WO2015073307A2 - Fc CONTAINING POLYPEPTIDES HAVING INCREASED BINDING TO HUMAN DC-SIGN - Google Patents

Fc CONTAINING POLYPEPTIDES HAVING INCREASED BINDING TO HUMAN DC-SIGN Download PDF

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Publication number
WO2015073307A2
WO2015073307A2 PCT/US2014/064458 US2014064458W WO2015073307A2 WO 2015073307 A2 WO2015073307 A2 WO 2015073307A2 US 2014064458 W US2014064458 W US 2014064458W WO 2015073307 A2 WO2015073307 A2 WO 2015073307A2
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Prior art keywords
containing polypeptide
antibody
mutations
glycans
host cell
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PCT/US2014/064458
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French (fr)
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WO2015073307A3 (en
Inventor
Natarajan Sethuraman
Hussam H. Shaheen
Terrance Andy STADHEIM
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Merck Sharp & Dohme Corp.
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Publication of WO2015073307A2 publication Critical patent/WO2015073307A2/en
Publication of WO2015073307A3 publication Critical patent/WO2015073307A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention is directed to methods and compositions for the production of Fc- containing polypeptides which are useful as human or animal therapeutic agents, and which comprise increased binding to human DC-SIGN.
  • IgG and IgE mediate their pro-inflammatory properties through the crosslinking of the Fc receptor (FcR) with the Fc portion of the antibodies.
  • IgG and IgE can also engage a second class of receptors, the evolutionarily related, C-type lectins Dendritic Cell-specified Intercellular adhesion molecule-3 -Grabbing Nonintegrin (DC-SIGN or CD209) and CD23 resulting in antiinflammatory and immunosuppressive responses.
  • DC-SIGN or CD209 Dendritic Cell-specified Intercellular adhesion molecule-3 -Grabbing Nonintegrin
  • DC-SIGN is a structurally homologous, calcium-dependent, carbohydrate-binding, type II lectin, displaying ligand specificity for mannose-containing glycoconjugates and fucose-containing Lewis antigens.
  • IVIG intravenous immunoglobulin
  • the invention comprises an Fc-containing polypeptide (such as antibody or an antibody fragment or immunoadhesin) comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R,
  • F241S/V263A/T335A/N389S E269G/N389S/S400P, N297D/S354P/K409R/S424P,
  • the invention also comprises an Fc-containing polypeptide (such as antibody or an antibody fragment or immunoadhesin), wherein the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of: Q295R/V302A/S344P/K414R , M252V/V303A/N315S, S239P/F241L/I377V, L351S/N384D/S426P/K447E,
  • Fc-containing polypeptide such as antibody or an antibody fragment or immunoadhesin
  • F241S/V263A/T335A/N389S and E269G/N389S/S400P wherein the numbering is according to the EU index as in Kabat, and wherein the Fc-containing polypeptide comprises N-glycans.
  • the invention also comprises an Fc-containing polypeptide (such as antibody or an antibody fragment or immunoadhesin) comprising a mutation in the Fc region selected from the group consisting of: 246, 276, 360, 389, 395, and 414, wherein the numbering is according to the EU index as in Kabat.
  • Fc-containing polypeptide such as antibody or an antibody fragment or immunoadhesin
  • the invention also comprises an Fc-containing polypeptide comprising a mutation in the Fc region selected from the group consisting of: F241L, F241S, K246E, E272G, N276D, N276S, R301G, S354P, K360E, N389S, N389D, P395L, P395S and K414R, wherein the numbering is according to the EU index as in Kabat, and wherein the Fc-containing polypeptide has enhanced binding to human DC-SIGN when compared to a parent Fc-containing
  • any of the Fc-containing polypeptides of the invention can further comprise one or more mutations at positions selected from the group consisting of: 241, 243, 246, 252, 254, 256, 264, 267, 272, 276, 301, 328, 339, 342, 354, 360, 389, 395, 414, 433 and 434 of the Fc region, wherein the numbering is according to the EU index as in Kabat.
  • any of the Fc-containing polypeptides of the invention can further comprise one or more mutations at positions selected from the group consisting of: F241L, F241S, K246E, E272G, N276D, N276S, R301G, S354P, K360E, N389S, N389D, P395L, P395S and K414R, wherein the numbering is according to the EU index as in Kabat.
  • any of the Fc-containing polypeptides of the invention can comprise N-glycans.
  • at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc- containing polypeptide comprise an N-linked oligosaccharide structure selected from the group consisting of SA(i_4)Gal(i_4)GlcNAc(2-4)Man3GlcNAc2.
  • the sialic acid residues in the sialylated N-glycans are attached via a-2,6 linkages.
  • the N- linked oligosaccharides recited above further comprise a core fucose.
  • the invention also comprises an Fc-containing polypeptide (such as antibody or an antibody fragment or immunoadhesin), wherein the Fc-containing polypeptide does not comprise N-glycans and wherein the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of: N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G, and V259A/N297S/N389S/G446D, wherein the numbering is according to the EU index as in Kabat.
