bacteria. The outer membrane may be completely resealed, but need not be completely resealed. For the methods of the present invention, partial resealing to a level that allows cell viability is sufficient. In some examples, the outer membrane is resealed when the membrane permeability barrier is restored to within 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% compared to a reference or wild-type bacterial outer membrane (e.g., the outer membrane of an E. coli 62A7 cell). In some examples, substantially resealing the outer membrane of the bacteria includes treating the bacteria with Mg. 2+ , Mn 2+ , Ca 2+ , Zn 2+ , Fe 2+ , Na + , and K + Salts such as, for example, SO 4 2- , F - , Cl 2 - , S.O. 3 2- , and P.O. 4 3- In some instances, substantially resealing the outer membrane of the bacterium involves contacting the bacterium with a salt of the aforementioned cation having an anion such as MgCl 2 In some embodiments, the method comprises contacting the MgCl 2 The concentration of is between about 1 mM and about 50 mM, for example, about 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM.

[0163] In any of the aforementioned methods, the step of contacting the bacteria with the detectably labeled target molecule may further include contacting the bacteria with a nucleic acid dye. In some examples, the nucleic acid dye is selected from the group consisting of Hoechst 33342 (also called Hoechst stain), 4',6-diamidino-2-phenylindole (DAPI), SYTO® 9, SYTO® 11, SYTO® 12, SYTO® 13, SYTO® 14, SYTO® 15, SYTO® 16, SYTO® 17, SYTO® 41, SYTO® 43, SYTO® 42, SYTO® 44, SYTO® 40, SYTO® 45, SYTO® 59, SYTO® 60, SYTO® 61, SYTO® 62, and SYTO® 63, SYTO® 64. In some embodiments, a nucleic acid dye is used that stains bacterial cells with an efficiency of at least about 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more). In some embodiments, a nucleic acid dye is used that stains bacterial cells with an efficiency of at least about 85%.

[0164] In any of the above methods, the detectably labeled target molecule may include a fluorescent label (also called a fluorophore). In some embodiments, any fluorescent label that is compatible with flow cytometry (e.g., FACS™) detection may be used. For example, any fluorescent label that has a quantum yield that allows sorting by flow cytometry (e.g., FACS™) may be used. In some examples, the fluorescent label includes a fluorescent dye or a fluorescent polypeptide. A variety of fluorescent labels are commercially available from a variety of vendors, such as Life Technologies. The fluorescent label may be selected based on its excitation and emission profile.

[0165] Non-limiting examples of fluorescent dyes that can be used include the ALEXA® dyes (e.g., ALEXA FLUOR® 488, ALEXA FLUOR® 514, ALEXA FLUOR® 532, ALEXA FLUOR® 555, ALEXA FLUOR® 546, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 610, ALEXA FLUOR® 633, ALEXA FLUOR® 647, ALEXA FLUOR® 660, ALEXA FLUOR® 680, ALEXA FLUOR® 700, ALEXA FLUOR® 750, and ALEXA FLUOR® 760). FLUOR® 790), DYLIGHT® dyes (e.g., DYLIGHT® 350, DYLIGHT® 405, DYLIGHT® 488, DYLIGHT® 550, DYLIGHT® 594, DYLIGHT® 633, DYLIGHT® 650, DYLIGHT® 680, DYLIGHT® 755, and DYLIGHT® 800), cyanine dyes (e.g., CY® 2, CY® 3, and CY® 5), BODIPY, TEXAS RED®, fluorescein and its derivatives (e.g., fluorescein isothiocyanate, FITC), and rhodamine and its derivatives (e.g., tetramethylrhodamine, TRITC). In some examples, the fluorescent dye is selected from the group consisting of ALEXA FLUOR® 488, ALEXA FLUOR® 647, and DYLIGHT® 649.

[0166] Exemplary fluorescent polypeptides that can be used include blue / UV fluorescent proteins (including TagBFP, mTagBFP2, Azurite, EBFP2, mKalama1, Sirius, Sapphire, and T-Sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, and mTFP1), green fluorescent proteins (e.g., GFP, EGFP, Emerald, Superfolder GFP, monomeric Azami, and the like). Green, TagGFP2, mUKG, mWasabi, Clover, and mNeonGreen), yellow fluorescent proteins (e.g., EYFP, Citrine, Venus, SYFP2, and TagYFP), orange fluorescent proteins (e.g., monomeric Kusabira-Orange, mKOk, mKO2, mOrange, and mOrange2), red fluorescent proteins (e.g., mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, RFP, Examples of fluorescent proteins include, but are not limited to, TagRFP, TagRFP-T, mApple, mRuby, and mRuby2), far-red fluorescent proteins (e.g., mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP), photoactivatable fluorescent proteins (e.g., PA-GFP, PAmCherry1, and PATagRFP), and photoconvertible fluorescent proteins (e.g., Kaede, KikGR1, PS-CFP2, mEos2, mEos3.2, and pSmOrange).

[0167] In some embodiments, the bacteria are contacted with the detectably labeled target molecule at a temperature of about 4° C. to about 25° C. (e.g., about 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 12° C., 14° C., 16° C., 18° C., 20° C., 22° C., 24° C., or 25° C.). In some embodiments, the temperature is about 4° C.

[0168] Any of the foregoing methods may further include at least one wash step (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 wash steps) involving resuspending the bacteria in a wash buffer. In some examples, the at least one wash step is performed after contacting the bacteria with the detectably labeled target molecule. In some examples, the wash buffer comprises a buffer, such as PBS. In some examples, the wash buffer comprises phosphate buffered saline. In some examples, the wash buffer comprises MgCl 2 In some examples, the wash buffer further comprises 1 mM to 50 mM MgCl 2 (e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 30 mM, 40 mM, or 50 mM MgCl 2 Includes.

[0169] In any of the foregoing methods, the selecting step may further include flow cytometry (e.g., FACS™). In some examples, the flow cytometry includes sorting single cells and sorting based on a signal of a detectably labeled target molecule. In some examples, the flow cytometry includes sorting based on a signal of a nucleic acid dye. In some examples, the flow cytometry includes, sequentially: (i) sorting based on a signal of a nucleic acid dye, (ii) sorting single cells, and (iii) sorting based on a signal of a detectably labeled target molecule. In some embodiments, the cells are about 1×10 5 ~Approx. 1×10 12 between 1 x 10 cells / ml, for example, 5 cells / ml, approximately 1 x 10 6 cells / ml, approximately 1 x 10 7 cells / ml, approximately 1 x 10 8 cells / ml, approximately 1 x 10 9 cells / ml, approximately 1 x 10 10 cells / ml, approximately 1 x 10 11 cells / ml, or approximately 1 x 1012 The concentration is in cells / ml.

[0170] In any of the foregoing methods, the bacterium may be a bacterium comprising an inner membrane, a periplasm, and an outer membrane. In some embodiments, the bacterium is a gram-negative bacterium. For example, in some instances, the bacterium is E. coli. Any suitable strain or genetic background of E. coli may be used. Commonly used strains and genetic backgrounds of E. coli are well known in the art. Exemplary E. coli strains include, but are not limited to, laboratory strains such as K-12 and its substrains (e.g., Clifton wild type, W3110, and DH5αE, etc.), and E. coli B and its substrains (e.g., BL21 and BL21(DE3)). In some embodiments, the E. coli is from strain 62A7. In some embodiments, the E. coli is from strain 33D3. In some embodiments, the E. coli is from strain 66C4.

[0171] In any of the foregoing methods, the binding polypeptide may be expressed in a soluble form within the periplasm of the bacteria.

[0172] In any of the aforementioned methods, the binding polypeptide may be expressed as a fusion protein in the bacterial periplasm. In some examples, the binding polypeptide is expressed as a fusion protein with a protein or fragment thereof that localizes to the outer surface of the inner membrane. It is understood that additional polypeptide sequences, such as linker polypeptides, may be added to the fusion protein. Such fusion proteins can be used to anchor the binding polypeptide to the outer surface of the inner membrane. In some embodiments, the leader peptide and the first six amino acids of an inner membrane lipoprotein (e.g., NlpA) may be included in the fusion protein. Such a fusion protein can serve as an N-terminal anchor of the binding polypeptide to the outer surface of the inner membrane. In some examples, the fusion protein is selected from the group consisting of Aas, AcrE (EnvC), AmiC, AmiD, ArtM, AraH, ArtQ, BtuC, CheY, CysT, CysW, DppB, DppC, DsbB, DsbD, ExbD, ExbB, FecC, FecD, FecR, FepD, FhuB, GlnP, HisM, HisQ, LacY, lipoprotein, LivH, LivM, LivA, LivE, MalF, MalG, MalC, MalD, MglC, ModB, NikB, NikC, NosY, OppB, phage-encoded celB, PhnM, PotB, PotC, PotH, PotI, ProW, PstA, PstC, K. pneumoniae pullulanase, RbsC, SecY, TatC, and TraB. In some embodiments, any lipoprotein or fragment thereof that includes a signal sequence and an aspartic acid at amino acid position 2 (either present in the wild-type protein or mutagenic (e.g., added by site-directed mutagenesis) may be included in the fusion protein. In some embodiments, a single transmembrane loop of any cytoplasmic protein may be used as a membrane anchor. The fusion protein may be an N-terminal or C-terminal fusion.

[0173] In some examples, the bacteria express a fusion protein that localizes to the outer surface of the inner membrane that contains a domain or a fragment thereof that can specifically bind to the binding polypeptide. For example, when the binding polypeptide is an antibody, the antibody binding domain or a fragment thereof can be included in a fusion protein with the inner membrane protein or a fragment thereof. In some examples, the antibody binding domain can be derived from the allergens Asp fl1, Asp fl2, or Asp fl3 of Aspergillus flavus; Fc-gammaRI, Fc-gammaRII, Fc-gammaRII-a, Fc-gammaRII-b, Fc-gammaRII-c, Fc-gammaRIII, Fc-gammaRIII-a, Fc-gammaRIII-b, FcRn, MRP proteins of S. aureus (spa); protein B of Streptococcus agalactiae; protein G of Streptococcus species group G (spg); protein H of Streptococcus pyogenes; protein sbi of S. aureus. In some examples, the domain may be derived from Protein A of S. aureus (e.g., the ZZ domain that binds to the Fc region of IgG. For other binding polypeptides, a protein-protein interaction domain and / or an epitope tag that binds to the binding polypeptide may be included in the fusion protein.

[0174] In any of the above methods, step (a) may include providing a plurality of bacteria, each of which comprises a library of nucleic acids encoding a candidate binding polypeptide. In some examples, the library of nucleic acids may comprise two or more nucleic acids encoding polypeptides that are substantially identical in sequence, preferably identical, except for the sequence of the variant at one or more specific positions. For example, in some examples, the library comprises a plurality of nucleic acids encoding candidate binding polypeptide variants having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or more) amino acid residue changes compared to a reference binding polypeptide.

[0175] For example, in any of the aforementioned methods, the library may comprise a plurality of nucleic acids encoding candidate antibody variants, each candidate antibody variant comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or more) amino acid residue changes compared to a reference antibody. The amino acid residue changes may be at any position of the antibody, e.g., the VH and / or VL and / or constant domains. For example, in some examples, the candidate antibody variant comprises one or more amino acid residue changes in the HVRs and / or FRs of the VH and / or VL. In some examples, the candidate antibody variant comprises one or more amino acid residue changes in the HVRs (e.g., HVR-H1, HVR-H2, and / or HVR-H3) and / or FRs (e.g., FR-H1, FR-H2, FR-H3, and / or FR-H4) of the VH. In some examples, the candidate antibody variant comprises one or more amino acid residue changes in the HVRs of the VH (e.g., HVR-H1, HVR-H2, and / or HVR-H3). In some examples, the candidate antibody variant comprises one or more amino acid residue changes in the FRs of the VH (e.g., FR-H1, FR-H2, FR-H3, and / or FR-H4). In other examples, the candidate antibody variant comprises one or more amino acid residue changes in the HVRs of the VL (e.g., HVR-L1, HVR-L2, and / or HVR-L3) and / or FRs (e.g., FR-L1, FR-L2, FR-L3, and / or FR-L4). In some examples, the candidate antibody variant comprises one or more amino acid residue changes in the HVRs of the VL (e.g., HVR-L1, HVR-L2, and / or HVR-L3). In some examples, the candidate antibody variants contain one or more amino acid residue changes in the FRs of the VL (e.g., FR-L1, FR-L2, FR-L3, and / or FR-L4). The amino acid residue changes may be in surface-exposed or buried residues.

[0176] In any of the above methods, the library may comprise a plurality of nucleic acids encoding candidate antibody variants, each of which comprises an amino acid residue change in the FR of VH or VL compared to a reference antibody, the amino acid residue change being (a) identified in a naturally occurring antibody having the same subtype as the reference antibody, and / or (b) in an amino acid residue predicted to be buried. For example, in some examples, the library may comprise a plurality of nucleic acids encoding candidate antibody variants, each of which comprises an amino acid residue change in the FR of VH or VL compared to a reference antibody, the amino acid residue change being (a) identified in a naturally occurring antibody having the same subtype as the reference antibody, and (b) in an amino acid residue predicted to be buried. In some examples, the amino acid residue change in a naturally occurring antibody is due to somatic hypermutation.

[0177] Amino acid residues can be predicted and / or determined to be buried (e.g., solvent inaccessible) or surface exposed (e.g., solvent accessible) using any method known in the art. For example, amino acid residues can be predicted to be buried using any of a number of algorithms known in the art. Preferably, solvent accessible positions are determined using the coordinates of a three-dimensional model of an antibody, preferably using a computer program such as the InsighfH Program (Accelrys, San Diego, Calif.). Buried positions can also be predicted using algorithms known in the art, such as (e.g., Lee et al. J. Mol. Biol. 55:379, 1971 and Connolly, J. Appl. Cryst. 16:548, 1983). Prediction of buried positions can be performed using software suitable for protein modeling and three-dimensional structural information obtained from the antibody. Software that can be utilized for these purposes includes SYBYL Biopolymer Module software (Tripos Associates). In general, when an algorithm (program) requires a user-input size parameter, the "size" of the probe used in the calculation is set to about 1.4 angstroms in radius or less. In addition, the determination of buried and / or surface-exposed regions, as well as area methods using software for personal computers, have been described by Pacios, Comput. Chem. 18(4):377-386, 1994 and J. Mol. Model. 1:46-53, 1995.

