BR112012030179B1 - FC-CONTAINING POLYPEPTIDE - Google Patents

Abstract

FC-CONTAINING POLYPEPTIDE, METHODS FOR PRODUCING AN FC-CONTAINING POLYPEPTIDE TO INCREASE THE ANTI-INFLAMMATORY PROPERTIES OR DECREASE THE CYTOXICITY OF AN FC-CONTAINING POLYPEPTIDE, AND USE OF THE FC-CONTAINING POLYPEPTIDE. The present invention relates to methods and compositions for producing Fc-containing polypeptides with improved properties, and comprising mutations at positions 243 and 264 of the Fc region.

Description

FIELD OF INVENTION

[001] The present invention relates to methods and compositions for producing glycosylated proteins (glycoproteins) and, specifically, Fc-containing polypeptides that are used as human or animal therapeutic agents.

FUNDAMENTALS OF THE INVENTION

[002] Monoclonal antibodies often achieve their therapeutic benefit through two binding events. First, the variable domain of the antibody binds to a specific protein on a target cell, e.g., CD20 on the surface of cancer cells. This is followed by the recruitment of effector cells, such as natural killer (NK) cells, which bind to the constant (Fc) region of the antibody and destroy cells to which the antibody is bound. This process, known as antibody-dependent cellular cytotoxicity (ADCC), depends on a specific N-glycosylation event at Asn 297 in the Fc domain of the heavy chain of IgGls, Rothman et al., Mol. Immunol. 26: 1113-1123 (1989). Antibodies lacking this N-glycosylation structure still bind antigen but cannot mediate ADCC, apparently as a result of the lower affinity of the antibody Fc domain for the Fc receptor FcYRIIIa on the surface of NK cells.

[003] The presence of N-glycosylation not only plays a role in the effector function of an antibody, the particular composition of the N-linked oligosaccharide is also important for its ultimate function. The lack of fucose or the presence of bifurcated N-acetyl glucosamine has been positively correlated with ADCC efficacy, Rothman (1989), Umana et al., Nat. Biotech. 17:176-180 (1999), Shields et al., J. Biol. Chem. 277:26733-26740 (2002), and Shinkawa et al., J. Biol. Chem. 278:3466-3473 (2003). There is also evidence that sialylation in the Fc region is positively correlated with the anti-inflammatory properties of intravenous immunoglobulin (IVIG). See, for example, Kaneko et al., Science, 313: 670-673, 2006; Nimmerjahn and Ravetch., J. Exp. Med., 204: 11-15, 2007.

[004] Given the utility of specific N-glycosylation in antibody function and efficacy, a method for modifying the composition of N-linked oligosaccharides and modifying antibody effector function would be desirable.

[005] Yeast and other fungal hosts are important production platforms for the generation of recombinant proteins. Yeast are eukaryotes and therefore share common evolutionary processes with higher eukaryotes, including many of the post-translational modifications that occur in the secretory pathway. Recent advances in glycoengineering have resulted in cell lines of the yeast strain Pichia pastoris with genetically modified glycosylation pathways that allow them to carry out a sequence of enzymatic reactions that mimic the glycosylation process in humans. See, for example, U.S. Patents 7,029,872, 7,326,681, and 7,449,308, which describe methods for producing a recombinant glycoprotein in a lower eukaryotic host cell that is substantially identical to its human counterparts. Human-type bi-antennated sialylated complex N-linked glycans, such as those produced in Pichia pastoris from the aforementioned methods, have demonstrated utility for the production of therapeutic glycoproteins. Thus, a method to further modify or enhance antibody production in yeasts such as Pichia pastoris would be desirable.

SUMMARY OF THE INVENTION

[006] The invention relates to an Fc-containing polypeptide comprising mutations at amino acid positions 243 and 264 of the Fc region, wherein the mutations at position 243 are selected from the group consisting of: F243A, F243G, F243S, F243T, F243V, F243L, F243I, F243D, F243Y, F243E, F243R, F243W and F243K and the mutations at position 264 are selected from the group consisting of: V264A, V264G, V264S, V264T, V264D, V264E, V264K, V264W, V264H, V264P, V264N, V264Q and V264L, wherein the numbering is according to the EU index as in Kabat. In another embodiment, the Fc-containing polypeptide comprises mutations F243A and V264A. In another embodiment, the Fc-containing polypeptide comprises mutations F243Y and V264G. In another embodiment, the Fc-containing polypeptide comprises mutations F243T and V264G. In another embodiment, the Fc-containing polypeptide comprises mutations F243L and V264A. In another embodiment, the Fc-containing polypeptide comprises mutations F243L and V264N. In another embodiment, the Fc-containing polypeptide comprises mutations F243V and V264G. In one embodiment, the Fc-containing polypeptide of the invention is an antibody or an antibody fragment. In one embodiment, the Fc-containing polypeptide of the invention is an antibody fragment comprising SEQ ID NO:18. In another embodiment, the Fc-containing polypeptide of the invention is an antibody fragment comprising SEQ ID NO:19. In another embodiment, the Fc-containing polypeptide of the invention is an antibody fragment consisting of (or consisting essentially of) SEQ ID NO:18 or SEQ ID NO:19.

[007] In one embodiment, the Fc-containing polypeptide is an antibody comprising the heavy chain amino acid sequence of SEQ ID NO:9 or a variant thereof, and the light chain amino acid sequence of SEQ ID NO:2 or a variant thereof. In one embodiment, the Fc-containing polypeptide is an antibody comprising the heavy chain amino acid sequence of SEQ ID NO:9, minus the last lysine (K) residue listed in SEQ ID NO:9.

[008] In one embodiment, the Fc-containing polypeptide is an antibody comprising the heavy chain amino acid sequence of SEQ ID NO:12 or a variant thereof, and the light chain amino acid sequence of SEQ ID NO:11 or a variant thereof.

[009] In one embodiment, the Fc-containing polypeptide is an antibody comprising the heavy chain amino acid sequence of SEQ ID NO:15 or a variant thereof, and the light chain amino acid sequence of SEQ ID NO:14 or a variant thereof.

[0010] In some embodiments, the Fc-containing polypeptides of the invention comprise N-glycans comprising sialic acid (including NANA, NGNA, and analogs and derivatives thereof). In one embodiment, the Fc-containing polypeptides of the invention comprise a mixture of α-2,3- and α-2,6-linked sialic acid. In another embodiment, the Fc-containing polypeptides of the invention comprise only α-2,6-linked sialic acid. In one embodiment, the Fc-containing polypeptides of the invention comprise α-2,6-linked sialic acid, and do not comprise any detectable level of α-2,3-linked sialic acid. In one embodiment, the sialic acid is N-acetylneuraminic acid (NANA) or N-glycolylneuraminic acid (NGNA) or a mixture thereof. In another embodiment, the sialic acid is an analog or derivative of NANA or NGNA with acetylation at the 9-position on the sialic acid. In one embodiment, the N-glycans on the Fc-containing polypeptides of the invention comprise NANA and no NGNA.

[0011] The N-glycans on the Fc-containing polypeptides of the invention may optionally comprise fucose. In one embodiment, the N-glycans on the Fc-containing polypeptides will comprise a mixture of fucosylated and non-fucosylated N-glycans. In another embodiment, the N-glycans on the Fc-containing polypeptides do not feature fucose.

[0012] In one embodiment, the Fc-containing polypeptide of the invention exhibits one or more of the following properties when compared to a parent Fc-containing polypeptide: (i) decreased effector function; (ii) improved anti-inflammatory properties; (iii) improved sialylation; (iv) improved bioavailability (absorption or exposure), and (v) decreased binding to FCYRI, FcYRIIa, FcYRIIb, FcyRIIIa (FcYRIIIa-V158 or FcrRIIIa-F158), and FcYRIIIb. In one embodiment, the Fc-containing polypeptide of the invention exhibits at least a 7-, 10-, 15-, 30-, 50-, 100-, 500-, or 1,000-fold reduction in effector function compared to a parent Fc-containing polypeptide. In one embodiment, the effector function is ADCC. In another embodiment, the effector function is CDC.

[0013] In one embodiment, the Fc-containing polypeptide of the invention exhibits decreased ADCC activity when compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide exhibits at least a 7-, 10-, 15-, 30-, 50-, 100-, 500-, or 1,000-fold reduction in ADCC activity. In another embodiment, the Fc-containing polypeptide exhibits at least a 100-fold reduction in ADCC activity. In another embodiment, the Fc-containing polypeptide exhibits at least a 500-fold reduction in ADCC activity. In another embodiment, the Fc-containing polypeptide exhibits at least a 1,000-fold reduction in ADCC activity.

[0014] In another embodiment, the Fc-containing polypeptide of the invention exhibits reduced CDC activity when compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide exhibits at least a 100-fold reduction in CDC activity.

[0015] In one embodiment, the Fc-containing polypeptide of the invention binds to FCYRI, FcYRIIa, FcYRIIb, FcYRIIIa (FcYRIIIa-V158 or FcYRIIIa-F158), and FcYRIIIb with lower affinity when compared to a parent Fc-containing polypeptide. In one embodiment, an Fc-containing polypeptide of the invention binds to FcYRIIa with at least a 50-fold lower affinity when compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide of the invention binds to FcYRIIb with at least a 20-fold lower affinity when compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide of the invention binds to FcYRIIIa LF with at least a 10-fold lower affinity when compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide of the invention binds to FcYRIIIa LV with at least a 1-, 2-, or 10-fold lower affinity compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide of the invention binds to FcYRIIb, FcYRIIIa LF, and FcYRIIIa LV with a lower affinity compared to a parent Fc-containing polypeptide.

[0016] In one embodiment, the Fc-containing polypeptide of the invention exhibits improved anti-inflammatory properties compared to a parent Fc-containing polypeptide.

[0017] In one embodiment, the Fc-containing polypeptide of the invention exhibits improved bioavailability (absorption or exposure) when injected parenterally, compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide of the invention exhibits improved bioavailability (absorption or exposure) when injected subcutaneously, compared to a parent Fc-containing polypeptide.

[0018] In one embodiment, the Fc-containing parental polypeptide comprises a natural Fc region. In another embodiment, the Fc-containing parental polypeptide comprises an F243A mutation. In another embodiment, the Fc-containing parental polypeptide comprises a V264A mutation.

[0019] The invention also comprises a method for producing an Fc-containing polypeptide in a host cell comprising: (i) providing a host cell that has been genetically modified to produce an Fc-containing polypeptide, wherein the host cell comprises a nucleic acid encoding mutations at amino acid positions 243 and 264 of the Fc region, wherein the mutations at positions 243 are selected from the group consisting of: F243A, F243G, F243S, F243T, F243V, F243L, F243I, F243D, F243Y, F243E, F243R, F243W, and F243K, and the mutations at position 264 are selected from the group consisting of: V264A, V264G, V264S, V264T, V264D, V264E, V264K, V264W, V264H, V264P, V264N, V264Q, and V264L, wherein the numbering is in accordance with the EU index as in Kabat; (ii) culturing the host cell under conditions that cause expression of the Fc-containing polypeptide; and (iii) isolating the Fc-containing polypeptide from the host cell. In one embodiment, the nucleic acid encodes the F243A and V264A mutations. In another embodiment, the nucleic acid encodes the F243Y and V264G mutations. In another embodiment, the nucleic acid encodes the 243T and V264G mutations. In another embodiment, the nucleic acid encodes the F243L and V264A mutations. In another embodiment, the nucleic acid encodes the F243L and V264N mutations. In another embodiment, the nucleic acid encodes the F243V and V264G mutations. In one embodiment, the Fc-containing polypeptide of the invention is an antibody or an antibody fragment. In one embodiment, the Fc-containing polypeptide is an antibody fragment comprising SEQ ID NO:18. In another embodiment, the Fc-containing polypeptide is an antibody fragment comprising SEQ ID NO:19. In another embodiment, the Fc-containing polypeptide is an antibody fragment consisting of (or consisting essentially of) SEQ ID NO:18 or SEQ ID NO:19.

[0020] In one embodiment, the method for producing an Fc-containing polypeptide is carried out in a mammalian cell. In another embodiment, the method for producing an Fc-containing polypeptide is carried out in a plant cell. In another embodiment, the method for producing an Fc-containing polypeptide is carried out in bacteria. In another embodiment, the method for producing an Fc-containing polypeptide is carried out in an insect cell. In another embodiment, the method for producing an Fc-containing polypeptide is carried out in a lower eukaryote cell. In another embodiment, the method for producing an Fc-containing polypeptide is carried out in a yeast cell. In one embodiment, the method for producing an Fc-containing polypeptide is carried out in Pichia pastoris.

[0021] In one embodiment, the Fc-containing polypeptide produced by the claimed method comprises N-glycans comprising sialic acid (including NANA, NGNA, and analogs and derivatives thereof). In one embodiment, the Fc-containing polypeptide produced by the claimed method has an N-glycan composition wherein at least 40 mole %, 70 mole %, or 90 mole % of the N-glycans on the Fc-containing polypeptide are sialylated (have a structure selected from SA(1-4)Gal(1-4)GlcNAc(2-4)Man3GlcNAc2 or SAGalGlcNAcMan5GlcNAc2). In one embodiment, less than 47 mole % of the N-glycans on the antibodies have the structure SA2Gal2GlcNAc2Man3GlcNAc2. In another embodiment, less than 47 mole % of the N-glycans on the antibodies have the structure NANA2Gal2GlcNAc2Man3GlcNAc2. In another embodiment, less than 66 mole % of the N-glycans on the antibodies have the structure SA2Gal2GlcNAc2Man3GlcNAc2. In another embodiment, less than 66 mole % of the N-glycans on the antibodies have the structure NANA2Gal2GlcNAc2Man3GlcNAc2. In one embodiment, the Fc-containing polypeptides produced by the claimed method comprise a mixture of α-2,3- and α-2,6-linked sialic acid. In another embodiment, the Fc-containing polypeptides comprise only α-2,6-linked sialic acid. In one embodiment, the Fc-containing polypeptides of the invention comprise α-2,6-linked sialic acid and do not comprise any detectable level of α-2,3-linked sialic acid. In one embodiment, the sialic acid is N-acetylneuraminic acid (NANA) or N-glycolylneuraminic acid (NGNA) or a mixture thereof. In another embodiment, the sialic acid is an analog or derivative of NANA or NGNA with acetylation at the 9-position on the sialic acid. In one embodiment, the N-glycans on the Fc-containing polypeptides produced by the claimed method comprise NANA and no NGNA.

[0022] The N-glycans on the Fc-containing polypeptides produced by the claimed method may optionally comprise fucose. In one embodiment, the N-glycans on the Fc-containing polypeptides produced by the claimed method comprise a mixture of fucosylated and non-fucosylated N-glycans. In one embodiment, the N-glycans on the Fc-containing polypeptides produced by the claimed method do not feature fucose.

[0023] In one embodiment, the Fc-containing polypeptide produced by the claimed method has an N-glycan composition wherein the amount and percentage of total sialylated N-glycans is greater relative to a parent Fc-containing polypeptide.

[0024] In some embodiments, the Fc-containing polypeptide produced by the claimed method exhibits one or more of the following properties when compared to a parent Fc-containing polypeptide: (i) decreased effector function; (ii) improved anti-inflammatory properties; (iii) improved sialylation; (iv) improved bioavailability (absorption or exposure), and (v) decreased binding to FCYRI, FcYRIIa, FcYRIIb, and FcYRIIIa. In one embodiment, the Fc-containing polypeptide of the invention exhibits at least a 7-, 10-, 15-, 30-, 50-, 100-, 500-, or 1,000-fold reduction in effector function relative to a parent Fc-containing polypeptide. In one embodiment, the effector function is ADCC. In another embodiment, the effector function is CDC.

[0025] In one embodiment, the Fc-containing polypeptide of the invention exhibits decreased ADCC activity when compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide exhibits at least a 7-, 10-, 15-, 30-, 50-, 100-, 500-, or 1,000-fold reduction in ADCC activity. In another embodiment, the Fc-containing polypeptide exhibits at least a 100-fold reduction in ADCC activity. In another embodiment, the Fc-containing polypeptide exhibits at least a 500-fold reduction in ADCC activity. In another embodiment, the Fc-containing polypeptide exhibits at least a 1,000-fold reduction in ADCC activity.

[0026] In another embodiment, the Fc-containing polypeptide produced by the claimed method exhibits reduced CDC activity when compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide exhibits at least a 100-fold reduction in CDC activity.

[0027] In one embodiment, the Fc-containing polypeptide produced by the claimed method has a 500-fold reduction in FCYRI, FcYRIIa, FcYRIIb, FcYRIIIa (FcYRIIIa-V158 or FcYRIIIa-F158), and FcYRIIIb with lower affinity when compared to an Fc-containing parent polypeptide. In one embodiment, the Fc-containing polypeptide of the invention binds to FcYRIIa with at least a 50-fold lower affinity when compared to an Fc-containing parent polypeptide. In one embodiment, the Fc-containing polypeptide of the invention binds to FcYRIIb with at least a 20-fold lower affinity when compared to an Fc-containing parent polypeptide. In one embodiment, the Fc-containing polypeptide of the invention binds to FcYRIIIa LF with at least a 10-fold lower affinity when compared to an Fc-containing parent polypeptide. In one embodiment, the Fc-containing polypeptide of the invention binds to FcYRIIIa LV with at least a 1-, 2-, or 10-fold lower affinity compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide of the invention binds to FcYRIIb, FcYRIIIa LF, and FcYRIIIa LV with a lower affinity compared to a parent Fc-containing polypeptide.

[0028] In one embodiment, the Fc-containing polypeptide produced by the claimed method exhibits improved anti-inflammatory properties relative to a parent Fc-containing polypeptide.

[0029] In one embodiment, the Fc-containing polypeptide produced by the claimed method exhibits improved bioavailability (absorption or exposure) when injected parenterally compared to an Fc-containing parent polypeptide. In one embodiment, the Fc-containing polypeptide produced by the claimed method exhibits improved bioavailability (absorption or exposure) when injected subcutaneously compared to an Fc-containing parent polypeptide.

[0030] In one embodiment, the Fc-containing parental polypeptide comprises a natural Fc region. In another embodiment, the Fc-containing parental polypeptide comprises an F243A mutation. In another embodiment, the Fc-containing parental polypeptide comprises a V264A mutation.

[0031] The invention also comprises a method of reducing the effector function of an Fc-containing polypeptide, comprising introducing mutations at positions 243 and 264 of a parent Fc-containing polypeptide, wherein said Fc-containing polypeptide has decreased effector function when compared to the parent Fc-containing polypeptide, wherein the numbering is according to the EU index as in Kabat. In one embodiment, the Fc-containing polypeptide comprises the mutations F243A and V264A. In another embodiment, the nucleic acid encodes the mutations F243Y and V264G. In another embodiment, the nucleic acid encodes the mutations 243T and V264G. In another embodiment, the nucleic acid encodes the mutations F243L and V264A. In another embodiment, the nucleic acid encodes the mutations F243L and V264N. In another embodiment, the nucleic acid encodes the mutations F243V and V264G. In one embodiment, the effector function is ADCC. In another embodiment, the effector function is CDC. In one embodiment, the Fc-containing polypeptide of the invention is an antibody or an antibody fragment. In one embodiment, the Fc-containing polypeptide is an antibody fragment comprising SEQ ID NO:18. In another embodiment, the Fc-containing polypeptide is an antibody fragment comprising SEQ ID NO:19. In another embodiment, the Fc-containing polypeptide is an antibody fragment consisting of (or consisting essentially of) SEQ ID NO:18 or SEQ ID NO:19. In one embodiment, the Fc-containing parent polypeptide comprises a natural Fc region. In another embodiment, the Fc-containing parent polypeptide comprises an F243A mutation. In another embodiment, the Fc-containing parent polypeptide comprises a V264A mutation.

[0032] The invention also comprises a method of increasing the anti-inflammatory properties of an Fc-containing polypeptide, comprising introducing mutations at positions 243 and 264 of a parent Fc-containing polypeptide, wherein the numbering is in accordance with the EU index as in Kabat, wherein said Fc-containing polypeptide exhibits increased anti-inflammatory activity when compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide comprises mutations F243A and V264A. In another embodiment, the Fc-containing polypeptide comprises mutations F243Y and V264G. In another embodiment, the Fc-containing polypeptide comprises mutations 243T and V264G. In another embodiment, the Fc-containing polypeptide comprises mutations F243L and V264A. In another embodiment, the Fc-containing polypeptide comprises mutations F243L and V264N. In another embodiment, the Fc-containing polypeptide comprises the mutations F243V and V264G. In one embodiment, the Fc-containing polypeptide of the invention is an antibody or an antibody fragment. In one embodiment, the Fc-containing polypeptide is an antibody or antigen-binding fragment thereof that binds to an antigen selected from the group consisting of: IL-25, IL-27, IL-33, CD2, CD4, CD11A, CD14, CD18, CD19, CD23, CD25, CD40, CD40L, CD20, CD52, CD64, CD80, CD147, CD200, CD200R, TSLP, TSLPR, PD-1, PDL1, CTLA4, VLA-4, VEGF, PCSK9, α4β7-integrin, E-selectin, Fact II, ICAM-3, beta2-integrin, IFNγ, C5, CBL, LCAT, CR3, MDL-1, GITR, ADDL, CGRP, TRKA, IGF1R, RANKL, GTC, or the receptor for any of the aforementioned molecules. In one embodiment, the Fc-containing polypeptide will bind to TNF-α. In another embodiment, the Fc-containing polypeptide will bind to Her2. In another embodiment, the Fc-containing polypeptide will bind to PCSK9. In one embodiment, the Fc-containing polypeptide of the invention is an antibody fragment comprising SEQ ID NO:18. In another embodiment, the Fc-containing polypeptide of the invention is an antibody fragment comprising SEQ ID NO:19. In another embodiment, the Fc-containing polypeptide of the invention is an antibody fragment consisting of (or consisting essentially of) SEQ ID NO:18 or SEQ ID NO:19. In one embodiment, the Fc-containing parent polypeptide comprises a naturally occurring Fc region. In another embodiment, the Fc-containing parent polypeptide comprises an F243A mutation. In another embodiment, the Fc-containing parent polypeptide comprises a V264A mutation.

[0033] The invention also comprises a method of increasing the anti-inflammatory properties of an Fc-containing polypeptide, comprising: selecting a parent Fc-containing polypeptide that is used to treat inflammation (e.g., an antibody or immunoadhesin that binds to an antigen that is involved in inflammation), and introducing mutations at positions 243 and 264 of the Fc region, wherein the numbering is according to the EU index as in Kabat, wherein the Fc-containing polypeptide exhibits greater anti-inflammatory activity when compared to the parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide comprises mutations F243A and V264A. In another embodiment, the Fc-containing polypeptide comprises mutations F243Y and V264G. In another embodiment, the Fc-containing polypeptide comprises mutations 243T and V264G. In another embodiment, the Fc-containing polypeptide comprises mutations F243L and V264A. In another embodiment, the Fc-containing polypeptide comprises mutations F243L and V264N. In another embodiment, the Fc-containing polypeptide comprises mutations F243V and V264G. In one embodiment, the Fc-containing polypeptide of the invention is an antibody or an antibody fragment. In one embodiment, the Fc-containing polypeptide is an antibody or antigen-binding fragment thereof that binds to an antigen selected from the group consisting of: IL-25, IL-27, IL-33, CD2, CD4, CD11A, CD14, CD18, CD19, CD23, CD25, CD40, CD40L, CD20, CD52, CD64, CD80, CD147, CD200, CD200R, TSLP, TSLPR, PD-1, PDL1, CTLA4, VLA-4, VEGF, PCSK9, α4β7-integrin, E-selectin, Fact II, ICAM-3, beta2-integrin, IFNγ, C5, CBL, LCAT, CR3, MDL-1, GITR, ADDL, CGRP, TRKA, IGF1R, RANKL, GTC, or the receptor for any of the aforementioned molecules. In one embodiment, the Fc-containing polypeptide will bind to TNF-α. In another embodiment, the Fc-containing polypeptide will bind to Her2. In another embodiment, the Fc-containing polypeptide will bind to PCSK9. In one embodiment, the Fc-containing polypeptide is an antibody fragment comprising SEQ ID NO:18. In another embodiment, the Fc-containing polypeptide is an antibody fragment comprising SEQ ID NO:19. In another embodiment, the Fc-containing polypeptide is an antibody fragment consisting of (or consisting essentially of) SEQ ID NO:18 or SEQ ID NO:19. In one embodiment, the Fc-containing parental polypeptide comprises a natural Fc region. In another embodiment, the Fc-containing parental polypeptide comprises an F243A mutation. In another embodiment, the Fc-containing parental polypeptide comprises a V264A mutation.