  • Fc-containing polypeptide such as antibody or an antibody fragment or immunoadhesin
  • the Fc-containing polypeptide of the invention is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide of the invention is an IgGl antibody or an antibody fragment.
  • the Fc-containing polypeptide of the invention is a human IgGl or a fragment of human IgGl that comprises or consists of any one of SEQ ID NOs: 11-33.
  • the Fc-containing polypeptide comprises N-glycans and comprises or consists essentially of any one of SEQ ID NOs: 11-29.
  • the Fc-containing polypeptide does not comprise N-glycans and comprises or consists essentially of any one of SEQ ID
  • the Fc-containing polypeptide of the invention has enhanced binding to human DC-SIGN when compared to a parent Fc-containing polypeptide.
  • the Fc-containing polypeptide of the invention has altered inflammatory properties when compared to a parent Fc-containing polypeptide.
  • the Fc-containing polypeptide of the invention has enhanced inflammatory properties when compared to a parent Fc-containing polypeptide.
  • the invention also comprises a method for producing an Fc-containing polypeptide in a host cell comprising: (a) providing a host cell comprising a nucleic acid encoding an Fc- containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R,
  • F241S/V263A/T335A/N389S E269G/N389S/S400P, N297D/S354P/K409R/S424P,
  • the host cell is capable of producing an Fc- containing polypeptide comprising N-glycans.
  • the Fc-containing polypeptide is an antibody or antibody fragment.
  • the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof.
  • the host cell is a lower eukaryotic host cell. In one embodiment, the host cell is Pichia pastoris. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce hybrid or complex type N-glycans. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce sialylated N-glycans.
  • the invention also comprises a method for producing an Fc-containing polypeptide in a host cell comprising: (a) providing a host cell comprising a nucleic acid encoding an Fc- containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R ,
  • the host cell is capable of producing an Fc-containing polypeptide comprising N-glycans.
  • the Fc-containing polypeptide is an antibody or antibody fragment.
  • the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof.
  • the host cell is a lower eukaryotic host cell.
  • the host cell is Pichia pastoris. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce hybrid or complex type N-glycans. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce sialylated N-glycans.
  • the invention also comprises a method for producing an Fc-containing polypeptide in a host cell comprising: (a) providing a host cell comprising a nucleic acid encoding an Fc- containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: N297D/S354P/K409R/S424P,
  • the method results in the production of an Fc-containing polypeptide that does not comprise N-glycans.
  • the Fc- containing polypeptide is an antibody or antibody fragment.
  • the Fc- containing polypeptide is a human IgGl antibody or a fragment thereof.
  • the host cell is a lower eukaryotic host cell. In one embodiment, the host cell is Pichia pastoris. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce hybrid or complex type N-glycans. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce sialylated N-glycans.
  • the invention also comprises a method for producing an Fc-containing polypeptide in a host cell comprising: (a) providing a host cell comprising a nucleic acid encoding an Fc- containing polypeptide comprising the amino acid sequence of any of SEQ ID NO: 11-33, (b) culturing the host cell under conditions which cause expression of the Fc-containing polypeptide; and (c) isolating the Fc-containing polypeptide from the host cell.
  • the host cell is capable of producing an Fc-containing polypeptide comprising N-glycans.
  • the host cell is a lower eukaryotic host cell.
  • the host cell is Pichia pastoris.
  • the host cell is a Pichia pastoris host cell that has been engineered to produce hybrid or complex type N-glycans. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce sialylated N-glycans.
  • the invention also comprises a method for producing an Fc-containing polypeptide in a host cell comprising: (a) providing a host cell comprising a nucleic acid encoding an Fc- containing polypeptide comprising one or more of mutations in the Fc region, at positions selected from the group consisting of: 241, 243, 246, 252, 254, 256, 264, 267, 272, 276, 301, 328, 339, 342, 354, 360, 389, 395, 414, 433 and 434 of the Fc region, wherein the numbering is according to the EU index as in Kabat (b) culturing the host cell under conditions which cause expression of the Fc-containing polypeptide; and (c) isolating the Fc-containing polypeptide from the host cell.
  • the host cell is capable of producing an Fc-containing polypeptide comprising N-glycans.
  • the Fc-containing polypeptide is an antibody or antibody fragment.
  • the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof.
  • the host cell is a lower eukaryotic host cell.
  • the host cell is Pichia pastoris.
  • the host cell is a Pichia pastoris host cell that has been engineered to produce hybrid or complex type N-glycans.
  • the host cell is a Pichia pastoris host cell that has been engineered to produce sialylated N-glycans.
  • the invention also comprises a method for producing an Fc-containing polypeptide in a host cell comprising: (a) providing a host cell comprising a nucleic acid encoding an Fc- containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: F241L, F241S, K246E, E272G, N276D, N276S, R301G, S354P, K360E, N389S, N389D, P395L, P395S and K414R, wherein the numbering is according to the EU index as in Kabat (b) culturing the host cell under conditions which cause expression of the Fc-containing polypeptide; and (c) isolating the Fc-containing polypeptide from the host cell.