[0178] In any of the foregoing methods, the library comprises from about 2 binding polypeptides to about 10 14 or more binding polypeptides (e.g., about 2, about 5, about 10, about 15, about 20, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 500, about 10 3 pieces, about 10 4 pieces, about 10 5 pieces, about 106 pieces, about 10 7 pieces, about 10 8 pieces, about 10 9 pieces, about 10 10 pieces, about 10 11 pieces, about 10 12 pieces, about 10 13 Pieces, or about 10 14 In some examples, the library may comprise about 10 8 The polypeptide may comprise a plurality of binding polypeptides.

[0179] Any of the foregoing methods may include identifying a candidate binding polypeptide as having an increased expression level based on the amount of labeled target molecule in the periplasm. In some examples, the increased expression level is determined by Western blot, IHC, mass spectrometry, enzyme-linked immunosorbent assay (ELISA), or other methods known in the art. In some examples, the candidate binding polypeptide has a 1-20 fold increase in expression, e.g., 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, or 20 fold increase in expression, compared to a reference or wild-type binding polypeptide. For example, the candidate binding polypeptide may have a 1-10 fold increase in expression, a 1-7 fold increase in expression, a 1-6 fold increase in expression, a 1-5 fold increase in expression, a 1-4 fold increase in expression, a 1-3 fold increase in expression, a 2-10 fold increase in expression, a 2-8 fold increase in expression, or a 2-6 fold increase in expression. In some embodiments, the candidate binding polypeptide has increased expression in both bacterial (e.g., E. coli) cells and mammalian cells. In some embodiments, the increase is compared to the expression level of a wild-type binding polypeptide. In other embodiments, the increase is compared to the expression level of a variant binding polypeptide.

[0180] Any of the foregoing methods may include identifying a candidate binding polypeptide as having increased stability based on the amount of labeled target molecules in the periplasm. In some examples, the increased stability is determined by differential scanning fluorimetry, circular dichroism, spectroscopy, or other methods known in the art. In some examples, the candidate binding polypeptide has an increased melting temperature (T) of between 1° C. and 20° C., e.g., 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 16° C., 17° C., 18° C., 19° C., or 20° C., compared to a reference or wild-type binding polypeptide. m In some examples, the candidate binding polypeptide has an increased T of between 1°C and 10°C, between 1°C and 9°C, between 1°C and 8°C, between 1°C and 7°C, between 1°C and 6°C, between 1°C and 5°C, between 1°C and 4°C, between 1°C and 3°C, between 1°C and 2°C, between 2°C and 10°C, between 2°C and 9°C, between 2°C and 8°C, between 2°C and 7°C, between 2°C and 6°C, between 2°C and 5°C, between 2°C and 4°C, or between 2°C and 3°C. m In some embodiments, the candidate binding polypeptide has increased stability in both bacterial (e.g., E. coli) cells and mammalian cells. In some embodiments, the candidate binding polypeptide has both increased stability and increased expression. In some embodiments, the increase is compared to the expression level of a wild-type binding polypeptide. In other embodiments, the increase is compared to the expression level of a variant binding polypeptide.

[0181] Any of the foregoing methods may include identifying a candidate binding polypeptide as having an increased binding affinity for the target molecule based on the amount of labeled target molecule in the periplasm. In some embodiments, the candidate binding polypeptide may have a 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20-fold increase in binding affinity for the target molecule compared to a reference binding polypeptide. For example, the method may include screening for antibodies with increased affinity compared to a reference antibody.

[0182] In any of the foregoing methods, the binding polypeptide may be an antibody. In some examples, the antibody of any of the above methods is a monoclonal antibody, including a chimeric, humanized, or human antibody. In some examples, the antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab') 2 In another example, the antibody is a full-length antibody, e.g., an intact IgG1 antibody, an intact IgG4 antibody, or other antibody class or isotype as defined herein. In another embodiment, the antibody is a half antibody, e.g., a knob-into-hole half antibody. For a description of knob-into-hole technology, see, e.g., U.S. Pat. No. 5,731,168. In any of the foregoing methods, the antibody may be a therapeutic antibody (e.g., a neutralizing antibody).

[0183] In any of the foregoing methods, the binding polypeptide may be an antibody mimic, such as an affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, designed ankyrin repeat protein (DARPin), finomer, Kunitz domain peptide, or monobody. In other embodiments, the binding polypeptide may be a bacterial protein that localizes to the periplasm. In other embodiments, the binding polypeptide may be a mammalian protein. For example, in some embodiments, the binding polypeptide may be a cell surface receptor, a cytokine, or a growth factor.

[0184] Any of the foregoing methods may further comprise isolating the nucleic acid after the selection step. In some instances, the method further comprises determining the sequence of the nucleic acid.

[0185] In some embodiments of any of the foregoing methods, the bacterium may comprise a mutation affecting a gene that affects expression of the binding polypeptide, e.g., a transcription regulator gene. In some examples, step (a) comprises providing a plurality of bacteria, the plurality of bacteria comprising a library of nucleic acids each encoding a variant of a transcription regulator gene. In some examples, the transcription regulator gene is selected from a transcription initiation factor, an anti-sigma factor, an RNA polymerase subunit, or a transcription factor. In some examples, the transcription initiation factor is a sigma factor. In some examples, the sigma factor is RpoD (σ 70 ), RpoE(σ 24 ), RpoH(σ 32 ), RpoS(σ 38 ), RpoN(σ 54 ), RpoF(σ 28 ), and FecI(σ 19 In a specific example, the sigma factor is selected from the group consisting of RpoD (σ 70 ). In some instances, the mutation affecting the transcriptional regulator gene is present on a synthetic plasmid. In other instances, the mutation affecting the transcriptional regulator gene is present in the endogenous bacterial genome. In some instances, the mutation affecting the transcriptional regulator gene is due to error-prone PCR mutagenesis, chemical mutagenesis, transposition mutagenesis, or targeted mutagenesis. In specific instances, the mutation affecting the transcriptional regulator gene is due to error-prone PCR mutagenesis.

[0186] Any of the methods described above can be used to identify bacteria having improved expression of the binding polypeptide. In some examples, the bacteria may have improved expression of the binding polypeptide by at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, or more. In some examples, the bacteria may have 1-10-fold improved expression, 1-7-fold improved expression, 1-6-fold improved expression, 1-5-fold improved expression, 1-4-fold improved expression, 1-3-fold improved expression, 2-10-fold improved expression, 2-8-fold improved expression, or 2-6-fold improved expression. In some embodiments, the improvement is compared to the expression level of the binding polypeptide in a wild-type bacterium, while in other embodiments, the improvement is compared to the expression level of the binding polypeptide in a mutant bacterium.

[0187] B. Library The present invention provides a library comprising a plurality of nucleic acids encoding binding polypeptides. For example, in some examples, the library comprises a plurality of nucleic acids encoding candidate binding polypeptide variants having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or more) amino acid residue changes compared to a reference binding polypeptide.

[0188] The candidate binding polypeptide may be a candidate antibody variant. In some examples, the candidate antibody variant is a monoclonal antibody, including a chimeric, humanized, or human antibody. In some examples, the candidate antibody variant is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab') 2In another example, the candidate antibody variant is a full length antibody, e.g., an intact IgG1 antibody, an intact IgG4 antibody, or other antibody class or isotype as defined herein. In another embodiment, the candidate antibody variant is a half antibody, e.g., a knob-into-hole half antibody. In any of the foregoing methods, the candidate antibody variant may be a therapeutic antibody (e.g., a neutralizing antibody).

[0189] In other examples, the candidate binding polypeptide may be an antibody mimic, such as an affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, designed ankyrin repeat protein (DARPin), finomer, Kunitz domain peptide, or monobody. In other embodiments, the binding polypeptide may be a bacterial protein that localizes to the periplasm. In other embodiments, the binding polypeptide may be a mammalian protein. For example, in some embodiments, the binding polypeptide may be a cell surface receptor, a cytokine, or a growth factor.

[0190] In some examples, the library may include a plurality of nucleic acids encoding candidate antibody variants, each candidate antibody variant comprising one or more amino acid residue changes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or more) compared to a reference antibody. The amino acid residue changes may be at any position of the antibody, e.g., in the VH and / or VL and / or constant domains. For example, in some examples, the candidate antibody variant comprises one or more amino acid residue changes in the HVRs and / or FRs of the VH and / or VL. In some examples, the candidate antibody variant comprises one or more amino acid residue changes in the HVRs (e.g., HVR-H1, HVR-H2, and / or HVR-H3) and / or FRs (e.g., FR-H1, FR-H2, FR-H3, and / or FR-H4) of the VH. In some examples, the candidate antibody variant comprises one or more amino acid residue changes in the HVRs of the VH (e.g., HVR-H1, HVR-H2, and / or HVR-H3). In some examples, the candidate antibody variant comprises one or more amino acid residue changes in the FRs of the VH (e.g., FR-H1, FR-H2, FR-H3, and / or FR-H4). In other examples, the candidate antibody variant comprises one or more amino acid residue changes in the HVRs of the VL (e.g., HVR-L1, HVR-L2, and / or HVR-L3) and / or FRs (e.g., FR-L1, FR-L2, FR-L3, and / or FR-L4). In some examples, the candidate antibody variant comprises one or more amino acid residue changes in the HVRs of the VL (e.g., HVR-L1, HVR-L2, and / or HVR-L3). In some examples, the candidate antibody variants contain one or more amino acid residue changes in the FRs of the VL (e.g., FR-L1, FR-L2, FR-L3, and / or FR-L4). The amino acid residue changes may be in surface-exposed or buried residues.

[0191] In some examples, the library may include a plurality of nucleic acids encoding candidate antibody variants, each of which comprises one or more amino acid residue changes in the FRs of VH and / or VL compared to a reference antibody, the amino acid residue changes being (a) identified in naturally occurring antibodies of the same subtype as the reference antibody, and / or (b) in amino acid residues predicted to be buried. In some examples, the library may include a plurality of nucleic acids encoding candidate antibody variants, each of which comprises one or more amino acid residue changes in the FRs of VH and / or VL compared to a reference antibody, the amino acid residue changes being (a) identified in naturally occurring antibodies of the same subtype as the reference antibody, and (b) in amino acid residues predicted to be buried. In some examples, each of the candidate antibody variants comprises one amino acid residue change compared to the reference antibody.

[0192] For example, in some examples, the library may include a plurality of nucleic acids encoding candidate antibody variants, each of which contains an amino acid residue change in a FR (e.g., FR-H1, FR-H2, FR-H3, or FR-H4) of the VH compared to a reference antibody, the amino acid residue change being (a) identified in a naturally occurring antibody having the same subtype as the reference antibody, and (b) in an amino acid residue predicted to be buried. In other examples, the library may include a plurality of nucleic acids encoding candidate antibody variants, each of which contains an amino acid residue change in a FR (e.g., FR-L1, FR-L2, FR-L3, or FR-L4) of the VL compared to a reference antibody, the amino acid residue change being (a) identified in a naturally occurring antibody having the same subtype as the reference antibody, and (b) in an amino acid residue predicted to be buried. In some examples, the amino acid residue change in the naturally occurring antibody is due to somatic hypermutation.

[0193] Naturally occurring antibodies having a VH and / or VL belonging to the same subtype as the reference antibody can be readily found in antibody databases, such as the Kabat database, IMGT® (International Immunogenetics Information System), Protein Data Bank (PDB), V BASE, or others known in the art.

[0194] In any of the foregoing embodiments, the library comprises from about 2 binding polypeptides to about 10 14 or more binding polypeptides (e.g., about 2 to about 10, about 2 to about 20, about 2 to about 30, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 70, about 2 to about 70, about 2 to about 80, about 2 to about 100, about 2 to about 10 14 pieces, about 2 to about 10 13 pieces, about 2 to about 10 12 pieces, about 2 to about 10 11 pieces, about 2 to about 10 10 pieces, about 2 to about 10 9 pieces, about 2 to about 10 8 pieces, about 20 to about 10 8 pieces, about 30 to about 10 8 pieces, about 40 to about 10 8 pieces, about 50 to about 10 8 pieces, about 60 to about 10 8 pieces, about 70 to about 10 8 pieces, about 80 to about 10 8 pieces, about 90 to about 10 8 pieces, about 100 to about 10 8 pieces, about 200 to about 10 8 400 to 10 8 pieces, about 600 to about 10 8 pieces, about 800 to about 10 8 pieces, about 10 3 ~about 10 8 pieces, about 10 4 ~about 10 8 pieces, about 10 5 ~about 10 8 Approximately 10 pieces 6 ~about 10 8 Pieces, or about 10 7 ~about 10 8 In some examples, the library may comprise about 108 The polypeptide may comprise a plurality of binding polypeptides.

[0195] C. Exemplary Anti-IL-13 Antibodies The present invention provides isolated antibodies that bind to IL-13. In some examples, the anti-IL-13 antibody may comprise at least one, two, or three HVRs selected from (a) HVR-H1 comprising the amino acid sequence of AYSVN (SEQ ID NO:7), (b) HVR-H2 comprising the amino acid sequence of MIWGDGKIVYNSALKS (SEQ ID NO:8), and (c) HVR-H3 comprising the amino acid sequence of DGYYPYAMDN (SEQ ID NO:9), or a combination of one or more of the above HVRs with one or more variants thereof having at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs:7-9.

[0196] In some examples, the anti-IL-13 antibody may further comprise one, two, three, or four of the following heavy chain framework regions: (a) FR-H1 comprising the amino acid sequence of QVTLRQSGPALVKPTQTLTLTCTVSGFSLS (SEQ ID NO: 18), QVTLRESGPALVKPTQTLTLTCTVSGLSLS (SEQ ID NO: 19), QVTLRQSGPALVKPTQTLTLTCTVSGLSLS (SEQ ID NO: 20), or QVTLRESGPALVKPTQTLTLTCTVSGFSLS (SEQ ID NO: 21); (b) FR-H2 comprising the amino acid sequence of WIRQPPGKALEWLA (SEQ ID NO: 22); (c) FR-H3 comprising the amino acid sequence of RLTISKDTSKNQVVLTMTNMDPVDTATYYCAG (SEQ ID NO: 23); and (d) FR-H4 comprising the amino acid sequence of WGQGSLVTVSS (SEQ ID NO: 24).