[0034] The invention also comprises a method of treating an inflammatory condition, in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of an Fc-containing polypeptide, comprising mutations at positions 243 and 264, wherein the numbering is in accordance with the EU index as in Kabat. In one embodiment, the Fc-containing polypeptide decreases the expression of a gene selected from the group consisting of: IL-1β, IL-6, RANKL, TRAP, ATP6v0d2, MDL-1, DAP12, CD11b, TIMP-1, MMP9, CTSK, PU-1, MCP1, MIPlα, Cxcl1-Groa, CXcl2-Grob, CD18, TNF, FCYRI, FcYRIIb, FCYRIII, and FCYRIV. In one embodiment, the Fc-containing polypeptide comprises mutations F243A and V264A. In another embodiment, the nucleic acid encodes the F243Y and V264G mutations. In another embodiment, the nucleic acid encodes the 243T and V264G mutations. In another embodiment, the nucleic acid encodes the F243L and V264A mutations. In another embodiment, the nucleic acid encodes the F243L and V264N mutations. In another embodiment, the nucleic acid encodes the F243V and V264G mutations. In one embodiment, the Fc-containing polypeptide is administered parenterally. In one embodiment, the Fc-containing polypeptide is administered subcutaneously. In one embodiment, the Fc-containing polypeptide is an antibody or antigen-binding fragment thereof. In one embodiment, the Fc-containing polypeptide is an antibody or antigen-binding fragment thereof that is used to treat an inflammatory condition. In one embodiment, the antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of: TNF-α, IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12, IL-15, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-23R, IL-25, IL-27, IL-33, CD2, CD4, CD11A, CD14, CD18, CD19, CD23, CD25, CD40, CD40L, CD20, CD52, CD64, CD80, CD147, CD200, CD200R, TSLP, TSLPR, PD-1, PDL1, CTLA4, VLA-4, VEGF, PCSK9, α4β7-integrin, E-selectin, Fact II, ICAM-3, beta2-integrin, IFNY, C5, CBL, LCAT, CR3, MDL-1, GITR, ADDL, CGRP, TRKA, IGF1R, RANKL, GTC, or the receptor for any of the aforementioned molecules. In one embodiment, the Fc-containing polypeptide will bind to TNF-α. In another embodiment, the Fc-containing polypeptide will bind to Her2. In another embodiment, the Fc-containing polypeptide will bind to PCSK9. In one embodiment, the Fc-containing polypeptide is an antibody fragment comprising SEQ ID NO:18. In another embodiment, the Fc-containing polypeptide is an antibody fragment comprising SEQ ID NO:19. In another embodiment, the Fc-containing polypeptide is an antibody fragment consisting of (or consisting essentially of) SEQ ID NO:18 or SEQ ID NO:19.

[0035] Another invention disclosed herein relates to a pharmaceutical composition comprising an Fc-containing polypeptide, wherein at least 70% of the N-glycans on the Fc-containing polypeptide comprise an oligosaccharide structure selected from the group consisting of SA(1-4)Gal(1-4)GlcNAc(2-4)Man3GlcNAc2 and SAGalGlcNAcMan5GlcNAc2, wherein the Fc-containing polypeptide comprises mutations at amino acid positions 243 and 264 of the Fc region, wherein the numbering is according to the EU index as in Kabat. In one embodiment, the mutations are F243A and V264A. In another embodiment, the mutations are F243Y and V264G. In another embodiment, the nucleic acid encodes mutations 243T and V264G. In another embodiment, the mutations are F243L and V264A. In another embodiment, the mutations are F243L and V264N. In another embodiment, the mutations are F243V and V264G. In one embodiment, at least 47 mole % of the N-glycans have the structure SA2Gal2GlcNAc2Man3GlcNAc2. In another embodiment, at least 47 mole % of the N-glycans have the structure NANA2Gal2GlcNAc2Man3GlcNAc2. In one embodiment, the sialylated N-glycans comprise a mixture of α-2,3- and α-2,6-linked sialic acid. In another embodiment, the sialylated N-glycans comprise only α-2,6-linked sialic acid. In another embodiment, the sialylated N-glycans comprise α-2,6-linked sialic acid and do not comprise any detectable level of α-2,3-linked sialic acid. In one embodiment, the sialic acid is N-acetylneuraminic acid (NANA) or N-glycolylneuraminic acid (NGNA) or a mixture thereof. In another embodiment, the sialic acid is an analog or derivative of NANA or NGNA with acetylation at the 9-position on the sialic acid. In one embodiment, the N-glycans on the Fc-containing polypeptides comprise NANA and no NGNA. In one embodiment, the Fc-containing polypeptide is an antibody fragment comprising SEQ ID NO:18. In another embodiment, the Fc-containing polypeptide is an antibody fragment comprising SEQ ID NO:19. In another embodiment, the Fc-containing polypeptide is an antibody fragment consisting of (or consisting essentially of) SEQ ID NO:18 or SEQ ID NO:19.

[0036] Another invention disclosed herein relates to a pharmaceutical composition comprising an Fc-containing polypeptide, wherein at least 70% of the N-glycans on the Fc-containing polypeptide comprise an oligosaccharide structure selected from the group consisting of SA(1-4)Gal(1-4)GlcNAc(2-4)Man3GlcNAc2 and SAGalGlcNAcMan5GlcNAc2, wherein the sialic acid residues are attached exclusively via an α-2,6 linkage, wherein the N-glycans do not feature fucose, and wherein the Fc-containing polypeptide comprises mutations at amino acid positions 243 and 264 of the Fc region, wherein the numbering is according to the EU index as in Kabat. In one embodiment, the mutations are F243A and V264A. In another embodiment, the mutations are F243Y and V264G. In another embodiment, the nucleic acid encodes mutations 243T and V264G. In another embodiment, the mutations are F243L and V264A. In another embodiment, the mutations are F243L and V264N. In another embodiment, the mutations are F243V and V264G. In one embodiment, at least 47 mole % of the N-glycans have the structure SA2Gal2GlcNAc2Man3GlcNAc2. In another embodiment, at least 47 mole % of the N-glycans have the structure NANA2Gal2GlcNAc2Man3GlcNAc2. In one embodiment, the sialylated N-glycans comprise a mixture of α-2,3- and α-2,6-linked sialic acid. In another embodiment, the sialylated N-glycans comprise only α-2,6-linked sialic acid. In another embodiment, the sialylated N-glycans comprise α-2,6-linked sialic acid and comprise no detectable level of α-2,3-linked sialic acid. In one embodiment, the sialic acid is N-acetylneuraminic acid (NANA) or N-glycolylneuraminic acid (NGNA) or a mixture thereof. In another embodiment, the sialic acid is an analog or derivative of NANA or NGNA with acetylation at the 9-position on the sialic acid. In one embodiment, the N-glycans on the Fc-containing polypeptides comprise NANA and no NGNA. In one embodiment, the Fc-containing polypeptide is an antibody fragment comprising SEQ ID NO:18. In another embodiment, the Fc-containing polypeptide is an antibody fragment comprising SEQ ID NO:19. In another embodiment, the Fc-containing polypeptide is an antibody fragment consisting of (or consisting essentially of) SEQ ID NO:18 or SEQ ID NO:19.

[0037] The invention also comprises an Fc-containing polypeptide comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:9 or a variant thereof, and the light chain comprises the amino acid sequence of SEQ ID NO:2 or a variant thereof, wherein the variant comprises one or more of the following properties when compared to an antibody comprising the heavy chain amino acid sequence of SEQ ID NO:1 and the light chain amino acid sequence of SEQ ID NO:2: lower effector function; improved anti-inflammatory properties; improved sialylation; improved bioavailability (absorption or exposure) when administered parenterally; and decreased binding to FCYRI, FcYRIIa, FcYRIIb, and FcYRIIIa. The invention also comprises an Fc-containing polypeptide comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:12 or a variant thereof, and the light chain comprises the amino acid sequence of SEQ ID NO:11 or a variant thereof, wherein the variant comprises one or more of the following properties when compared to an antibody comprising the heavy chain amino acid sequence of SEQ ID NO:10 and the light chain amino acid sequence of SEQ ID NO:11: lower effector function, improved anti-inflammatory properties, improved sialylation, improved bioavailability (absorption or exposure) when administered parenterally, and decreased binding to FcYRI, FcYRIIa, FcYRIIb, FcYRIIIa, and FcYRIIIb. The invention also comprises an Fc-containing polypeptide comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:15 or a variant thereof, and the light chain comprises the amino acid sequence of SEQ ID NO:14 or a variant thereof, wherein the variant comprises one or more of the following properties when compared to an antibody comprising the heavy chain amino acid sequence of SEQ ID NO:13 and the light chain amino acid sequence of SEQ ID NO:14: decreased effector function, improved anti-inflammatory properties, improved sialylation, improved bioavailability (absorption or exposure) when administered parenterally, and decreased binding to FcYRI, FcYRIIa, FcYRIIb, FcYRIIIa, and FcYRIIIb. In one embodiment, the variant comprises up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative or non-conservative amino acid substitutions. In one embodiment, the variant comprises at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the claimed sequence.

[0038] The invention also comprises a method of increasing the anti-inflammatory properties of an Fc-containing polypeptide, comprising introducing a mutation at position 243 or a mutation at position 264 of a parent Fc-containing polypeptide, wherein the numbering is according to the EU index as in Kabat, wherein said Fc-containing polypeptide presents improved anti-inflammatory function when compared to the parent Fc-containing polypeptide. The invention also comprises a method of increasing the anti-inflammatory properties of an Fc-containing polypeptide, which comprises: selecting a parent Fc-containing polypeptide that is used to treat inflammation (e.g., an antibody or immunoadhesin that binds to an antigen that is involved in inflammation), and introducing a mutation at position 243 or a mutation at position 264 of a parent Fc-containing polypeptide, wherein the numbering is according to the EU index as in Kabat, wherein the Fc-containing polypeptide presents improved anti-inflammatory properties when compared to the parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide comprises the F243A mutation. In another embodiment, the Fc-containing polypeptide comprises the V264A mutation. In one embodiment, the parental Fc-containing polypeptide comprises a natural Fc region.

[0039] The invention also comprises a method of treating an inflammatory condition, in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of a polypeptide containing Fc a mutation at position 243 or a mutation at position 264 of a parent polypeptide containing Fc, wherein the numbering is in accordance with the EU index as in Kabat. In one embodiment, the Fc-containing polypeptide is administered parenterally. In one embodiment, the Fc-containing polypeptide is administered subcutaneously. In one embodiment, the Fc-containing polypeptide comprises the F243A mutation. In another embodiment, the Fc-containing polypeptide comprises the V264A mutation. In one embodiment, the parent polypeptide containing Fc comprises a natural Fc region.

[0040] In any of the preceding embodiments, an increase in anti-inflammatory activity can be detected using any method known in the art. In one embodiment, an increase in anti-inflammatory activity is detected by measuring a decrease in the expression of a gene selected from the group consisting of: IL-1β, IL-6, RANKL, TRAP, ATP6v0d2, MDL-1, DAP12, CD11b, TIMP-1, MMP9, CTSK, PU-1, MCP1, MIP1α, Cxcl1-Groa, CXcl2-Grob, CD18, TNF, FCYRI, FeyRIIb, FCYRIII, and FCYRIV.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a graphical representation of pGLY3483, the F243A and V264A dual mutein expression plasmid. Both the heavy and light chains were under the control of a methanol-inducible promoter, AOX1. The PpTrp2 gene was the locus applied to integrate the entire cassette. With the exception of the mutations in the heavy chain, the structure of the expression plasmid was the same as that of the wild-type (parental), F243A single mutein, V264A single mutein, and double mutein expression plasmids.

[0042] Figure 2 is a representation of gels from an SDS-PAGE analysis characterizing non-reduced (NR) and reduced (R) antibodies produced by the materials and methods herein. Lane 1 contains an anti-Her2 monoclonal antibody, Her2; Lane 2 contains a single Fc mutein, F243A; lane 3 contains a single Fc mutein, V264A; and lane 4 contains a dual Fc mutein, F243A/V264A.

[0043] Figure 3 illustrates the affinity of antigen for various antibodies produced by the materials and methods herein, determined by a cell-based assay using a SK-BR3 cell line, a human breast cancer cell line overexpressing Her2. ■ - Her2; ♦ - F243A/V264A GS6.0 glycosylation; ▲ - F243A with GS6.0 glycosylation; ▼ - V264A with GS6.0 glycosylation; ◊ - control IgG; X - Pichia pastoris Her2 produced with GFI 5.0 glycosylation.

[0044] Figure 4 illustrates a MALDI-TOF MS analysis of N-glycans of a Pichia pastoris Her2 antibody produced in strain GFI 5.0 YDX477. The peaks are Man5GlcNAc2, 1261.24 (GS2.0), GlcNAc2Man3GlcNAc2, 1343.50 (G0) (predominant), GalGlcNAc2Man3GlcNAc2, 1505.97 (G1), and Gal2GlcNAc2Man3GlcNAc2, 1668.47 (G2). Figure 5 illustrates a MALDI-TOF MS analysis of N-glycans of a single Fc mutein, F243A, antibody produced in strain GFI 5.0 YDX551. The peaks are Man5GlcNAc2, 1261.05, GlcNAc2Man3GlcNAc2, 1343.71, GalGlcNAc2Man3GlcNAc2, 1506.62, and Gal2GlcNAc2Man3GlcNAc2, 1668.97 (predominant).

[0045] Figure 6 illustrates a MALDI-TOF MS analysis of N-glycans of a single Fc mutein, V264A, antibody produced in GFI 5.0 strain YDX551. The peaks are Man5GlcNAc2, 1261.98, GalGlcNAc2Man3GlcNAc2, 1505.45, and Gal2GlcNAc2Man3GlcNAc2, 1668.85 (predominant).

[0046] Figure 7 illustrates a MALDI-TOF MS analysis of N-glycans of a dual Fc mutein, F243A/V264A, antibody produced in the GFI 5.0 YDX557 strain. The major peak corresponds to Gal2GlcNAc2Man3GlcNAc2, 1668.39.

[0047] Figure 8 illustrates a MALDI-TOF MS analysis of N-glycans of a dual Fc mutein, F243A/V264A, antibody produced in GFI 6.0 strain YGLY4563. The double peaks at 2224.28 and 2245.83 (predominant) correspond to NANAGal2GlcNAc2Man3GlcNAc2 and NANA2Gal2GlcNAc2Man3GlcNAc2, respectively.

[0048] Figures 9A-9D are graphical representations of the binding of FCYR to various antibodies produced by the materials and methods described in Example 11: FcYRIIIaLF (Figure 9A); FCYRI (Figure 9B); FcYRIIb/c (Figure 9C); and FcYRIIIaLV (Figure 9D). For Figures 9A-9D: ■ - Her2; ▲ - F243A produced in GFI 6.0; ▼ - V264A produced in GFI 6.0; ♦ - F243A/V264A produced in GFI 6.0; • - F243A/V264A produced in GFI 6.0 and treated with PNGase.

[0049] Figure 10 is a graphical representation of C1q binding with various antibodies produced by the materials and methods described in Example 12: ◊ - anti-CD20 antibody, a positive control; ■ - Her2; ▲ - F243A produced with GS6.0 glycosylation; ▼- V264A produced with GS6.0 glycosylation; ♦ - F243A/V264A produced with GS6.0 glycosylation; • - F243A/V264A with GS6.0 glycosylation and PNGase.

[0050] Figure 11 is a graphical representation of the ADCC response to the various antibodies produced by the materials and methods described in Example 13: ■ - Her2; ▲ - F243A produced with GS6.0 glycosylation; ▼ - V264A produced with GS6.0 glycosylation; ♦ - F243A/V264A produced with GS6.0 glycosylation; ◊ - NK killer cell without antibody; X - Her2 produced in GFI2.0.

[0051] Figure 12 is a graphical representation of serum monoclonal antibody concentration over time for mice injected with: ■ - Her2; X - Her2 produced in GFI 5.0; ♦ - F243A/V264A produced in GFI 6.0, in the manner described in Example 14.

[0052] Figures 13A-13E are graphical representations of the binding of FCYR to various antibodies described in Example 15.

[0053] Figure 14 is a graphical representation of the ADCC response to various antibodies produced by the materials and methods described in Example 16. The results in Figures 14A and 14B were from experiments using F/V heterozygous effector cells. The results in Figures 14C and 14D were from experiments using F/F effector cells. The results in Figure 14E were from an experiment using V/V effector cells.

[0054] Figure 15 is a graphical representation of the ADCC activity of the anti-Her2 Fc double mutant compared to the assumed additional reference curve of each of the single mutants, as described in Example 17.

[0055] Figure 16 is a graphical representation of the ADCC response to various antibodies produced by the materials and methods described in Example 18.

[0056] Figure 17 is a graphical representation of the CDC response to various antibodies produced by the materials and methods described in Example 19.

[0057] Figure 18 is a graphical representation of the effect of the Fc muteins of the invention on an AIA model described in Example 20.

[0058] Figure 19 is a graphical representation of the effect of the Fc muteins of the invention on an AIA model described in Example 21.

[0059] Figure 20 is a graphical representation of the binding of FCYR to the anti-TNFα antibodies described in Example 22.

[0060] Figure 21 is a graphical representation of the binding of FCYR to the anti-TNFα antibodies described in Example 23.

[0061] Figure 22 is a graphical representation of the effect of the Fc muteins of the invention on an AIA model described in Example 24.

DETAILED DESCRIPTION OF THE INVENTION Definitions

[0062] The term “G0” when used herein refers to a complex bi-antennated oligosaccharide lacking galactose or fucose, GlcNAc2Man3GlcNAc2.

[0063] The term “G1” when used herein refers to a complex bi-antennated oligosaccharide lacking fucose and containing a galactosyl residue, GalGlcNAc2Man3GlcNAc2.

[0064] The term “G2” when used herein refers to a complex bi-antennated oligosaccharide lacking fucose and containing two galactosyl residues, Gal2GlcNAc2Man3GlcNAc2.

[0065] The term “G0F” when used herein refers to a complex bi-antennated oligosaccharide containing a fucose core and no galactose, GlcNAc2Man3GlcNAc2F.

[0066] The term “G1F” when used herein refers to a complex bi-antennated oligosaccharide containing a fucose core and a galactosyl residue, GalGlcNAc2Man3GlcNAc2F.

[0067] The term “G2F” when used herein refers to a complex bi-antennated oligosaccharide containing a fucose core and two galactosyl residues, Gal2GlcNAc2Man3GlcNAc2F.

[0068] The term “Man5” when used herein refers to the oligosaccharide structure shown as:

[0069] The term “GFI 5.0” when used herein refers to glycoengineered Pichia pastoris strains that produce glycoproteins predominantly with Gal2GlcNAc2Man3GlcNAc2 N-glycans.

[0070] The term “GFI 6.0” when used herein refers to glycoengineered Pichia pastoris strains that produce glycoproteins predominantly with NANA2Gal2GlcNAc2Man3GlcNAc2 N-glycans.

[0071] The term “GS5.0” when used herein refers to the N-glycosylation structure of Gal2GlcNAc2Man3GlcNAc2.

[0072] The term “GS5.5” when used herein refers to the N-glycosylation structure of NANAGal2GlcNAc2Man3GlcNAc2, which when produced in Pichia pastoris strains in which α2,6 sialyl transferase has been glycoengineered, results in α2,6-linked sialic acid, and which when produced in Pichia pastoris strains in which α2,3 sialyl transferase has been glycoengineered, results in α2,3-linked sialic acid.

[0073] The term “GS6.0” when used herein refers to the N-glycosylation structure of NANA2Gal2GlcNAc2Man3GlcNAc2, which when produced in strains of Pichia pastoris in which α2,6 sialyl transferase has been glycoengineered, results in α2,6-linked sialic acid, and which when produced in strains of Pichia pastoris in which α2,3 sialyl transferase has been glycoengineered, results in α2,3-linked sialic acid.

[0074] The term “wild type” or “wt”, when used herein in conjunction with a strain of Pichia pastoris, refers to a naturally occurring strain of Pichia pastoris that has not undergone genetic modification to control glycosylation.

[0075] The term “antibody” when used herein refers to an immunoglobulin molecule capable of binding to a specific antigen through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term includes not only intact polyclonal or monoclonal antibodies, which consist of four polypeptide chains, i.e., two identical pairs of polypeptide chains, each pair having a "light" (LC) chain (about 25 kDa) and a "heavy" (HC) chain (about 50-70 kDa), but also fragments thereof, such as Fab, Fab', F(ab')2, Fv, single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site and at least the CH2 domain portion of the heavy chain immunoglobulin constant region, comprising an N-linked glycosylation site of the CH2 domain or a variant thereof. As used herein, the term includes an antibody of any class, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgM, IgA, IgD, and IgE, respectively.

[0076] The term “CH2 consensus sequence” when used herein refers to the amino acid sequence of the CH2 domain of the heavy chain constant region containing an N-linked glycosylation site, which was derived from the most common amino acid sequences observed in the CH2 domains of a variety of antibodies.

[0077] The term “Fc region” is used to define a C-terminal region of an immunoglobulin heavy chain. The “Fc region” may be a naturally occurring sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of a human IgG heavy chain is generally defined to extend from an amino acid residue at position Cys226, or from Pro230, to the carboxyl terminus thereof. The Fc region of an immunoglobulin comprises two constant domains, CH2 and CH3, and may optionally comprise a hinge region. In one embodiment, the Fc region comprises the amino acid sequence of SEQ ID NO:18. In one embodiment, the Fc region comprises the amino acid sequence of SEQ ID NO:19. In another embodiment, the Fc region comprises the amino acid sequence of SEQ ID NO:18, with the addition of a lysine (K) residue at the 5’ end. The Fc region contains a unique N-linked glycosylation site in the Ch2 domain, which corresponds to the Asn297 site of a full-length heavy chain of an antibody.

[0078] The term “Fc-containing polypeptide” refers to a polypeptide, such as an antibody or immunoadhesin, that comprises an Fc region. This term includes polypeptides comprising or consisting of (or consisting essentially of) an Fc region. Polypeptides comprising an Fc region can be generated by papain digestion of antibodies or by recombinant DNA technology.

[0079] The term “parental antibody,” “parental immunoglobulin,” or “parental Fc-containing polypeptide,” when used herein, refers to an antibody or Fc-containing polypeptide that lacks the Fc region mutations disclosed herein. A parental Fc-containing polypeptide may comprise a naturally occurring Fc region or an Fc region with a pre-existing sequence of amino acid modifications. A naturally occurring Fc region comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Natural sequence Fc regions include the natural sequence human IgG1 Fc region, the natural sequence human IgG2 Fc region, the natural sequence human IgG3 Fc region, and the natural sequence human IgG4 Fc region, as well as naturally occurring variants thereof. When used as a comparator, a parental antibody or a parental Fc-containing polypeptide may be expressed in any cell. In one embodiment, the parental antibody or a parental Fc-containing polypeptide is expressed in the same cell as the Fc-containing polypeptide of the invention.

[0080] As used herein, the term “immunoadhesin” refers to antibody-like molecules that combine the “binding domain” of a heterologous “adhesin” protein (e.g., a receptor, ligand, or enzyme) with an immunoglobulin constant domain. Structurally, immunoadhesins comprise a fusion of the adhesin amino acid sequence with the desired binding specificity, which is other than the antigen recognition and binding site (antigen combining site) of an antibody (i.e., is “heterologous”), and an immunoglobulin constant domain sequence. The term “ligand binding domain” as used herein refers to any natural cell surface receptor, or any region or derivative thereof, that retains at least a qualitative ligand binding capability of a corresponding natural receptor. In a specific embodiment, the receptor is a cell surface polypeptide with an extracellular domain that is homologous to a member of the immunoglobulin supergene family. Other receptors, which are not members of the immunoglobulin superfamily but are nevertheless specifically covered by this definition, are receptors for cytokines and, in particular, receptors with tyrosine kinase activity (tyrosine kinase receptors), elements of hematopoietin and nerve growth factor that predispose the mammal to the disorder in question. In one embodiment, the disorder is cancer. Methods of preparing immunoadhesins are well known in the art. See, for example, WO00/42072.

[0081] The term “Her2” or “Her2 antibody” when used herein refers to an antibody with an amino acid sequence similar to that for a commercially available Her2 antibody produced in mammalian cells, i.e. CHO cells, known as trastuzumab.

[0082] The term “Pichia Her2 antibody” or “Pichia anti-Her2 antibody” when used herein refers to an antibody with an amino acid sequence similar to that of a commercially available Her2 antibody (trastuzumab) produced in glycoengineered Pichia pastoris.

[0083] The term “Fc mutein antibody” when used herein refers to an antibody comprising one of the single Fc muteins or the double Fc mutein described herein.

[0084] The term “Fc mutein” when used herein refers to an Fc-containing polypeptide in which one or more point mutations have been made in the Fc region.

[0085] The term “Fc mutation” when used herein refers to a mutation made in the Fc region of an Fc-containing polypeptide. Examples of such a mutation include the F243A or V264A mutations described herein.

[0086] The term “single Fc mutein” when used herein refers to an Fc-containing polypeptide that incorporates a mutation at position 243 or 264 of the Fc region. The term “F243A” refers to a mutation from F (wild-type) to A at position 243 of the Fc region of an Fc-containing polypeptide. The term “V264A” refers to a mutation from V (wild-type) to A at position 264 of the Fc region of an Fc-containing polypeptide. Positions 243 and 264 represent amino acid positions in the CH2 domain of the Fc region of an Fc-containing polypeptide.