  • the host cell is capable of producing an Fc- containing polypeptide comprising N-glycans.
  • polypeptide is an antibody or antibody fragment.
  • the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof.
  • the host cell is a lower eukaryotic host cell.
  • the host cell is Pichia pastoris.
  • the host cell is a Pichia pastoris host cell that has been engineered to produce hybrid or complex type N-glycans.
  • the host cell is a Pichia pastoris host cell that has been engineered to produce sialylated N-glycans.
  • the invention also comprises a method of altering the inflammatory properties of an Fc- containing polypeptide comprising: introducing one or more of mutations in the Fc region of the Fc-containing polypeptide, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R, M252V/V303A/N315S, S239P/F241L/I377V,
  • L351S/N384D/S426P/K447E F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274R/K360E/Y373H/P387S,
  • T289A/E345G/E357V/Q386R/N434S A231V/P232S/S267F/S354P, R301G/K360E, K246E/M252T/E272G/S375P, D249G, F241S/V263A/T335A/N389S, E269G/N389S/S400P, N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G and
  • the Fc-containing polypeptide is an antibody or antibody fragment.
  • the Fc-containing polypeptide is a human IgGl antibody or an antibody fragment thereof.
  • the Fc-containing polypeptide comprises or consists essentially of the amino acid sequence of any of SEQ ID NOs: l l-33.
  • the invention also comprises a method of altering the inflammatory properties of an Fc- containing polypeptide comprising: introducing one or more of mutations in the Fc region of the Fc-containing polypeptide, at positions selected from the group consisting of: 241, 243, 246, 252, 254, 256, 264, 267, 272, 276, 301, 328, 339, 342, 354, 360, 389, 395, 414, 433 and 434 of the Fc region, wherein the numbering is according to the EU index as in Kabat, and wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc- containing polypeptide.
  • the Fc-containing polypeptide is an antibody or antibody fragment.
  • the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof.
  • the invention also comprises a method of altering the inflammatory properties of an Fc- containing polypeptide comprising: introducing one or more of mutations in the Fc region of the Fc-containing polypeptide, wherein the mutations are selected from the group consisting of: F241L, F241S, K246E, E272G, N276D, N276S, R301G, S354P, K360E, N389S, N389D, P395L, P395S and K414R, wherein the numbering is according to the EU index as in Kabat, and wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc-containing polypeptide.
  • the Fc-containing polypeptide is an antibody or antibody fragment.
  • the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof.
  • the invention also comprises a method of increasing the anti-inflammatory properties of an Fc-containing polypeptide comprising: introducing one or more of mutations in the Fc region of the Fc-containing polypeptide, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R, M252V/V303A/N315S, S239P/F241L/I377V, L351S/N384D/S426P/K447E, F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K27
  • K246E/M252T/E272G/S375P D249G, F241S/V263A/T335A/N389S, E269G/N389S/S400P, N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G and
  • the Fc-containing polypeptide is an antibody or antibody fragment.
  • the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof.
  • the Fc-containing polypeptide comprises or consists essentially of the amino acid sequence of any of SEQ ID NOs: 11-33.
  • the invention also comprises a method of increasing the anti-inflammatory properties of an Fc-containing polypeptide, wherein the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of: Q295R/V302A/S344P/K414R ,
  • the Fc-containing polypeptide comprises N-glycans, and wherein the Fc-containing polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc-containing polypeptide.
  • the Fc-containing polypeptide is an antibody or antibody fragment.
  • the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof.
  • the Fc- containing polypeptide comprises or consists essentially of the amino acid sequence of any of SEQ ID NOs: 11-29.
  • the invention also comprises a method of increasing the anti-inflammatory properties of an Fc-containing polypeptide, wherein the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of: N297D/S354P/K409R/S424P,
  • the Fc-containing polypeptide is an antibody or antibody fragment.
  • the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof.
  • the Fc-containing polypeptide comprises or consists essentially of the amino acid sequence of any of SEQ ID NOs:30-33.
  • the invention also comprises a method of increasing the anti-inflammatory properties of an Fc-containing polypeptide comprising: introducing one or more of mutations in the Fc region of the Fc-containing polypeptide at positions selected from the group consisting of: 241, 243, 246, 252, 254, 256, 264, 267, 272, 276, 301, 328, 339, 342, 354, 360, 389, 395, 414, 433 and 434 of the Fc region, wherein the numbering is according to the EU index as in Kabat, and wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc- containing polypeptide.
  • the Fc-containing polypeptide is an antibody or antibody fragment.
  • the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof.
  • the invention also comprises a method of increasing the anti-inflammatory properties of an Fc-containing polypeptide comprising: introducing one or more of mutations in the Fc region of the Fc-containing polypeptide, wherein the mutations are selected from the group consisting of: F241L, F241S, K246E, E272G, N276D, N276S, R301G, S354P, K360E, N389S, N389D, P395L, P395S and K414R, wherein the numbering is according to the EU index as in Kabat, and wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc-containing polypeptide.