[0197] In some examples, the anti-IL-13 antibody may further comprise at least one, two, or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of RASKSVDSYGNSFMH (SEQ ID NO: 10); (b) HVR-L2 comprising the amino acid sequence of LASNLES (SEQ ID NO: 11); and (c) HVR-L3 comprising the amino acid sequence of QQNNEDPRT (SEQ ID NO: 12); or a combination of one or more of the above HVRs with one or more variants thereof having at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 10-12.

[0198] In some examples, the anti-IL-13 antibody may further comprise one, two, three, or four of the following light chain framework regions: (a) FR-L1 comprising the amino acid sequence of DIVLTQSPDSLSVSLGERATINC (SEQ ID NO: 13) or DIVMTQSPDSLSVSLGERATINC (SEQ ID NO: 14); (b) FR-L2 comprising the amino acid sequence of WYQQKPGQPPKLLIY (SEQ ID NO: 15); (c) FR-L3 comprising the amino acid sequence of GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 16); and (d) FR-L4 comprising the amino acid sequence of FGGGTKVEIK (SEQ ID NO: 17).

[0199] In some examples, the anti-IL-13 antibody comprises (a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or having the sequence of, any one of SEQ ID NOs:2-5, (b) a light chain variable (VL) domain comprising an amino acid sequence having at least 90% sequence (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or having the sequence of, SEQ ID NO:1 or SEQ ID NO:6, or (c) a VH domain as in (a) and a VL domain as in (b). For example, in some examples, the antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:2 and a VL domain comprising the amino acid sequence of SEQ ID NO:6. In some examples, the antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:3 and a VL domain comprising the amino acid sequence of SEQ ID NO:6. In some examples, the antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:3 and a VL domain comprising the amino acid sequence of SEQ ID NO:6. In some examples, the antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:4 and a VL domain comprising the amino acid sequence of SEQ ID NO:6. In some examples, the antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:5 and a VL domain comprising the amino acid sequence of SEQ ID NO:1. In some examples, the antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:4 and a VL domain comprising the amino acid sequence of SEQ ID NO:1. In some examples, the antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:3 and a VL domain comprising the amino acid sequence of SEQ ID NO:1. In some examples, the antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO:2 and a VL domain comprising the amino acid sequence of SEQ ID NO:1.

[0200] In a further aspect of the invention, the anti-IL-13 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the anti-IL-13 antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab') 2In another embodiment, the antibody is a full length antibody, e.g., an intact IgG1 antibody, an intact IgG4 antibody, or other antibody class or isotype as defined herein. In another embodiment, the antibody is a half antibody, e.g., a knob-into-hole half antibody. See, e.g., U.S. Patent No. 5,731,168 for a description of knob-into-hole technology.

[0201] In a further aspect, an anti-IL-13 antibody according to any of the above embodiments may incorporate any of the features described in Sections 1-7 below, either alone or in combination.

[0202] 1. Antibody affinity In certain embodiments, the antibodies provided herein have a dissociation constant (K D ) (e.g., 10 -6 M or less, e.g., 10 -6 M~10 -9 M or less, e.g., 10 -9 M~10 -13 M or less).

[0203] In one embodiment, K D is measured by radiolabeled antigen binding assay (RIA). In one embodiment, an RIA is performed with a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of a Fab for an antigen is determined by binding the Fab to the lowest concentration ( 125I) is measured by equilibrating with labeled antigen and then capturing bound antigen with anti-Fab antibody-coated plates (see, e.g., Chen et al. J. Mol. Biol. 293:865-881, 1999). To establish the conditions for the assay, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% bovine serum albumin (w / v) in PBS at room temperature (approximately 23° C.) for 2-5 hours. In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al. Cancer Res. 57:4593-4599, 1997). The Fab of interest is then incubated overnight; however, incubation can be continued for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl of scintillant (MICROSCINT-20™, Packard) is added per well and the plate is counted in a TOPCOUNT™ gamma counter (Packard) for 10 minutes. The concentration of each Fab that results in 20% or less of maximum binding is selected for use in the competitive binding assay.

[0204] According to another embodiment, K Dis measured using a BIACORE® surface plasmon resonance assay. For example, assays using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25° C. with an immobilized antigen CM5 chip of about 10 response units (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / mL (about 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μL / min to obtain about 10 response units (RU) of coupled protein. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in phosphate-buffered saline (PBS) containing 0.05% polysorbate 20 (TWEEN® 20) surfactant (PBST) at 25° C. at a flow rate of approximately 25 μl / min. The association rate (k オン ) and dissociation rate (k オフ ) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). D ) to k オフ / k オン For example, see Chen et al. (J. Mol. Biol. 293:865-881, 1999). If the on-rate by the surface plasmon resonance assay described above is 10 6 M -1 s -1Above 100 nm, the on-rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation=295 nm, emission=340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25° C. in the presence of increasing antigen concentrations as measured with a spectrophotometer equipped with stopped flow (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) equipped with a stirred cuvette.

[0205] 2. Antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include Fab, Fab', Fab'-SH, F(ab') 2 Antibody fragments include, but are not limited to, Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994), see also WO 93 / 16185, and U.S. Patent Nos. 5,571,894 and 5,587,458. Fab and F(ab') fragments that contain salvage receptors that bind epitope residues and have increased in vivo half-life. 2 For a discussion of fragments, see US Pat. No. 5,869,046.

[0206] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404,097, WO 1993 / 01161, Hudson et al. Nat. Med. 9:129-134, 2003, and Hollinger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993. Triabodies and tetrabodies are also described in Hudson et al. Nat. Med. 9:129-134, 2003.

[0207] A single domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (see, e.g., U.S. Patent No. 6,248,516 B1).

[0208] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phages), as described herein.

[0209] 3. Chimeric and humanized antibodies In certain embodiments, the antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567, and Morrison et al. Proc. Natl. Acad. Sci. USA, 81:6851-6855, 1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or a non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0210] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to retain the specificity and affinity of the parent non-human antibody while reducing immunogenicity to humans. In general, a humanized antibody comprises one or more variable domains, in which the HVRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody will also optionally comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with the corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0211] Humanized antibodies and methods for making them are reviewed, for example, in Almagro et al. Front. Biosci. 13:1619-1633, 2008, and are described, for example, in Riechmann et al. Nature 332:323-329, 1988, Queen et al. Proc. Natl. Acad. Sci. USA 86:10029-10033, 1989, U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, Kashmiri et al. Methods 36:25-34, 2005 (describing specificity determining region (SDR) grafting), Padlan, Mol. Immunol. 28:489-498, 1991 (describing "resurfacing"), Dall'Acqua et al. Methods 36:43-60, 2005 (describing "FR shuffling"), as well as Osbourn et al. Methods 36:61-68, 2005 and Klimka et al. Br. J. Cancer, 83:252-260, 2000 (describing a "guided selection" approach to FR shuffling).

[0212] Human framework regions that can be used for humanization include framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296, 1993), framework regions derived from consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285, 1992, and Presta et al. J. Immunol., 151:2623, 1993), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro et al. Front. Biosci. 13:1619-1633, 2008), and framework regions derived from screening of FR libraries (see, e.g., Baca et al. J. Biol. Chem. 272:10678-10684, 1997, and Rosok et al. J. Immunol. 272:10678-10684, 1997). al. J. Biol. Chem. 271:22611-22618, 1996), but are not limited to these.

[0213] 4. Human antibodies In certain embodiments, the antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk et al. Curr. Opin. Pharmacol. 5:368-74, 2001 and Lonberg, Curr. Opin. Immunol. 20:450-459, 2008.

[0214] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace endogenous immunoglobulin loci or are extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat.Biotech.23:1117-1125,2005. See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology, U.S. Patent No. 5,770,429, which describes HUMAB® technology, U.S. Patent No. 7,041,870, which describes KM MOUSE® technology, and U.S. Patent Application Publication No. US2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions derived from intact antibodies produced by such animals may be further modified, for example, by combining with a different human constant region.

[0215] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J.Immunol.133:3001,1984; Brodeur et al.Monoclonal Antibody Production Techniques and Applications,pp.51-63(Marcel Dekker,Inc.,New York,1987); and Boerner et al.J.Immunol.147:86,1991). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al.Proc.Natl.Acad.Sci.USA,103:3557-3562,2006. Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268, 2006 (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers et al. Histology and Histopathology 20(3):927-937, 2005 and Vollmers et al. Methods and Findings in Experimental and Clinical Pharmacology 27(3):185-91, 2005.

[0216] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0217] 5. Library-derived Antibodies Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with the desired activity(ies). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and also described, for example, in McCafferty et al. Nature 348:552-554, 1990, Clackson et al. Nature 352:624-628, 1991, Marks et al. J. Mol. Biol. 222:581-597, 1992, Marks et al. in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003), 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. Natl. Acad. Sci. USA 101(34):12467-12472, 2004; and Lee et al. J. Immunol. Methods 284(1-2):119-132, 2004.

[0218] In one particular phage display method, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phages, as described in Winter et al. Ann. Rev. Immunol., 12:433-455, 1994. Phages typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries derived from immunized sources provide high affinity antibodies to immunogens without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self antigens and also self antigens without any immunization, as described in Griffiths et al. EMBO J. 12:725-734, 1993. Finally, as described by Hoogenboom et al. J. Mol. Biol., 227:381-388, 1992, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells, using PCR primers containing random sequences to encode the hypervariable HVR3 region, and achieving rearrangement in vitro. Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373, and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0219] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0220] 6. Multispecific antibodies In certain embodiments, the antibody provided herein is a multispecific antibody, for example, a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity for at least two different sites. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0221] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein et al. Nature 305:537, 1983; WO93 / 08829; and Traunecker et al. EMBO J. 10:3655, 1991), and "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by engineering electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO 2009 / 089004 A1), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al. Science, 229:81, 1985), using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al. J. Immunol., 148(5):1547-1553, 1992), using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993), using single-chain Fv (scFv) dimers (see, e.g., Gruber et al. al. J. Immunol. 152:5368, 1994), as well as, for example, by preparing trispecific antibodies as described in Tutt et al. J. Immunol. 147:60, 1991.

[0222] Engineered antibodies with three or more functional antigen-binding sites, including "octopus antibodies," are also included herein (see, eg, US 2006 / 0025576 A1).

[0223] The antibodies or fragments herein also include "dual acting Fabs" or "DAFs" that contain antigen binding sites that bind to IL-13 or VEGF and another different antigen (see, e.g., US2008 / 0069820).

[0224] 7. Antibody variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has the desired characteristics, e.g., antigen binding, expression, and / or stability.

[0225] a) Substitution, insertion, and deletion variants In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". More substantial changes are shown in Table 1 under the heading of "exemplary substitutions", as further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the subject antibody and the product screened for the desired activity, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. TIFF2025069126000001.tif169170 Amino acids can be grouped according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0226] Non-conservative substitutions will entail the exchange of a member of one of these classes for another class.

[0227] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have a modified (e.g., improved) biological property compared to the parent antibody (e.g., increased affinity, reduced immunogenicity) and / or will have a particular biological property of the parent antibody substantially retained. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0228] Changes (e.g., substitutions) can be made in HVRs to, for example, improve antibody affinity. Such changes may be made in HVR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196, 2008) and / or residues that contact the antigen, and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by construction of and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al. ed., Human Press, Totowa, NJ, 2001). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, HVR-H3 and HVR-L3 are often targeted.

[0229] In certain embodiments, substitutions, insertions, or deletions may be made in one or more HVRs, so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in the HVRs. Such changes may, for example, be outside of the antigen-contacting residues in the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unaltered or contains no more than one, two, or three amino acid substitutions.

[0230] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is that described in Cunningham et al. Science 244:1081-1085, 1989, called "alanine scanning mutagenesis." In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted with neutral or negatively charged amino acids (e.g., Ala or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact and adjacent residues can be targeted or eliminated as candidates for substitution. The variants can be screened to determine whether they contain the desired properties.

[0231] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a range of 100 or more residues in length, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0232] b) Glycosylation variants In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0233] If the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Natural antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide, generally linked to Asn297 of the CH2 domain of the Fc region by an N-linkage. See, for example, Wright et al. TIBTECH 15:26-32, 1997. The oligosaccharide may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention may be made to generate antibody variants with certain improved properties.

[0234] In one embodiment, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65% or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid and high mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 (Eu numbering of Fc region residues) in the Fc region, however, Asn297 can also be located about ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variations within the antibody. Such fucosylation variants can have improved ADCC function. See, e.g., U.S. Patent Publication Nos. 2003 / 0157108 and 2004 / 0093621. Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 013214, 0, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J. Mol. Biol. 336: 1239-1249, 2004, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614, 2004.Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545, 1986, US2003 / 0157108, and WO2004 / 056312A1, especially Example 11), as well as knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614, 2004, Kanda et al. Biotechnol. Bioeng. 94(4):680-688, 2006, and WO2003 / 085107).

[0235] Further provided are antibody variants having bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878, U.S. Patent No. 6,602,684, and US2005 / 0123546. Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described in WO1997 / 30087, WO1998 / 58964, and WO1999 / 22764.

[0236] c) Fc domain variants In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0237] In certain embodiments, the present invention contemplates antibody variants that possess some, but not all, effector functions, making them desirable candidates for applications in which antibody half-life in vivo is important, but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only Fc(RIII, while monocytes express Fc(RI, Fc(RII, and Fc(RIII). FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch et al. Annu. Rev. Immunol. 9:457-492, 1991. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest include those described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom et al. Proc. Natl. Acad. Sci. USA 83:7059-7063, 1986 and Hellstrom et al. Proc. Natl. Acad. Sci. USA 82:1499-1502, 1985); U.S. Pat. No. 5,821,337 (Bruggemann et al. al. J. Exp. Med. 166:1351-1361, 1987). Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA, and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci. USA 95:652-656, 1998. C1q binding assays can also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro et al. J. Immunol. Methods 202:163, 1996; Cragg et al. Blood 101:1045-1052, 2003; and Cragg et al. Blood 103:2738-2743, 2004). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova et al. Intl. Immunol. 18(12):1759-1769, 2006).

[0238] Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327 (including, for example, the so-called "DANA" Fc variants with substitutions of residues 265 and 297 to alanine) (U.S. Patent No. 7,332,581).

[0239] Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al. J. Biol. Chem. 9(2):6591-6604, 2001).

[0240] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0241] In some embodiments, changes are made within the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184, 2000.