[0087] The term “dual Fc mutein” when used herein refers to an Fc-containing polypeptide comprising mutations at positions 243 and 264 of the Fc region. The term “F243A/V264A” refers to a double Fc mutein comprising the two specified mutations.

[0088] Throughout this specification and claims, the numbering of residues in an immunoglobulin heavy chain, or an Fc-containing polypeptide, is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), expressly incorporated herein by reference. The “EU index as in Kabat” refers to the residue numbering of the human IgG1 antibody EU.

[0089] The term “effector function” as used herein refers to a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Exemplary “effector functions” include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions can be assessed using various assays known in the art.

[0090] The term “glycoengineered Pichia pastoris” when used herein refers to a strain of Pichia pastoris that has been genetically altered to express human-type N-glycans. For example, the strains GFI 5.0, GFI 5.5, and GFI 6.0 described above.

[0091] The terms “N-glycan”, “glycoprotein”, and “glycoform”, when used herein, refer to an N-linked oligosaccharide, e.g., one that is attached by an asparagine-N-acetylglucosamine bond to an asparagine residue of a polypeptide. The predominant sugars found in glycoproteins are glucose, galactose, mannose, fucose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and sialic acid (SA, including NANA, NGNA, and derivatives and analogues thereof, including acetylated NANA or acetylated NGNA). In glycoengineered Pichia pastoris, the sialic acid is exclusively N-acetyl-neuraminic acid (NANA) (Hamilton et al., Science 313 (5792): 1441-1443 (2006)). The N-glycans have a common pentasaccharide core of Man3GlcNAc2, where “Man” refers to mannose, “Glc” refers to glucose, “NAc” refers to N-acetyl, and GlcNAc refers to N-acetylglucosamine. N-glycans differ with respect to the number of branches (antennas) comprising peripheral sugars (e.g., GlcNAc, galactose, fucose, and sialic acid), which are added to the core structure of Man3GlcNAc2 (“Man3”), which is also referred to as the “trimannose core”, the “pentasaccharide core”, or the “paucimannose core”. N-glycans are classified according to their branching constituents (e.g., high mannose, complex, or hybrid).

[0092] As used herein, the term “sialic acid” or “SA” refers to any member of the sialic acid family, including without limitation: N-acetylneuraminic acid (Neu5Ac or NANA), N-glycolylneuraminic acid (NGNA), and any analogue or derivative thereof (including those arising from acetylation at any position in the sialic acid molecule). Sialic acid is a generic name for a group of about 30 naturally occurring acidic carbohydrates that are essential components of a large number of glycoconjugates. Schauer, Biochem. Society Transactions, 11, 270-271 (1983). Sialic acids are generally the terminal residue of oligosaccharides. N-acetylneuraminic acid (NANA) is the most common form of sialic acid, and N-glycolylneuraminic acid (NGNA) is the second most common form. Schauer, Glycobiology, 1, 449-452 (1991). NGNA is dispersed throughout the animal kingdom and, depending on the species and tissue, often constitutes a significant proportion of the glycoconjugate-bound sialic acid. Certain species such as chicken and man are exceptions, since they do not have NGNA in normal tissues. Corfield, et al., Cell Biology Monographs, 10, 5-50 (1982). In human serum samples, the percentage of sialic acid in the form of NGNA is reported to be 0.01% of the total sialic acid. Schauer, "Sialic Acids as Antigenic Determinants of Complex Carbohydrates", found in The Molecular Immunology of Complex Carbohydrates, (Plenum Press, New York, 1988).

[0093] The term “human-type N-glycan,” as used herein, refers to N-linked oligosaccharides that closely resemble oligosaccharides produced by non-genetically modified human felba-like cells. For example, wild-type Pichia pastoris and other lower eukaryotic cells typically produce proteins hypermannosylated at N-glycosylation sites. The host cells described herein produce glycoproteins (e.g., antibodies) comprising human-type N-glycans that are not hypermannosylated. In some embodiments, the host cells of the present invention are capable of producing human-type N-glycans with hybrid and/or complex N-glycans. The specific type of “human-type” glycans present on a particular glycoprotein produced from a host cell of the invention will depend on the specific glycoengineering steps that are performed in the host cell.

[0094] The term “high mannose” N-glycan when used herein refers to an N-glycan having five or more mannose residues.

[0095] The term “complex” N-glycan when used herein refers to an N-glycan with at least one GlcNAc attached to the 1,3-mannose arm, and at least one GlcNAc attached to the 1,6-mannose arm of a “trimannose” core. Complex N-glycans may also feature galactose (“Gal”) or N-acetylgalactosamine (“GalNAc”) residues, which are optionally modified with sialic acid or derivatives (e.g., “NANA” or “NeuAc,” where “Neu” refers to neuraminic acid and “Ac” refers to acetyl). Complex N-glycans may also feature interchain substitutions comprising “biantenated” GlcNAc and a fucose (“Fuc”) core. As an example, when an N-glycan comprises a biantennated GlcNAc on the trimannose core, the structure can be represented as Man3GlcNAc2(GlcNAc) or Man3GlcNAc3. When an N-glycan comprises a fucose core attached to the trimannose core, the structure can be represented as Man3GlcNAc2(Fuc). Complex N-glycans can also exhibit multiple antennae on the “trimannose core,” often referred to as “multiantennae glycans.”

[0096] The term “hybrid” N-glycan when used herein refers to an N-glycan with at least one GlcNAc at the terminus of the 1,3-mannose arm of the trimannose core, and zero or more than one mannose on the 1,6-mannose arm of the trimannose core.

[0097] When referring to the “mole percent” of a glycan present in a glycoprotein preparation, the term means the mole percent of a particular glycan present in the N-linked oligosaccharide cluster released when the protein preparation is treated with PNGase, and then quantified by a method that is not affected by glycoform composition, (e.g., labeling a PNGase-released glycan cluster with a fluorescent label, such as 2-aminobenzamide, and then separating by high-performance liquid chromatography or capillary electrophoresis, and then quantifying the glycans by fluorescence intensity). For example, 50 mole percent NANA2 Gal2GlcNAc2Man3GlcNAc2 means that 50 percent of the released glycans are NANA2 Gal2GlcNAc2Man3GlcNAc2, and the remaining 50 percent are comprised of other N-linked oligosaccharides.

[0098] The term “anti-inflammatory antibody,” as used herein, refers to an antibody intended for use to treat inflammation. The anti-inflammatory properties of an Fc-containing polypeptide can be measured using any method known in the art. In one embodiment, the anti-inflammatory properties of an Fc-containing polypeptide are measured using an animal model, such as the models described in Kaneko et al., Science 313:670-673 (2006), Anthony et al., Science 320:373-376 (2008), and Examples 20-21 herein. In another embodiment, the anti-inflammatory properties of an Fc-containing polypeptide are measured by determining the level of an inflammation-related biomarker (including without limitation: CRP, pro-inflammatory cytokines such as tumor necrosis factor (TNF-alpha), interferon-gamma, interleukin 6 (IL-6, IL-8, IL-10, chemokines, the coagulation marker D-dimer, sCD14, intestinal fatty acid binding peptide (IFABP), and hyaluronic acid). In one embodiment, the anti-inflammatory properties of an Fc-containing polypeptide are measured by determining the level of C-reactive protein (CRP) using a method known in the art. A decrease in the level of C-reactive protein indicates that the Fc-containing polypeptide exhibits anti-inflammatory properties.

[0099] “Conservatively modified variants” or “conservative substitution” refers to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that the changes can be made frequently without altering the biological activity of the protein. Those skilled in the art recognize that, in general, single amino acid substitutions in nonessential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)). Furthermore, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are listed below:

[00100] Glycosylation of immunoglobulin G (IgG) in the Fc region, Asn297 (according to the EU numbering system), has been shown to be a requirement for optimal recognition and activation of effector pathways, including antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), Wright & Morrison, Trends in Biotechnology, 15: 26-31 (1997), Tao & Morrison, J. Immunol., 143(8):2595- 2601 (1989). As such, genetic modification by glycosylation in the IgG constant region becomes an active area of research for the development of therapeutic monoclonal antibodies (mAbs). The presence of N-linked glycosylation at Asn297 can be established as important for mAb activity in assays of immune effector function, including ADCC, Rothman (1989), Lifely et al., Glycobiology, 5:813-822 (1995), Umana (1999), Shields (2002), and Shinkawa (2003), and complement-dependent cytotoxicity (CDC), Hodoniczky et al., Biotechnol. Prog., 21(6): 1644-1652 (2005), and Jefferis et al., Chem. Immunol., 65: 111-128 (1997). This effect on function has been attributed to the specific conformation adopted by the glycosylated Fc domain, which appears to be absent when glycosylation is absent. More specifically, IgG lacking glycosylation in the Fc CH2 domain does not bind to FCYR, including FCYRI, FcyRII, and FCYRIII, Rothman (1989).

[00101] Not only does the presence of glycosylation appear to play a role in the effector function of an antibody, the particular composition of the N-linked oligosaccharide is also important. For example, the presence of fucose shows a marked effect on FcyRIIIa binding in vitro and ADCC in vitro, Rothman (1989), and Li et al., Nat. Biotechnol. 24(2): 2100-215 (2006). Recombinant antibodies produced by mammalian cell culture, such as CHO or NS0, contain N-linked oligosaccharides that are predominantly fucosylated, Hossler et al., Biotechnology and Bioengineering, 95(5):946-960 (2006), Umana (1999), and Jefferis et al., Biotechnol. Prog. 21:11-16 (2005). Additionally, there is evidence that sialylation in the Fc region may impart anti-inflammatory properties to antibodies. Intravenous immunoglobulin (IVIG) purified by a lectin column to enrich the sialylated form showed a distinct anti-inflammatory effect limited to the sialylated Fc fragment and was linked to an increase in the expression of the inhibitory receptor FcYRIIb, Nimmerjahn and Ravetch., J. Exp. Med. 204:11-15 (2007).

[00102] Glycosylation at the Fc region of an antibody derived from mammalian cell lines typically consists of a heterogeneous mixture of glycoforms, with the predominant forms typically being comprised of the complex fucosylated glycoforms: G0F, G1F, and to a lesser extent, G2F. Possible conditions that result in incomplete galactose transfer to the G0F structure include, but are not limited to, suboptimal galactose transfer machinery such as β-1,4 galactosyl transferase, poor UDP-galactose transport in the Golgi apparatus, suboptimal cell culture and protein expression conditions, and steric hindrance by amino acid residues surrounding the oligosaccharide. While each of these conditions can modulate the final degree of terminal galactose, subsequent transfer of sialic acid to the Fc oligosaccharide is known to be inhibited by the closed pocket configuration of the CH2 domain. See, for example, Figure 1, Jefferis, R., Nature Biotech., 24 (10): 1230–1231, 2006. Without the correct terminal monosaccharide, specifically galactose, or with insufficient terminal glycosylated forms, there is little chance of producing a sialylated form capable of acting as a therapeutic protein, even when produced in the presence of sialyl transferase. Protein genetic modification and structural analysis of human IgG-Fc glycoforms have shown that glycosylation profiles are affected by Fc conformation, such as the finding that increased levels of galactose and sialic acid in oligosaccharides derived from CHO-produced IgG3 can be achieved when specific amino acids in the Fc pocket are mutated to an alanine, including F241, F243, V264, D265, and R301. Lund et al., J. Immunol. 157(11); 4963-4969 (1996). It is further noted that certain mutations had some effect on cell-mediated superoxide generation and complement-mediated red cell lysis, which are used as surrogate markers for FCYRI and C1q binding, respectively.

[00103] Yeast has been reported to have been genetically engineered to produce host strains capable of secreting glycoproteins with highly uniform glycosylation. Choi et al., PNAS, USA 100(9): 5022-5027 (2003) describe the use of libraries of α1,2 mannosidase catalytic domains and N-acetylglucosaminyltransferase I catalytic domains, in combination with a library of fungal type II membrane protein core sequences, to localize the catalytic domains in the secretory pathway. In this manner, the strains that were isolated produced glycoproteins in vivo with uniform Man5GlcNAc2 or GlcNAcMan5GlcNAc2 N-glycan structures. Hamilton et al., Science 313 (5792): 1441-1443 (2006) described the production of a glycoprotein, erythropoietin, produced in Pichia pastoris as a glycan composition that consisted predominantly of a bisialylated, GS6.0, NANA2Gal2GlcNAc2Man3GlcNAc2 (90.5%), and monosialylated, GS5.5, NANAGal2GlcNAc2 Man3GlcNAc2 (7.9%) glycan structure. However, an antibody produced in a similar strain will have a markedly lower level of sialylated N-glycan because of the relatively low level of terminal galactose substrate on the antibody, as seen in Figure 4. It has also been recently observed that sialylation of an Fc oligosaccharide imparts anti-inflammatory properties to therapeutic intravenous gamma globulin and its Fc fragments, Kaneko et al., Science 313(5787): 670-673 (2006), and that anti-inflammatory activity is dependent on the α2,6-linked, but not the α2,3-linked, form of sialic acid, Anthony et al., Science, 320: 373-376 (2008).

Host organisms and cell lines

[00104] The Fc-containing polypeptides of this invention can be prepared in any host organism or cell line. In one embodiment, an Fc-containing polypeptide of the invention is prepared in a host cell that is capable of producing sialylated N-glycans.

[00105] In one embodiment, an Fc-containing polypeptide of the invention is prepared in a mammalian cell, where either endogenously or by genetic or engineering process the cell produces glycoproteins containing a mixture of each α2-6 and α2-3 terminal sialic acid, or only the α2-6 terminal sialic acid. Propagation of mammalian cells in culture (tissue culture) has become a routine procedure. Examples of mammalian host cell lines used are SV40-transformed monkey kidney line CV1 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); newborn hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO); mouse Sertoli cells (TM4,); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; hybridoma cell lines; NS0; SP2/0; and a human hepatoma cell line (Hep G2).

[00106] In one embodiment, an Fc-containing polypeptide of the invention can be prepared in a plant cell that is genetically modified to produce sialylated N-glycans. See, e.g., Cox et al., Nature Biotechnology (2006) 24, 1591 - 1597 (2006) and Castilho et al., J. Biol. Chem. 285(21): 15923-15930 (2010).

[00107] In one embodiment, an Fc-containing polypeptide of the invention can be prepared in an insect cell that is genetically modified to produce sialylated N-glycans. See, e.g., Harrison and Jarvis, Adv. Virus Res. 68:159-91 (2006).

[00108] In one embodiment, an Fc-containing polypeptide of the invention can be prepared in a bacterial cell that is genetically engineered to produce sialylated N-glycans. See, e.g., Lizak et al., Bioconjugate Chem. 22:488-496 (2011).

[00109] In one embodiment, an Fc-containing polypeptide of the invention can be prepared in a lower eukaryotic host cell or organism. Recent developments allow for the production of fully humanized therapeutics in lower eukaryotic host organisms, yeasts, and filamentous fungi, such as Pichia pastoris, Gerngross et al., U.S. Patent 7,029,872 and U.S. Patent 7,449,308, the disclosures of which are incorporated herein by reference. See also Jacobs et al., Nature Protocols 4(1):58-70 (2009). Applicants have further developed herein modified Pichia pastoris host organisms and cell lines capable of expressing antibodies comprising two mutations at amino acid positions 243 and 264 in the Fc region of the heavy chain. Antibodies with these mutations had increased levels and a more homogeneous composition of α2,6-linked sialylated glycans when compared to a parent antibody. Applicants also surprisingly observed that mutations in amino acids at positions 243 and 264 in the Fc region of the heavy chain resulted in an antibody that had decreased binding to all FCY receptors, and decreased C1q binding, the former being a surrogate for ADCC, which was independent of the increased levels of α2,6-linked sialic acid. Thus, based on the increased level and more homogeneity of terminal sialic acid linked to α2,6 N-glycan, those skilled in the art can recognize and appreciate that materials and methods described herein can be used to produce recombinant glycosylated antibodies in cells of lower eukaryotes, such as yeast and filamentous fungi, and in particular Pichia pastoris, that had improved anti-inflammatory properties when compared to a parent antibody.

[00110] Because of the increased binding of FCYR and C1q, the materials and methods described herein can be used to produce recombinant glycosylated antibodies with less effector function when compared to a parental antibody. Antibodies so produced in Pichia pastoris by the methods of the invention were produced in high yield, with less effector function, and had a predominant glycoprotein species with a terminal sialic acid attached to the α2,6 residue, compared to antibodies produced in glycoengineered Pichia pastoris cells that lack the specific Fc mutations, or in Pichia pastoris host cells that retain their endogenous glycosylation machinery.

[00111] In one embodiment, an Fc-containing polypeptide of the invention is prepared in a host cell, more preferably a yeast or filamentous fungal host cell, that has been genetically engineered to produce glycoproteins with a predominant N-glycan comprising a terminal sialic acid. In one embodiment of the invention, the predominant N-glycan is the α2,6-linked form of SA2Gal2GlcNAc2Man3GlcNAc2, produced in glycoengineered strains with α2,6 sialyl transferase, which do not produce any α2,3-linked sialic acid. In other embodiments, the strain will be genetically modified to express an α2,3 sialyl transferase alone or in combination with an α2,6 sialyl transferase, resulting in α2,3-linked sialic acid or a combination of α2,6- and α2,3-linked sialic acid as the predominant N-glycans.

[00112] The cell lines to be used to prepare the Fc-containing polypeptides of the invention may be any cell line, in particular cell lines capable of producing one or more sialylated glycoproteins. Those skilled in the art will recognize and appreciate that the materials and methods described herein are not limited to the specific strain of Pichia pastoris provided herein as an example, but may include any strain of Pichia pastoris or other strains of yeast or filamentous fungus, in which N-glycans with one or more terminal galactose, such as Gal2GlcNAc2Man3, are produced. The terminal galactose acts as a substrate for the production of α2,6-linked sialic acid, resulting in the N-glycan structure NANA2Gal2GlcNAc2Man3GlcNAc2. Examples of suitable strains are described in U.S. Patent 7,029,872, U.S. 2006-0286637 and Hamilton et al., Science 313 (5792): 1441-1443 (2006), the disclosures of which are incorporated herein as if presented at full length.

[00113] In general, lower eukaryotes such as yeast are used for the expression of proteins, particularly glycoproteins, because they can be economically cultivated, provide high yields, and, when appropriately modified, are capable of adequate glycosylation. Yeast particularly offer established genetic characteristics that allow rapid transformations, tested protein localization strategies, and simple gene knockout techniques. Suitable vectors feature expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences, and the like, if desired.

[00114] Although the invention is demonstrated here using the methylotrophic yeast Pichia pastoris, other lower eukaryotic host cells used include Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichiamembraneefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporiumi lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum and Neurospora crassa. Several yeasts, such as K. lactis, Pichia pastoris, Pichia methanolica and Hansenula polymorpha, are particularly suitable for cell culture because they are capable of growing at high cell densities and secreting large amounts of recombinant protein. Likewise, filamentous fungi such as Aspergillus niger, Fusarium sp., Neurospora crassa and others can be used to produce glycoproteins of the invention on an industrial scale.

[00115] Lower eukaryotes, particularly yeasts and filamentous fungi, can be genetically modified to express glycoproteins in which the glycosylation pattern is human-like or humanized. As indicated above, the term “human-like N-glycan” as used herein refers to N-linked oligosaccharides that closely resemble oligosaccharides produced by wild-type, non-genetically modified human cells. In preferred embodiments of the present invention, the host cells of the present invention are capable of producing human-like glycoproteins with hybrid and/or complex N-glycans; i.e., “human-like N-glycosylation”. The specific “human-like” glycans present predominantly on glycoproteins produced from the host cells of the invention will depend on the specific genetically modified steps that are performed. In this manner, glycoprotein compositions can be produced in which a specific desired glycoform is predominant in the composition. This may be achieved by deleting selected endogenous glycosylation enzymes, and/or by genetically modifying the host cells, and/or by providing exogenous enzymes to mimic all or part of the mammalian glycosylation pathway, in the manner described in U.S. Patent 7,449,308. If desired, further genetic modification of glycosylation may be carried out, such that the glycoprotein may be produced with or without core fucosylation. The use of lower eukaryotic host cells is additionally advantageous, with the advantage that these cells are capable of producing very homogeneous glycoprotein compositions, such that the predominant glycoform of the glycoprotein may be present as greater than thirty mole percent of the glycoprotein in the composition. In particular aspects, the predominant glycoform may be present in greater than forty mole percent, fifty mole percent, sixty mole percent, seventy mole percent, and most preferably greater than eighty mole percent of the glycoprotein present in the composition.

[00116] Lower eukaryotes, particularly yeast, can be genetically modified to express glycoproteins in which the glycosylation pattern is human-like or humanized. This can be achieved by deleting selected endogenous glycosylation enzymes and/or providing exogenous enzymes, as described by Gerngross et al., U.S. Patent 7,449,308. For example, a host cell can be selected or genetically modified to be depleted in α1,6-mannosyl transferase activity, which can otherwise add mannose residues to the N-glycan on a glycoprotein.

[00117] In one embodiment, the host cell further includes an α1,2-mannosidase catalytic domain fused to a cell targeting signal peptide, which is not normally associated with the catalytic domain, and selected to target α1,2-mannosidase activity in the ER or Golgi complex of the host cell. Passage of a recombinant glycoprotein through the ER or Golgi complex of the host cell produces a recombinant glycoprotein comprising a glycoform of Man5GlcNAc2, e.g., a recombinant glycoprotein composition comprising predominantly a glycoform of Man5GlcNAc2. For example, U.S. patents 7,029,872 and 7,449,308 and published U.S. patent application 2005/0170452 disclose lower eukaryotic host cells capable of producing a glycoprotein comprising a glycoform of Man5GlcNAc2.

[00118] In a further embodiment, the immediately preceding host cell further includes a GlcNAc transferase I (GnT I) catalytic domain fused to a cell targeting signal peptide, not generally associated with the catalytic domain, and selected to target GlcNAc transferase I activity in the ER or Golgi complex of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi complex of the host cell produces a recombinant glycoprotein comprising a glycoform of GlcNAcMan5GlcNAc2, e.g., a recombinant glycoprotein composition comprising predominantly a glycoform of GlcNAcMan5GlcNAc2. U.S. Patents 7,029,872 and 7,449,308 and published U.S. Patent Application 2005/0170452 disclose lower eukaryotic host cells capable of producing a glycoprotein comprising a glycoform of GlcNAcMan5GlcNAc2. The glycoprotein produced in the foregoing cells can be treated in vitro with a hexosaminidase to produce a recombinant glycoprotein comprising a glycoform of Man5GlcNAc2.

[00119] In a further embodiment, the immediately preceding host cell further includes a mannosidase II catalytic domain fused to a cell targeting signal peptide, not normally associated with the catalytic domain, and selected to target mannosidase II activity to the ER or Golgi complex of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi complex of the host cell produces a recombinant glycoprotein comprising a glycoform of GlcNAcMan3GlcNAc2, e.g., a recombinant glycoprotein composition comprising predominantly a glycoform of GlcNAcMan3GlcNAc2. U.S. Patent 7,029,872 and published U.S. Patent Application 2004/0230042 disclose lower eukaryotic host cells that express mannosidase II enzymes, and are capable of producing glycoproteins predominantly with a GlcNAcMan3GlcNAc2 glycoform. The glycoprotein produced in the foregoing cells can be treated in vitro with a hexosaminidase to produce a recombinant glycoprotein comprising a Man3GlcNAc2 glycoform.

[00120] In a further embodiment, the immediately preceding host cell further includes the catalytic domain of GlcNAc transferase II (GnT II), fused to a cell-targeting signal peptide, not normally associated with the catalytic domain, and selected to target GlcNAc transferase II activity in the ER or Golgi complex of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi complex of the host cell produces a recombinant glycoprotein comprising a glycoform of GlcNAc2Man3GlcNAc2, e.g., a recombinant glycoprotein composition comprising predominantly a glycoform of GlcNAc2Man3GlcNAc2. U.S. Patents 7,029,872 and 7,449,308 and published U.S. Patent Application 2005/0170452 disclose lower eukaryotic host cells capable of producing a glycoprotein comprising a GlcNAc2Man3GlcNAc2 glycoform. The glycoprotein produced in the foregoing cells can be treated in vitro with a hexosaminidase to produce a recombinant glycoprotein comprising a Man3GlcNAc2 glycoform.

[00121] In a further embodiment, the immediately preceding host cell further includes a galactosyltransferase catalytic domain fused to a cell targeting signal peptide, not normally associated with the catalytic domain, and selected to target galactosyltransferase activity to the ER or Golgi complex of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi complex of the host cell produces a recombinant glycoprotein comprising a GalGlcNAc2Man3GlcNAc2 or Gal2GlcNAc2Man3GlcNAc2 glycoform, or mixture thereof, e.g., a recombinant glycoprotein composition comprising predominantly a GalGlcNAc2Man3GlcNAc2 glycoform or Gal2GlcNAc2Man3GlcNAc2 glycoform, or mixture thereof. U.S. Patent 7,029,872 and published U.S. Patent Application 2006/0040353 disclose lower eukaryotic host cells capable of producing a glycoprotein comprising a Gal2GlcNAc2Man3GlcNAc2 glycoform. The glycoprotein produced in the foregoing cells can be treated in vitro with a galactosidase to produce a recombinant glycoprotein comprising a GlcNAc2Man3 GlcNAc2 glycoform, e.g., a recombinant glycoprotein composition comprising predominantly a GlcNAc2Man3GlcNAc2 glycoform.