  • the Fc-containing polypeptide is an antibody or antibody fragment.
  • the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof.
  • the invention also comprises a method of treating a subject in need thereof comprising: administering to the subject a therapeutically effective amount of an Fc-containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R, M252V/V303A/N315S,
  • F241S/V263A/T335A/N389S E269G/N389S/S400P, N297D/S354P/K409R/S424P,
  • the Fc-containing polypeptide is an antibody or antibody fragment.
  • the Fc-containing polypeptide of is a human IgGl antibody or a fragment thereof.
  • the Fc-containing polypeptide comprises or consists essentially of the amino acid sequence of any of SEQ ID NOs: 11-33.
  • the method further comprises the administration of a vaccine.
  • the method further comprises the administration of an anti-inflammatory compound.
  • the method further comprises the administration of an agent that binds to FcyRIIB.
  • the subject has an inflammatory condition.
  • the invention also comprises a method of treating a subject in need thereof comprising: administering to the subject a therapeutically effective amount of an Fc-containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R , M252V/V303A/N315S,
  • the Fc-containing polypeptide is an antibody or antibody fragment.
  • the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof.
  • the Fc-containing polypeptide comprises or consists essentially of the amino acid sequence of any of SEQ ID NOs: 11-29.
  • the method further comprises the administration of a vaccine. In one embodiment, the method further comprises the administration of an anti-inflammatory compound. In one embodiment, the method further comprises the administration of an agent that binds to FcyRIIB. In one embodiment, the subject has an inflammatory condition.
  • the invention also comprises a method of treating a subject in need thereof comprising: administering to the subject a therapeutically effective amount of an Fc-containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G and V259A/N297S/N389S/G446D, wherein the numbering is according to the EU index as in Kabat, wherein the polypeptide does not comprise N-glycans, and wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc-containing polypeptide.
  • the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof. In one embodiment, the Fc-containing polypeptide comprises or consists essentially of the amino acid sequence of any of SEQ ID NOs:30-33. In one embodiment, the method further comprises the administration of a vaccine. In one embodiment, the method further comprises the administration of an anti-inflammatory compound. In one embodiment, the method further comprises the administration of an agent that binds to FcyRIIB. In one embodiment, the subject has an inflammatory condition.
  • the invention also comprises a method of treating a subject in need thereof comprising: administering to the subject a therapeutically effective amount of an Fc-containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are at positions selected from the group consisting of: 241, 243, 246, 252, 254, 256, 264, 267, 272, 276, 301, 328, 339, 342, 354, 360, 389, 395, 414, 433 and 434, wherein the numbering is according to the EU index as in Kabat and wherein the polypeptide comprises enhanced binding to human DC- SIGN when compared to a parent Fc-containing polypeptide.
  • the Fc- containing polypeptide is an antibody or antibody fragment.
  • the Fc- containing polypeptide is a human IgGl antibody or a fragment thereof.
  • the method further comprises the administration of a vaccine.
  • the method further comprises the administration of an anti-inflammatory compound.
  • the method further comprises the administration of an agent that binds to FcyRIIB.
  • the subject has an inflammatory condition.
  • the invention also comprises a method of treating a subject in need thereof comprising: administering to the subject a therapeutically effective amount of an Fc-containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: F241L, F241S, K246E, E272G, N276D, N276S, R301G, S354P, K360E, N389S, N389D, P395L, P395S and K414R, wherein the numbering is according to the EU index as in Kabat, and wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc-containing polypeptide.
  • the method further comprises the administration of a vaccine. In one embodiment, the method further comprises the administration of an anti-inflammatory compound. In one embodiment, the method further comprises the administration of an agent that binds to FcyRIIB. In one embodiment, the subject has an inflammatory condition.
  • the invention also comprises a pharmaceutical formulation comprising: an Fc-containing polypeptide as described above and a pharmaceutically acceptable carrier.
  • the invention comprises a pharmaceutical formulation comprising: (a) an Fc-containing polypeptide (such as antibody or an antibody fragment or immunoadhesin) comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R, M252V/V303A/N315S, S239P/F241L/I377V,
  • an Fc-containing polypeptide such as antibody or an antibody fragment or immunoadhesin
  • L351S/N384D/S426P/K447E F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274R/K360E/Y373H/P387S,
  • K246E/M252T/E272G/S375P D249G, F241S/V263A/T335A/N389S, E269G/N389S/S400P, N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G and
  • V259A/N297S/N389S/G446D wherein the numbering is according to the EU index as in Kabat, and (b) a pharmaceutically acceptable carrier.
  • polypeptide is an antibody or antibody fragment.
  • the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof.
  • the Fc- containing polypeptide is a human IgGl or a fragment of human IgGl that comprises or consists of any one of SEQ ID NOs: 11-33.