[0242] Antibodies with increased half-life and improved binding to the fetal Fc 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), are described in US2005 / 0014934. These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0243] See also Duncan et al. Nature 322:738-40, 1988, U.S. Patent Nos. 5,648,260 and 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0244] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues. In certain embodiments, the replaced residues occur at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby placed at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates as further described herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0245] e) Antibody derivative In certain embodiments, the antibodies provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Suitable moieties for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages during manufacturing due to its stability in water. The polymers can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

[0246] In another embodiment, a complex of an antibody and a nonproteinaceous moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam et al. Proc. Natl. Acad. Sci. USA 102:11600-11605, 2005). The radiation can be of any wavelength, including but not limited to wavelengths that are not harmful to normal cells but heat the nonproteinaceous moiety to a temperature that kills cells proximal to the antibody-nonproteinaceous moiety.

[0247] D. Recombinant Methods and Compositions The antibodies may be produced using recombinant methods and compositions described, for example, in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid is provided that encodes an anti-IL-13 antibody described herein. In another embodiment, an isolated nucleic acid is provided that encodes an anti-VEGF antibody described herein. Such a nucleic acid may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In such an embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell, a 293 cell, or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In one embodiment, a method of making an anti-IL-13 antibody is provided, the method comprising culturing a host cell comprising nucleic acid encoding an antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0248] For recombinant production of an anti-IL-13 or anti-VEGF antibody, for example, nucleic acid encoding the above-mentioned antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0249] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction or further purified.

[0250] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross Nat. Biotech. 22:1409-1414, 2004 and Li et al. Nat. Biotech. 24:210-215, 2006.

[0251] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plants and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0252] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0253] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7) transformed by SV40, human embryonic kidney lines (e.g., 293 or 293 cells, described in Graham et al. J. Gen Virol. 36:59, 1977), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells, described in Mather Biol. Reprod. 23:243-251, 1980), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK, buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT060562), e.g., Mather et al., Annals Other useful mammalian host cell lines include TRI cells, MRC5 cells, and FS4 cells, described in NYAcad.Sci.383:44-68, 1982. -These include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al. Proc. Natl. Acad. Sci. USA 77:4216, 1980), and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki et al. Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268, 2003.

[0254] E. Assay Any of the antibodies described herein (e.g., anti-IL-13 antibodies or anti-VEGF antibodies) may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0255] 1. Binding and other assays In one embodiment, any of the antibodies described herein (eg, anti-IL-13 antibodies or anti-VEGF antibodies) is tested for its antigen binding activity by known methods such as, for example, ELISA, Western blot, and the like.

[0256] In another embodiment, a competition assay can be used to identify antibodies that compete with the anti-IL-13 antibodies of the present invention for binding to IL-13. In another embodiment, a competition assay can be used to identify antibodies that compete with the anti-VEGF antibodies described herein for binding to VEGF. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or conformational epitope) as the anti-IL-13 antibodies of the present invention bind. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or conformational epitope) as the anti-VEGF antibodies described herein bind. Details of exemplary methods for mapping epitopes to which antibodies bind are provided in Morris "Epitope Mapping Protocols," in Methods in Molecular Biology Vol. 66 (Humana Press, Totowa, NJ), 1996.

[0257] In an exemplary competitive assay, immobilized IL-13 is incubated in a solution containing a first labeled antibody that binds to IL-13 and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to IL-13. The second antibody may be present in a hybridoma supernatant. As a control, immobilized IL-13 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to IL-13, excess unbound antibody is removed and the amount of label associated with immobilized IL-13 is measured. If the amount of label associated with immobilized IL-13 is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody competes with the first antibody for binding to IL-13. See Harlow et al. Antibodies: A Laboratory Manual Ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), 1988.

[0258] In another exemplary competitive assay, immobilized VEGF is incubated in a solution containing a first labeled antibody that binds to VEGF and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to VEGF. The second antibody may be present in a hybridoma supernatant. As a control, immobilized VEGF is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to VEGF, excess unbound antibody is removed and the amount of label associated with immobilized VEGF is measured. If the amount of label associated with immobilized VEGF is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody competes with the first antibody for binding to VEGF.

[0259] 2. Activity Assay In one aspect, an assay is provided to identify the anti-IL-13 antibody having biological activity. Biological activity can include, for example, binding to IL-13 (e.g., IL-13 in the bloodstream) or a peptide fragment thereof, either in vivo, in vitro (e.g., presented in the periplasm of bacteria), or ex vivo. In other embodiments, biological activity can include blocking or neutralizing IL-13, or preventing IL-13 from binding to a ligand, e.g., a receptor (e.g., interleukin-13 receptor). Antibodies having such biological activity in vivo and / or in vitro are also provided. In certain embodiments, the antibodies of the present invention are tested for such biological activity.

[0260] In another aspect, an assay is provided for identifying the anti-VEGF antibody having biological activity. Biological activity can include, for example, binding to VEGF (e.g., VEGF in the bloodstream) or peptide fragments thereof, either in vivo, in vitro (e.g., presented in the periplasm of bacteria), or ex vivo. In other embodiments, biological activity can include blocking or neutralizing VEGF, or preventing VEGF from binding to its ligand, e.g., receptor (e.g., Tie2, KDR, and / or Flt-1). Antibodies having such biological activity in vivo and / or in vitro are also provided. In certain embodiments, the antibodies of the present invention are tested for such biological activity.

[0261] F. Immune complex The present invention also provides immunoconjugates that include any of the antibodies described herein (e.g., an anti-IL-13 antibody or an anti-VEGF antibody) conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope.

[0262] In one embodiment, the immunoconjugate is selected from the group consisting of maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent EP 0425235 B1); auristatins, such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al. Cancer Res. 53:3336-3342, 1993, and Lode et al. Cancer Res. 58:2925-2928, 1998); anthracyclines such as daunomycin or doxorubicin (Kratz et al. Current Med. Chem. 13:477-523, 2006; Jeffrey et al. Bioorganic & Med. Chem. Letters 16:358-362, 2006; Torgov et al. Bioconj. Chem. 16:717-721, 2005; Nagy et al. Proc. Natl. Acad. Sci. USA 97:829-834, 2000; Dubowchik et al. Bioorg. & Med. Chem. Letters 12:1529-1532, 2002; King et al. al. J. Med. Chem. 45:4336-4343, 2002, and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and antibody-drug conjugates (ADCs), in which an antibody is conjugated to one or more drugs, including, but not limited to, CC1065.

[0263] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, aleurone proteins, diansin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), bitter melon inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0264] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu. When a radioactive complex is used for detection, it is preferably a radioactive atom, such as technetium-99m (tc99m) or I for scintigraphy studies. 123 , or spin labels (again, such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron) for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri).

[0265] Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al. Science 238:1098,1987. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethyleneaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO94 / 11026. The linker may be a "cleavable linker" that facilitates the release of the cytotoxic drug inside the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers (see, for example, Chari et al. Cancer Res. 52:127-131,1992, U.S. Patent No. 5,208,020) can be used.

[0266] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, conjugates prepared with crosslinker reagents, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), which is commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA).

[0267] G. Methods and Compositions for Diagnostics and Detection In certain embodiments, any of the anti-IL-13 antibodies provided herein are useful for detecting the presence of IL-13 in a biological sample. As used herein, the term "detecting" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample contains cells or tissues such as T helper type 2 (Th2) cells, mast cells, B cells, monocytes, macrophages, and non-hematopoietic cells (including smooth muscle cells, epithelial cells, endothelial cells, or fibroblasts).

[0268] In one embodiment, an anti-IL-13 antibody is provided for use in a method of diagnosis or detection. In a further aspect, a method for detecting the presence of IL-13 in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-IL-13 antibody described herein under conditions that allow binding of the anti-IL-13 antibody to IL-13, and detecting whether a complex is formed between the anti-IL-13 antibody and IL-13. Such a method may be an in vitro or in vivo method. In one embodiment, the anti-IL-13 antibody is used to select subjects suitable for therapy with an anti-IL-13 antibody, for example, when IL-13 is a biomarker for patient selection.

[0269] Exemplary disorders that may be diagnosed using the anti-IL-13 of the present invention include IL-13 mediated disorders or IgE mediated disorders. In some examples, IL-13 mediated disorders include, for example, allergies, asthma (e.g., allergic asthma, atopic asthma, severe asthma, and non-allergic (intrinsic) asthma), autoimmune diseases (e.g., psoriasis, rheumatoid arthritis, juvenile rheumatoid arthritis, and inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), inflammatory diseases, allergic rhinitis (including seasonal allergic rhinitis), atopic dermatitis, allergic conjunctivitis, eczema, urticaria, food allergies, food intolerances, and chronic obstructive pulmonary disease (COPD). Allergic reactions, abdominal diseases, immune-mediated skin diseases (e.g., bullous dermatitis, erythema multiforme, contact dermatitis), scleroderma, fibrosis, hepatic fibrosis, endomyocardial fibrosis, chronic bronchitis, bronchiectasis, idiopathic interstitial pneumonia, goblet cell metaplasia, pulmonary inflammatory disorders, inflammatory and fibrotic lung diseases (e.g., cystic fibrosis, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease (COPD)), gluten-sensitive enteropathy, Whipple's disease, immune disorders of the lungs (e.g., eosinophilic pneumonia (including chronic eosinophilic pneumonia), idiopathic pulmonary fibrosis, allergic bronchopulmonary aspergillosis, and hypersensitivity pneumonitis), Churg-Strauss syndrome (periarteritis nodosa plus atopy), eosinophilic myalgia syndrome, hypereosinophilic syndrome, edematous reactions (e.g., episodic angioedema, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic enteritis, and eosinophilic colitis), nasal micropolyposis and polyposis, RSV infection, uveitis, osteoporosis, and malignancies including cancers and tumors associated with aberrant expression of IL-13 (e.g., Hodgkin's disease, In some embodiments, the disorder is allergic asthma, non-allergic (intrinsic) asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, eczema, urticaria, food allergy, chronic obstructive pulmonary disease, ulcerative colitis, RSV infection, uveitis, scleroderma, or osteoporosis. In some examples, the IgE-mediated disorder is bronchial asthma, allergic rhinitis, allergic dermatitis, urticaria, anaphylaxis, or atopic dermatitis.

[0270] In certain embodiments, the present invention provides a method for diagnosing a disorder associated with VEGF (e.g., increased expression of VEGF). In certain embodiments, the method includes contacting a test cell with an anti-VEGF antibody described herein, determining (either quantitatively or qualitatively) the level of expression of VEGF by the test cell by detecting binding of the anti-VEGF antibody to VEGF, and comparing the level of expression of VEGF by the test cell to the level of expression of VEGF by a control cell (e.g., a normal cell originating from the same tissue as the test cell, or a cell expressing VEGF at a level comparable to such a normal cell), where a higher level of expression of VEGF by the test cell compared to the control cell indicates the presence of a disorder associated with increased expression of VEGF. In certain embodiments, the test cell is obtained from an individual suspected of having a disorder associated with increased expression of VEGF. In certain embodiments, the disorder is a tumor, a cancer, and / or a cell proliferative disorder.

[0271] Exemplary disorders that can be diagnosed using the anti-VEGF antibodies described herein include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumors, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer or renal cancer. These include, but are not limited to, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer and various types of head and neck cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentigo melanoma, nodular melanoma, B-cell lymphoma, chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; post-transplant lymphoproliferative disorder (PTLD), abnormal blood vessel growth associated with phacomatosis, edema associated with brain tumors, and Meigs' syndrome.

[0272] In another aspect, the invention provides a method for detecting the presence of VEGF in a biological sample, hi certain embodiments, the method comprises contacting the biological sample with an anti-VEGF antibody under conditions that permit binding of the anti-VEGF antibody to VEGF, and detecting whether a complex is formed between the anti-VEGF antibody and VEGF.

[0273] Anti-VEGF antibodies can be used to detect VEGF in any one of several well-known detection assay methods. For example, biological samples can be assayed for VEGF by obtaining a sample from a desired source, mixing the sample with an anti-VEGF antibody to allow the antibody to form an antibody / VEGF complex with any VEGF present in the mixture, and detecting any antibody / VEGF complexes present in the mixture. Biological samples can be prepared for assay by methods known in the art that are suitable for a particular sample. The method of mixing the sample with the antibody and the method of detecting the antibody / VEGF complex are selected according to the type of assay used.

[0274] Labeled anti-IL-13 or anti-VEGF antibodies are described herein. Labels include, but are not limited to, labels or moieties that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties such as enzymes or ligands that are indirectly detected, for example, through enzymatic reactions or molecular interactions. Exemplary labels include radioisotopes, 32 P, 14 C. 125 I, 3 H, and 131Examples of suitable fluorophores include, but are not limited to, I, fluorophores (such as rare earth chelates or fluorescein and its derivatives), rhodamine and its derivatives, dansyl, umbelliferone, luceriferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases (such as uricase and xanthine oxidase) coupled with enzymes (such as HRP, lactoperoxidase, or microperoxidase) that oxidize dye precursors using hydrogen peroxide, biotin / avidin, spin labels, bacteriophage labels, and stable free radicals.

[0275] H. Pharmaceutical Preparations Pharmaceutical formulations of any of the antibodies described herein (e.g., anti-IL-13 or anti-VEGF antibodies) are prepared in the form of a lyophilized formulation or aqueous solution by mixing such antibodies having the desired degree of purity with any one or more pharma- ceutically acceptable carriers (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin; Examples of suitable pharmacopoeitic carriers include, but are not limited to, proteins such as amine, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin); chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacopoeitic carriers herein further include intercalating drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0276] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Water-soluble antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0277] The compositions herein may also contain two or more active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide additional therapeutic agents (e.g., chemotherapeutic agents, cytotoxic agents, growth inhibitory agents, and / or antihormonal agents, such as those cited herein above). Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

[0278] The active ingredient can be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980.

[0279] 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.

[0280] Formulations to be used for in vivo administration are generally sterile. Sterility is readily accomplished, for example, by filtration through sterile filtration membranes.

[0281] I. Treatment Methods and Compositions Any of the antibodies described herein (eg, anti-IL-13 or anti-VEGF antibodies) can be used in the methods of treatment.

[0282] In one aspect, an anti-IL-13 antibody is provided for use as a pharmaceutical. In a further aspect, an anti-IL-13 antibody is provided for use in treating an IL-13-mediated disorder. In certain embodiments, an anti-IL-13 antibody is provided for use in a method of treatment. In certain embodiments, the invention provides an anti-IL-13 antibody for use in a method of treating an individual having an IL-13-mediated disorder, the method comprising administering to the individual an effective amount of an anti-IL-13 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. An "individual" according to any of the above embodiments is preferably a human.