[00122] In a further embodiment, the immediately preceding host cell further includes a sialyltransferase catalytic domain fused to a cell targeting signal peptide, not normally associated with the catalytic domain, and selected to target sialyltransferase activity in the ER or Golgi complex of the host cell. In a preferred embodiment, the sialyltransferase is an alpha2,6-sialyltransferase. Passage of the recombinant glycoprotein through the ER or Golgi complex of the host cell produces a recombinant glycoprotein comprising predominantly a NANA2Gal2GlcNAc2Man3GlcNAc2 glycoform or NANAGal2GlcNAc2Man3GlcNAc2 glycoform or mixture thereof. For lower eukaryotic host cells, such as yeast and filamentous fungi, it is known that the host cell additionally includes a means of providing CMP-sialic acid for N-glycan transfer. Published U.S. patent application 2005/0260729 discloses a method for genetically engineering lower eukaryotes to have a CMP-sialic acid synthesis pathway, and published U.S. patent application 2006/0286637 discloses a method for genetically engineering lower eukaryotes to produce sialylated glycoproteins. To increase the amount of sialylation, it may be advantageous to engineer the host cell to include two or more copies of the CMP-sialic acid synthesis pathway, or two or more copies of sialyltransferase. The glycoprotein produced in the above cells can be treated in vitro with a neuraminidase to produce a recombinant glycoprotein comprising predominantly a Gal2GlcNAc2Man3GlcNAc2 glycoform, or GalGlcNAc2Man3GlcNAc2 glycoform, or mixture thereof.

[00123] Any of the foregoing host cells can additionally include one or more GlcNAc transferases selected from the group consisting of GnT III, GnT IV, GnT V, GnT VI, and GnT IX, to produce glycoproteins with biantennary (GnT III) and/or multiantennary (GnT IV, V, VI, and IX) N-glycan structures, such as those disclosed in published U.S. patent applications 2005/0208617 and 2007/0037248. Additionally, the foregoing host cells can produce recombinant glycoproteins (e.g., antibodies) comprising SA(1-4)Gal(1- 4)GlcNAc(2-4)Man3GlcNAc2, including antibodies comprising NANA(1-4)Gal(1-4)GlcNAc(2-4)Man3GlcNAc2, NGNA(1-4)Gal(1- 4)GlcNAc(2-4)Man3GlcNAc2, or a combination of NANA(1-4)Gal(1- 4)GlcNAc(2-4)Man3GlcNAc2 and NGNA(1-4)Gal(1-4)GlcNAc(2-4)Man3GlcNAc2. In one embodiment, the recombinant glycoprotein will comprise N-glycans comprising a structure selected from the group consisting of SA(1-4)Gal(1-4)GlcNAc(2-4)Man3GlcNAc2, and lacking any α2-3 linked AS.

[00124] In additional embodiments, the host cell producing glycoproteins, which predominantly display GlcNAcMan5GlcNAc2 N-glycans, further includes a galactosyltransferase catalytic domain fused to a cell targeting signal peptide, not normally associated with the catalytic domain, and selected to target galactosyltransferase activity to the ER or Golgi complex of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi complex of the host cell produces a recombinant glycoprotein comprising predominantly the GalGlcNAcMan5GlcNAc2 glycoform.

[00125] In a further embodiment, the immediately preceding host cell that produced glycoproteins, which predominantly display GalGlcNAcMan5GlcNAc2 N-glycans, further includes a sialyltransferase catalytic domain fused to a cell-targeting signal peptide, not normally associated with the catalytic domain, and selected to target sialyltransferase activity to the ER or Golgi complex of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi complex of the host cell produces a recombinant glycoprotein comprising a glycoform of SAGalGlcNAcMan5GlcNAc2 (e.g., NANAGalGlcNAcMan5GlcNAc2 or NGNAGalGlcNAcMan5GlcNAc2, or a mixture thereof).

[00126] Any of the host cells may additionally include one or more sugar transporters, such as UDP-GlcNAc transporters (e.g., Kluyveromyces lactis and Mus musculus UDP-GlcNAc transporters), UDP-galactose transporters (e.g., Drosophila melanogaster UDP-galactose transporters), and CMP-sialic acid transporter (e.g., human sialic acid transporter). Because lower eukaryotic host cells, such as yeast and filamentous fungi, lack the above transporters, it is preferred that the lower eukaryotic host cells, such as yeast and filamentous fungi, be genetically engineered to include the above transporters.

[00127] Additionally, any of the foregoing host cells may be further engineered to increase N-glycan maintenance. See, for example, Gaulitzek et al., Biotechnol. Bioengin. 103:11641175 (2009); Jones et al., Biochim. Biospyhs. Acta 1726:121-137 (2005); WO2006/107990. In one embodiment, any of the foregoing host cells can be further genetically engineered to comprise at least one nucleic acid molecule encoding a heterologous single subunit oligosaccharyltransferase (e.g., Leishmania sp. STT3A protein, STT3B protein, STT3C protein, STT3D protein, or combinations thereof), and a nucleic acid molecule encoding the heterologous glycoprotein, and wherein the host cell expresses the endogenous genes of the host cell that encode proteins comprising the endogenous OTase complex. In one embodiment, any of the foregoing host cells can be further genetically engineered to comprise at least one nucleic acid molecule encoding a Leishmania sp. STT3D protein, and a nucleic acid molecule encoding the heterologous glycoprotein, and wherein the host cell expresses the endogenous genes of the host cell that express proteins comprising the endogenous OTase complex.

[00128] Host cells additionally include lower eukaryotic cells (e.g., yeast such as Pichia pastoris), which are genetically modified to produce glycoproteins that lack α-mannosidase-resistant N-glycans. This can be achieved by deleting or disrupting one or more of the β-mannosyltransferase genes (e.g., BMT1, BMT2, BMT3, and BMT4) (see published U.S. patent application 2006/0211085), and glycoproteins with phosphomannose residues by deleting or disrupting one or both of the phosphomannosyl transferase genes, PNO1 and MNN4B (See, e.g., U.S. Patents 7,198,921 and 7,259,007), which in additional aspects can also include deleting or disrupting the MNN4A gene. Disruption includes disrupting the open reading frame encoding particular enzymes, or disrupting expression of the open reading frame or abrogating translation of RNAs encoding one or more of the β-mannosyltransferases and/or phosphomannosyltransferases using RNA interference, antisense RNA, or the like. Additionally, cells can produce glycoproteins with α-mannosidase-resistant N-glycans by the addition of chemical inhibitors, or by modifying the cell culture conditions. These host cells can be further modified, in the manner described above, to produce particular N-glycan structures.

[00129] Host cells further include lower eukaryotic cells (e.g., yeast such as Pichia pastoris), which are genetically engineered to control glycoprotein O-glycosylation by deleting or disrupting one or more of the protein O-mannosyltransferase (Dol-P-Man:Protein (Ser/Thr) Mannosyl Transferase) (PMTs) genes (See U.S. Patent 5,714,377), or growth in the presence of Pmtp inhibitors and/or an α-mannosidase, in the manner disclosed in published international application WO 2007/061631, or both. Disruption includes disrupting the open reading frame encoding the Pmtp, or disrupting expression of the open reading frame, or abrogating translation of RNAs encoding one or more of the Pmtps using RNA interference, antisense RNA, or the like. Host cells may additionally include any of the previously mentioned host cells modified to produce particular N-glycan structures.

[00130] Pmtp inhibitors include, but are not limited to, a benzylidene thiazolidinedione. Examples of benzylidene thiazolidinediones that may be used are 5-[[3,4-bis(phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic acid; 5-[[3-(1-Phenylethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic acid, and 5-[[3-(1-Phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic acid.

[00131] In particular embodiments, the function or expression of at least one endogenous PMT gene is reduced, disrupted, or eliminated. For example, in particular embodiments, the function or expression of at least one endogenous PMT gene selected from the group consisting of the PMT1, PMT2, PMT3, and PMT4 genes is reduced, disrupted, or eliminated; or the host cells are cultured in the presence of one or more PMT inhibitors. In additional embodiments, the host cells include one or more PMT gene deletions or disruptions, and the host cells are cultured in the presence of one or more Pmtp inhibitors. In particular aspects of these embodiments, the host cells further express a secreted α-1,2-mannosidase.

[00132] PMT deletions or disruptions and/or Pmtp inhibitors control O-glycosylation by reducing the maintenance of O-glycosylation, i.e., by reducing the total number of O-glycosylation sites on the glycoprotein that are glycosylated. The addition of an additional α-1,2-mannosidase, which is secreted by the cell, controls O-glycosylation by reducing the mannose chain size of the O-glycans that are on the glycoprotein. Thus, combining PMT deletions or disruptions, and/or Pmtp inhibitors, with the expression of a secreted α-1,2-mannosidase controls O-glycosylation by reducing chain utilization and chain size. In particular circumstances, the particular combination of PMT, Pmtp, and α-1,2-mannosidase deletions or disruptions is determined empirically as particular heterologous glycoproteins (e.g., Fabs and antibodies) may be expressed and transported through the Golgi complex with varying degrees of efficiency and thus may require a particular combination of PMT, Pmtp, and α-1,2-mannosidase deletions or disruptions. In another aspect, genes encoding one or more endogenous mannosyltransferase enzymes are deleted. This deletion(s) may be in conjunction with the provision of secreted α-1,2-mannosidase and/or PMT inhibitors, or may be in place of the provision of secreted α-1,2-mannosidase and/or PMT inhibitors.

[00133] Thus, control of O-glycosylation can be used to produce particular glycoproteins in the host cells disclosed herein, in improved overall yield or in yield of properly assembled glycoprotein. Reduction or elimination of O-glycosylation appears to have a beneficial effect on the assembly and transport of full-length antibodies and Fab fragments as they traverse the secretory pathway and are transported to the cell surface. Thus, in cells in which O-glycosylation is controlled, the yield of properly displayed antibodies or Fab fragments is greater than the yield obtained in host cells in which O-glycosylation is not controlled.

[00134] To reduce or eliminate the likelihood of N-glycans and O-glycans with β-linked mannose residues that are resistant to α-mannosidases, recombinant and glycoengineered Pichia pastoris host cells are genetically modified to eliminate glycoproteins with α-mannosidase-resistant N-glycans by deleting or disrupting one or more of the β-mannosyltransferase genes (e.g., BMT1, BMT2, BMT3, and BMT4) (See, U.S. Patent 7,465,577 and U.S. Patent 7,713,719). Deletion or disruption of BMT2 and one or more of BMT1, BMT3, and BMT4 also reduces or eliminates detectable cross-reactivity of antibodies against the host cell protein.

[00135] Glycoprotein yield may, in some situations, be improved by overexpression of nucleic acid molecules encoding mammalian or human chaperone proteins, or by replacing the genes encoding one or more endogenous chaperone proteins with nucleic acid molecules encoding one or more mammalian or human chaperone proteins. Furthermore, expression of mammalian or human chaperone proteins in the host cell also appears to control O-glycosylation in the cell. Thus, additionally included herein are host cells in which the function of at least one endogenous gene encoding a chaperone protein has been reduced or eliminated, and a vector encoding at least one mammalian or human homologue of the chaperone protein is expressed in the host cell. Also included are host cells in which both the endogenous chaperones of the host cell and the mammalian or human chaperone proteins are expressed. In further aspects, the lower eukaryotic host cell is a yeast or filamentous fungal host cell. Examples of the use of host cell chaperones, into which human chaperone proteins are introduced to improve the yield and reduce or control O-glycosylation of recombinant proteins, have been disclosed in published international applications WO 2009105357 and WO2010019487 (the disclosures of which are incorporated herein by reference). As before, further included are lower eukaryotic host cells in which, in addition to replacing the genes encoding one or more of the endogenous chaperone proteins with nucleic acid molecules encoding one or more mammalian or human chaperone proteins, or overexpressing one or more mammalian or human chaperone proteins in the manner described above, the function or expression of at least one endogenous gene encoding a protein O-mannosyltransferase (PMT) is reduced, disrupted or eliminated. In particular embodiments, the function of at least one endogenous PMT gene, selected from the group consisting of the PMT1, PMT2, PMT3, and PMT4 genes, is reduced, disrupted, or eliminated.

[00136] Furthermore, O-glycosylation may have an effect on an antibody or Fab fragment's affinity and/or avidity for an antigen. This may be particularly significant when the final host cell for production of the antibody or Fab is not the same host cell that was used to select the antibody. For example, O-glycosylation may interfere with an antibody's or Fab's affinity for an antigen, so that an antibody or Fab fragment must otherwise exhibit high affinity for an antigen that cannot be identified because O-glycosylation may interfere with the ability of the antibody or Fab fragment to bind to the antigen. In other cases, an antibody or Fab fragment that exhibits high avidity for an antigen may not be identified because O-glycosylation interferes with the antibody or Fab fragment's avidity for the antigen. In both of these cases, an antibody or Fab fragment that may be particularly effective when produced in a mammalian cell line may not be identified because the host cells for identifying and selecting the antibody or Fab fragment are from another cell type, e.g., a yeast or fungal cell (e.g., a Pichia pastoris host cell). It is well known that O-glycosylation in yeast can be significantly different from O-glycosylation in mammalian cells. This is particularly relevant when comparing wild-type yeast O-glycosylation with mammalian mucin-type or dystroglycan-type O-glycosylation. In particular cases, O-glycosylation may enhance the affinity or avidity of the antibody or Fab fragment for an antigen, rather than interfering with antigen binding. This effect is undesirable when the production host cell must be different from the host cell used to identify and select the antibody or Fab fragment (e.g., identification and selection are performed in yeast and the production host is a mammalian cell), because the O-glycosylation in the production host is no longer of the type that caused the best affinity or avidity for the antigen. Therefore, controlling O-glycosylation may enable the use of the materials and methods herein to identify and select antibodies or Fab fragments with specificity for a particular antigen, based on the affinity or avidity of the antibody or Fab fragment for the antigen, without identifying and selecting the antibody or Fab fragment that is influenced by the O-glycosylation system of the host cell. Thus, controlling O-glycosylation further improves the utility of yeast or fungal host cells for identifying and selecting antibodies or Fab fragments that will ultimately be produced in a mammalian cell line.

[00137] Those skilled in the art can further appreciate and understand how to utilize the methods and materials described herein in combination with other Pichia pastoris and yeast cell lines that have been genetically engineered to produce specific N-glycans or sialylated glycoproteins, such as, but not limited to, the host organisms and cell lines described above that have been genetically engineered to produce specific galactosylated or sialylated forms. See, e.g., U.S. 2006-0286637, Production of Sialylated N-Glycans in Lower Eukaryotes, in which the pathway for galactose uptake and utilization as a carbon source has been genetically engineered, the disclosure of which is incorporated herein in the manner set forth at length.

[00138] Additionally, the methods herein can be used to produce the previously described recombinant Fc-containing polypeptides in other lower eukaryotic cell lines that have been genetically engineered to produce human-like and human-like glycoproteins that lack α2,6 sialyltransferase activity. The methods can also be used to produce the previously described recombinant Fc-containing polypeptides in eukaryotic cell lines in which the production of sialylated N-glycans is an innate trait.

[00139] The levels of α2,3- and α2,6-linked sialic acid on Fc-containing polypeptides can be measured using well-known techniques, including nuclear magnetic resonance (NMR), normal phase high-performance liquid chromatography (HPLC), and high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

Biological properties of Fc muteins

[00140] For many Fc-containing polypeptides, the absence or significant decrease in effector function, as shown by decreased FCYR and C1q binding, Idusogie et al., J. Immunology, 164(8): 4178-84 (2000) and Shields et al., J. Biol. Chem., 276: 6591-6604 (2001), and improved anti-inflammatory properties, would be desirable features. Applicants have discovered that specific modifications of amino acid positions 243 and 264 in the Fc region of an IgG can convey an absence, or a significant decrease, in effector function independent of the presence of sialylation at the terminal glycan positions. Specifically, Applicants have observed that modification of residues F243 and V264 in the Fc region to alanine resulted in an antibody with decreased binding to the FCY and C1q receptors. It is notable that antibodies produced with these modifications in the Fc region exhibited less binding to FCY receptors, regardless of whether they were shown to have α2,6-linked sialic acid as the terminal glycan.

[00141] As such, applicants have developed a dual Fc mutein, F243A/V264A, that will produce Fc-containing polypeptides with the aforementioned desired characteristics. Examples herein comprise transforming a host cell with a polynucleotide vector encoding an Fc-containing polypeptide comprising mutations at positions 243 and 264 of the Fc region, and culturing the transformed host cell to produce the Fc-containing polypeptide.

Production of Fc-containing polypeptides

[00142] The Fc-containing polypeptides of the invention can be prepared according to any method known in the art for generating polypeptides comprising an Fc region. In one embodiment, the Fc-containing polypeptide is an antibody or an antibody fragment (including, without limitation, a polypeptide consisting of or consisting essentially of the Fc region of an antibody). In another embodiment, the Fc-containing polypeptide is an immunoadhesin. Methods of preparing antibodies and antibody fragments are well known in the art. Methods of introducing point mutations into a polypeptide, e.g., site-directed mutagenesis, are also well known in the art.

[00143] In the examples disclosed herein, an IgG1 heavy and light chain containing a CH2 consensus sequence and the double Fc mutants described herein were expressed in two different glycoengineered Pichia pastoris strains. In the manner described in the following examples, the heavy and light chain gene sequences were under the control of a methanol-inducible promoter, AOX1, and incorporated a bleomycin (Zeocin) selection marker. This strategy integrates the complete expression cassette into the Trp2 locus by homologous DNA recombination.

[00144] Secreted antibodies were captured from the fermentation broth by protein A affinity chromatography, followed by a Source 30S cation exchange purification step. The purified antibodies were characterized by SDS-PAGE (Figure 2), and size exclusion chromatography (SEC), and reversed-phase HPLC to assess proper assembly. As seen in Figure 2, antibodies produced by the materials and methods herein exhibited a purity profile on SDS-PAGE that was similar to that for a mammalian cell (CHO) that produced Her2 antibody. Analysis of IgG by SEC and reversed-phase HPLC further demonstrated that antibodies prepared from the glycoengineered strains with Fc mutein assembled properly, and were similar to antibodies produced by the mammalian cell in terms of assembly (data not shown). The affinity of the antigen to the various antibodies prepared by the materials and methods herein was determined by a cell-based assay using a SK-BR3 cell line, which in this example was a human breast cancer cell line overexpressing Her2. As expected, all antibodies, including the Fc muteins, bound equally well to the SK-BR3 cell line (Figure 3).

N-Glycan analysis of Fc muteins

[00145] For many glycoproteins, including certain antibodies, sialylation of the N-linked terminal glycan of an IgG Fc region is essential to produce glycoproteins and antibodies that have the correct conformation to convey therapeutic activity. See, for example, Anthony et al., Science, 320: 373-376 (2008), where terminal sialylation was correlated with anti-inflammatory activity for an IVIG preparation. Sialylation requires the presence of a penultimate galactose, on which sialyl transferase acts to form the sialylated glycan. Thus, glycoproteins lacking one or more terminal galactose glycoforms cannot produce antibodies with the α2,6-linked sialic acid composition associated with anti-inflammatory activity.

[00146] Typically, antibodies produced in mammalian cell culture, such as CHO cells, have a glycoform composition comprising: G0F (37%), G1F (43%), G2F (9%), G0 (4%), G1 (3%) and Man5 (3%), which have little or no terminal galactose to act as a substrate for sialic acid transfer. In the case of CHO cell production, the terminal glycan produced is the α2,3-linked form; CHO cells do not express an α2,6 sialyl transferase necessary to produce the α2,6-linked sialic acid form, which is associated with anti-inflammatory activity. However, overexpression of a specific α2,6 sialyltranferase in CHO can give rise to a mixture of α2,3-linked and α2,6-linked sialic acid (Bragonzi et al., BBA 1474:273-282 (2000); Biochem. Biophys. Res. Comm. 289:243-249 (2001)). Glycoengineered Pichia pastoris strains GFI5.0, which are capable of producing high levels of non-antibody-type galactosylated proteins such as erythropoietin (Hamilton et al., Science, 313:1441-1443 (2006)), produce antibodies with relatively low amounts of a terminal galactose that can act to synthesize the α2,6-linked sialylated form (Figure 4). Antibodies produced in such Pichia pastoris strains typically have a composition including glycoforms G0 (60%), G1 (17%), G2 (4%), and Man5 (8%). Even antibodies produced in Pichia pastoris strains GFI6.0, which have a glycan composition comprising G0 (43.5%), G1 (20.8%), G2 (2.7%), NANAGalGlcNAcMan5GlcNAc2 (5.5%), and NANAGal2GlcNAc2 Man3GlcNAc2 (4.9%), have relatively low levels of the α2,6-linked sialylated form. Thus, antibodies produced in strains GFI5.0 and 6.0 have much lower levels of galactosylation and sialylation compared to non-antibody-like proteins (such as erythropoietin) produced in the same strains.

[00147] The N-glycan composition of the Her2 antibody and corresponding Fc mutein antibodies produced here in glycoengineered Pichia pastoris strains GFI5.0 and GFI6.0 was analyzed by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry, following release of the N-peptide F-glycan antibody (Figures 4-8). The carbohydrate composition released from the Her2 and Fc mutein antibodies was quantified by HPLC on an Allentech Prevail carbo column (Alltech Associates, Deerfield IL).

[00148] The glycoforms of the Pichia pastoris Her2 antibody from strain GFI5.0 included biantennary G0, G1, and G2 and other neutral glycans, with G0 as the dominant glycoform (Figure 4). This glycan composition is consistent with that produced commercially in CHO cells, with the exception that the Pichia pastoris-derived antibody does not inherently contain fucose. Conversely, the glycoforms of the Fc mutein antibodies produced in GFI5.0 have dissimilar glycan compositions. The single Fc mutein antibodies, both F243A and V264A, exhibited an increase in galactosylated N-glycans, which may function as the substrate for α2,6 sialyl transferase, as observed from the highest peak in MALDI-TOF mass spectrometry (Figures 5 and 6), while the N-glycan composition for the dual Fc mutein antibody, F243A/V264A, exhibited an even greater increase with over 80% of the total N-glycans galactosylated (Figure 7). The level of G2 (bi-galactosylated N-glycan) present on the dual Fc mutein antibody represented the highest proportion of G2 observed for any antibody evaluated by us. When these same Fc mutein antibodies were expressed in strain GFI6.0, α2,6-linked sialic acid was added to either the G1 (mono-galactosylated) or G2 (bi-galactosylated) N-glycans. For the single Fc mutein antibody, nearly all of the G2 N-glycans were converted to a sialylated glycan (data not shown). While antibodies produced from the single Fc muteins exhibited a much higher level of α2,6-linked sialylation (40–51%, see Table 3), the level was lower than that for antibodies produced from the double Fc mutein (Figure 8 and Table 3), where 74% sialylation was achieved with bioreactor fermentation and 91% or greater sialylation was achieved with small-scale fermentation.

[00149] Without wishing to be bound by any theory, Applicants believe that the transfer of sialic acid to the Fc oligosaccharide is enhanced by the more open pocket configuration of the CH2 domain imparted by the double Fc mutein compared to the single Fc mutein. It is also noted that the α2,6-form of sialic acid produced from a GFI6.0 strain is the same form produced in humans, and differs from the sialic acid linked to the α2,3-form present in antibodies produced in CHO cell lines, Jassal et al., Biochem. Biophys. Res. Comm. 289:243-249, 2001.

FCYR binding to Fc muteins

[00150] Using an ELISA-based assay, Applicants compared the binding of Fc gamma receptor (FCYR) to the Pichia pastoris Her2 antibody, single and double Fc mutein antibodies, and the Her2 antibody. As shown in the experiments described in Examples 11 and 15, the double Fc mutein exhibited a decrease in affinity for FcyRI. Since FcyRI is a receptor that has been shown to stimulate an immune response upon binding to the antibody, these data suggest that double mutein antibodies will be less able to elicit an immune response.

[00151] With respect to FcyRIIb, a receptor that exhibits less affinity for the antibody compared to FcyRI and that has been shown to inhibit an immune response, the dual Fc mutein binds with lower affinity. With respect to FcyRIIa, the dual Fc mutein also appears to bind with lower affinity. These data suggest that the conformational structure of the dual mutein antibodies is altered such that the ability to inhibit an immune response via FcyRIIa and FcyRIIb/c is significantly reduced or eliminated.

[00152] FcyRIIIa-F158 and FcyRIIIa-V158 are polymorphisms of a receptor known to stimulate an immune response, but exhibits less affinity for antibody compared to FcyRI. The dual Fc mutein exhibited little affinity for FcYRIIIa-F158, while maintaining some affinity for FcYRIIIa-V158. Taken together, these data suggest that the dual Fc mutein is less likely to activate and recruit immune cells, such as macrophages, monocytes, and natural killer cells, in a manner compared to the parent antibody.