  • the invention also comprises a pharmaceutical formulation comprising: (a) an Fc- containing polypeptide (such as antibody or an antibody fragment or immunoadhesin) comprising N-glycans, wherein the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of: Q295R/V302A/S344P/K414R ,
  • the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc- containing polypeptide is a human IgGl antibody or a fragment thereof. In one embodiment, the Fc-containing polypeptide is a human IgGl or a fragment of human IgGl that comprises or consists of any one of SEQ ID NOs: 11 -29.
  • At least 30%, 40%, 50%, 60%, 70%), 80%) or 90%) of the N-glycans on the Fc-containing polypeptide comprise an N-linked oligosaccharide structure selected from the group consisting of SA(i_4)Gal(i_4)GlcNAc(2- 4)Man3GlcNAc2-
  • the sialic acid residues in the sialylated N-glycans are attached via a-2,6 linkages.
  • the invention also comprises a pharmaceutical formulation comprising: (a) an Fc- containing polypeptide (such as antibody or an antibody fragment or immunoadhesin) comprising a mutation in the Fc region selected from the group consisting of: 246, 276, 360, 389, 395, and 414, wherein the numbering is according to the EU index as in Kabat, and (b) a pharmaceutically acceptable carrier.
  • the Fc-containing polypeptide is an antibody or antibody fragment.
  • the Fc-containing polypeptide is a human IgGl antibody or an antibody fragment thereof.
  • the invention also comprises a pharmaceutical formulation comprising: (a) an Fc- containing polypeptide (such as antibody or an antibody fragment or immunoadhesin) comprising a mutation in the Fc region selected from the group consisting of: F241L, F241S, K246E, E272G, N276D, N276S, R301G, S354P, K360E, N389S, N389D, P395L, P395S; and K414R, wherein the numbering is according to the EU index as in Kabat, and (b) a pharmaceutical formulation comprising: (a) an Fc- containing polypeptide (such as antibody or an antibody fragment or immunoadhesin) comprising a mutation in the Fc region selected from the group consisting of: F241L, F241S, K246E, E272G, N276D, N276S, R301G, S354P, K360E, N389S, N389D, P3
  • the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or an fragment thereof.
  • the invention also comprises a pharmaceutical formulation comprising: (a) an Fc- containing polypeptide (such as antibody or an antibody fragment or immunoadhesin), wherein the Fc-containing polypeptide does not comprise N-glycans and wherein the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of:
  • N297D/S354P/K409R/S424P N297S/N390D/P395L, N297S/R301G, and
  • V259A/N297S/N389S/G446D wherein the numbering is according to the EU index as in Kabat, and (b) a pharmaceutically acceptable carrier.
  • polypeptide is an antibody or antibody fragment.
  • the Fc-containing polypeptide is a human IgGl antibody or an fragment thereof.
  • the Fc- containing polypeptide is a human IgGl or a fragment of human IgGl that comprises or consists of any one of SEQ ID NOs:30-33.
  • FIGURE 1 illustrates the plasmid designated pGLYl 1714 containing anti-PC SK9 Lc bait cassette.
  • FIGURE 2 is a schematic representation of the Lc-Sedlp antibody display system described in Example 1.
  • the DNA sequence comprising the IgG light chain (Lc) is fused through a flexible linker to Sedlp.
  • the Lc portion of the anchored fusion heterodimerizes in the ER with the heavy chain (He) region of the IgG molecule, forming disulfide bridges.
  • FIGURE 3 illustrates the plasmid designated pGLYl 1576.
  • FIGURE 4 illustrates the plasmid designated pGLY4464.
  • FIGURE 5 is a schematic representation of the method used to isolate the novel IgGl Fc variants of the invention having improved binding to human DC-SIGN.
  • FIGURE 6 is an alignment of the IgGl Fc variants isolated as described in Example 2 and of the consensus sequence for the Fc region of human IgGl . (Residue number 1 in the alignment corresponds to amino acid position 215 according to EU numbering system as in Kabat).
  • FIGURE 7 shows the binding affinity of the IgGl Fc variants of the invention towards DC-SIGN and various FcyRs.
  • 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.
  • the term encompasses not only intact polyclonal or monoclonal antibodies, consisting of four polypeptide chains, i.e. two identical pairs of polypeptide chains, each pair having one "light” chain (LC) (about 25 kDa) and one "heavy” chain (HC) (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 that comprises an antigen recognition site and at least the portion of the Cpf2 domain of the heavy chain immunoglobulin constant region which comprises an N-linked glycosylation site of the Cpf2 domain, or a variant thereof.
  • the term includes an antibody of any class, such as IgG (for example, IgGl, IgG2, IgG3 or IgG4), IgM, IgA, IgD and IgE, respectively.
  • the term "Fc region” is used to define a C-terminal region of an immunoglobulin heavy chain.
  • the "Fc region” may be a native sequence Fc region or a variant Fc region.
  • the human IgG heavy chain Fc region is usually defined to stretch 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 can optionally comprise a hinge region.