[0283] In a further aspect, the invention provides for the use of an anti-IL-13 antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of an IL-13 mediated disorder. In a further embodiment, the medicament is for use in a method of treating an IL-13 mediated disorder, comprising administering an effective amount of the medicament to an individual having an IL-13 mediated disorder. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. An "individual" according to any of the above embodiments may preferably be a human.

[0284] In a further aspect, the present invention relates to a method for treating a variety of conditions, including, for example, allergies, asthma (e.g., allergic asthma, atopic asthma, severe asthma, and non-allergic (intrinsic) asthma), autoimmune diseases (e.g., psoriasis, rheumatoid arthritis, juvenile rheumatoid arthritis, and inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), inflammatory diseases, allergic rhinitis (including seasonal allergic rhinitis), atopic dermatitis, allergic conjunctivitis, eczema, urticaria, food allergies, food hypersensitivity, celiac disease, immune-mediated skin diseases. (e.g., bullous dermatoses, erythema multiforme, contact dermatitis), scleroderma, fibrosis, hepatic fibrosis, endomyocardial fibrosis, chronic bronchitis, bronchiectasis, idiopathic interstitial pneumonia, goblet cell metaplasia, pulmonary inflammatory disorders, inflammatory and fibrotic lung diseases (e.g., cystic fibrosis, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease (COPD)), gluten-sensitive enteropathy, Whipple's disease, lung immunological disorders (e.g., eosinophilic pneumonia (including chronic eosinophilic pneumonia), idiopathic pulmonary fibrosis, allergic bronchopulmonary aspergillosis, and hypersensitivity pneumonitis), Churg-Schmidt's disease, Methods are provided for treating IL-13 mediated disorders including Trauss syndrome (periarteritis nodosa plus atopy), eosinophilic myalgia syndrome, hypereosinophilic syndrome, edematous reactions (e.g., episodic angioedema, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic enteritis and eosinophilic colitis), nasal micropolyposis and polyposis, RSV infection, uveitis, osteoporosis, and malignancies including cancers and tumors associated with aberrant expression of IL-13 (e.g., Hodgkin's lymphoma). In embodiments, the disorder is allergic asthma, non-allergic (intrinsic) asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, eczema, urticaria, food allergy, chronic obstructive pulmonary disease, ulcerative colitis, RSV infection, uveitis, scleroderma, or osteoporosis.In some embodiments, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent as described below.The "individual" according to any of the above embodiments may preferably be a human.

[0285] In some examples, the invention provides a method of treating a patient suffering from asthma symptoms, comprising administering an amount of any of the anti-IL-13 antibodies of the invention effective to reduce the asthma symptoms.

[0286] In some examples, the invention provides a method for inhibiting IgE antibody production in a patient, comprising administering to the patient an effective amount of any of the anti-IL-13 antibodies of the invention that inhibits IgE antibody production. In some examples, the inhibition of IgE antibody production is intended for the prevention of bronchial asthma, the prevention of allergic rhinitis, the prevention of allergic dermatitis, the treatment of bronchial asthma, the treatment of allergic rhinitis, the treatment of urticaria, the prevention of anaphylaxis, or the treatment of atopic dermatitis.

[0287] In other examples, the invention provides methods of treating an IgE-mediated disorder in a patient, comprising administering to the patient an effective amount of an anti-IL-13 antibody of the invention, hi some examples, the IgE-mediated disorder is bronchial asthma, allergic rhinitis, allergic dermatitis, urticaria, anaphylaxis, or atopic dermatitis.

[0288] In a further aspect, the invention provides a pharmaceutical formulation comprising any of the anti-IL-13 antibodies provided herein, e.g., for use in any of the above-described methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the anti-IL-13 antibodies provided herein and a pharma- ceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-IL-13 antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

[0289] The anti-IL-13 antibodies of the present invention can be used in therapy, either alone or in combination with other agents. For example, the anti-IL-13 antibodies of the present invention can be advantageously used in combination with other monoclonal or chimeric antibodies, or with lymphokine or hematopoietic growth factors (e.g., IL-2, IL-3, IL-5, IL-7, IFN, etc.), which serve, for example, to increase the number or activity of effector cells that interact with the antibody. The antibodies of the present invention can be administered alone or in combination with other types of treatments (immunotherapy, bronchodilators, anti-IgE molecules, antihistamines, or anti-leukotrienes, etc.).

[0290] Such combination therapy as described above encompasses combined administration (wherein two or more therapeutic agents are included in the same or separate formulations) and separate administration, where administration of the antibody of the invention can occur prior to, concurrently with, and / or after administration of the additional therapeutic agent(s). In one embodiment, administration of the anti-IL-13 antibody and administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other. The antibodies of the invention can also be used in combination with radiation therapy.

[0291] In one aspect, described herein is an anti-VEGF antibody for use as a medicament. In a further aspect, described herein is an anti-VEGF antibody for use in treating a disorder associated with pathological angiogenesis. In certain embodiments, described herein is an anti-VEGF antibody for use in a method of treatment. In certain embodiments, described herein is an anti-VEGF antibody for use in a method of treating an individual having a disorder associated with pathological angiogenesis, the method comprising administering to the individual an effective amount of an anti-VEGF antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. An "individual" according to any of the above embodiments is preferably a human.

[0292] In a further aspect, the use of an anti-VEGF antibody in the manufacture or preparation of a medicament is described herein. In one embodiment, the medicament is for the treatment of a disorder associated with pathological angiogenesis. In a further embodiment, the medicament is for use in a method of treating a disorder associated with pathological angiogenesis, comprising administering an effective amount of the medicament to an individual having a disorder associated with pathological angiogenesis. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. The "individual" according to any of the above embodiments may preferably be a human.

[0293] In a further aspect, the invention provides a pharmaceutical formulation comprising any of the anti-VEGF antibodies described herein, e.g., for use in any of the above methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the anti-VEGF antibodies described herein and a pharma- ceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-VEGF antibodies described herein and at least one additional therapeutic agent, e.g., as described below.

[0294] In another aspect, the present invention provides a method of reducing or inhibiting angiogenesis, e.g., in a subject having a disorder associated with pathological angiogenesis, comprising administering to the subject an effective amount of an anti-VEGF antibody described herein, thereby reducing or inhibiting angiogenesis in the subject. In certain embodiments, the disorder associated with pathological angiogenesis is non-neoplastic. In certain embodiments, the disorder associated with pathological angiogenesis is cancer. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional agent, e.g., as described below.

[0295] In another aspect, the invention provides a method of treating a tumor, cancer, or cell proliferative disorder comprising administering an effective amount of an anti-VEGF antibody described herein, hi one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional agent, e.g., as described below.

[0296] The anti-VEGF antibodies described herein can be used in therapy, either alone or in combination with other agents. For example, the anti-VEGF antibodies can be co-administered with at least one additional agent. In certain embodiments, the additional agent is a chemotherapeutic agent, a cytotoxic agent, an antiangiogenic agent, an immunosuppressant, a prodrug, a cytokine, a cytokine antagonist, a cytotoxic radiation therapy, a corticosteroid, an antiemetic agent, a cancer vaccine, an analgesic agent, a growth inhibitor, an apoptotic agent, an antitubulin agent, or another agent, such as an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), a HER1 / EGFR inhibitor (e.g., erlotinib (TARCEVA™), a platelet-derived growth factor inhibitor (e.g., GLEEVEC™ (imatinib mesylate)), a COX-2 inhibitor (e.g., celecoxib), an interferon, a cytokine, an antibody that binds to a target other than VEGF, or another bioactive or organic chemical agent.

[0297] Such combination therapy as described above encompasses combined administration (wherein two or more agents are contained in the same or separate compositions) and separate administration, where administration of the antibody of the invention can occur prior to, concurrently with, and / or after administration of the additional agent(s). In one embodiment, administration of the anti-VEGF antibody and administration of the additional agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other. The anti-VEGF antibodies described herein can also be used in combination with radiation therapy.

[0298] The anti-IL-13 or anti-VEGF antibodies (and any additional therapeutic agents) described herein can be administered by any suitable means, including intramuscularly, intravenously, intradermally, transdermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intravitreally, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, intraorbitally, orally, topically, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by localized perfusion directly bathing the target cells, by catheter, by lavage, in a capsule, or in a lipid composition. The compositions utilized in the methods described herein can also be administered systemically or locally. The method of administration can vary depending on a variety of factors, such as the compound or composition being administered and the severity of the condition, disease, or disorder being treated.

[0299] The antibodies will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to the physician. The antibodies need not be formulated with one or more agents currently used to prevent or treat the disorder in question, but are optionally so formulated. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These will generally be used in the same dosages and routes of administration as described herein, or at about 1-99% of the dosages described herein, or at any dosage and via any route empirically / clinically determined to be appropriate.

[0300] For the prevention or treatment of disease, the appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of the antibody may be an initial candidate dosage for administration to the patient, whether, for example, in one or more separate administrations or by continuous infusion. One typical daily dosage may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or more depending on the condition, treatment will generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody would be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, weekly, every two weeks, every three weeks, or every four weeks (e.g., such that the patient receives about two to about twenty, or, for example, about six doses of the antibody). For example, a dose may be administered once a month (e.g., by subcutaneous injection). A higher initial loading dose, followed by one or more lower doses, may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0301] It will be understood that any of the above formulations or methods of treatment may be practiced using the immunoconjugates of the invention in place of, or in addition to, anti-IL-13 or anti-VEGF antibodies.

[0302] In some embodiments, the method may further include an additional therapy. The additional therapy may be radiation therapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the above. The additional therapy may be in the form of adjuvant therapy or neoadjuvant therapy. In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of the treatment, such as an antiemetic agent). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy may be a separate administration of one or more of the above-mentioned therapeutic agents.

[0303] J.Manufactured products In another aspect of the invention, an article of manufacture is provided that contains materials useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds the composition, either by itself or in combination with another composition that is effective for the treatment, prevention, and / or diagnosis of a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat a selected condition. Additionally, the article of manufacture may include (a) a first container having a composition therein that includes an antibody of the invention, and (b) a second container having a composition therein that includes an additional cytotoxic or other therapeutic agent. The article of manufacture of this embodiment of the invention may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container containing a pharma- ceutically 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.

[0304] It will be understood that any of the above articles of manufacture may contain the immunoconjugate of the invention in place of, or in addition to, an anti-IL-13 antibody or an anti-VEGF antibody.

[0305] K. Engineered bacteria The invention provides engineered bacteria, which can be used, for example, in any of the methods of the invention (e.g., the methods described above in Section A or in the Examples).

[0306] In one example, the invention provides a bacterium comprising (i) a mutation that allows diffusion of a membrane-impermeable molecule across the outer membrane into the periplasm, and (ii) an expression construct encoding a binding polypeptide.

[0307] In some instances, the mutation that allows diffusion of membrane impermeable molecules across the outer membrane into the periplasm is in a gene encoding an outer membrane protein and / or a protein that affects lipopolysaccharide (LPS) composition. In some instances, the mutation is in a gene encoding an outer membrane protein, e.g., the major outer membrane lipoprotein Lpp (Lpp), a porin, or TolC. In some instances, the outer membrane protein is Lpp. In some embodiments, the mutation is in a gene encoding a protein that affects LPS composition, e.g., an Env protein (e.g., EnvA, EnvB, and EnvM), RfaD, RfaE, RfaH, RfaRd, TolA, TolB, and TolD.

[0308] In some examples, the binding polypeptide can be an antibody. In some examples, the antibody of any of the above methods is a monoclonal antibody, including a chimeric, humanized, or human antibody. In some examples, the antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab') 2 In another example, the antibody is a full-length antibody, e.g., an intact IgG1 antibody, an intact IgG4 antibody, or other antibody class or isotype as defined herein. In another embodiment, the antibody is a half antibody, e.g., a knob-into-hole half antibody. For a description of knob-into-hole technology, see, e.g., U.S. Pat. No. 5,731,168. In any of the foregoing methods, the antibody may be a therapeutic antibody (e.g., a neutralizing antibody).

[0309] In other examples, the binding polypeptide may be an antibody mimic, such as an affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, designed ankyrin repeat protein (DARPin), finomer, Kunitz domain peptide, or monobody. In other embodiments, the binding polypeptide may be a bacterial protein that localizes to the periplasm. In other embodiments, the binding polypeptide may be a mammalian protein. For example, in some embodiments, the binding polypeptide may be a cell surface receptor, a cytokine, or a growth factor.

[0310] For example, the invention provides a bacterium comprising (i) a loss-of-function mutation in a gene encoding the major outer membrane lipoprotein Lpp (Lpp) and (ii) an expression construct encoding an antibody. In some examples, the antibody is a full-length antibody. In some examples, the antibody is an IgG antibody. In some examples, the antibody is a half antibody. In some examples, the antibody is an antibody fragment. In some examples, the antibody fragment is a Fab, Fab'-SH, Fv, scFv, and (Fab') 2 The nucleic acid is selected from the group consisting of:

[0311] Any of the above bacteria may contain any suitable mutation (e.g., loss-of-function mutation) in the gene encoding Lpp. Exemplary lpp mutations include, but are not limited to, lpp-1; lpp-21; lpp-3; lpp5508; lpp-6; lpp::Tn5KAN-2; lppD70E, G, or S; lppK75S or T; lppK78R; lppR77D or L; lppS22D; lppY76C, D, E, G, N, P, or S; lppY76F, H, I, or L; Δlpp, Δlpp-254, and Δlpp-752::kan, or those described herein. Such lpp mutations can be obtained from a repository, such as, for example, The Coli Genetic Stock Center (CGSC). In other examples, mutations in the gene encoding Lpp can be made using standard genetic approaches. In some examples, the loss-of-function mutation in the gene encoding Lpp is selected from a point mutation, an insertion mutation, or a deletion mutation. In some examples, the loss-of-function mutation is a deletion mutation.

[0312] Any of the above bacteria may include an inner membrane, a periplasm, and an outer membrane. In some examples, the bacteria is a gram-negative bacterium. For example, in some examples, the bacteria is E. coli. Any suitable strain or genetic background of E. coli may be used. Commonly used strains and genetic backgrounds of E. coli are well known in the art. Exemplary E. coli strains include, but are not limited to, laboratory strains such as K-12 and its substrains (e.g., Clifton wild type, W3110, and DH5αE, etc.), and E. coli B and its substrains (e.g., BL21 and BL21(DE3)). In some embodiments, the E. coli is from strain 62A7. In some embodiments, the E. coli is from strain 33D3. In some embodiments, the E. coli is from strain 66C4.