C1q binding of Fc muteins

[00153] Antibody-C1q binding is an important parameter for complement-dependent cytotoxicity. The binding activity (affinity) of an immunoglobulin (IgG) molecule for C1q can be determined by a cell-based assay, such as that provided in Example 13 herein. Those skilled in the art can recognize and appreciate that the disclosed assay can be readily adapted for use with any IgG molecule.

ADCC effects on Fc muteins

[00154] It is well established that the FcYRIIIa receptor (CD16) is responsible for antibody-dependent cell-mediated cytotoxicity (ADCC) (Daeron et al., Annu. Rev. Immunol. 15:203-234, 1997). Applicants observed that antibodies produced from the dual Fc mutein described herein exhibited less binding to FcYRIIIa (Figure 9C and D; Tables 4 and 5) and as such likely lost the ability to perform FcYRIIIa-mediated ADCC.

[00155] Example 13 provides an in vitro assay for measuring B cell depletion and fluorescence released by ADCC.

Bioavailability of Fc muteins

[00156] Bioavailability refers to the degree and rate at which an active moiety, whether a drug or metabolite, enters the human circulation and thereby reaches the site of action. The bioavailability of a drug is primarily affected by the properties of the dosage form, as opposed to the physicochemical properties of the drug, which in turn may determine its absorption potential. Chemical equivalence suggests that drug products contain the same active ingredient(s) and in the same amount, although other inactives may differ. Similarly, bioequivalence suggests that two drug products, when given to the same patient in the same dosage regimen, will produce equivalent concentrations of drug in the circulation and tissues. Conversely, two drug products that are not identical may be capable of producing the same therapeutic effect (and adverse effects) when given to the same patient. Thus, the bioavailability of a drug is related to a determination of any equivalence in which it may be possible to achieve therapeutic equivalence, even when the bioavailabilities differ. For drugs with a narrow therapeutic index, that is, the ratio of the minimum toxic concentration to the mean effective concentration, differences in bioavailability may result in therapeutic non-equivalence.

[00157] Applicants have surprisingly observed that dual Fc mutein antibodies produced in Pichia pastoris by the materials and methods herein, when injected subcutaneously, exhibited better bioavailability (absorption or exposure) than a comparable antibody produced in CHO cells by known commercial methods. In general, subcutaneous administration is preferable versus intravenous administration, in which a subcutaneous formulation can be self-administered by the patient. As shown in Figure 12, serum concentrations from mice treated (as set forth in Example 14) with a dual Fc mutein antibody increased by about 30% compared to serum concentrations for a counterpart produced in CHO, and were greater than those for an antibody produced in Pichia pastoris that does not exhibit the increased α2,6-linked sialylated form.

Production of human glycosylated antibodies with increased levels of sialylation

[00158] In light of the findings herein, Applicants have developed a method of producing antibodies in which they increase α2,6-linked sialylation by modifying the Fc region of the antibody, such that a strain is created that can produce antibodies in vivo in which they exhibit increased galactosylation and sialylation at the Asn297 position. Without wishing to be bound by theory, Applicants propose that the absence of sialic acid and the low level of galactose present on the N-glycan at the Asn297 position of an antibody is due not to a low efficiency of the respective glycosyl transferase, but rather to the steric hindrance inherent in the structure of the Fc region. Applicants believe that the structure of the Fc region prevents the addition of galactose and sialic acid during the short period of time when the antibody passes through the secretory pathway. Previous reports suggest that high levels of galactosylation of a CD20 mAb are possible in vitro when incubation times no longer than those typically used in vivo are used in conjunction with galactosyltransferase, Li et al., Nat. Biotechnol. 24(2): 210–215 (2006). Galactose transfer is known to occur when the antibody is in an open configuration with less steric hindrance. The structural or conformational changes that result from the double mutation, i.e., double Fc mutein, result in a permanently open conformation, allowing much greater transfer of galactose and sialic acid.

[00159] From studies of the replicants herein, it appears that individual mutation of the amino acid F243 or V264 had a similar impact on increasing galactosylation and sialylation of the Fc region of an IgG1 molecule. Lund et al., J. Immunology 157(11): 4963-4969 (1996) reported a moderate increase in sialylated IgG3 produced in a CHO-K1 cell line by altering single amino acids in the Fc region, Phe241, Phe243, Val264, Aps265, or Tyr296, resulting in levels of 12-42% monosialylated form and 4-31% bisialylated form. As reported herein, antibodies produced by the materials and methods herein showed a significant increase in sialylation when specific sites, F243 or V264, were altered to alanine. Furthermore, levels of α2,6-silylated species in excess of 91% were achieved when both sites were mutated simultaneously (Table 3).

Biological targets

[00160] It may be noted that, although in the following examples applicants exemplify the materials and methods of the invention using IgG1 antibodies with sequences similar to those for commercially available anti-Her2 and anti-TNF antibodies, the invention is not limited to the antibodies disclosed. Those skilled in the art may recognize and understand that the materials and methods herein may be used to produce any Fc-containing polypeptide, for which the characteristics of improved anti-inflammatory activity or decreased effector function may be desirable. It may be further noted that there is no restriction on the type of Fc-containing polypeptide or antibody thus produced by the invention. The Fc region of the Fc-containing polypeptide may be from an IgA, IgD, IgE, IgG, or IgM. In one embodiment, the Fc region of the Fc-containing polypeptide is from an IgG, including IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc region of the Fc-containing polypeptide is from an IgG1. In specific embodiment, the antibodies or antibody fragment produced by the materials and methods herein may be humanized, chimeric, or human antibodies.

[00161] In some embodiments, the Fc-containing polypeptides of the invention will bind to a biological target that is involved in inflammation.

[00162] In some embodiments, the Fc-containing polypeptide of the invention will bind to a pro-inflammatory cytokine. In some embodiments, the Fc-containing polypeptide of the invention will bind to a molecule selected from the group consisting of: 27, IL-33, CD2, CD4, CD11A, CD14, CD18, CD19, CD23, CD25, CD40, CD40L, CD20, CD52, CD64, CD80, CD147, CD200, CD200R, TSLP, TSLPR, PD-1, PDL1, CTLA4, VLA-4, VEGF, PCSK9, α4β7-integrin, E-selectin, Fact II, ICAM-3, beta2-integrin, IFNY, C5, CBL, LCAT, CR3, MDL-1, GITR, ADDL, CGRP, TRKA, IGF1R, RANKL, GTC, αBLys, or the receptor for any of the aforementioned molecules. In one embodiment, the Fc-containing polypeptide of the invention will bind to TNF-α. In another embodiment, the Fc-containing polypeptide of the invention will bind to Her2. In another embodiment, the Fc-containing polypeptide of the invention will bind to PCSK9. In another embodiment, the Fc-containing polypeptide of the invention will bind to TNFR. In another embodiment, the Fc-containing polypeptide of the invention will bind to LCAT. In another embodiment, the Fc-containing polypeptide of the invention will bind to TSLP. In another embodiment, the Fc-containing polypeptide of the invention will bind to PD-1. In another embodiment, the Fc-containing polypeptide of the invention will bind to IL-23.

[00163] In some embodiments, the Fc-containing polypeptides of the invention will be specific for an antigen selected from autoimmune antigens, allergens, MHC molecules, or Rhesus factor D antigen. See, for example, the antigens listed in Table 1 of WO2010/10910, which is incorporated herein by reference.

Methods of increasing anti-inflammatory properties or decreasing effector function/cytotoxicity

[00164] The invention also comprises a method of enhancing the anti-inflammatory properties of an Fc-containing polypeptide comprising: selecting a parent Fc-containing polypeptide that is used to treat an inflammatory condition (e.g., an antibody or immunoadhesin that binds to an antigen that is involved in inflammation), and introducing mutations at positions 243 and 264 of the Fc-containing polypeptide, wherein the numbering is in accordance with the EU index as in Kabat, wherein the Fc-containing polypeptide exhibits improved anti-inflammatory properties when compared to the parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide comprises mutations F243A and V264A. In another embodiment, the Fc-containing polypeptide comprises mutations F243Y and V264G. In another embodiment, the Fc-containing polypeptide comprises mutations F243T and V264G. In another embodiment, the Fc-containing polypeptide comprises mutations F243L and V264A. In another embodiment, the Fc-containing polypeptide comprises mutations F243L and V264N. In another embodiment, the Fc-containing polypeptide comprises mutations F243V and V264G. In one embodiment, the Fc-containing parent polypeptide is an antibody, antibody fragment, or immunoadhesin that binds to an antigen that is involved in inflammation. In one embodiment, the Fc-containing parent polypeptide is an antibody, antibody fragment, or immunoadhesin that is already marketed or in development for the treatment of inflammatory conditions. In another embodiment, the parent Fc-containing polypeptide is an antibody selected from the group consisting of: Muromonab-CD3 (anti-CD3 receptor antibody), Abciximab (anti-CD41 7E3 antibody), Rituximab (anti-CD20 antibody), Daclizumab (anti-CD25 antibody), Basiliximab (anti-CD25 antibody), Palivizumab (anti-RSV (respiratory syncytial virus) antibody), Infliximab (anti-TNFα antibody), Trastuzumab (anti-Her2 antibody), Gemtuzumab ozogamicin (anti-CD33 antibody), Alemtuzumab (anti-CD52 antibody), Ibritumomab tiuxeten (anti-CD20 antibody), Adalimumab (anti-TNFα antibody), Omalizumab (anti-IgE antibody), Tositumomab-131I (iodinated derivative of an anti-CD20 antibody), Efalizumab (anti-CD11a antibody), Cetuximab (anti-EGF receptor antibody), Golimumab (anti-TNFα antibody), Bevacizumab (anti VEGF-A antibody), Natalizumab (anti α4 integrin), Efalizumab (anti CD11a), Ketolizumab (anti-TNFα antibody), Tocilizumab (anti-IL-6R), Ustenkinumab (anti IL-12/23), alemtuzumab (anti CD52) and natalizumab (anti α4 integrin), and variants thereof. In another embodiment, the Fc-containing parent polypeptide is an Fc fusion protein selected from the group consisting of: Arcalyst/rilonacept (IL1R-Fc fusion), Orencia/abatacept (CTLA-4-Fc fusion), Amevive/alefacept (LFA-3-Fc fusion), Anakinra-Fc fusion (IL-1Ra-Fc fusion protein), etanercept (TNFR-Fc fusion protein), FGF-21-Fc fusion protein, GLP-1-Fc fusion protein, RAGE-Fc fusion protein, ActRIIA-Fc fusion protein, ActRIIB-Fc fusion protein, glucagon-Fc fusion protein, oxyntomodulin-Fc fusion protein, GM-CSF-Fc fusion protein, EPO-Fc fusion protein, insulin-Fc fusion protein, proinsulin-Fc fusion protein, and insulin precursor-Fc fusion protein, and analogs thereof. variants of these.

[00165] The invention also comprises a method of reducing the effector function of an Fc-containing polypeptide, comprising introducing mutations at positions 243 and 264 of a parent Fc-containing polypeptide, wherein said Fc-containing polypeptide has decreased effector function when compared to the parent Fc-containing polypeptide, wherein the numbering is according to the EU index as in Kabat. In one embodiment, the Fc-containing polypeptide comprises mutations F243A and V264A. In one embodiment, the Fc-containing polypeptide is an antibody or antigen-binding fragment thereof. In one embodiment, the effector function is ADCC. In another embodiment, the effector function is CDC.

[00166] The invention also comprises a method of decreasing the cytotoxicity of an Fc-containing polypeptide comprising: selecting a parent Fc-containing polypeptide that is used to treat an inflammatory condition (e.g., an antibody or immunoadhesin that binds to an antigen that is involved in inflammation) that binds to an antigen that is involved in inflammation, and introducing mutations at positions 243 and 264 of the Fc-containing polypeptide, wherein the numbering is in accordance with the EU index as in Kabat, wherein the Fc-containing polypeptide has decreased cytotoxicity when compared to the parent Fc-containing polypeptide.

[00167] In one embodiment, the Fc-containing parental polypeptide comprises a natural Fc region. In another embodiment, the Fc-containing parental polypeptide comprises an F243A mutation. In another embodiment, the Fc-containing parental polypeptide comprises a V264A mutation.

Treatment Methods

[00168] The invention also comprises a method of treating an inflammatory condition in a subject in need thereof comprising: administering to the subject a therapeutically effective amount of an Fc-containing polypeptide comprising mutations at positions 243 and 264, wherein the numbering is in accordance with the EU index as in Kabat. In one embodiment, the Fc-containing polypeptide comprises mutations F243A and V264A. In another embodiment, the Fc-containing polypeptide comprises mutations F243Y and V264G. In another embodiment, the Fc-containing polypeptide comprises mutations F243T and V264G. In another embodiment, the Fc-containing polypeptide comprises mutations F243L and V264A. In another embodiment, the Fc-containing polypeptide comprises mutations F243L and V264N. In another embodiment, the Fc-containing polypeptide comprises mutations F243V and V264G. The Fc-containing polypeptide of the invention may be administered by any route. In one embodiment, the Fc-containing polypeptide is administered parenterally. In one embodiment, the Fc-containing polypeptide is administered subcutaneously.

[00169] In one embodiment, the inflammatory condition is unwanted inflammatory immune reactions.

[00170] In one embodiment, the inflammatory condition is an autoimmune disease. In one embodiment, the inflammatory condition will be multiple sclerosis. In one embodiment, the inflammatory condition is systemic lupus erythematosus. In one embodiment, the inflammatory condition is type I diabetes.

[00171] In one embodiment, the inflammatory condition is a primary immunodeficiency syndrome, including congenital agammaglobulinemia and hypogammaglobulinemia, common variable immunodeficiency, severe combined immunodeficiency, or Wiskott Aldrich syndrome.

[00172] In one embodiment, the inflammatory condition is a secondary immunodeficiency syndrome, including B-cell lymphocytic leukemia, HIV infection, or an allogeneic bone marrow transplant.

[00173] In one embodiment, the inflammatory condition is idiopathic thrombocytopenic purpura.

[00174] In one embodiment, the inflammatory condition is multiple myeloma.

[00175] In one embodiment, the inflammatory condition is Guillain-Barre syndrome.

[00176] In one embodiment, the inflammatory condition is Kawasaki disease.

[00177] In one embodiment, the inflammatory condition is chronic inflammatory demyelinating polyneuropathy (CIDP).

[00178] In one embodiment, the inflammatory condition is autoimmune neutropenia.

[00179] In one embodiment, the inflammatory condition is hemolytic anemia.

[00180] In one embodiment, the inflammatory condition is anti-Factor VIII autoimmune disease.

[00181] In one embodiment, the inflammatory condition is multifocal neuropathy.

[00182] In one embodiment, the inflammatory condition is systemic vasculitis (ANCA positive).

[00183] In one embodiment, the inflammatory condition is polymyositis.

[00184] In one embodiment, the inflammatory condition is dermatomyositis.

[00185] In one embodiment, the inflammatory condition is antiphospholipid syndrome.

[00186] In one embodiment, the inflammatory condition is septic syndrome.

[00187] In one embodiment, the inflammatory condition is graft-vs-host disease.

[00188] In one embodiment, the inflammatory condition is allergy.

[00189] In one embodiment, the inflammatory condition is an anti-Rhesus factor D reaction.

[00190] In one embodiment, the inflammatory condition is an inflammatory condition of the cardiovascular system. The Fc-containing polypeptides of the invention can be used to treat atherosclerosis, atherothrombosis, coronary artery hypertension, acute coronary syndrome, and heart failure, all of which are associated with inflammation.

[00191] In one embodiment, the inflammatory condition is an inflammatory condition of the central nervous system. In another embodiment, the inflammatory condition will be an inflammatory condition of the peripheral nervous system. For example, the Fc-containing polypeptides of the invention can be used for the treatment of, for example, Alzheimer's disease, amyotrophic lateral sclerosis (known as ALS; Lou Gehrig's disease), ischemic brain injury, prion-associated diseases, and HIV-associated dementia.

[00192] In one embodiment, the inflammatory condition is an inflammatory condition of the gastrointestinal tract. For example, the Fc-containing polypeptides of the invention can be used to treat inflammatory bowel disorders, e.g., Crohn's disease, ulcerative colitis, celiac disease, and irritable bowel syndrome.

[00193] In one embodiment, the inflammatory condition is psoriasis, atopic dermatitis, arthritis including rheumatoid arthritis, osteoarthritis, and psoriatic arthritis.

[00194] In one embodiment, the inflammatory condition is steroid-dependent atopic dermatitis.

[00195] In one embodiment, the inflammatory condition is cachexia.

[00196] Examples of other inflammatory disorders that may be treated using the Fc-containing polypeptides of the invention also include: acne vulgaris, asthma, autoimmune diseases, chronic prostatitis, glomerulonephritis, hypersensitivities, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, vasculitis, interstitial cystitis, and myopathies.

[00197] In one embodiment, the Fc-containing polypeptide of the invention will be administered at a dose between 1 to 100 milligrams per kilogram of body weight. In one embodiment, the Fc-containing polypeptide of the invention will be administered at a dose between 0.001 to 10 milligrams per kilogram of body weight. In one embodiment, the Fc-containing polypeptide of the invention will be administered at a dose between 0.001 to 0.1 milligrams per kilogram of body weight. In one embodiment, the Fc-containing polypeptide of the invention will be administered at a dose between 0.001 to 0.01 milligrams per kilogram of body weight.

Pharmaceutical formulations

[00198] The invention also encompasses pharmaceutical formulations comprising an Fc-containing polypeptide of the invention and a pharmaceutically acceptable carrier.

[00199] In one embodiment, the invention relates to a pharmaceutical composition comprising an Fc-containing polypeptide, wherein at least 70% of the N-glycans on the Fc-containing polypeptide comprise an oligosaccharide structure selected from the group consisting of NANA(1-4)Gal(1-4)GlcNAc(2-4)Man3GlcNAc2, wherein the Fc-containing polypeptide comprises mutations at amino acid positions 243 and 264 of the Fc region, wherein the numbering is according to the EU index as in Kabat. In one embodiment, the mutations are F243A and V264A. In one embodiment, at least 47 mole % of the N-glycans have the structure NANA2Gal2GlcNAc2Man3GlcNAc2. In one embodiment, the sialic acid residues on the sialylated N-glycans are attached via an α-2,6 linkage. In one embodiment, the sialic acid residues on the sialylated N-glycans are attached via an α-2,6 linkage and there is no detectable level of an α-2,3-linked sialic acid. In one embodiment, the sialylated N-glycans will not comprise any N-glycolylneuraminic acid (NGNA).

[00200] As used herein, the term "pharmaceutically acceptable" means a nontoxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s), approved by a regulatory agency of the federal government, or a state government, or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals and, more particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic substance is administered and includes, but is not limited to, sterile liquids such as water and oils. The characteristics of the carrier will depend on the route of administration.

[00201] Pharmaceutical formulations of therapeutic and diagnostic agents can be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions (see, for example, Hardman et al. (2001) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[00202] The mode of administration may vary. Suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intraarterial.

[00203] In certain embodiments, the Fc-containing polypeptides of the invention can be administered by an invasive route, such as by injection (see previously). In some embodiments of the invention, the Fc-containing polypeptides of the invention, or pharmaceutical composition thereof, are administered intravenously, subcutaneously, intramuscularly, intra-arterially, intra-articularly (e.g., into arthritic joints), intratumorally, or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., orally; e.g., in a pill, capsule, or tablet) is also within the scope of the present invention.

[00204] In certain embodiments, the Fc-containing polypeptides of the invention can be administered by an invasive route, such as by injection (see previously). In some embodiments of the invention, the Fc-containing polypeptides of the invention, or pharmaceutical composition thereof, are administered intravenously, subcutaneously, intramuscularly, intra-arterially, intra-articularly (e.g., into arthritic joints), intratumorally, or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., orally; e.g., in a pill, capsule, or tablet) is also within the scope of the present invention.

[00205] The compositions may be administered with medical devices known in the art. For example, a pharmaceutical composition of the invention may be administered by injection with a hypodermic needle including, for example, a pre-filled syringe or autoinjector.

[00206] The pharmaceutical compositions of the invention may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Patents 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.

[00207] The pharmaceutical compositions of the invention may also be administered by infusion. Examples of well-known implants and modules for administering the pharmaceutical compositions include: U.S. Patent 4,487,603, which discloses an implantable microinfusion pump for dispensing medication at a controlled rate; U.S. Patent 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent 4,447,224, which discloses an implantable variable-flow infusion apparatus for continuous drug delivery; U.S. Patent 4,439,196, which discloses an osmotic drug delivery system with multi-chamber compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[00208] Alternatively, the antibody can be administered at a site other than systemically, e.g., by injecting the antibody directly into an arthritic joint, often in a depot or sustained release formulation. Furthermore, the antibody can be administered in a targeted drug delivery system, e.g., in a liposome coated with a tissue-specific antibody that targets, e.g., the arthritic joint, or pathogen-induced lesion characterized by immunopathology. The liposomes will be selectively targeted and taken up by the affected tissue.

[00209] The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic antibody to effect improvement in the target disease state while minimizing unwanted side effects. Accordingly, the amount of biological product delivered depends in part on the particular therapeutic antibody and the severity of the condition being treated. Guidelines on selecting appropriate doses of therapeutic antibodies are available (see, for example, Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert, et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J Med 342:613-619; Ghosh et al. (2003) New Engl. J Med 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602).

[00210] Determination of the appropriate dose is performed by the physician, for example, using parameters or factors known or suspected in the art to perform the treatment. In general, the dose starts with an amount slightly less than the optimal dose and increases in small amounts from there until the desired or optimal effect is achieved with respect to any negative side effects. Important diagnostic measures include those of symptoms, for example, of inflammation or level of inflammatory cytokines produced. Preferably, a biological substance to be used is derived from the same species as the animal targeted for treatment, thereby minimizing any immune response to the reagent. In the case of human subjects, for example, chimeric, humanized and fully human Fc-containing polypeptides are preferred.

[00211] The Fc-containing polypeptides can be delivered by continuous infusion, or by doses administered, e.g., daily, 1-7 times per week, weekly, bi-weekly, monthly, bi-monthly, quarterly, semi-annually, annually, etc. The doses can be delivered, e.g., intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. A total weekly dose is generally at least 0.05 μg/kg of body weight, more generally at least 0.2 μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.25 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 5.0 mg/mL, 10 mg/kg, 25 mg/kg, 50 mg/kg, or more (see, e.g., Yang et al., New Engl. J. Med. 349:427-434 (2003); Herold et al., New Engl. J. Med. 346:1692-1698 (2002); Liu et al., J. Neurol. Neurosurg. Psych. 67:451-456 (1999); Portielji et al., Cancer Immunol. Immunother. 52:133-144 (2003). In other embodiments, an Fc-containing polypeptide of the present invention is administered subcutaneously or intravenously on a weekly, biweekly, “every 4 weeks,” monthly, bimonthly, or quarterly basis at 10, 20, 50, 80, 100, 200, 500, 1,000, or 2,500 mg/subject basis.

[00212] As used herein, the terms “therapeutically effective amount,” “therapeutically effective dose,” and “effective amount” refer to an amount of an Fc-containing polypeptide of the invention that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective in causing a measurable improvement in one or more symptoms of a disease or condition, or the progression of such a disease or condition. A therapeutically effective dose further refers to that amount of the Fc-containing polypeptide sufficient to result in at least partial improvement of symptoms, e.g., treatment, healing, prevention, or amelioration of the relevant medical condition, or an increase in the rate of treatment, healing, prevention, or amelioration of such conditions. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied in a combination, a therapeutically effective dose refers to the combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously. An effective amount of a therapeutic agent will result in an improvement in a diagnostic measure or parameter by at least 10%; generally by at least 20%; preferably at least about 30%; more preferably by at least 40%, and most preferably by at least 50%. An effective amount may also result in an improvement in a subjective measure, in cases where subjective measures are used to assess disease severity.

EXAMPLE 1 Strains and reagents

[00213] Escherichia coli strains TOP10 or DH5α (Invitrogen, CA) were used for recombinant DNA run. Restriction endonucleases, DNA modification enzymes, and PNGase F were obtained from New England Biolabs, Ipswich, MA. Oligonucleotides were ordered from Integrated DNA Technologies, Coralville, IA.

EXAMPLE 2 Construction of anti-Her 2 IgG1 Fc muteins and recombinant Pichia pastoris expression vector

[00214] Preparation of single and double Fc muteins of the Her2 IgG1 monoclonal antibody in Pichia pastoris was performed using the sequences and protocols listed below.

A. Heavy and light chains

[00215] The heavy and light chain sequences, SEQ ID NOS: 1 and 2, respectively, used for the preparation of the Her2 IgG1 monoclonal antibody, are as follows. The amino acid sequence of the anti-Her2 double mutein antibody heavy chain is shown in SEQ ID NO:9. The heavy and light chains were codon optimized according to the codon usage of Pichia pastoris, and were synthesized by GeneArt AG (Josef-Engert-Str. 11, D-93053 Regensburg, Germany) and cloned into pUC19.