  • the Fc region comprises or consists essentially of the amino acid sequence of SEQ ID NO: 1 (or a variant thereof comprising point mutations).
  • the Fc region comprises or consists essentially of the amino acid sequence of SEQ ID NO:2 (or a variant thereof comprising point mutations). In another embodiment, the Fc region comprises or consists essentially of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 with the addition of a lysine (K) residue at the 3' end.
  • the Fc region contains a single N-linked glycosylation site in the CH2 domain that corresponds to the Asn297 site of a full-length heavy chain of an antibody, wherein the numbering is according to the EU index as in Kabat.
  • Fc-containing polypeptide refers to a polypeptide, such as an antibody or immunoadhesin, which comprises an Fc region or fragment of an Fc region which retains the N- linked glycosylation site in the CH2 domain and retains the ability to recruit immune cells.
  • This term encompasses polypeptides comprising or consisting of (or consisting essentially of) an Fc region either as a monomore or dimeric species.
  • Polypeptides comprising an Fc region can be generated by papain digestion of antibodies or by recombinant DNA technology.
  • parent antibody when used herein refers to an antibody or Fc-containing polypeptide which lacks the Fc region mutations disclosed herein.
  • a parent Fc-containing polypeptide may comprise a native sequence Fc region or an Fc region with pre-existing amino acid sequence modifications.
  • a native sequence Fc region comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature.
  • Native sequence Fc regions include the native sequence human IgGl Fc region, the native sequence human IgG2 Fc region, the native sequence human IgG3 Fc region and the native sequence human IgG4 Fc region as well as naturally occurring variants thereof.
  • a parent antibody or a parent Fc- containing polypeptide can be expressed in any cell. In one embodiment, the parent antibody or a parent Fc-containing polypeptide is expressed in the same cell as the Fc-containing polypeptide of the invention.
  • immunoadhesin designates antibody-like molecules which combine the "binding domain" of a heterologous "adhesin” protein (e.g. a receptor, ligand or enzyme) with an immunoglobulin constant domain.
  • adhesin protein e.g. a receptor, ligand or enzyme
  • the 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.
  • ligand binding domain refers to any native cell-surface receptor or any region or derivative thereof retaining at least a qualitative ligand binding ability of a corresponding native receptor.
  • the receptor is from a cell-surface polypeptide having an extracellular domain that is homologous to a member of the immunoglobulin supergenefamily.
  • Other receptors which are not members of the immunoglobulin supergenefamily but are nonetheless specifically covered by this definition, are receptors for cytokines, and in particular receptors with tyrosine kinase activity (receptor tyrosine kinases), members of the hematopoietin and nerve growth factor which predispose the mammal to the disorder in question.
  • receptor tyrosine kinases members of the hematopoietin and nerve growth factor which predispose the mammal to the disorder in question.
  • the disorder is cancer.
  • Methods of making immunoadhesins are well known in the art. See, e.g., WO00/42072.
  • Fc mutein or “Fc mutein antibody” when used herein refers to an Fc- containing polypeptide in which one or more point mutations have been made to the Fc region.
  • Fc mutation when used herein refers to a mutation made to the Fc region of an Fc-containing polypeptide.
  • the numbering of the 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 IgGl EU antibody.
  • effector function 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 Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); Clq binding; complement dependent cytotoxicity (CDC) phagocytosis; down regulation of cell surface receptors (e. g. B cell receptor; BCR), etc.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement dependent cytotoxicity
  • phagocytosis e. g. B cell receptor; BCR
  • Fc receptor or “FcR” refer to a receptor that binds to the Fc region of an antibody.
  • the preferred Fc receptor is a native sequence of human FcR.
  • a preferred FcR is one that binds an IgG antibody - an FcyR - and includes receptors FcyRI, FcyRII, and FcyRIII, including allelic variants and alternatively spliced forms of these receptors.
  • the term also includes the neonatal receptor, FcRn, that is responsible for the transfer of maternal IgGs to the fetus.
  • FcyRIIB refers to a polyeptide en coded by the human FcRIIB gene, and includes, but is not limited to FcyRIIBl (GenBank Accession No. NP 003992), FcyRIIB2 (GenBank Accession No. NP 001002273), FcyRIIB3 GenBank Accession No.
  • DC-SIGN refers to a Dendritic Cell-Specific Adhesion Molecule-3 -Grabbing
  • Nonintegrin [CD209].
  • the human DC-SIGN receptor identified by Geijtenbeek, et al, Cell 100:575-585 (2000); US 6,391,507) is a C-type lectin (calcium-dependent) receptor which is expressed at least on dendritic cells, macrophages and activated B cells.
  • a polynucleotide sequence encoding human DC-SIGN can be found using GenBank Accession No. NM 021155.
  • a mouse ortholog to human DC-SIGN is SIGN-R1. Method of detecting binding between a compound and DC-SIGN are well known in the art. See, e.g. WO2011/031736 and
  • glycoengineered Pichia pastoris when used herein refers to a strain of Pichia pastoris that has been genetically altered to express human- like N-glycans.