[0313] In some examples, any of the aforementioned bacteria can include a mutation that affects a transcriptional regulator gene. In some examples, the transcriptional regulator gene is selected from a transcription initiation factor, an anti-sigma factor, an RNA polymerase subunit, or a transcription factor. In some examples, the transcription initiation factor is a sigma factor. In some examples, the sigma factor is RpoD (σ 70 ), RpoE(σ 24 ), RpoH(σ 32 ), RpoS(σ 38 ), RpoN(σ 54 ), RpoF(σ 28 ), and FecI(σ 19 In a specific example, the sigma factor is selected from the group consisting of RpoD (σ 70 In some instances, the mutation affecting the transcriptional regulator gene is present on a synthetic plasmid. In other instances, the mutation affecting the transcriptional regulator gene is present in the endogenous bacterial genome. In some instances, the mutation affecting the transcriptional regulator gene is due to error-prone PCR mutagenesis, chemical mutagenesis, transposition mutagenesis, or targeted mutagenesis. In some instances, the mutation affecting the transcriptional regulator gene is due to error-prone PCR mutagenesis.

[0314] In some examples, the invention provides a plurality of any of the aforementioned bacteria. In some embodiments, the plurality of bacteria can comprise a library of the invention, such as any of the libraries described in Section B above or in the Examples herein. In some examples, the plurality of bacteria can each express a variant of a transcriptional regulator gene (e.g., a sigma factor, e.g., RpoD (σ 70 The method may include a library of nucleic acids encoding the polypeptides.

[0315] Any of the foregoing bacteria may have improved expression of a binding polypeptide (e.g., an antibody) by at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, or more. In some examples, the bacteria may have 1-10-fold improved expression, 1-7-fold improved expression, 1-6-fold improved expression, 1-5-fold improved expression, 1-4-fold improved expression, 1-3-fold improved expression, 2-10-fold improved expression, 2-8-fold improved expression, or 2-6-fold improved expression. In some embodiments, the improvement is compared to the expression level of the binding polypeptide in a wild-type bacterium, while in other embodiments, the improvement is compared to the expression level of the binding polypeptide in a mutant bacterium. III. Examples

[0316] Below are examples of methods and compositions of the present invention. Given the general overview provided above, it will be understood that various other embodiments may be practiced.

[0317] Example 1: Development of a live cell bacterial antibody display system compatible with full-length polypeptides We have developed a method for live cell bacterial antibody display that is compatible with full length binding polypeptides and ligands, such as full length antibodies and antigens. To overcome the limitations of previous bacterial display systems, we have developed a novel method for delivering ligands (e.g., antigens bound by antibodies) across the bacterial outer membrane and into the periplasm without substantially compromising the viability of the bacterial cell.

[0318] Deletion of the major outer membrane protein (murein lipoprotein, Lpp) renders the outer membrane semi-permeable. EDTA was used to further permeabilize the outer membrane (Leive et al. J. Biol. Chem. 243:6384-6391, 1968; Hancock, Annu. Rev. Microbiol. 38:237-264, 1984). The Δlpp genetic background was used to allow leakage of proteins from the periplasmic space into the medium to facilitate purification (Kanamori et al. Gene 66:295-300, 1988). However, this genetic background could also in principle be used to allow diffusion of ligands normally excluded by the outer membrane permeability barrier into the periplasm.

[0319] To demonstrate the functionality of this system, anti-IL-13 IgG (Spiess et al. J. Biol. Chem. 288:26583-26593, 2013) was expressed in a Δlpp background and fluorescence microscopy was used to determine whether cells treated with EDTA could bind exogenously added, fluorescently labeled IL-13 cytokine. Only cells expressing the antibody retained the antigen (Figure 1A), indicating that membrane tethering is not required to efficiently retain the antibody in the cell. The IL-13 antigen has a molecular weight of approximately 15 kDa, while anti-Fc F(ab') 2 Staining with EDTA indicates that antigens having a size of at least 100 kDa can efficiently enter and be retained in the periplasm of permeabilized cells. In other experiments, cells were successfully stained with full-length antibodies, which showed that molecules having a size of at least 150 kDa can efficiently enter and be retained in the periplasm of permeabilized cells. However, the use of EDTA to permeabilize cells resulted in a decrease in cell viability. To overcome the decrease in viability, MgCl was added after staining with antigen and EDTA. 2 , which restored viability to levels seen before EDTA treatment (Figure 1B).

[0320] Next, other antibody formats were tested to determine if they could be efficiently retained within the periplasm to bind antigen. Similar to full-length antibodies, both Fab and half-antibody formats could be expressed and stained with antigen without affecting viability (Figure 1B). While empty vector control (pBR322) cells showed autofluorescence by flow cytometry that contributed to background signal, all tested antibody formats showed antigen-specific signals above this background, allowing efficient sorting (Figure 1C). Half-antibodies with knob-into-hole Fc technology are of interest because they can be used to generate full-length bispecific antibodies (Spiess et al. Nat. Biotechnol. 31:753-758, 2013). For all the following presented experiments, half-antibody constructs were utilized.

[0321] As described below, this technology (sometimes referred to herein in a broad and non-limiting sense as bacterial antibody display or "BAD") overcomes the limitations of current bacterial display by delivering full-length antigens to live cells and allowing rapid round-by-round progression using flow cytometry (e.g., FACS™) selection (FIG. 1D). It is understood that BAD can be used not only with antibodies, but also with any suitable binding polypeptide. In a first step, enrichment of binding antibodies from a pool of non-binders was tested using BAD. Cells expressing anti-IL-13 antibodies were spiked into a pool of cells expressing anti-VEGF antibodies. To track the ratio of each antibody in the mixture, a plasmid conferring only carbenicillin resistance was used to express anti-VEGF, while the plasmid used to express anti-IL-13 confers both carbenicillin and tetracycline resistance. 1:10 5 and 1:10 6The accuracy of spiking was first determined by spot plating serial dilutions of the mixed culture at a ratio of 1:10 onto carbenicillin or tetracycline-containing plates, which confirmed that anti-IL-13 antibodies were represented in the pool at the expected frequency (Figure 2A). For more accurate quantification of subsequent selection experiments, larger volumes were plated, colony counts were performed, and the ratio of anti-IL-13 to anti-VEGF in each sample was calculated. 6 Starting with a ratio of anti-IL-13:anti-VEGF, two rounds of BAD were performed by sorting for anti-IL-13 positive cells, achieving a 235,000-fold enrichment (Figure 2B). To ensure that the antigen / antibody pair could be enriched, the reverse experiment was performed, resulting in a 19,500-fold enrichment of binding anti-VEGF antibodies after two rounds of FACS™ (Figure 2B). This enrichment corresponded to a large VEGF-positive shift in the population after a second round of FACS™ (Figure 2C). These experiments confirmed that binding clones could be rapidly and highly enriched by the BAD system.

[0322] Next, the BAD system was tested for its ability to enrich for the best expressing or folding variants of the same antibody. To test the resolution of the system, three variants with different expression levels were used along with half antibodies based on anti-VEGF.2 antibodies. The variants contained different combinations of light chain (L4M, S24R, F71Y) and heavy chain (E6Q, M34I) mutations. Non-reduced soluble samples were detected with anti-human Fc antibody by Western blot, and expression levels were normalized to anti-VEGF.2 half antibody wild type (WT) (Figure 3A). Anti-VEGF.2 wild type had the lowest functional expression, while L4M.E6Q.M34I, F71Y.E6Q.M34I, and S24R.E6Q.M34I had 3.7-, 5.6-, and 7.2-fold increases in expression levels, respectively. To ensure concordance of expression and FACS™ shifts, BAD was used to determine the individual FACS™ shifts of anti-VEGF.2 wild type and variants stained with fluorescently labeled VEGF (Figure 3B). FACS™ mean fluorescence intensity was in the same order as expression levels, with L4M.E6Q.M34I, F71Y.E6Q.M34I, and S24R.E6Q.M34I having 1.25+ / -0.2-fold, 1.37+ / -0.2-fold, and 1.4+ / -0.3-fold increases compared to wild type anti-VEGF.2. Thus, these antibodies were good candidates to test whether BAD could select the best expressing antibodies.

[0323] A BAD screen combining anti-VEGF.2 wild type with two variants was performed. Two rounds of FACS™ were performed to select the top 1% VEGF binding clones in each round. The FACS™ shift of the four combined anti-VEGF.2 antibodies was monitored over two rounds of sorting (Figure 3C). A bimodal distribution of cells was observed in some of the samples, which was due to partial cell permeabilization and background autofluorescence of E. coli. The VEGF binding signal of the pooled variants was superimposed on the best expressing variant S24R.E6Q.M34I after two rounds of sorting, suggesting that better expressing antibodies were enriched. Sequencing of the inputs shows that all four anti-VEGF.2 antibodies were present at the start of the sorting, while anti-VEGF.2 wild type and L4M.E6Q.M34I were partially depleted after the first round (Figure 3D). After the second round, anti-VEGF.2 wild type and L4M.E6Q.M34I were not detected, and only F71Y.E6Q.M34I and S24R.E6Q.M34I were enriched. These data demonstrate that BAD can successfully enrich for the best expressing antibodies despite small expression differences.

[0324] The above-mentioned method is useful in many applications. With regard to antibody engineering, it can be used with larger libraries for Fc engineering, antibody discovery, or affinity synthesis. Beyond antibodies, the BAD system can be applicable as a general high-throughput tool to study protein export, folding, or localization within the E. coli periplasm, since it provides a true readout in the cellular host environment. The main strength of this system is the untethered approach, which allows the use of native protein formats and obviates the need for fusion partners, reporter proteins, or membrane tethering. Furthermore, the live cell setting approach allows for exceptionally rapid screening for desired host characteristics, a significant improvement over previous display techniques.

[0325] material Plasmid construction and expression Antibodies were cloned into E. coli expression vectors (Simmons et al. J. Immunol. Methods 263:133-147, 2002; Carter et al. Biotechnology (NY) 10:13-167, 1992) or mammalian expression vectors (Eaton et al. Biochemistry 25:8343-8347, 1986) as previously described by standard molecular biology techniques. Antibody expression was carried out using 50 mL E. coli cell cultures (Spiess et al. J. Biol. Chem. 288:26583-26593, 2013) or 30 mL transient transfection cultures of CHO cells (Wong et al. Biotechnol. Bioeng. 106:751-763, 2010) or HEK293T (Bos et al. J. Biotechnol. 180:10-16, 2014) cells as previously described.

[0326] Antibody purification To purify Fab proteins from E. coli, proteins were expressed in strain 64B4, which has a chromosomal lpp gene deletion (Reilly et al. Antibody Engineering, Springer Berlin Heidelberg, pp. 331-344, 2010), as previously described (Spiess et al. Nat. Biotechnol. 31:753-758, 2013). 500 mL CRAP cultures were grown at 30°C for 24 h, after which EDTA at pH 8.0 was added to a final concentration of 10 mM. Incubation continued for 1 h at 30°C, 200 rpm, followed by addition of MgCl 2was added to a final concentration of 20 mM. Cell debris was removed by centrifugation (20 min, 4,500 rcf, 4°C), after which 400 μg of DNase I (Sigma-Aldrich, USA) was added and the supernatant was filtered through a GF / F filter (Whatman, England) and a 0.2μ PES filter (Thermo Fischer Scientific, USA). 1.5 mL of Protein G SEPHAROSE® slurry (GE Healthcare, USA) was added to 50 mL of lysate and incubated overnight at room temperature. Unbound proteins were removed by washing with PBS, and Fab proteins were eluted with 4 ml of 50 mM phosphate (pH 3), neutralized with 20× PBS, and concentrated with an AMICON® Ultra-4 (10,000 MW cutoff) (Merck Millipore, Ireland).

[0327] Human IgG1 was purified from mammalian culture supernatant by MABSELECT SURE™ (GE Healthcare, USA) according to the manufacturer's protocol. To purify human Fab from mammalian culture supernatant, a FLAG tag was added to the C-terminus of the heavy chain, and Fab was purified with anti-FLAG resin according to the manufacturer's protocol (Sigma-Aldrich, USA). Protein yield was quantified by determining absorbance at 280 nm and using the protein-specific extinction coefficient.

[0328] antigen Human VEGF 8-109 Expression and purification of Unizyme-tagged human IL-13 has been described previously (Muller et al. Proc. Natl. Acad. Sci. USA 94:7192-7197k 1997). 33-146was expressed in E. coli, purified by Ni-NTA chromatography (Qiagen, Germany) under denaturing conditions, and refolded. Proteins were fluorescently labeled with ALEXA FLUOR® 488 or ALEXA FLUOR® 647 succinimidyl ester (Invitrogen, USA) according to the manufacturer's protocol. Goat anti-human IgG Fc F(ab') labeled with DYLIGHT® 649 2 -fragment was obtained from Jackson Immuno Research, USA.

[0329] Bacterial antibody display and flow cytometry For bacterial display, antibodies were expressed in the Lpp deletion (Δlpp) of strain 62A7. 62A7 was derived from parent strain 33D3 by curing kanamycin resistance of strain 33D3 (Simmons et al. J. Immunol. Methods 263:133-147, 2002). For spiking experiments or experiments with individual framework variants, cells were individually transformed with each plasmid, grown as overnight cultures at 30° C., and used to inoculate 50 mL CRAP cultures at the ratios indicated or combined equally by volume and at a dilution of 1:100. After 24 hours at 30° C., 1 OD aliquots were harvested and pelleted by centrifugation (4 min, 6500 rcf). Cells were resuspended in 100 μL of PBS with 2% BSA and 5 mM EDTA (pH 7.4) and incubated at 4° C. for 30 min. After the initial incubation, SYTO® 41 or 9 nucleic acid stains (Molecular Probes, USA) were added to a final dilution of 1:100 and incubated with ALEXA 488 or ALEXA 647 Antigen labeled with 1-2 μM was added to a final concentration of 1-2 μM. Incubation continued for 1 h at 4°C in the dark, at which point MgCl 2 to a final concentration of 10-20 mM. 2 Unbound proteins were removed by washing three times with 500 mM NaCl. Stained cells were diluted at 1 × 107 to a final concentration of 1 x 10 cells / ml, and 1 x 10 for sorting using a BD FACSARIA™ II Flow Cytometer. 6 The cells were resuspended in SOC medium (New England Biolabs, USA) to a final concentration of 1000 cells / ml. The FACS™ gating strategy included cells that were SYTO® dye positive. A doublet discrimination gate was used to remove doublets and finally sort antigen positive cells. Cells were sorted into SOC medium, allowed to recover overnight at 30° C. and used to re-seed expression cultures or frozen as glycerol stocks.