[00216] Fc mutations to alanine at amino acid position F243 were performed using a QuikChange® site-directed mutagenesis kit (Strategene, CA) using the sense and reverse oligonucleotide primers, FcF243A-F (SEQ ID NO: 3) and FcF243A-R (SEQ ID NO: 4), respectively. Similarly, mutations at amino acid position V264 were performed using the sense and reverse oligonucleotide primers, V254A-F (SEQ ID NO: 5) and V264A-R (SEQ ID NO: 6), respectively. Double mutation was performed using enzymatic digestion between the F243A and V264A sites of each of the single mutein plasmids followed by ligation.

B. Signal sequence

[00217] The signal sequence of an α-combining factor predomain was fused in alignment to the 5' end of the light or heavy chain by fusion PCR. The sequence was codon optimized as previously described. An AAACG Kozak sequence was added to the 5' end of the methionine, and an EcoR1 site was added before the Kozak sequence for cloning purposes. The DNA sequence (SEQ ID NO: 7) and amino acid translation (SEQ ID NO: 8) are as shown below.

C. Recombinant plasmids for expression of IgG1 and IgG1 Fc muteins

[00218] Heavy and light chains with the fused IgG1 signal sequence and their muteins were cloned downstream of the Pichia pastoris AOX1 promoter and in front of the S. cerevisiae Cyc terminator, respectively. The expression cassette of the complete heavy and light chains was placed together with the final expression vector. Genomic insertion into Pichia pastoris was achieved by linearization of the vector with Spe1 and targeted integration at the Trp2 site.

[00219] A summary of the plasmids used herein is provided below in Table 1. A graphical representation of the final expression plasmid for the Her2 dual Fc mutein is shown in Figure 1.TABLE 1

EXAMPLE 3 Pichia GFI5.0 and GFI6.0, glycoengineered hosts, to produce anti-Her2 and its Fc muteins

[00220] Two different glycoengineered Pichia hosts have been applied in this invention, GFI5.0 and GFI 6.0. Following the procedures disclosed in Gerngross U.S. 7,029,872 and Gerngross U.S. 7,449,308, one can construct vectors that are used to genetically modify lower eukaryotic host cells such that they are capable of expressing a desired polypeptide with a desired N-glycoform as the predominant species. Strains GFI 5.0 and GFI6.0 were genetically modified from NRRL11430 (American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, USA) according to the methods described in Hamilton et al., Science, 313: 1441–1443 (2006) and Hamilton U.S. 2006/0286637. The genetically modified Pichia pastoris strain GFI5.0 is capable of producing proteins with a biantennary galactose-terminal N-glycan structure. The genotype of the GFI5.0 strain used here, RDP697, is as follows: ura5Δ::ScSUC2 ochlΔ::lacZ bmt2 Δ::lacZ/KIMNN2-2 mnn4L1 Δ::lacZ/MmSLC35A3 pnol Δ::lacZADE1::lacZ/FB8/NA10/MmSLC35A3 his1::lacZ-URA5-lacZ/XB33/SpGALE/DmUGT arg1::HIS1/KD53/TC54. The genotype of the genetically modified Pichia pastoris GFI 6.0 strain, YGLY3582, is as follows: ura5Δ::ScSUC2 och1Δ::lacZ, bmt2Δ::lacZ/KlMNN2-2 mnn4L1Δ::lacZ/MmSLC35A3, pno1Δmnn4Δ::lacZ met16Δ::lacZ,his1Δ::lacZ/ScGAL10/XB33/DmUGT, arg1Δ::HIS1/KD53/TC54,ADE1::lacZ/NA10/MmSLC35A3/FB8, PRO1::lacZ-URA5-lacZ/TrMDS1, TRP2:ARG1/MmCST/HsGNE/HsCSS/HsSPS/MmST6-33Y. Strain GFI 6.0 is capable of producing proteins with a biantennary N-glycan structure, in which the terminal α2,6-linked sialic acid is attached to galactose.

[00221] Abbreviations used to describe genotypes are commonly known and understood by those skilled in the art, and include the following abbreviations:ScSUC2 S. cerevisiae invertaseOCH1 Alpha-1,6-mannosyltransferaseKlMNN2-2 K. lactis UDP-GlcNAc transporterBMT1 Beta-mannose transfer (beta-mannose elimination)BMT2 Beta-mannose transfer (beta-mannose elimination)BMT3 Beta-mannose transfer (beta-mannose elimination)BMT4 Beta-mannose transfer (beta-mannose elimination)MNN4L1 MNN4 type 1 (charge elimination)MmSLC35A3 Mouse homolog of UDP-GlcNAc transporterPNO1 Phosphomannosylation of N-glycans (charge elimination)MNN4 Mannosyltransferase (charge elimination) cargo)ScGAL10 UDP-glucose 4-epimeraseXB33 HsGalT1 truncated and fused to majorScKRE2DmUGT UDP-Galactose transporterKD53 DmMNSII truncated and fused to majorScMNN2TC54 RnGNTII truncated and fused to majorScMNN2NA10 HsGNTI truncated and fused to majorPpSEC12FB8 MmMNS1A truncated and fused to majorScSEC12TrMDS1 Secreted MNS1 from T. reseeiADE1 N-succinyl-5-aminoimidazole-4-carboxamideribotide (SAICAR) synthetaseMmCST Mouse CMP-sialic acid transporterHsGNE Human UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinaseHsCSS Human CMP-sialic acid synthaseHsSPS N-acetylneuraminate-9-phosphate synthase of human MmST6-33 Mouse alpha-2,6-salyl transferase truncated and fused to major ScKRE2LmSTT3d Catalytic subunit of oligosaccharyltransferase of Leishmania major

EXAMPLE 4 Yeast transformation and selection

[00222] Glycoengineered strains GFI5.0 and GS6.0 were grown in rich YPD medium (1% yeast extract, 2% peptone, and 2% dextrose), harvested in logarithmic phase by centrifugation, and washed three times with ice-cold 1 M sorbitol. One to five μg of a Spe1-digested plasmid was mixed with competent yeast cells, and electroporated using a Bio-Rad Gene Pulser XcellTM preset Pichia pastoris electroporation program (Bio-Rad, 2000 Alfred Nobel Drive, Hercules, CA 94547). After one hour in rich recovery medium at 24 °C, cells were plated on a plate with minimal dextrose medium (1.34% YNB, 0.0004% biotin, 2% dextrose, 1.5% agar) containing 300 μg/mL zeocin and incubated at 24 °C until transformants appeared.

[00223] To evaluate high titer strains, 96 transformants were inoculated in buffered glycerol complex medium (BMGY) and grown for 72 hours, followed by a 24-hour induction in buffered methanol complex medium (BMMY). Antibody secretion was assessed by a protein A microbead assay as follows. Fifty microliters of supernatant from 96-well plate cultures was diluted 1:1 with 50 mM Tris pH 8.5 in a 96-well plate assay without binding. For each 96-well plate, 2 mL of BioMag protein A magnetic microbead suspension (Qiagen, Valencia, CA) was plated in a tube maintained on a magnetic rack. After 2-3 minutes, when the microbeads were collected in the tube, the buffer was allowed to decant. The beads were washed three times with a volume of wash buffer equal to the original volume (100 mM Tris, 150 mM NaCl, pH 7.0) and resuspended in the same wash buffer. Twenty μL of beads were added to each well of the assay plate containing diluted samples. The plates were covered, vortexed gently, and then incubated at room temperature for 1 h, vortexing every 15 min. After incubation, the sample plate was placed on a magnetic plate that induced the beads to collect on one side of each well. In the Biomek NX Liquid Handler (Beckman Coulter, Fullerton, CA), the plate supernatant was removed to a waste container. The sample plate was then removed from the magnet, and the beads were washed with 100 μL of wash buffer. The plate was placed back on the magnet before the wash buffer was removed by aspiration. Twenty μL of filling buffer (Invitrogen E-PAGE gel filling buffer containing 25 mM NEM (Pierce, Rockford, IL)) was added to each well, and the plate was vortexed briefly. After centrifugation at 500 rpm in a Beckman Allegra 6 centrifuge, samples were incubated at 99 °C for five minutes and then run on a precast high-throughput E-PAGE gel (Invitrogen, Carlsbad, CA). Gels were overlaid with gel staining solution (0.5 g Coomassie brilliant blue G250, 40% MeOH, 7.5% acetic acid), heated in a microwave for 35 seconds, and then incubated at room temperature for 30 minutes. Gels were destained in distilled water overnight. High titer colonies were further selected by Sixfors fermentation selection, described in detail in Example 5. A summary of the strains producing wild-type (parental) IgG1 and Fc mutein is provided below in Table 2.TABLE 2

EXAMPLE 5 Bioreactor Selection (Sixfors)

[00224] Fermentation in the bioreactor selection was performed as follows: fed-batch fermentations of glycoengineered Pichia pastoris were carried out in 0.5 L bioreactors (Sixfors multifermentation system, ATR Biotech, Laurel, MD) under the following conditions: pH 6.5, 24 °C, 300 mL air flow/min, and an initial agitation speed of 550 rpm, with an initial working volume of 350 mL (330 mL BMGY medium [100 mM potassium phosphate, 10 g/L yeast extract, 20 g/L peptone (BD, Franklin Lakes, NJ), 40 g/L glycerol, 18.2 g/L sorbitol, 13.4 g/L YNB (BD, Franklin Lakes, NJ), 4 mg/L biotin] and inoculum of 20 mL). The IRIS multifermenter software (ATR Biotech, Laurel, MD) was used to increase the agitation speed from 550 rpm to 1200 rpm linearly between hours 1 and 10 of fermentation. Consequently, the dissolved oxygen concentration varied naturally during fermentation. Fermentation was carried out in batch mode until the initial glycerol charge (40 g/L) was consumed (typically 18–24 hours). A second batch phase was initiated by adding 17 mL of a glycerol feed solution to the bioreactor (50% [w/w] glycerol, 5 mg/L biotin, and 12.5 mL/L PTM1 salts (65 g/L FeSO4.7H2O, 20 g/L ZnCl2, 9 g/L H2SO4, 6 g/L CuSO4.5H2O, 5 g/L H2SO4, 3 g/L MnSO4.7H2O, 500 mg/L CoCl2.6H2O, 200 mg/L NaMoO4.2H2O, 200 mg/L biotin, 80 mg/L NaI, 20 mg/L H3BO4). Fermentation was again performed in batch mode until the added glycerol was consumed (typically 6–8 h). The induction phase was initiated by feeding a methanol solution (100% [w/w] methanol, 5 mg/L biotin, and 12.5 mL/L PTM1 salts) at 0.6 g/h, typically for 36 h prior to harvest. The total volume was removed from the reactor and centrifuged in a Sorvall Evolution RC centrifuge equipped with an SLC-6000 rotor (Thermo Scientific, Milford, MA) for 30 min at 8,500 rpm. The cell mass was discarded, and the supernatant was kept for purification and analysis. Glycan quality was assessed by MALDI time-of-flight (TOF) spectrometry and 2-aminobenzidine (2-AB) labeling according to Li et al., Nat. Biotech. 24(2): 210–215 (2006). Glycans were released from the antibody by PNGase-F treatment and analyzed by MALDI-TOF to confirm the glycan structures. To quantify the related amounts of charged and neutral glycans present, glycans released by N-glycosidase F were labeled with 2-AB and analyzed by HPLC.

EXAMPLE 6 Bioreactor cultivations

[00225] Fermentations were performed in 3 L (Applikon, Foster City, CA) and 15 L (Applikon, Foster City, CA) glass bioreactors in place of a 40 L stainless steel steam bioreactor (Applikon, Foster City, CA). Seed cultures were prepared by inoculating BMGY medium directly with frozen stock vials at a volumetric ratio of 1%. Seed vials were incubated at 24 °C for 48 h to obtain an optical density (OD600) of 20 ± 5 to ensure that cells were exponentially grown upon transfer. The culture medium contained 40 g of glycerol, 18.2 g of sorbitol, 2.3 g of K2HPO4, 11.9 g of KH2PO4, 10 g of yeast extract (BD, Franklin Lakes, NJ), 20 g of peptone (BD, Franklin Lakes, NJ), 4 x 10-3 g of biotin, and 13.4 g of yeast nitrogen base (BD, Franklin Lakes, NJ) per liter. The bioreactor was inoculated with a 10% volumetric ratio of seed to starting medium. Cultivations were performed in batch mode under the following conditions: temperature adjusted to 24 ± 0.5 °C, pH controlled at 6.5 ± 0.1 with NH4OH, dissolved oxygen was maintained at 1.7 ± 0.1 mg/L by cascade agitation rate upon addition of O2. The air flow rate was maintained at 0.7 vvm. After the initial glycerol load (40 g/L) was depleted, a 50% glycerol solution containing 12.5 mL/L PTM1 salts was fed exponentially at 50% of the maximum growth rate for eight hours until 250 g/L dry cell weight was reached. Induction was initiated after a thirty-minute starvation phase, when methanol was fed exponentially to maintain a specific growth rate of 0.01 h-1. When an oxygen uptake rate of 150 mM/L/h was reached, the methanol feed rate was kept constant to avoid oxygen limitation.

EXAMPLE 7 Antibody purification

[00226] Purification of secreted antibody can be performed by those skilled in the art using available published methods, e.g., Li et al., Nat. Biotech. 24(2):210-215 (2006), wherein antibodies are captured from the fermentation supernatant by protein A affinity chromatography, and further purified using hydrophobic interaction chromatography with a phenyl sepharose fast flow resin.

EXAMPLE 8 MALDI-TOF analysis of glycans

[00227] N-glycans were analyzed as described in Choiet al., Proc. Natl. Acad. Sci. USA 100:5022-5027 (2003) and Hamilton et al., Science 301:1244-1246 (2003). After the glycoproteins were reduced and carboxymethylated, the N-glycans were released by treatment with peptide-N-glycosidase F. The released oligosaccharides were recovered after precipitation of the protein with ethanol. Molecular weights were determined using a Voyager PRO linear MALDI-TOF mass spectrometer (Applied Biosystems) with slow extraction according to the manufacturer's instructions. The results of N-glycan analysis of the anti-Her2 antibodies produced according to the previous examples are shown in Figures 4-8.

EXAMPLE 9 HPLC analysis of N-linked glycan

[00228] To quantify the relative amount of each glycoform, glycans released by N-glycosidase F were labeled with 2-aminobenzidine (2-AB) and analyzed by HPLC as described in Choi et al., Proc. Natl. Acad. Sci. USA 100:5022–5027 (2003) and Hamilton et al., Science 313:1441–1443 (2006). The amounts of sialylated anti-Her2 antibody produced in a GFI 6.0 strain for the single and double muteins produced in 3 L bioreactors, and for wild-type and double mutein produced in a small-scale 0.5 L bioreactor are shown in Table 3. TABLE 3

EXAMPLE 10 Antigen affinity assay

[00229] Mammalian cells expressing the antigen were harvested by trypsinization, filtered through a 40 μm cell strainer, and suspended in 1% fetal bovine serum (FBS) in phosphate buffered saline (PBS) in a 96 deep-well plate. Serial dilutions of the purified antibody were added to the cells at final concentrations ranging from 10 to 0.01 μg mL-1. The antibody and cell mixture was incubated for 45 minutes on ice. The cells were washed in ice-cold PBS and stained with 2 μg mL-1 anti-human IgG-AlexaFluor488 (Invitrogen, Carlsbad, CA) in 1% FBS in PBS for 45 minutes on ice in the dark. Cells were washed again in ice-cold PBS, suspended in 1% FBS in PBS, and transferred to a 96-well U-bottom plate (USA Scientific, Ocala, FL). Mean fluorescence intensity (MFI) was detected on a Guava ExpressPlus (Millipore, Billerica, MA) using an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The results are shown in Figure 3.

EXAMPLE 11 FCYR binding assay

[00230] FCY receptor binding assays were performed as described in Shields et al., J. Biol. Chem. 276:6591–6604 (2001), with minor modifications. Protein-rich 96-well plates (Corning Costar, Lowell, MA) were coated with 100 μL per well of FCY receptor solutions in PBS at the following concentrations: 1 μg/mL for FcyRI (R&D Systems) and Rc/RIIa (produced by Pichia pastoris), 2 μg/mL for FcyRIIb/c (produced by P. pastoris), 0.4 μg/mL for FcYRIIIa-V158, and 0.8 μg/mL for FcYRIIIa-F158 (both produced by P. pastoris). The FcYRIIIa-V158 and FcYRIIIa-F158 receptors were expressed using P. pastoris in the manner described in Li et al., Nat. Biotech. 24:210-215(2006).

[00231] FcYRIIa was also expressed in glycoengineered Pichia using a similar method described in Li et al. The FcYRIIa extracellular domain was PCR amplified from human cDNA and cloned into the pCR2.1 topo vector. Fc gamma receptors were cloned into the Pichia expression vector using the S. cerevisiae combining factor alpha pre-pro domain, and downstream of the AOX1 promoter. The final strain, yGLY4665, was generated by transforming pGLY3249 into yGLY638 (GFI2.0 host).

[00232] FcYRIIb/c was expressed and produced using glycoengineered Pichia YGLY638 (host GFI2.0). The DNA sequence of the extracellular domain of the human Fc gamma IIb/c receptor (NP_003992) carrying its C-terminal His-tag 9 was codon optimized from Pichia and determined in pAS197 (GeneArt, Germany). The amino acid sequence of the histidine-tagged FcYRIIb/c extracellular domain can be found in SEQ ID NO:17. For the construction of plasmid pGLY3246, the codon-optimized hFcYRIIb/c (AfeI/KpnI) and αMFprepro from Saccharomyces cerevisiae (EcoRI/blunt) were cloned into pGLY2219, at the EcoRI and KpnI sites. The resulting plasmid pGLY3246 was transformed into yGLY638 to generate yGLY4653. YGLY4653 was fermented and purified according to Li et al.

[00233] For FcYRI, the antibody was coated in assay diluent (1% BSA, PBS, 0.05% Tween20) in monomeric form. For all other receptors, the antibody was coated after dimerization with alkaline phosphatase-conjugated anti-human IgG F(ab')2 (Jackson ImmunoResearch, West Grove, PA) for one hour at room temperature. Antibody bound to FCYRI was also detected using F(ab')2, and all plaques were quantified by measuring excitation at 340 nm and emission at 465 nm after an 18-hour incubation with SuperPhos (Virolabs, Chantilly, VA).

[00234] The results are shown in Figure 9. As shown in Figure 9A, the single Fc muteins (▲ and ^) displayed binding to FcyRI (Fc receptor gamma chain I, CD64) similar to both the Her2 antibody (■) (Figure 9A) and Pichia pastoris Her2 (data not shown), whereas the double Fc mutein (♦) displayed an approximately fourteen-fold decrease in affinity for FcyRI (Figure 9A).

[00235] For FcyRIIb/c, the single Fc muteins (▲ and ^) demonstrated a tenfold decrease in receptor binding properties compared to the Her2 antibody (■) (Figure 9B), while the double Fc mutein does not appear to bind to FcyRIIb/c.

[00236] FcyRIIIa-F158 and FcyRIIIa-V158, both single Fc muteins (▲ and ▼), bind twenty-fold better than the commercial antibody Herceptin (■) to FcyRIIIa-F158 (Figure 9C), but bind FcyRIIIa-V158 only slightly better than the commercial antibody Herceptin (Figure 9D). The double Fc mutein (♦) showed little affinity (50-fold lower) for FcyRIIIa-F158 (Figure 9C), although it maintained some affinity for FcyRIIIa-V158, although thirty-fold weaker than the commercial antibody Herceptin (■) and the Her2 antibody from Pichia pastoris (data not shown) from GS5.0 (Figure 9D).

[00237] Furthermore, the affinity of the double Fc mutein for both FcyRIIIa polymorphisms does not appear to change upon release of sialic acid by neuraminidase (data not shown). Thus, without wishing to be bound by theory, Applicants attribute the decrease in affinity for both FcyRIIIa polymorphisms to structural or conformational changes resulting from the double mutation, i.e., double Fc mutein, and not to increased levels of α2,6-linked sialylated N-glycans.

EXAMPLE 12 C1q binding assay for anti-Her2 antibodies and their Fc muteins

[00238] The C1q binding assay was performed using the methods described by Idusogie et al., J. Immunology, 164: 4178-4184 (2000). Serially diluted antibodies were coated at 100 μL per well in 50 mM Na2HCO3 pH9.0 to highlight highly bound plaques. Human complement C1q (US Biological, Swampscott, MA) was coated with 2 μg/mL assay diluent (0.1% bovine gelatin, PBS, 0.05% Tween20) for two hours. C1q was detected with HRP-conjugated sheep polyclonal anti-human C1q antibody (AbDSerotec), and quantified by measuring OD450.

[00239] The results are shown in Figure 10. As shown in Figure 10, the single and dual Fc mutein antibodies produced by the materials and methods described herein exhibited less binding to C1q relative to those produced in mammalian cell culture or non-sialylated Pichia pastoris strains. C1q binding to antibodies produced from the single Fc muteins (▲ or ▼) was decreased 5-10-fold relative to the Her2 antibody, although C1q binding to the dual Fc mutein (♦) was virtually eliminated.

[00240] Both the Pichia pastoris Her2 antibody produced in a GFI5.0 strain (data not shown) and the Her2 antibody showed similar affinity to C1q.

EXAMPLE 13 Antibody-dependent cellular toxicity to anti-Her2 and its Fc muteins

[00241] Antibody-dependent cellular toxicity (ADCC) was measured using a europium incorporation assay. The human ovarian adenocarcinoma cell line SKOV3 was cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS). Peripheral blood mononuclear cells (PBMCs) were obtained from Leuco-paks. PBMCs were subjected to Ficoll-Hypaque density centrifugation, washed in PBS supplemented with 2% FBS, and resuspended in McCoy's 5A medium with 10% FBS. The FCYRIIIA F158V genotype was determined for each individual donor. SKOV3 cells were labeled with EuDTPA. Cells were added to 96-well tissue culture plates at 5,000/well, and effector cells (PBMC) were added at 300,000/well (E:T 60:1). Antibodies were added at different concentrations and diluted across the plate. Controls included background, target, and effector baseline counts, and 100% lysed wells. PBMC mixtures with and without antibody were incubated for four hours at 37 °C. EuDTPA release from lysed PBMC was determined by mixing 20 μL of the supernatant with DELPHIA enhancement solution (Perkin Elmer, cat# 1244-105) to form a highly fluorescent chelate, which is measured using time-resolved fluorometry. Triplicate wells were established for each antibody dilution, and percent lysis was calculated according to the formula: ((experimental release - background)/(maximal release - background)) - ((natural cytotoxicity - background)/(maximal release - background)). Terms are defined as follows: experimental release represents the mean count for target cells in the presence of effector cells and antibody, background represents the mean for the final wash supernatant after labeling target cells, maximum release represents the mean for target cells incubated with DELPHIA lysis buffer (Perkin Elmer, cat# 4005-0010), and natural cytotoxicity represents the mean count for target cells in the presence of effector cells. Data points were fit to a four-parameter logistic regression model using Prism 5.0 software. All curves were forced to share the same maximum, minimum, and slope.

[00242] Those skilled in the art can recognize and understand that the assays in Examples 10-12 can be readily adapted to the requirements pertaining to any immunoglobulin molecule. Furthermore, an in vivo ADCC assay in an animal model can be adapted to any specific IgG using the methods of Borchmann et al., Blood, 102:3737-3742 (2003); Niwa et al., Cancer Research, 64:2127-2133 (2004).

[00243] The results are shown in Figure 11. The single Fc muteins showed an ADCC activity profile similar to that of the Her2 antibody, and approximately five-fold lower than the Pichia pastoris Her2 antibody produced in a GFI 5.0 host. As illustrated in Figure 11, ADCC activity is virtually absent for the antibody produced from the double Fc mutein, as expected from the FcYRIIIa binding data.

EXAMPLE 14 Subcutaneous PK study for anti-Her2 and its Fc muteins

[00244] The subcutaneous PK study was performed in C57B6 mice. Antibody samples were administered subcutaneously at a dose of 1 mg/kg (n=3). Ten μL of blood was collected with a capillary tube at the collection time points of 1, 6, 24, 53, 77, 120, 192, 240, 288, and 360 hours, and transferred to a microcentrifuge tube containing 90 μL of PBS without calcium and magnesium and 1.8 mg/mL K2EDTA. Human IgG levels were determined on a Gyros® Bioaffy workstation (Uppsala, Sweden) using a sandwich immunoassay. 100 μg/mL biotinylated mouse monoclonal anti-human kappa chain (BD Pharmingen, San Diego, CA) was used as the capture antibody, and 12.5 nM ALEXA-647-labeled mouse monoclonal anti-human Pan IgG Fc (Southern Biotech, Birmingham, AL) was used as the detection antibody. The entire immunoassay was automated, from capture antibody immobilization and analyte addition, to detection antibody addition and washing steps. Standards and QC were prepared in 5% mouse control plasma (EDTA) as a 20x stock, and diluted 1:20 in 5% mouse plasma in assay buffer (PBS, 0.01% Tween) prior to analysis. The linear range for accurate determination of IgG concentration was established with QC highlighted and noted as 5–5,000 ng/mL. Acceptable limits of accuracy and precision were +/- 20% with a lower limit of quantitation (LLOQ) of +/- 25%. Standards and QC could be frozen and thawed three times without significant loss of signal. Standards, QC, and study samples were stored at -70°C. Study samples were thawed and diluted 1:20 in 5% mouse plasma in assay buffer, and further diluted 1:10 if levels were outside the linear assay range. All standards, QC, and study samples were assayed in duplicate and mean results were reported. Concentrations were determined using a 5th parameter logistic curve fitting. Pharmacokinetic parameters were calculated for each animal with WinNonlin using noncompartmental analysis of serum mAb concentration-time data (WinNonlin Enterprise Version 5.01, Pharsight Corp, Mountain View, CA).