  • GFI 5.0, GFI 5.5 and GFI 6.0 strains The term “GFI 5.0” when used herein refers to glycoengineered Pichia pastoris strains that produce glycoproteins having predominantly Gal2GlcNAc2Man3GlcNAc2 N-glycans.
  • GFI 6.0 when used herein refers to glycoengineered Pichia pastoris strains that produce glycoproteins having predominantly NANA2Gal2GlcNAc2Man3GlcNAc2 N- glycans.
  • GS5.0 when used herein refers to the N-glycosylation structure
  • NANAGal2GlcNAc2Man3GlcNAc2 which when produced in Pichia pastoris strains to which a 2,6 sialyl transferase has been glycoengineered result in a-2,6-linked sialic acid and which when produced in Pichia pastoris strains to which a-2,3 sialyl transferase has been glycoengineered result in a-2,3-linked sialic acid.
  • GS6.0 when used herein refers to the N-glycosylation structure
  • NANA2Gal2GlcNAc2Man3GlcNAc2 which when produced in Pichia pastoris strains to which a-2,6 sialyltransferase has been glycoengineered result in a-2,6-linked sialic acid and which when produced in Pichia pastoris strains to which a-2,3 sialyl transferase has been
  • wild type or wt when used herein in connection to a Pichia pastoris strain refers to a native Pichia pastoris strain that has not been subjected to genetic modification to control glycosylation.
  • wild type or wt when used herein in connection to an FcR refers to a an FcR polypeptode or amino acid that comprises the a native nucleotide or amino acid sequence - a sequence that has not been subject to any genetic modification.
  • N-glycan refers to an N- linked oligosaccharide, e.g., one that is attached by an asparagine-N-acetylglucosamine linkage to an asparagine residue of a polypeptide.
  • Predominant sugars found on glycoproteins are glucose, galactose, mannose, fucose, ⁇ -acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialic acid (SA, including NANA, NGNA and derivatives and analogs thereof, including acetylated NANA or acetylated NGNA).
  • N-acetyl-neuraminic acid In glycoengineered Pichia pastoris, sialic acid is exclusively N-acetyl-neuraminic acid (NANA) (Hamilton et al., Science 313 (5792): 1441-1443 (2006)). N-glycans have a common pentasaccharide core of Man3GlcNAc2, wherein “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 (antennae) comprising peripheral sugars (e.g., GlcNAc, galactose, fucose and sialic acid) that are added to the Man3GlcNAc2 ("Man3") core structure which is also referred to as the "trimannose core", the "pentasaccharide core” or the "paucimannose core”.
  • branches comprising peripheral sugars (e.g., GlcNAc, galactose, fucose and sialic acid) that are added to the Man3GlcNAc2 (“Man3") core structure which is also referred to as the "trimannose core", the "pentasaccharide core” or the "paucimannose core”.
  • Man3 Man3GlcNAc2
  • N-glycans are classified according to their branched constituents (e.g., high mannose, complex or hybrid).
  • sialic acid refers to any member of the sialic acid family, including without limitation: N-acetylneuraminic acid (Neu5Ac or NANA), N- glycolylneuraminic acid (NGNA) and any analog or derivative thereof (including those arising from acetylation at any position on 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 usually the terminal residue of the oligosaccharides.
  • N-acetylneuraminic acid is the most common sialic acid form and N-glycolylneuraminic acid (NGNA) is the second most common form.
  • NGNA N-glycolylneuraminic acid
  • human-like N-glycan refers to the N-linked oligosaccharides which closely resemble the oligosaccharides produced by non-engineered, wild-type human cells.
  • wild-type Pichia pastoris and other lower eukaryotic cells typically produce hypermannosylated proteins at N-glycosylation sites.
  • the host cells described herein produce glycoproteins (for example, antibodies) comprising human-like N-glycans that are not hypermannosylated.
  • the host cells of the present invention are capable of producing human-like N-glycans with hybrid and/or complex N-glycans.
  • the specific type of "human-like" glycans present on a specific glycoprotein produced from a host cell of the invention will depend upon the specific glycoengineering steps that are performed in the host cell.
  • high mannose type N-glycan when used herein refers to an N-glycan having five or more mannose residues.
  • complex N-glycan when used herein refers to an N-glycan having 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 have galactose (“Gal”) or N- acetylgalactosamine (“GalNAc”) residues that 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 have intrachain substitutions comprising "bisecting" GlcNAc and core fucose ("Fuc").
  • GlcNAc core fucose
  • the structure when a N-glycan comprises a bisecting GlcNAc on the trimannose core, the structure can be represented as Man3GlcNAc2(GlcNAc) or Man3GlcNAc3.
  • the structure may be represented as Man3GlcNAc2(Fuc).
  • Complex N-glycans may also have multiple antennae on the "trimannose core,” often referred to as “multiple antennary glycans.”
  • hybrid N-glycan when used herein refers to an N-glycan having at least one GlcNAc on the terminal 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.