[0330] Microscopy Expression and staining of cells for fluorescence microscopy was carried out similarly to the bacterial display protocol described above. 2 After a PBS washing step with 20 mM MgCl 2 Slides were prepared by resuspending cells in 100 μL of PBS with 0.1% NaCl and mixing with 1% gelatin at 37° C. Fluorescence microscopy was performed using a Zeiss Axio Imager A1.

[0331] Immunoblotting Immunoblotting was performed as previously described (Spiess et al. Nat. Biotechnol. 31:753-758, 2013), except that antibodies were expressed in a derivative line of 33D3 that was cured of kanamycin resistance.

[0332] Protein stability measurement by differential scanning fluorimetry Protein stability was determined in a Biorad CFX96 Real-Time System (Bio-Rad, USA) with a final dilution of 1:500 of SYPRO® Orange dye stock (Molecular Probes, USA). Fluorescence of 25 μL samples in PBS was recorded from 20 to 100 °C (0.2 °C increments, 10 s hold per step).

[0333] Periplasmic extract To generate periplasmic extracts for Biacore analysis, 40 mL of E. coli expression broth from bacterial antibody display strains was pelleted by centrifugation at 5,000 rpm for 5 min. The pellet was resuspended in 1.5 mL of 50 mM cold Tris (pH 8.0), 1 mM EDTA, and 500 mM sucrose and incubated on ice with agitation for 30 min. Samples were centrifuged at 9000 rpm for 10 min, after which the supernatant was collected as the periplasmic extract.

[0334] Surface plasmon resonance Surface plasmon resonance was used to measure the binding kinetics of anti-IL-13 and anti-VEGF antibodies on a BIACORE™ 3000 or T200 instrument (GE Healthcare), respectively. All kinetic experiments were performed at a flow rate of 30 μL / min, 25° C., and a running buffer of 10 mM HEPES (pH 7.4), 150 mM NaCl, and 0.005% Tween-20. For anti-IL-13, Fab binders from the Human Fab Capture Kit (GE Healthcare) were immobilized on a CM5 sensor chip via amine-based coupling to capture anti-IL-13 half-antibodies from E. coli periplasmic extracts. Human IL-13 binding to the antibodies was measured using a 2-fold concentration series of cytokines ranging from 1.56 to 50 nM. Sensorgrams of IL-13 binding were recorded using a 120 second injection time followed by a 1000 second dissociation time and regeneration of the surface between cycles with glycine (pH 2.1). For anti-VEGF, human VEGF was immobilized on a CM5 sensor chip via amine-based coupling. Binding to VEGF was analyzed using a 3-fold concentration series of anti-VEGF Fab ranging from 6.17 to 500 nM. Single cycle kinetic sensorgrams were recorded using a 120 second injection time followed by a 300 second dissociation time and regeneration of the surface between cycles with 20 mM HCl. All observed sensorgrams for antigen binding to antibody were analyzed using a 1:1 Langmuir binding model to calculate kinetics and binding constants.

[0335] Example 2: Optimization of cell sorting using bacterial antibody display To maximize cell viability during the sorting process and promote the speed and efficiency of sorting using BAD per round, the viability of different wild-type E. coli strains after expression of antibodies was compared. E. coli expression strains had different viability after expression of antibodies, for example, wild-type 64B4 cells had about a 1-fold decrease in cell viability compared to other wild-type strains including 62A7 cells (Figure 8). Furthermore, 62A7 cells with a deletion of Lpp (62A7Δlpp) had a greater viability than 64B4Δlpp cells (Figure 8). 62A7Δlpp cells remained viable after FACS™ sorting after antibody expression and incubation with labeled antigen, and were able to undergo multiple rounds of labeling and sorting. Thus, the choice of strain background is one factor that can be used to optimize the cell sorting process in BAD. In the method using Δlpp deletion, the method is compatible with any wild-type genetic background that can express antibodies.

[0336] Flow cytometry sorting conditions were also optimized to increase the amount of cells recovered after sorting. Various nucleic acid dyes were tested for their ability to improve the detection of E. coli cells during flow cytometry. A panel of six blue nucleic acid dyes (SYTO® dyes 40-45) was tested for staining efficiency of cells expressing anti-VEGF antibodies compared to an unstained control (Figure 9). Each of the nucleic acid dyes was used to detect dye-positive cells. Of the stains tested, SYTO® 41 showed the highest staining efficiency (approximately 90% of treated cells). When this nucleic acid dye was used as part of a selection strategy during flow cytometry, the amount of cells recovered by sorting was increased. SYTO® 9 is a non-limiting example of a dye that stains with comparable efficiency to SYTO® 41. To facilitate enrichment of antibodies with differences in expression levels and / or stability, loss of antigen-positive cells during sorting should be minimized. The presence of cell duplexes, two cells that are attached to each other but detected as single cells by flow cytometry, could in principle impair the enrichment of antigen-positive cells after sorting. In this scenario, antigen-positive cells may reduce enrichment by adhering to cells that are antigen-negative. To test whether cell duplexes occur under our sorting conditions, cells expressing the anti-VEGF antibody, anti-VEGF.V48L, were labeled with VEGF-ALEXA FLUOR® 488, while cells expressing anti-VEGF.A97T were labeled with VEGF-ALEXA FLUOR® 647. These cells were then equally combined and subjected to flow cytometry. 1×10 8 At a sorting concentration of 1 × 10 cells / ml, cell doublets were present, indicated by cells positive for both antigens (Figure 10). 6 A lower cell concentration of 10 cells / mL was used, which was sufficient to eliminate duplexes detected by flow cytometry analysis.

[0337] Use of the 62A7Δlpp strain and 1 × 10 6A sorting strategy was tested that combines sorting at a sorting cell concentration of 10000 cells / ml, flow cytometry, including sorting based on the signal of a nucleic acid stain to facilitate the localization of E. coli cells, standard doublet discrimination gates, and selection of antigen-positive cells (Figure 11). To confirm the success of this sorting strategy, a single round of BAD was performed using 62A7Δlpp E. coli cells expressing anti-VEGF.V48L or anti-VEGF.A97T (described in Example 4 below) in equal combinations, labeled with VEGF-ALEXA FLUOR® 488 or VEGF-ALEXA FLUOR® 647, respectively. Cells were sorted based on single antigen positive binding, double positive binding, or double negative binding to the separately labeled antigens (Figure 12A). Equal combinations of both anti-VEGF variants were detected from the sequencing of the input clones. After one round of sorting, approximately 93-95% of cells sorted as positive for a single antigen expressed the expected anti-VEGF variant, co...

Claims

1. 1. A method for identifying a bacterium that comprises a binding polypeptide that specifically binds to a target molecule, comprising: (a) providing a bacterium having an outer membrane that is permeable to molecules having a molecular weight greater than 10 kDa, said bacterium expressing a nucleic acid encoding a candidate binding polypeptide, said candidate binding polypeptide being present within the periplasm of said bacterium; (b) contacting the bacterium with a detectably labeled target molecule; (c) identifying the bacteria as containing a binding polypeptide by the presence of the labeled target molecule in the periplasm, wherein the bacteria remain viable after step (c).

2. 1. A method for identifying bacteria having improved expression of a binding polypeptide that specifically binds to a target molecule, comprising: (a) providing a bacterium having an outer membrane that is permeable to molecules having a molecular weight greater than 10 kDa, said bacterium expressing a nucleic acid encoding a binding polypeptide, said binding polypeptide being present within the periplasm of said bacterium; (b) contacting the bacterium with a detectably labeled target molecule; (c) identifying the bacterium as having improved expression of the binding polypeptide by the presence of the labeled target molecule in the periplasm, wherein the bacterium remains viable after step (c).

3. The method of claim 1 or 2, wherein the target molecule has a molecular weight of less than 250 kDa.

4. The method of claim 3 , wherein the target molecule has a molecular weight of less than 150 kDa.

5. 1. A method for identifying a bacterium that comprises a binding polypeptide that specifically binds to a target molecule, comprising: (a) providing a bacterium comprising a mutation that allows diffusion of a membrane impermeable molecule across an outer membrane into the periplasm, said bacterium expressing a nucleic acid encoding a candidate binding polypeptide, said candidate binding polypeptide being present in the periplasm of said bacterium; (b) contacting the bacterium with a detectably labeled target molecule; (c) identifying the bacteria as containing a binding polypeptide by the presence of the labeled target molecule in the periplasm, wherein the bacteria remain viable after step (c).

6. 1. A method for identifying bacteria having improved expression of a binding polypeptide that specifically binds to a target molecule, comprising: (a) providing a bacterium comprising a mutation that allows diffusion of a membrane impermeable molecule across an outer membrane into the periplasm, said bacterium expressing a nucleic acid encoding a candidate binding polypeptide, said candidate binding polypeptide being present in the periplasm of said bacterium; (b) contacting the bacterium with a detectably labeled target molecule; (c) identifying the bacterium as having improved expression of the binding polypeptide by the presence of the labeled target molecule in the periplasm, wherein the bacterium remains viable after step (c).

7. The method of any one of claims 1 to 6, further comprising, prior to step (b), subjecting the bacterium to conditions that permeabilize the outer membrane of the bacterium.

8. 8. The method of claim 7, further comprising resealing the outer membrane of the bacterium after contacting the bacterium with a detectably labeled target molecule.

9. 1. A method for identifying a bacterium that comprises a binding polypeptide that specifically binds to a target molecule, comprising: (a) providing a bacterium expressing a nucleic acid encoding a candidate binding polypeptide, wherein said candidate binding polypeptide is present within the periplasm of said bacterium; (b) subjecting the bacterium to conditions that permeabilize the outer membrane of the bacterium; (c) contacting the bacterium with a detectably labeled target molecule; (d) after step (c), resealing the outer membrane of the bacterium; (e) identifying the bacterium as containing a binding polypeptide by the presence of the labeled target molecule in the periplasm.

10. 1. A method for identifying bacteria having improved expression of a binding polypeptide that specifically binds to a target molecule, comprising: (a) providing a bacterium expressing a nucleic acid encoding a candidate binding polypeptide, wherein said candidate binding polypeptide is present within the periplasm of said bacterium; (b) subjecting the bacterium to conditions that permeabilize the outer membrane of the bacterium; (c) contacting the bacterium with a detectably labeled target molecule; (d) after step (c), resealing the outer membrane of the bacterium; (e) identifying the bacterium as having improved expression of the binding polypeptide by the presence of the labeled target molecule within the periplasm.

11. The method of any one of claims 1 to 10, further comprising repeating said steps of the method at least once without the intervening step of isolating said nucleic acid.

12. 12. The method of claim 11, further comprising incubating the bacteria in a growth medium before repeating the method.

13. 13. The method of any one of claims 1 to 4 and 7 to 12, wherein the bacterium comprises a mutation that allows diffusion of membrane impermeable molecules across the outer membrane into the periplasm.

14. 14. The method of any one of claims 5, 6 and 13, wherein the mutation is in a gene encoding an outer membrane protein or a protein that affects lipopolysaccharide (LPS) composition.

15. 15. The method of claim 14, wherein the outer membrane protein is the major outer membrane lipoprotein Lpp (Lpp), a porin, or TolC.

16. 15. The method of claim 14, wherein the protein that affects LPS composition is selected from EnvA, RfaD, RfaE, RfaH, RfaRd, TolA, TolB, and TolD.

17. 17. The method of any one of claims 7 to 16, wherein subjecting the bacterium to conditions that permeabilize the outer membrane of the bacterium comprises treating the bacterium with a permeabilizing agent.

18. The permeabilizing agent may be a divalent cation chelator, NaCl, sucrose, antibiotics, detergents, lysozyme, Tris, Tris-EDTA, ascorbate, polylysine, benzalkonium chloride, protamine, bactericidal / permeability enhancing protein (BPI), serum, complement, and Ca. ２＋ 20. The method of claim 17, selected from the group consisting of:

19. 20. The method of claim 18, wherein the divalent cation chelator is EDTA.

20. 20. The method of claim 18, wherein the antibiotic is selected from the group consisting of aminoglycosides, polymyxin B, deacylated polymyxins, octapeptins, and benzylpenicillins.

21. Resealing the outer membrane of the bacterium comprises treating the bacterium with Mg ２＋ , Mn ２＋ , Ca ２＋ , Zn ２＋ , Fe ２＋ , Na ＋ , or K ＋ A method according to any one of claims 8 to 20, comprising contacting with a salt of a cation selected from:

22. The cation is Mg ２＋ 22. The method of claim 21 , wherein

23. The Mg ２＋ The salt of MgCl ２ 23. The method of claim 22, wherein:

24. 24. The method of any one of claims 1 to 23, wherein the contacting step further comprises contacting the bacteria with a nucleic acid dye.

25. 25. The method of claim 24, wherein the nucleic acid dye is selected from the group consisting of SYTO® 9, SYTO® 41, SYTO® 43, SYTO® 42, SYTO® 44, SYTO® 40, and SYTO® 45.

26. 26. The method of claim 25, wherein the nucleic acid dye is SYTO® 9 or SYTO® 41.

27. The method of any one of claims 1 to 26, wherein the detectably labeled target molecule comprises a fluorescent label.

28. 28. The method of claim 27, wherein the fluorescent label comprises a fluorescent label or a fluorescent polypeptide.

29. 29. The method of claim 28, wherein the fluorescent dye is selected from the group consisting of an ALEXA® dye or a DYLIGHT® dye.

30. 30. The method of claim 29, wherein the ALEXA® dye is ALEXA FLUOR® 488 or ALEXA FLUOR® 647.

31. 30. The method of claim 29, wherein the DYLIGHT® dye is DYLIGHT® 649.

32. 32. The method of any one of claims 1 to 31, further comprising at least one washing step comprising resuspending the bacteria in a wash buffer after contacting the bacteria with the detectably labeled target molecule.