[00245] As shown in Figure 12, mice treated with glycosylated Pichia pastoris Her2 and GFI5.0 Her2 exhibited similar t 1/2 and serum concentrations, while mice treated with the dual Fc mutein Her2 produced with GFI6.0 glycosylation exhibited a higher Cmax (maximum concentration) and approximately a 30% increase in serum concentration compared to mice treated with CHO cell-produced Her2. Thus, of the three subcutaneously injected samples, the dual Fc mutein antibody exhibited better bioavailability (absorption or exposure) based on higher Cmax and serum concentrations.

EXAMPLE 15

[00246] Another set of FCY receptor binding assays for the anti-Her2 antibody of the invention and its Fc muteins was performed in the manner described in Example 11, and the results are shown in Figure 13; and Tables 4 and 5. TABLE 4 Comparison of FCYR binding affinity to Her 2 antibody Ratio calculation: STD EC50/anti-antigen mAbRatio > 1.0 higher affinity than Her2 AbRatio < 1.0 lower affinity than Her 2 AbTABLE 5Comparison of FcyR binding affinity on Pichia Her 2 antibody Ratio calculation: STD EC50/anti-antigen mAbRatio > 1.0 higher affinity than Pichia Her2 AbRatio < 1.0 lower affinity than Pichia Her 2 Ab

EXAMPLE 16 ADCC evaluation of anti-HER2 antibodies and Fc muteins

[00247] ADCC analysis was performed using SKOV3 target cells (ATCC, Cat# HTB-77). The day before the assay, primary effector NK cells (Biological Specialty, Cat# 215-11-10) were precipitated at 1,000 rpm for 15 minutes, and resuspended at 1 x 106 cells/mL in RPMI without phenol red medium (Invitrogen, catalog# 11835-030) supplemented with 10% FBS (Cellgro, Cat# 35-016-CV). Resuspended NK cells were incubated overnight at 37°C in 5% CO2.

[00248] On the day of the assay, a vial with adherent SKOV3 target cells was washed with PBS, and the cells were detached using 3 mL of trypsin (Cellgro, Cat# 25-053-CI) and incubated for 2–5 minutes at 37 °C. Cells were harvested with 23 mL of phenol red medium without RPMI, 10% FBS, and pipetted up and down to break up clumps. Harvested cells were centrifuged at 1,800 rpm for 5 minutes and resuspended at a concentration of 1 x 107 cells/mL with phenol red medium without RPMI, 10% FBS. Target cells (1 x 107 cells) were labeled with 100 μCi of chromium-51 (5 mCi of sodium chromate in normal saline, Perkin Elmer, Cat# NEZ03005MC). Target cells were incubated at 37 °C for 1 h with shaking every 15 min. Cells were centrifuged at 1,800 rpm for 2 min and resuspended in 1 mL phenol red medium without RPMI, 10% FBS. Cells were washed twice more with 1 mL phenol red medium without RPMI, 10% FBS, with centrifugation at 1,800 rpm for 2 min between each wash. After the final wash, labeled target cells were resuspended in phenol red medium without RPMI, 10% FBS, at a final concentration of 2.5 × 105 cells/mL.

[00249] The test antibodies used in these assays were anti-HER2 mAb produced in GFI 5.0, anti-HER2 mAb F243A produced in GFI 6.0 (GS6.0/F243A), anti-HER2 mAb V264A produced in GFI 6.0 (GS6.0/V264A), and anti-HER2 mAb F243A/V264A produced in GFI 6.0 (GS6.0/F243A/V264A). Although the SKOV3 target cells were labeled, the test antibodies were diluted using a 3-fold serial titration (starting at 1 μg/mL) in phenol red medium without RPMI, 10% FBS, in a 96-well polystyrene plate (Costar, Cat# 353077). To the wells of a separate 96-well assay plate, 100 μL of Cr-51-labeled SKOV3 target cells (= 25,000 cells) were transferred. After the antibody dilution plate was prepared, 10 μL of each dilution was transferred to the 96-well assay plate containing the labeled target cells. For controls, 10 μL of Triton-X100 (10% stock, Fluka Analytical, Cat # 93443) or 10 μL of medium was added to the “maximum lysis” or “spontaneous release” control wells, respectively. Each antibody dilution was tested in duplicate, while 6 replicates of each control were tested.

[00250] Primary NK cells were precipitated at 1,200 rpm for 5 minutes and gently resuspended at 2.5 x 106 cells/mL in phenol red medium without RPMI, 10% FBS. In all sample wells, and in the “no antibody” control wells (i.e., excluding the “spontaneous release” and “maximal lysis” controls), 100 μL of NK cells (= 250,000 cells) were added at an effector:target ratio of 10:1. The assay plate was incubated at 37 °C in 5% CO2 for four hours. After incubation, the assay plate was centrifuged at 300 rpm for 5 minutes. Thirty μL of the supernatants were added to 250 μL of Microscint 20 (Perkin Elmer, Cat # 6013621) in a 96-well picoplate (Perkin Elmer, Cat # 6005185). Cr-51 release was measured in a Packard Top Count scintillation counter.

[00251] Percent lysis was calculated as: ((experimental ADCC release - spontaneous release) / (maximal release - spontaneous release)) * 100

[00252] The results of these assays are presented in Figure 14. As shown in Figure 14, the single mutein antibody F243A and the single mutein antibody V264A showed a 5-10 fold reduction in ADCC activity when compared to the parent (wild type) antibody; while the double mutein antibody F243A/V264A showed a greater than 100 fold reduction in ADCC activity when compared to the parent (wild type) antibody.

EXAMPLE 17 Statistical analysis of ADCC data of anti-HER2 antibody and its Fc muteins

[00253] The objective of the study was to determine whether the percent lysis for the double mutant variant is synergistic with respect to the two single mutant variants. The determination is performed by comparing the ED50 (antibody level corresponding to 50% lysis) between the double mutant variant (group 4) and the additional reference curve evaluated (shown in figure 15 and table 6). Since the lower limit of the ED50 for the double mutant curve is below the upper level of antibody assayed, and is well below the upper limit of the ED50 for the additional reference curve, it is concluded that the effect of the double mutation is much greater than that of the additional one.

Statistical methods and results:

[00254] The objective was to determine whether the double mutation variant of the Herceptin antibody (GS6.0/F243A/V264A) demonstrates a synergistic effect, compared to the two single mutation variants (GS6.0/F243A and GS6.0/V264A). There are four groups of antibody: Group 1 (GS5.0), Group 2 (GS6.0/F243A), Group 3 (GS6.0/V264A) and Group 4 (GS6.0/F243A/V264A).

[00255] Data from three donors are run in separate 96-well plates. Observations from each plate are normalized by converting the values to percent lysis as follows: where:Orc is the response observed in row r3 and column ca of each plate.

[00256] ON is the mean response of the negative controls for each plate.

[00257] OP is the mean response of the positive controls for each plate.

[00258] Averages of the duplicate % lysis values for each antibody level and group are then performed.

[00259] Data from all four groups and all three donors are modeled together. The “response-antibody level” relationship is assumed to follow a Hill sigmoid equation (Eq. 1). (100% minus % lysis) is used as the response variable (Y) and log antibody levels as the explanatory variable (X). In order to make comparisons about the efficacy of the various mutations and to fit the models, some assumptions are made about the model parameters for each group.

[00260] The shape of the model used simulates: 1. The space (a) and the plateau (d) for all 4 treatment groups are the same. 2. The same slope for all treatment groups, except the GS5.0 group which naturally presents a different slope. 3. The EC50 parameters (γ) are different for the different groups. The Hill equation: With the constraints: γ1= 0, β1=0 and β2 = β3 = β4= β Here: Y ij is the i th response (100-% lysis) for the j th treatment X ij is the antibody level (in log scale) that corresponds to Y ij j = 1 for GS5.0 j = 2 for GS6.0/F243A j = 3 for GS6.0/V264A j = 4 for GS6.0/F243A/V264A

[00261] It is further accepted that if the two single mutants (GS6.0/F243A and GS6.0/V264A) are added to the equation, the combined effects may be: with parameters a, d, γ, γ2, γ3, β and β0 being the same as in Eq. 1.

[00262] The models (Eq. 1 and Eq. 2) are fitted using PROC NLIN in SAS v9.2. The observed data values and fitted curves for the four groups, as well as the additional accepted reference curve for the two single mutation groups combined, are displayed in Figure 15. The ED50 along with their confidence intervals (on the original scale) are given in Table 6.TABLE 6 * Estimated value is greater than the highest antibody level in the experiment.

[00263] In order to evaluate the effects of GS6.0/F243A/V264A compared to the two single mutations, GS6.0/F243A and GS6.0/V264A, the ED50 of group 4 is compared to the ED50 of the additional reference curve. If the confidence intervals do not overlap, then it can be concluded that the two quantities are statistically different.

[00264] The 95% confidence interval for the ED50 of the additional reference curve is (0.3031, 0.5455), while the estimated and confidence intervals for the ED50 of group 4 are all greater than 1 (the estimated values are outside the observed range of antibody levels). The lower 95% confidence limit for the ED50 of the double mutation is >1, which is much greater than the upper 95% confidence limit for the ED50 of the additional reference curve (groups 2 and 3 combined). Consequently, it can be concluded that the two quantities are significantly different.

[00265] The above comparison of ED50 confidence intervals is equivalent to comparing γ4 versus the sum (γ2 + γ3), i.e. testing the hypothesis that Ho: γ4 = (γ2 + γ3), versus the alternative that Ha: γ4 > (γ2 + γ3).

[00266] It is understood that reducedSSE and fullSSE are the residual sum of squares of the reduced model and the full model, respectively. The full model is obtained by fitting Eq. 1 to the data, while the reduced model is obtained by replacing γ4 in Eq. 1 with (γ2 + γ3). The hypothesis: The statistical test: The p-value:

[00267] Since the p-value is <0.0001, H0 can be rejected and it is concluded that γ4 > (γ2 + γ3).

[00268] The data and model-based comparisons indicate that the GS6.0/F243A/V264A double mutation has a significantly lower % lysis than the hypothetical combined effects of the two single mutations. The effect of the double mutation is much greater than any additive effect of the single mutations.References related to this example:[1] SAS (r) Proprietary Software 9.2 (TS2M2), SAS Institute Inc., Cionadasary, NC, USA.[2] ‘Application of the Four-Parameter Logistic Model to Bioassay: Comparison with Slope Ratio and Parallel Line Models’; Aage V0lund, Biometrics, Vol. 34, No. 3 (Sep. 1978), pp. 357-365.

EXAMPLE 18 Construction of anti-TNFα Fc muteins and ADCC evaluation

[00269] Preparation of dual Fc muteins (F243A/V264A) of an anti-TNF monoclonal antibody in Pichia pastoris was performed using the sequences and protocols listed below. The heavy and light chain sequences of the parental (wild-type) anti-TNFα antibody are set forth in SEQ ID NOs:10 and 11. The heavy chain sequence of the anti-TNFα double mutein antibody is set forth in SEQ ID NO:12. The light chain sequence of the wt and anti-TNFα double mutein antibodies are identical.

[00270] The signal sequence of an alpha combining factor predomain (SEQ ID NO:8) was fused in alignment to the end of the light chain or fusion chain by PCR. The sequence was codon optimized and synthesized by Genscript (GenScript USA Inc., 860 Centennial Ave. Piscataway, NJ 08854, USA). Both the heavy chain and light chain were cloned into the antibody expression vector in a manner similar to the construction of anti-HER2 IgG1 and its Fc muteins.

[00271] The signal sequence-fused heavy and light chains of IgG1 and their muteins were cloned downstream of the Pichia pastoris AOX1 promoter and in front of the S. cerevisiae Cyc terminator, respectively. The expression cassette of the complete heavy and light chains was placed together with the final expression vector. Genomic insertion into Pichia pastoris was achieved by linearization of the vector with Spe1 and targeted integration at the Trp2 site. Plasmid pGLY6964 encodes the wild-type IgG1 anti-TNFα antibody. Plasmid pGLY7715 encodes the anti-TNFα IgG1 double mutein F243A/V264A. Pichia GFI5.0 YGLY8316 and GFI6.0 YGLY 22834 glycoengineered hosts to produce their anti-TNFα Fc muteins.

[00272] The genotype of strain GFI5.0 YGLY16786, used for the expression of anti-TNFα antibodies and their Fc muteins, is as follows: ura5 Δ::ScSUC2 ochl Δ::lacZ bmtl Δ::lacZbmt2 Δ::lacZbmt3 Δ::lacZbmt4 Δ::lacZ/KIMNN2-2mnn4L1 Δ::lacZ/MmSLC35A3 pnol Δ::lacZADE1::lacZ/FB8/NA10/MmSLC35A3 his1::lacZ-URA5-lacZ/XB33/SpGALE/DmUGT arg1::HIS1/KD53/TC54 PRO1::ARG1/AOX1- ScMFpreTrMNS1PRO1::ARG1/AOX1-ScMFpreTrMNS1.

[00273] The genotype of the genetically modified Pichia pastoris GFI 6.0 strain, YGLY23423, used for the expression of the anti-TNFα Fc DM mutein is as follows: ura5Δ::ScSUC2 och1Δ::lacZbmt2Δ::lacZ/KlMNN2-2 mnn4L1Δ::lacZ/MmSLC35A3 pno1Δ mnn4Δ::lacZ ADE1:lacZ/NA10/MmSLC35A3/FB8his1Δ::lacZ/ScGAL10/XB33/DmUGT arg1Δ::HIS1/KD53/TC54bmt4Δ::lacZ bmt1Δ::lacZ bmt3Δ::lacZTRP2:ARG1/MmCST/HsGNE/HsCSS/HsSPS/MmST6- 33ste13Δ::lacZ/TrMDS1 dap2Δ::NatRTRP5:HygRMmCST/HsGNE/HsCSS/HsSPS/MmST6-33 Vps10-1Δ::AOX1p_LmSTT3d.

[00274] Cells were transformed, selected and purified as indicated in Examples 4-7.

[00275] Evaluation of Antibody-Dependent Cellular Cytotoxicity (ADCC) of Anti-TNFα Fc Muteins

[00276] ADCC analysis was performed using Jurkat FlpIn target cells stably expressing a non-cleavable variant of TNFα that has the first 12 amino acids removed - Jurkat FlpIn TNFα target cells (Δ1-12). These cells were prepared by transfecting human T-cell leukemia cells, Jurkat FlpIn, (Invitrogen) with a non-secretable cell surface mutant of TNFα (Δ1-12 TNFα). Perez et al., Cell 63;251-258 (1990). The Δ1-12 TNFα DNA was cloned into the pcDNA5 vector (Invitrogen) for use in transfection. Cell surface expression of TNFα was confirmed by flow cytometry.

[00277] The day before the assay, primary effector NK cells (Biological Specialty, Cat# 215-11-10) were precipitated at 1,200 rpm for 12 minutes, and gently resuspended at approximately 1 - 1.5 x 106 cells/mL in phenol red-free RPMI medium (Invitrogen, Cat# 11835030) supplemented with 10% heat-inactivated FBS (Sigma, Cat# F4135), 10 mM Hepes (Gibco, Cat# 15630), 2 mM L-glutamine (Cellgro, Cat# 25-005-CI), and 1X penicillin/streptomycin (Cellgro, Cat# 30-002-CI). Resuspended NK cells were incubated overnight at 37 °C in 5% CO2.

[00278] On the day of the assay, Jurkat FlpIn TNFα (Δ1-12) target cells were centrifuged at 1,200 rpm for 5 minutes, and the cell pellet was gently resuspended at a concentration of 2 x 106 cells/mL in phenol red medium without RPMI, supplemented with 5% heat-inactivated FBS. Cells were labeled with 25 μL of DELFIA BATDA labeling reagent (from DELFIA EuTDA Cytotoxicity Kit, Perkin Elmer, Cat# AD0116). Cells were gently mixed and incubated at 37 °C for 20 minutes with gentle agitation every 10 minutes. Cell volume was adjusted to 30 mL with DPBS containing 1.5 mM probenecid (Invitrogen, Cat# P36400), and centrifuged at 1,200 rpm for 5 min. Cells were washed three times with 30 mL of DPBS containing 1.5 mM probenecid, with centrifugation at 1,200 rpm for 5 min between each wash. After the final wash, labeled target cells were resuspended in phenol red medium without RPMI, supplemented with 5% heat-inactivated FBS, and 1.5 mM probenecid at a final concentration of 2.5 × 105 cells/mL.

[00279] The test antibodies used in the assay were: wild-type anti-TNF IgG1 antibody (termed “GFI774”) produced in GFI 5.0, non-sialylated anti-TNF IgG1 antibody (GFI774) F243A/V264A produced in GFI 5.0, and sialylated anti-TNF IgG1 antibody (GFI774) F243A/V264A produced in GFI 6.0. The non-sialylated anti-TNF IgG1 antibodies, F243A/V264A, produced in GFI 5.0 comprised the following glycoforms: 84% G2, 2% G1, and 14% hybrid N-glycans. The sialylated anti-TNF antibodies IgG1, F243A/V264A, produced in GFI 6.0 comprise the following glycoforms: 27% A1, 50.6% A2 and 5.7% hybrid A1 (overall, more than 83% of the N-glycans are sialylated).

[00280] While Jurkat FlpIn TNFα (Δ1-12) target cells were being labeled, 2X concentrations of antibodies were diluted using a 3-fold serial titration (starting at 40 μg/mL) in phenol red medium without RPMI, supplemented with 5% heat-inactivated FBS, and 1.5 mM probenecid in a 96-well round bottom polypropylene plate (Costar, Cat # 5699; lids - Costar Cat # 3931). After the dilution plate was prepared, 100 μL of each 2X dilution was transferred to a new 96-well round bottom polypropylene plate (only 100 μL of medium was transferred for “no antibody” controls). Phenol red medium without RPMI, supplemented with 5% heat-inactivated FBS, and 1.5 mM probenecid were also transferred to the 96-well plate for the “spontaneous release” and “maximal lysis” controls (150 μL per well) and the “background” controls (200 μL per well). Each antibody dilution was tested in duplicate, while each control was tested in quadruplicate.

[00281] In all wells except “background” controls, 50 μL of europium-labeled Jurkat FlpIn TNFα (Δ1-12) cells (= 12,500 cells) were added and mixed gently. Primary NK cells were precipitated at 1,200 rpm for 12 minutes, and gently resuspended at 2.5 x 106 cells/mL in phenol red-free RPMI medium supplemented with 5% heat-inactivated FBS and 1.5 mM probenecid. In all sample wells (i.e., excluding “spontaneous release”, “maximal lysis”, and “background” controls), 50 μL of NK cells (= 125,000 cells) were added at an effector:target ratio of 10:1. The samples were mixed gently and the assay plate was incubated at 37 °C for two hours. After 1 hour and 15 minutes, 10 μL of 20% Triton-X100 (Pierce Surfact-Amps X-100, Cat # 28314) or 10 μL of medium was added to the “maximum lysis” or “spontaneous release” control wells, respectively. The plate was incubated at 37 °C for an additional 45 minutes (total incubation time was 2 hours). While the plates were incubating, the DELFIA europium solution (from the DELFIA EuTDA Cytotoxicity Kit) was equilibrated to room temperature. After the two-hour incubation, the assay plate was centrifuged at 1,500 rpm for 5 minutes. Twenty μL of the supernatants were transferred to a clear, flat-bottomed white plate (from the DELFIA EuTDA Cytotoxicity kit or Costar, Cat# 3632), taking care not to introduce bubbles. To each well, 200 μL of DELFIA europium solution was added and the plate was covered with an aluminum foil seal. The assay plate was incubated at room temperature for 15 minutes with gentle shaking. Fluorescence was measured using a Perkin Elmer Envision instrument set to read europium.

[00282] Percent lysis was calculated as follows: 1) the mean of the background control was subtracted from all raw values, 2) % ADCC activity was calculated as: ((Experimental ADCC value - Spontaneous release) / (Maximum lysis - Spontaneous release)) * 100, and 3) final % lysis values were reported as % ADCC activity from step 2 minus the "no antibody" control. The results of these assays are presented in Figure 16. As shown in Figure 16, the unsialylated and sialylated F243A/V264A anti-TNFα IgG1 double mutein exhibited a greater than 1,000-fold reduction in ADCC compared to the parental (wild-type) polypeptide produced in GS5.0.

EXAMPLE 19

[00283] Evaluation of complement-dependent cytotoxicity (CDC) of anti-TNFα dual Fc muteins.

[00284] CDC analysis was performed using HEK293 FlpIn cells that stably express a non-cleavable TNFα variant that has the first 12 amino acids removed. HEK293 FlpIn TNFα (Δ1-12) cells were grown in tissue culture flasks in Dulbecco's minimum essential medium (DMEM) lacking phenol red (Gibco, Cat# 21063) supplemented with 10% heat-inactivated FBS (Sigma, Cat# F4135), 100 ug/mL hygromycin B (Cellgro, Cat# 30-240-CR), and L-glutamine (Cellgro, Cat# 25-005-CI) to 70% confluence. Cells were treated with 2 mL trypsin (Cellgro, Cat# MT25-053-CI), harvested with 8 mL phenol red medium without DMEM, 10% heat-inactivated FBS, and centrifuged at 1,200 rpm for 10 min. The supernatant was removed, and the cell pellet was resuspended in phenol red medium without DMEM, 10% heat-inactivated FBS at a concentration of 4 × 105 cells/mL. Cells were plated at 100 μL (40,000 cells) per well in a clear, dark-bottom 96-well plate (Costar, Cat# 3603), and incubated overnight at 37 °C in 5% CO2.

[00285] The test antibodies used in the assay were: anti-TNF IgG1 antibody (termed “GFI774”) produced in GFI 5.0, anti-TNF IgG1 antibody (GFI774) F243A/V264A produced in GFI 5.0, and anti-TNF IgG1 antibody (GFI774) F243A/V264A produced in GFI 6.0. (These antibodies are described in Example 18.) On the day of the assay, the medium was aspirated with a multichannel pipettor from the wells of the 96-well plate and replaced with 50 μL of phenol red medium without DMEM, penicillin/streptomycin 1X. A 2-fold serial titration of test antibody (starting at 30 μg/mL) was prepared in phenol red-free DMEM containing 1X penicillin/streptomycin, 10 μg/mL mouse anti-human CD55 IgG1 monoclonal antibody (IBGRL Research Products, Clone BRIC216, Cat # 9404P), and 10 μg/mL mouse anti-human CD59 IgG2b monoclonal antibody (IBGRL Research Products, Clone BRIC 229, Cat # 9409P). 50 μL of the diluted antibody was added to the appropriate assay plate wells. Assay negative controls were assay medium alone and human IgG (Jackson ImmunoResearch, Cat # 009-000-003) diluted in phenol red-free DMEM assay medium (containing CD55 and CD59 antibodies). The lysis control in the assay was 0.25% Triton X100 (10% stock, Fluka, Cat# 93443) diluted in phenol red assay medium without DMEM (containing CD55 and CD59 antibodies). Each test antibody concentration was assayed in duplicate, while assay controls were assayed in triplicate. The assay plate containing the test samples was mixed by gentle tapping, and the plate was incubated for approximately 10 minutes at 37 °C while human complement serum was being prepared. Three mL of human complement (QUIDEL, Cat# A113) was diluted 1:2 with 3 mL of phenol red assay medium without DMEM containing 1X penicillin/streptomycin, and 50 μL of the diluted complement was added to the wells of the assay plate. The assay plate was mixed by gentle tapping, and the plate was incubated for 4 hours at 37 °C in 5% CO2. A 40% alamar blue solution (Biosource, Cat# DAL1100) was prepared by diluting 100% stock with DMEM-free phenol red medium containing 1X penicillin/streptomycin, and 50 μL of the diluted alamar blue solution was added to the assay plate wells (= 10% final). The assay plate was mixed by gently tapping, and the plate was incubated overnight (15–20 hours) at 37 °C in 5% CO2. The next day, the assay plate was incubated for 10 minutes on a shaker at room temperature, and fluorescence was read at 544 nm excitation, 590 nm emission. The percent CDC was calculated as: (1 – (raw sample fluorescence unit (RFU) – Triton RFU) / (medium RFU – Triton RFU)) * 100

[00286] The results of these assays are presented in Figure 17. As shown in Figure 17, the unsialylated and sialylated anti-TNFα IgG1 double mutein F243A/V264A showed an approximately 10-fold reduction in CDC activity compared to the parental (wild-type) antibody.

EXAMPLE 20

[00287] Effect of anti-TNFα antibody and its Fc muteins in an antibody-collagen-induced arthritis (AIA) model.

[00288] INDUCTION MODEL: AIA (Antibody-induced arthritis) is induced with a commercial arthritogenic monoclonal antibody Arthrogen-CIA® (obtained from Chondrex), which consists of a cocktail of 5 monoclonal antibodies, clone A2-10 (IgG2a), F10-21 (IgG2a), D8-6 (IgG2a), D1-2G(IgG2b), and D2-112 (IgG2b), which recognizes epitopes conserved in several species of type II collagen.

[00289] ANIMALS: Ten-week-old male B10.RIII mice, which are susceptible to arthritis induction without the addition of costimulatory factors, were used. These animals were obtained from the Jackson Laboratory.

[00290] CLINICAL GRAding: Paw swelling was measured daily after arthritis induction. Disease severity was graded on a scale of 0-3 per paw as follows: 0, normal; 1, swelling of one digit; 2, swelling of two or more digits; 3, total paw swelling. The maximum clinical score per mouse is 12.

[00291] STUDY DESIGN: Arthritis was induced by passive transfer of 3 mg of pathogen anti-CII mAb cocktail IV on day 0.

[00292] Groups of mice were treated subcutaneously with the following reagents: Group n=5 for all groups, except groups A and H which have n=3.

[00293] An IgG1 isotype antibody was used as a control. The antibody bound to mouse anti-hexon and displayed the determination “27F11”.

[00294] The sample identified as “anti-TNF non-sialylated” corresponds to the anti-TNF antibody, as described in Example 18, produced in GFI 5.0 comprising the F243A/V264A mutations. The sample identified as “anti-TNF α2,6 sialylated” corresponds to the anti-TNF antibody, described in Example 18, produced in GFI 6.0 comprising the F243A/V264A mutations. The anti-TNF IgG1 non-sialylated antibodies, F243A/V264A, produced in GFI 5.0 comprise the following glycoforms: 84% G2, 2% G1, and 14% hybrid N-glycans. The sialylated anti-TNF IgG1 antibodies, F243A/V264A, produced in GFI 6.0 comprise the following glycoforms: 27% A1, 50.6% A2, and 5.7% hybrid A1 (overall, more than 83% of the N-glycans are sialylated).

[00295] mTNFR-Ig is an immunoadhesin (anti-TNF Ig receptor fusion protein) comprising the extracellular domain of mTNFR2 connected to the mIgG1 Fc, starting at the hinge and spanning the CH2 and CH3 regions, and comprising the amino acid sequence of SEQ ID NO:16 produced in CHO cells.

[00296] GAMMAGARD liquid was obtained from Baxter Corp.

[00297] HUMIRA was obtained from Abbott Labs. HUMIRA comprises non-sialylated N-glycans with very little terminal galactose.

[00298] All groups of mice were dosed on day pre-1 and day 7, with the exception of mTNFR-Ig, which received a total of three doses on days pre-1, +3, and +7. Clinical scoring was monitored for 14 days.

[00299] The results of these experiments are shown in Figures 18A and 18B. In this study, GAMMAGARD did not demonstrate clinical efficacy. Mu-TNF-Ig showed good protection against disease in 4 of 5 mice. HUMIRA showed disease kinetics very similar to the control IgG1. Non-sialylated anti-TNF showed some containment of disease. Sialylated anti-TNF α 2,6 showed good protection against disease in 5 of 5 mice, and scores were compared to anti-TNF therapy.

[00300] GENE EXPRESSION ANALYSIS: The expression of inflammatory and bone remodeling genes was determined by RT/PCR analysis of the hind paws of mice treated with the a2,6-silated mAb isotype or mTNFR-Ig (n=4 per group). To perform quantitative PCR, total RNA was isolated from the hind paws using RNA STAT-60 (Tel-Test, Friendswood, TX, USA). Total RNA (5 μg) was subjected to DNase treatment (Roche). DNase-treated total RNA was reverse transcribed using Superscript II (Gibco/BRL). Oligonucleotide primers were determined using Primer Express (PE Biosystems), or were obtained commercially from Applied Biosystems.

[00301] Real-time quantitative PCR on 10 ng of cDNA from each sample was performed using one of two methods. In the first method, two unlabeled gene-specific primers were used at 400 nM in a Perkin Elmer SYBR green real-time quantitative PCR assay using an ABI 5700 instrument. In the second method, two unlabeled primers, at 900 nM each, were used with 250 nM of FAM-labeled probe (Applied Biosystems) in a TAQMAN™ real-time quantitative PCR reaction on an ABI 7700 sequence detection system. The absence of genomic DNA contamination was confirmed using primers that recognize genomic regions of the CD4 promoter; samples with DNA contamination detectable by real-time PCR were excluded from the study. Ubiquitin levels were measured in a separate reaction and used to normalize the data by the Δ - Δ Ct method, using the mean cycle threshold (Ct) value for ubiquitin and the genes of interest for each sample; equation 1.8 and (ubiquitin Ct - gene of interest Ct) x 104 was used to obtain the normalized values. The results are shown in Table 7. The data shown represent fold increases in inflammatory/bone remodeling gene expression relative to those of gene expression in hindlimbs of wild-type mice. TABLE 7 Fold induction in wild-type mice

EXAMPLE 21

[00302] Effect of anti-TNFα antibody or its Fc muteins in an antibody-collagen-induced arthritis (AIA) model

[00303] The experiment described in Example 20 was repeated in the manner described herein, with the exception that GAMMAGARD was administered intravenously (while all other reagents were administered subcutaneously). STUDY DESIGN: Arthritis was induced in the manner described in Example 20.

[00304] All groups of mice were dosed on day 1, with the exception of mTNFR-Ig which received a total of two doses on days 1 and +3. Clinical scoring was monitored for 7 days.

[00305] Groups of mice were treated with the following reagents: Group n=5 for all groups

[00306] The reagents: “IgG1 isotype”, “anti-non-sialylated TNF”, “anti- TNF α2,6 sialylated Anti-TNF, mTNFR-Ig, GAMMAGARD and HUMIRA were described in example 20.

[00307] The reagents identified as “α2,3 sialylated anti-TNF” correspond to the anti-TNF antibody described in example 18, produced in GFI 6.0 comprising F243A/V264A, which was treated in vitro with neuraminidase to eliminate α2,6-linked sialic acid, and further treated in vitro with α2,3 sialyltransferase. Briefly, purified antibody (4-5 mg/mL) was in formulation buffer comprising 6.16 mg sodium chloride, 0.96 mg sodium phosphate monobasic dihydrate, 1.53 mg sodium phosphate dibasic dihydrate, 0.30 mg sodium citrate, 1.30 mg citric acid monohydrate, 12 mg mannitol, 1.0 mg polysorbate 80 per 1 mL adjusted to pH 5.2. Neuraminidase (10 mU/mL) was added to the antibody mixture and incubated at 37 °C for at least 5 hours, or until desialylation was complete. The desialylated material was applied to a CaptoMMC purification column (GE Healthcare) to remove neuraminidase, and was reformulated in sialyltransferase buffer (50 mM Hepes pH 7.2, 150 mM NaCl, 2.5 mM CaCl2, 2.5 mM MgCl2, 2.5 mM MnCl2) at 4 mg/mL. Recombinant mouse α2,3 sialyltransferase enzyme, expressed in Pichia and purified via his-tag, was used for α2,3 sialic acid extension. The enzyme mixture was formulated in PBS in the presence of protease inhibitor cocktail (Roche™, cat # 11873580001) at 1.2 mg/mL. Prior to the sialylation reaction, pepstatin (50 μg/mL), chymostatin (2mg/mL), and 10mM CMP-sialic acid were added to the enzyme mixture, followed by sterilization through a 0.2 μm filter. One mL of the enzyme mixture was added to 10 mL of the desialylated material. The reaction was carried out at 37 °C for 8 h. The sialylation yield was confirmed by ESI-Q-TOF mass determination. The final material was purified using MabSelect (GE Healthcare) and formulated in the buffer described above, and sterilized by filtration (0.2 μm membrane).

[00308] The reagent identified as “18 ADX PNG Humira” corresponds to the anti-TNF antibody described in Example 18, produced in GFI 6.0 comprising F243A/V264A, which was treated in vitro with PNGase F to remove all N-glycans. PNGase F enzyme was obtained from Prozyme, Inc., and used with a stoichiometry of 2 μL of commercial enzyme to 4 mg of IgG1 at pH 7.4. The digested material was purified using Mabselect (GE Healthcare). The final material was formulated and sterilized by filtration (0.2 μm membrane).

[00309] The reagent identified as “20 ADX No Glyco” corresponds to the wild-type anti-TNF antibody described in Example 18, comprising a single mutation at position 297, which does not result in any glycosylation, produced in a GFI5.0 YGLY8316. The strain producing this antibody was clarified by centrifugation for 15 minutes at 13,000 g in a Sorvall Evolution RC (kendo, Asheville, NC). The capture step was performed using MabSelect (GE Healthcare), followed by a polishing step using Capto MMC (GE Healthcare). The final material was formulated and sterilized by filtration (0.2 μm membrane).

[00310] The results of these experiments are shown in Figures 19A and 19B.

[00311] GENE EXPRESSION ANALYSIS: The expression of inflammatory and bone remodeling genes was determined by RT/PCR analysis. To perform quantitative PCR, total RNA was isolated from hind paws using RNA STAT-60 (Tel-Test, Friendswood, TX, USA). Total RNA (5 μg) was subjected to DNase treatment (Roche). DNase-treated total RNA was reverse transcribed using Superscript II (Gibco/BRL). Oligonucleotide primers were determined using Primer Express (PE Byosistems), or were obtained commercially from Applied Biosystems.

[00312] Real-time quantitative PCR on 10 ng of cDNA from each sample was performed using one of two methods. In the first method, two unlabeled gene-specific primers were used at 400 nM in a Perkin Elmer SYBR green real-time quantitative PCR assay using an ABI 5700 instrument. In the second method, two unlabeled primers, at 900 nM each, were used with 250 nM of FAM-labeled probe (Applied Biosystems) in a TAQMAN™ real-time quantitative PCR reaction on an ABI 7700 sequence detection system. The absence of genomic DNA contamination was confirmed using primers that recognize genomic regions of the CD4 promoter; samples with DNA contamination detectable by real-time PCR were excluded from the study. Ubiquitin levels were measured in a separate reaction and used to normalize the data by the Δ - Δ Ct method, using the mean cycle threshold (Ct) value for ubiquitin and the genes of interest for each sample; equation 1.8 and (ubiquitin Ct - gene of interest Ct) × 104 was used to obtain the normalized values. The results are shown in Table 8. The data shown are the fold increase in inflammatory/bone remodeling gene expression relative to that of gene expression in hindlimbs of wild-type mice. TABLE 8 Fold Induction in Wild-type Mice

EXAMPLE 22 FCYR binding assays of anti-TNF antibodies and muteins

[00313] FCY receptor binding assays were performed as described in Example 1 using the anti-TNF antibodies described in Example 18. The results are shown in Figure 20 and Tables 9-10.TABLE 9

[00314] Reduction in FCY receptor binding compared to wild-type (parental) antibody produced in GS 5.0 ↓ indicates decrease in affinity↑ indicates increase in affinityTABLE 10

[00315] Reduction in Fcy receptor binding compared to commercial HUMIRA ↓ indicates decrease in affinity↑ indicates increase in affinity

EXAMPLE 23

[00316] Construction of additional anti-Her2 dual Fc muteins (at positions 243/264) and their N-glycan composition

[00317] The additional anti-Her2 IgG1 antibody dual Fc muteins described in Example 2 were constructed using a Stratagene QuikChange® site-directed mutagenesis kit, Catalog #200518 (30 reactions), following the protocol provided for the saturation mutagenesis process with degenerate oligonucleotide primers. The signal sequence of an alpha-combining factor predomain was fused in alignment to the 5' end of the light chain and heavy chain. The resulting heavy and light chains with the fused IgG1 signal sequence and their muteins were cloned into the Pichia pastoris AOX1 promoter and in front of the S. cerevisiae Cyc terminator, respectively. The expression cassette of the complete heavy and light chains was placed together in the final expression vector.

[00318] Vectors were expressed in glycoengineered Pichia GFI6.0, YGLY3582, and YGLY22812 host cells.TABLE 11

[00319] The genotype of the GFI6.0 strain YGLY3582 strain is described in Example 4 (same for YGLY4563).

[00320] The genotype of the genetically modified Pichia pastoris GFI 6.0 strain YGLY22812 used for the expression of the anti-TNFα DM Fc mutein is as follows: ura5Δ::ScSUC2 och1Δ::lacZ bmt2Δ::lacZ/KlMNN2-2 mnn4L1Δ::lacZ/MmSLC35A3 pnolΔ mnn4Δ::lacZ ADE1:lacZ/NA10/MmSLC35A3/FB8his1Δ::lacZ/ScGAL10/XB33/DmUGT arg1Δ::HIS1/KD53/TC54bmt4Δ::lacZ bmt1Δ::lacZ bmt3Δ::lacZTRP2:ARG1/MmCST/HsGNE/HsCSS/HsSPS/MmST6-33ste13Δ::lacZ/TrMDS1 dap2Δ::NatRTRP5:HygRMmCST/HsGNE/HsCSS/HsSPS/MmST6-33 Vps10-1Δ::AOX1p_LmSTT3d. YGLY23294, YGLY23280, YGLY23301, YGLY25259 and YGLY23305 have the same genotype except that they express different muteins listed in Table 11.

[00321] Strains were grown in 500 mL shake flasks with 300 mL of 2% BMGY medium and shaken at 24 °C for 3 days.

[00322] Shake flask induction protocol: Collect the total volume of each culture (300 mL) in falcon tubes and centrifuge at 2,500 rpm for 5 minutes. Discard the supernatant and resuspend the cell pellets in a final volume of 150 mL in 2% BMMY and 360 uL PMTi4 (stock concentration at 0.65 mg/mL). Transfer to a new 500 mL shake flask at 24 °C for 2 days. Centrifuge the induced cultures and collect the supernatant in new falcon tubes.

[00323] Secreted antibodies were purified by protein A column using STREAMLINE rProtein A (catalog no. 17-1281-01) from GE Healthcare and BioRad poly-prep chromatography columns (10 mL) (catalog no. 731-1550). The following buffers were used:- Wash Buffer #1: 20mM Tris pH 7.0, 1M NaCl- Wash Buffer #2: 20mM Tris pH 7.0- Neutralization Buffer: 1M Tris pH 8.0- pH 9.0- Elution Buffer: 100 mM or 50 mM Sodium Citrate pH 3.0- Cleaning Solution: 6M urea in water.

[00324] The purification protocol is as follows:- Add 500 uL of STREAMLINErProtein A microspheres to each BioRad column. The microspheres can be in 20% ethanol. The microsphere slurry composition can be 50% microspheres, 50% liquid.- Once the Protein A microspheres are on the column, they can be washed with 5 mL of Wash Buffer #2 (discard flow-through).- Add 10 mL of supernatant to the BioRad column. During this step, antibodies will bind to the Protein A microspheres. (discard flow-through).- Wash away excess unwanted proteins by adding 5 mL of Wash Buffer #1 to the column. (discard flow-through).

[00325] Wash the column again by adding 5 mL of wash buffer #2 (discard flow-through).

[00326] Add 1 mL of neutralization buffer to the 15 mL protein collection tube.- Place the BioRad column in the 15 mL collection tube.- Add 3 mL of elution buffer to the BioRad column. This will remove the desired antibodies from the protein A microspheres.- Collect the eluted protein in the 15 mL protein collection tube.- Determine the concentration of the eluted protein by the Bradford assay (use 10 uL of protein for the Bradford assay).

[00327] To quantify the relative amount of each glycoform, glycans released by N-glycosidase F were labeled with 2-aminobenzidine (2-AB) and analyzed by HPLC as described in Choi et al., Proc. Natl. Acad. Sci. USA 100:5022-5027 (2003) and Hamilton et al., Science 313:1441-1443 (2006). Table 12 shows the glycan profile of the antibodies produced (NQP = no quantification possible).TABLE 12

[00328] Binding of the above mutants to the various FCY receptors was determined using the method described in Example 11. The results are shown in Figure 21 and Tables 13 and 14.TABLE 13 TABLE 14Binding affinity compared to commercially available Herceptin

EXAMPLE 23 Parental anti-PCKS9 antibody and Fc muteins

[00329] An anti-PCSK9 antibody having the heavy chain amino acid sequence of SEQ ID NO:13 and the light chain amino acid sequence of SEQ ID NO:14 was also constructed, and the heavy chain mutations F243A and V264A were introduced in the manner described in Example 2. The heavy chain amino acid sequence of the anti-PCSK9 double mutein is shown in SEQ ID NO:15. The wild-type and double mutein forms of the antibody were expressed in glycoengineered Pichia GFI5.0 and GFI6.0, and purified according to the procedures described in Examples 3-7, and their ability to bind to various FCY receptors was determined using the procedures described in Example 11. The anti-PCSK9 double mutein antibody (produced in GFI6.0, comprising the heavy chain amino acid sequence of SEQ ID NO:15 and the light chain amino acid sequence of SEQ ID NO:14), when compared to the parental antibody (produced in GFI5.0), showed approximately a 4-fold reduction in binding to FCYRI, an 8-60-fold reduction in binding to FcYRIIIa LF, a 2-6-fold reduction in binding to FcYRIIIa LV, and no detectable binding to FcyRIIa or FcyRIIb/c.

EXAMPLE 24

[00330] Effect of anti-Her2 antibody and its Fc muteins in an antibody-collagen-induced arthritis (AIA) model

[00331] INDUCTION MODEL: AIA (Antibody-induced arthritis) is induced with a commercial arthritogenic monoclonal antibody Arthrogen-CIA® (obtained from Chondrex), which consists of a cocktail of 5 monoclonal antibodies, clone A2-10 (IgG2a), F10-21 (IgG2a), D8-6 (IgG2a), D1-2G(IgG2b), and D2-112 (IgG2b), which recognizes epitopes conserved in several species of type II collagen.

[00332] ANIMALS: Ten-week-old male B10.RIII mice, which are susceptible to arthritis induction without the addition of costimulatory factors, were used. These animals were obtained from the Jackson Laboratory.

[00333] CLINICAL GRAding: Paw swelling was measured daily after arthritis induction. Disease severity was graded on a scale of 0-3 per paw as follows: 0, normal; 1, swelling of one digit; 2, swelling of two or more digits; 3, total paw swelling. The maximum clinical score per mouse is 12.

[00334] STUDY DESIGN: Arthritis was induced by passive transfer of 3 mg of pathogen anti-CII mAb cocktail IV on day 0.

[00335] All groups of mice were dosed the day before. Clinical scoring was monitored for 7 days.

[00336] Groups of mice were treated subcutaneously with the following reagents: All groups n=5, except group E which has n=4

[00337] An IgG1 isotype antibody was used as a control. The antibody bound to mouse anti-hexon and displayed the determination “27F11”.

[00338] The sample identified as “Anti-Her2 unsialylated” corresponds to the anti-Her2 antibody comprising the F243A/V264A mutations produced in the GFI 5.0 YGLY19709 strain. The YGLY19709 strain was derived from the anti-Her2 antibody producing strain YGLY13979, which was transformed with plasmid pGLY6301 to express LsSTT3d. The YGLY13979 strain and the pGLY6301 plasmid are described in patent application: WO 2010/099186.

[00339] The sample identified as “sialylated anti-Her2 α2,6” corresponds to the anti-Her2 antibody comprising the F243A/V264A mutations produced in the GFI 6.0 YGLY4563 strain (see example 4).

[00340] mTNFR-Ig is an immunoadhesin (anti-TNF Ig receptor fusion protein) comprising the extracellular domain of mTNFR2 linked to the mIgG1 Fc, starting at the hinge and spanning the CH2 and CH3 regions, and comprising the amino acid sequence of SEQ ID NO:16 produced in CHO cells. The results of these experiments are shown in Figure 22. SEQUENCE LISTING

[00341] Although the present invention is described herein with reference to the illustrated embodiments, it is to be understood that the invention is not limited thereto. Those skilled in the art with access to the teachings herein will recognize additional modifications and embodiments within the scope hereof.

Claims (22)

1. An Fc-containing polypeptide comprising the amino acid sequence of SEQ ID NO: 18 and further comprising mutations at amino acid positions 243 and 264 of the Fc region, wherein the Fc-containing polypeptide is an antibody or antibody fragment comprising sialylated N-glycans on the Fc region of the Fc-containing polypeptide, wherein at least 70 molar % of the N-glycans are sialylated N-glycans with sialic acid residues attached via α-2,6 linkages, wherein the sialylated N-glycans do not comprise detectable α-2,3-linked sialic acid, and wherein the mutations are F243A and V264A, wherein the numbering is in accordance with the EU index as in Kabat. 2. The Fc-containing polypeptide of claim 1, wherein at least 83 molar % of the N-glycans are sialylated N-glycans with sialic acid residues linked via α-2,6 bonds. 3. The Fc-containing polypeptide of claim 1 or 2, wherein at least 70 molar % of the N-glycans on the Fc-containing polypeptide have a structure selected from the group consisting of SA(1-4)Gal(1-4)GlcNAc((2-4)Man3GlcNAc2. 4. The Fc-containing polypeptide of claim 1 or 2, wherein at least 90 molar % of the N-glycans on the Fc-containing polypeptide have a structure selected from the group consisting of SA(1-4)Gal(1-4)GlcNAc((2-4)Man3GlcNAc2. 5. The Fc-containing polypeptide of claim 1, wherein the sialic acid is N-acetylneuraminic acid (NANA) or N-glycolylneuraminic acid (NGNA) or a mixture thereof. 6. The Fc-containing polypeptide of claim 1, wherein the sialic acid is an analogue or derivative of NANA or NGNA with acetylation at position 9 on the sialic acid. 7. The Fc-containing polypeptide of claim 1, wherein the N-glycans on the Fc-containing polypeptide comprise NANA and no NGNA. 8. The Fc-containing polypeptide of claim 1, wherein at least 83 molar % of the N-glycans are sialylated N-glycans with sialic acid residues linked via α-2,6 bonds. 9. The Fc-containing polypeptide of claim 1, wherein the sialylated N-glycans comprise NANA2Gal2GlcNAc2Man3GlcNAc2 and NANAGal2GlcNAc2Man3GlcNAc2 structures. 10. The Fc-containing polypeptide of claim 1, wherein the N-glycans on the Fc-containing polypeptide comprise about 50 mole % NANA2Gal2GlcNAc2Man3GlcNAc2, about 27 mole % NANAGal2GlcNAc2Man3GlcNAc2MancNAc2, and about 5.7 mole % NANAGal2GlcNAc2Man3GlcNAcNA3GlCNAc2MancNAc2. 11. The Fc-containing polypeptide of claim 1, wherein the sialylated N-glycans comprise NANA2Gal2GlcNAc2Man3GlcNAc2 and NANAGal2GlcNAc2Man3GlcNAc2 structures. 12. The Fc-containing polypeptide of claim 1, wherein the N-glycans on the Fc-containing polypeptide comprise about 50 mole % NANA2Gal2GlcNAc2Man3GlcNAc2, about 27 mole % NANAGal2GlcNAc2Man3GlcNAc2MancNAc2, and about 5.7 mole % NANAGal2GlcNAc2Man3GlcNAcNA3GlCNAc2MancNAc2. 13. The Fc-containing polypeptide of claim 1, wherein at least 47 mole % of the sialylated N-glycans on the Fc-containing polypeptide have a NANA2Gal2GlcNAc2Man3GlcNAc2 structure. 14. The Fc-containing polypeptide of claim 1, wherein at least 66 molar % of the sialylated N-glycans on the Fc-containing polypeptide have a NANA2Gal2GlcNAc2Man3GlcNAc2 structure. 15. The Fc-containing polypeptide of claim 1, wherein the Fc-containing polypeptide is an antibody or an Fc fragment. 16. The Fc-containing polypeptide of claim 1, wherein the Fc-containing polypeptide is an immunoadhesion. 17. The Fc-containing polypeptide of claim 1, wherein at least 90 molar % of the sialylated N-glycans are sialylated. 18. The Fc-containing polypeptide of claim 1, wherein the sialic acid is N-acetylneuraminic acid (NANA) or N-glycolylneuraminic acid (NGNA) or a mixture thereof. 19. The Fc-containing polypeptide of claim 1 or 2, wherein at least 66 molar % of the N-glycans on the Fc-containing polypeptide have a NANA2Gal2GlcNAc2Man3GlcNAc2 structure. 20. The Fc-containing polypeptide of any one of claims 1 to 18, wherein the Fc-containing polypeptide has one or more of the following properties when compared to a parent Fc-containing polypeptide: a) reduced effector function, b) increased anti-inflammatory properties, c) increased sialylation, d) increased bioavailability when administered parenterally, and e) reduced binding to FCYRI, FcYRIIa, FcYRIIb, and FcyRIIIa. 21. The Fc-containing polypeptide of claim 1, wherein the Fc-containing polypeptide is produced from a host cell that has been genetically modified to produce glycoproteins with predominantly sialylated N-glycans in which sialic acid residues are linked via α-2,6 linkages and in which no sialic acid residues are linked via α-2,3 linkages. 22. The Fc-containing polypeptide of claim 21, wherein the host cell is Pichia pastoris.