33. The method of any one of claims 1 to 32, wherein the identifying step comprises flow cytometry.

34. 34. The method of claim 33, wherein the flow cytometry comprises sorting single cells and sorting based on the signal of the detectably labeled target molecule.

35. 35. The method of claim 34, further comprising sorting based on a nucleic acid dye signal.

36. 36. The method of claim 35, wherein the flow cytometry sequentially comprises: (i) sorting based on a signal of a nucleic acid dye; (ii) sorting single cells; and (iii) sorting based on the signal of the detectably labeled target molecule.

37. The method according to any one of claims 1 to 36, wherein the bacterium is a gram-negative bacterium.

38. 38. The method of claim 37, wherein the gram negative bacterium is an E. coli bacterium.

39. 39. The method of any one of claims 1 to 38, wherein the binding polypeptide is expressed in a soluble form within the periplasm of the bacterium.

40. The method of any one of claims 1 to 39, wherein the binding polypeptide is an antibody.

41. 41. The method of claim 40, wherein the antibody is a full-length antibody.

42. 42. The method of claim 41, wherein the full-length antibody is an IgG antibody.

43. The method of any one of claims 40 to 42, wherein the antibody is a half antibody.

44. 41. The method of claim 40, wherein the antibody is an antibody fragment.

45. The antibody fragments include Fab, Fab'-SH, Fv, scFv, and (Fab') ２ 45. The antibody of claim 44, selected from the group consisting of:

46. 46. ​​The method of any one of claims 1 to 45, wherein step (a) comprises providing a plurality of bacteria, said plurality of bacteria comprising a library of nucleic acids each encoding a candidate binding polypeptide.

47. 47. The method of claim 46, wherein the library comprises a plurality of nucleic acids encoding candidate antibody variants, each candidate antibody variant comprising an amino acid residue change in a FR of VH or VL compared to a reference antibody, the amino acid residue change being in an amino acid residue that is (a) identified in a naturally occurring antibody having the same subtype as the reference antibody and (b) predicted to be buried.

48. The method of claim 47, wherein the amino acid residue changes in the naturally occurring antibody are due to somatic hypermutation.

49. 49. The method of any one of claims 46 to 48, further comprising identifying candidate binding polypeptides as having increased expression levels based on the amount of the labeled target molecule within the periplasm.

50. 50. The method of any one of claims 46 to 49, further comprising identifying candidate binding polypeptides as having increased stability based on the amount of said labeled target molecule within the periplasm.

51. The method of any one of claims 1 to 50, further comprising isolating said nucleic acid after said identifying step.

52. 52. The method of any one of claims 1 to 51, wherein the bacterium comprises a mutation affecting a transcriptional regulator gene.

53. 53. The method of claim 52, wherein step (a) comprises providing a plurality of bacteria, said plurality of bacteria comprising a library of nucleic acids encoding variants of said transcriptional regulator gene.

54. 54. The method of claim 52 or 53, wherein the transcriptional regulatory gene is selected from a transcription initiation factor, an anti-sigma factor, an RNA polymerase subunit, or a transcription factor.

55. 55. The method of claim 54, wherein the transcription initiation factor is a sigma factor.

56. The sigma factor is RpoD (σ ７０ ), RpoE(σ ２４ ), RpoH(σ ３２ ), RpoS(σ ３８ ), RpoN(σ ５４ ), RpoF(σ ２８ ), and FeC I (σ １９ 56. The method of claim 55, wherein said compound is selected from the group consisting of:

57. The sigma factor is RpoD (σ ７０ 57. The method of claim 56, wherein

58. 58. The method of any one of claims 52 to 57, wherein the mutation affecting the transcriptional regulator gene is present on a synthetic plasmid.

59. 58. The method of any one of claims 52 to 57, wherein the mutation affecting the transcriptional regulator gene is present in an endogenous bacterial genome.

60. 60. The method of any one of claims 52 to 59, wherein the mutation affecting the transcriptional regulatory gene is due to error-prone PCR mutagenesis, chemical mutagenesis, translocation mutagenesis, or targeted mutagenesis.

61. 61. The method of claim 60, wherein the mutation affecting the transcriptional regulatory gene is due to error-prone PCR mutagenesis.

62. 62. The method of any one of claims 52 to 61, wherein the method identifies bacteria having improved expression of the binding polypeptide.

63. A bacterium comprising (i) a loss-of-function mutation in a gene encoding Lpp, and (ii) an expression construct encoding an antibody.

64. The bacterium described in claim 63, wherein the antibody is a full-length antibody.

65. The bacterium described in claim 64, wherein the full-length antibody is an IgG antibody.

66. The bacterium according to any one of claims 63 to 65, wherein the antibody is a half-antibody.

67. The bacterium described in claim 63 , wherein the antibody is an antibody fragment.

68. The antibody fragments include Fab, Fab'-SH, Fv, scFv, and (Fab') ２ 68. The bacterium of claim 67, selected from the group consisting of:

69. The bacterium according to any one of claims 63 to 68, wherein the bacterium is a gram-negative bacterium.

70. 70. The bacterium of claim 69, wherein the gram-negative bacterium is an E. coli bacterium.

71. The bacterium according to any one of claims 63 to 70, wherein the antibody is expressed in a soluble form within the periplasm of the bacterium.

72. The bacterium according to any one of claims 63 to 71, wherein the loss-of-function mutation is a point mutation, an insertion mutation, or a deletion mutation.

73. The bacterium of claim 72, wherein the loss-of-function mutation is a deletion mutation.

74. The bacterium according to any one of claims 63 to 73, wherein the expression construct comprises a promoter operably linked to a gene encoding the antibody.

75. The bacterium of any one of claims 63 to 74, wherein the expression construct comprises a synthetic plasmid.

76. 76. The bacterium of any one of claims 63 to 75, further comprising a mutation affecting a transcriptional regulatory gene.

77. 1. A bacterium comprising: (i) a mutation that allows diffusion of a membrane-impermeable molecule across the outer membrane into the periplasm; (ii) an expression construct encoding a binding polypeptide; and (iii) a mutation that affects a transcriptional regulatory gene.

78. 78. The bacterium of claim 76 or 77, wherein the transcriptional regulatory gene is selected from a transcription initiation factor, an anti-sigma factor, an RNA polymerase subunit, or a transcription factor.

79. The bacterium of claim 78, wherein the transcription initiation factor is a sigma factor.

80. The sigma factor is RpoD (σ ７０ ), RpoE(σ ２４ ), RpoH(σ ３２ ), RpoS(σ ３８ ), RpoN(σ ５４ ), RpoF(σ ２８ ), and FeC I (σ １９ 80. The bacterium of claim 79, selected from the group consisting of:

81. The sigma factor is RpoD (σ ７０ 81. The bacterium described in claim 80,

82. The bacterium according to any one of claims 76 to 81, wherein the mutation affecting the transcriptional regulatory gene is present on a synthetic plasmid.

83. The bacterium according to any one of claims 76 to 81, wherein the mutation affecting the transcriptional regulator gene is present in the endogenous bacterial genome.

84. The bacterium according to any one of claims 76 to 83, wherein the mutation affecting the transcriptional regulatory gene is caused by error-prone PCR mutagenesis, chemical mutagenesis, transposition mutagenesis, or targeted mutagenesis.

85. 85. The bacterium of claim 84, wherein the mutation affecting the transcriptional regulatory gene is due to error-prone PCR mutagenesis.

86. The bacterium of any one of claims 77 to 85, wherein the binding polypeptide is an antibody.

87. The bacterium described in claim 86, wherein the antibody is a full-length antibody.

88. The bacterium described in claim 87, wherein the full-length antibody is an IgG antibody.

89. The bacterium according to any one of claims 86 to 88, wherein the antibody is a half-antibody.

90. The bacterium described in claim 86, wherein the antibody is an antibody fragment.

91. 91. The bacterium of any one of claims 77 to 90, wherein the mutation that allows diffusion of membrane impermeable molecules across the outer membrane into the periplasm is in a gene encoding an outer membrane protein or a protein that affects LPS composition.

92. 92. The bacterium of claim 91, wherein the outer membrane protein is the major outer membrane lipoprotein Lpp (Lpp), a porin, or TolC.

93. 93. The bacterium of claim 92, wherein the outer membrane protein is Lpp.

94. 92. The bacterium of claim 91, wherein the protein that affects LPS composition is selected from EnvA, RfaD, RfaE, RfaH, RfaRd, TolA, TolB, and TolD.

95. An isolated antibody that specifically binds to interleukin-13 (IL-13), the antibody comprising: (a) a light chain variable region comprising the amino acid sequence of SEQ ID NO:1; and (b) a heavy chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

5.

96. An isolated antibody that specifically binds to IL-13, the antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2; and (b) a light chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

6.

97. An isolated antibody that specifically binds to IL-13, the antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3; and (b) a light chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

6.

98. An isolated antibody that specifically binds to IL-13, the antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4; and (b) a light chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

6.

99. An isolated antibody that specifically binds to IL-13, the antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

5.

100. An isolated antibody that specifically binds to IL-13, the antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

4.

101. An isolated antibody that specifically binds to IL-13, the antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:6 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

2.

102. An isolated antibody that specifically binds to IL-13, the antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:6 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

3.

103. An isolated antibody that specifically binds to IL-13, the antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:6 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

4.

104. The antibody comprises the following six HVRs: (a) HVR-H1 comprising the amino acid sequence of AYSVN (SEQ ID NO: 7); (b) HVR-H2 comprising the amino acid sequence of MIWGDGKIVYNSALKS (SEQ ID NO: 8); and (c) HVR-H3 comprising the amino acid sequence of DGYYPYAMDN (SEQ ID NO: 9); (d) HVR-L1 comprising the amino acid sequence of RASKSVDSYGNSFMH (SEQ ID NO: 10); and (e) HVR-L2 comprising the amino acid sequence of LASNLES (SEQ ID NO: 11); and (f) an HVR-L3 comprising the amino acid sequence of QQNNEDPRT (SEQ ID NO: 12),

105. The antibody has a K Ｄ The isolated antibody of any one of claims 95 to 104, which specifically binds to human IL-13 at

106. The antibody has a K Ｄ 106. The isolated antibody of claim 105, which specifically binds to human IL-13 at

107. The antibody has a K Ｄ 107. The isolated antibody of claim 106, which specifically binds to human IL-13 at

108. The antibody of any one of claims 95 to 107, wherein the antibody is monoclonal, human, humanized, or chimeric.

109. The antibody of any one of claims 95 to 108, wherein the antibody is a full-length antibody.

110. The antibody of claim 109, wherein the full length antibody is an IgG antibody.

111. The antibody of any one of claims 95 to 110, wherein the antibody is a half antibody.

112. The antibody of any one of claims 95 to 108, wherein the antibody is an antibody fragment that binds to IL-13.

113. The antibody fragments include Fab, Fab'-SH, Fv, scFv, and (Fab') ２ The antibody of claim 112, selected from the group consisting of fragments.

114. The antibody of any one of claims 95 to 113, wherein the antibody is a monospecific antibody.

115. The antibody of any one of claims 95 to 113, wherein the antibody is a multispecific antibody.

116. The antibody of claim 115, wherein the multispecific antibody is a bispecific antibody.

117. An isolated nucleic acid encoding an antibody according to any one of claims 95 to 116.

118. A vector comprising the isolated nucleic acid of claim 117.

119. A host cell comprising the vector described in claim 118.

120. 120. The host cell of claim 119, wherein the host cell is a mammalian cell.

121. The host cell of claim 119, wherein the host cell is a prokaryotic cell.

122. 122. The host cell of claim 121, wherein the prokaryotic cell is an E. coli cell.

123. A method for producing an antibody according to any one of claims 95 to 116, comprising culturing a host cell according to claim 114 in a culture medium.

124. 124. The method of claim 123, wherein the method further comprises recovering the antibody from the host cell or the culture medium.

125. A composition comprising an antibody according to any one of claims 95 to 116.

126. 126. The composition of claim 125, further comprising a pharma- ceutically acceptable carrier, excipient, or diluent.

127. The composition of claim 126, wherein the composition is a pharmaceutical composition.

128. The antibody according to any one of claims 95 to 116 for use as a medicament.

129. The antibody of any one of claims 95 to 116 for use in the treatment of an IL-13 mediated disorder.

130. 130. A method of treating a patient suffering from an asthma condition, comprising administering to said patient an effective amount of an antibody according to any one of claims 95 to 116, thereby reducing said asthma symptoms.

131. A method for inhibiting IgE antibody production in a patient, comprising administering to said patient an effective amount of an antibody according to any one of claims 95 to 116.

132. 132. The method of claim 131, wherein said inhibition of IgE antibody production is intended to treat bronchial asthma, allergic rhinitis, allergic dermatitis, bronchial asthma, allergic rhinitis, urticaria, anaphylaxis, or atopic dermatitis.

133. A method of treating an IL-13 mediated disorder in a patient, comprising administering to the patient an effective amount of an antibody of any one of claims 95-116.

134. 134. The method of claim 133, wherein the disorder is allergic asthma, non-allergic (intrinsic) asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, eczema, urticaria, food allergy, chronic obstructive pulmonary disease, ulcerative colitis, RSV infection, uveitis, scleroderma, or osteoporosis.

135. 117. A method of treating an IgE-mediated disorder in a patient, comprising administering to the patient an effective amount of an antibody according to any one of claims 95 to 116.

136. 136. The method of claim 135, wherein the IgE-mediated disorder is bronchial asthma, allergic rhinitis, allergic dermatitis, urticaria, anaphylaxis, or atopic dermatitis.

137. The method of claims 130-136, wherein the antibody inhibits binding of IL-13 to its receptor and inhibits one or more functions associated with binding of IL-13 to its receptor.

138. The method of claims 130-137, wherein the antibody is administered by one or more of the routes selected from the group consisting of intravenous, intraperitoneal, inhalation, intramuscular, subcutaneous, and oral.

139. 139. The method of claim 138, wherein the route is subcutaneous.

140. The method of claims 130 to 139, wherein the patient is a human.

141. A library comprising a plurality of nucleic acids encoding candidate antibody variants, each candidate binding polypeptide variant comprising an amino acid residue change in a FR of VH or VL compared to a reference antibody, the amino acid residue change being (a) in an amino acid residue that is identified in a naturally occurring antibody having the same subtype as the reference antibody and (b) that is predicted to be buried.

142. The library of Claim 141, wherein the amino acid residue changes in the naturally occurring antibodies are due to somatic hypermutation.

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