  • mole percent of a glycan present in a preparation of a glycoprotein means the molar percent of a particular glycan present in the pool of N-linked oligosaccharides released when the protein preparation is treated with PNGase and then quantified by a method that is not affected by glycoform composition, (for instance, labeling a PNGase released glycan pool with a fluorescent tag such as 2-aminobenzamide and then separating by high performance liquid chromatography or capillary electrophoresis and then quantifying glycans by fluorescence intensity).
  • a fluorescent tag such as 2-aminobenzamide
  • Gal2GlcNAc2Man3GlcNAc2 means that 50 percent of the released glycans are NANA2
  • Gal2GlcNAc2Man3GlcNAc2 and the remaining 50 percent are comprised of other N-linked oligosaccharides.
  • mole percent and “percent” are used interchangeably.
  • anti-inflammatory antibody refers to an antibody intended to be used to treat inflammation.
  • the anti-inflammatory properties of an Fc-containing polypeptide can be measured using any method known in the art.
  • 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
  • the antiinflammatory properties of an Fc-containing polypeptide are measured by determining the level of a biomarker related to inflammation (including without limitation: CRP, pro-inflammatory cytokines such as tumor necrosis factors (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.
  • a biomarker related to inflammation including without limitation: CRP, pro-inflammatory cytokines such as tumor necrosis factors (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.
  • the anti-inflammatory properties of an Fc-containing polypetpide is 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 has anti-inflammatory properties.
  • CRP C-reactive protein
  • Constantly 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 frequently be made without altering the biological activity of the protein.
  • Those of skill in this 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.)).
  • substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are listed below:
  • IgG immunoglobulin G
  • ADCC antibody dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • mAbs monoclonal antibodies
  • IgG which lacks glycosylation in the Fc Cpf2 domain does not bind to FcyR, including FcyRI, FcyRII, and FcyRIII, Rothman (1989).
  • FcyRI FcyRI
  • FcyRII FcyRII
  • FcyRIII Rothman (1989).
  • the particular composition of the N-linked oligosaccharide is also important.
  • fucose shows a marked effect on in vitro FcyRIIIa binding and in vitro ADCC, 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 predominately fucosylated, Hossler et al, Biotechnology and
  • IVIG Intravenous immunoglobulin
  • Glycosylation in the Fc region of an antibody derived from mammalian cell lines typically consists of a heterogeneous mix of glycoforms, with the predominant forms typically being comprised of the complex fucosylated glycoforms: G0F, GIF, and, to a lesser extent, G2F.
  • Possible conditions resulting in incomplete galactose transfer to the G0F structure include, but are not limited to, non-optimized galactose transfer machinery, such as ⁇ -1,4 galactosyl transferase, and poor UDP-galactose transport into the Golgi apparatus, suboptimal cell culture and protein expression conditions, and steric hindrance by amino acid residues neighboring the oligosaccharide.
  • Yeast 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) describes the use of libraries of a 1,2 mannosidase catalytic domains and N- acetylglucosaminyltransferase I catalytic domains in combination with a library of fungal type II membrane protein leader sequences to localize the catalytic domains to the secretory pathway. In this way, strains were isolated that produced in vivo glycoproteins with uniform
  • Man5GlcNAc2 or GlcNAcMan5GlcNAc2 N-glycan structures were described the production of a glycoprotein, erythropoietin, produced in Pichia pastoris, as having a glycan composition that consisted predominantly of a bisialylated glycan structure, GS6.0, NANA2Gal2GlcNAc2Man3GlcNAc2 (90.5%) and monosialylated, GS5.5, NANAGal2GlcNAc2 Man3GlcNAc2 (7.9%).
  • WO201//149999 describes the production of sialyulated antibodies in Pichia pastoris.
  • the Fc-containing polypeptides of this invention can be made in any host organism or cell line.
  • an Fc-containing polypeptide of the invention is made in a host cell which is capable of producing proteins comprising N-glycans.
  • an Fc-containing polypeptide of the invention is made in a host cell which is capable of producing proteins comprising sialylated N-glycans.
  • an Fc-containing polypeptide of the invention is made in a mammalian cell where the cell either endogenously or through genetic or process manipulation produces glycoproteins containing either a mixture of terminal a2-6 and a2-3 sialic acid, or only terminal a2-6 sialic acid.
  • the propagation of mammalian cells in culture has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney
  • CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby 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 3 A, 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;
  • Hep G2 a human hepatoma line
  • an Fc-containing polypeptide of the invention can be made in a plant cell which is engineered to produce sialylated N-glycans. See, e.g., Cox et al, Nature
  • an Fc-containing polypeptide of the invention can be made in an insect cell which is engineered to produce sialylated N-glycans. See, e.g., Harrison and Jarvis, Adv. Virus Res. 68: 159-91 (2006).
  • an Fc-containing polypeptide of the invention can be made in a bacterial cell which is engineered to produce sialylated N-glycans. See, e.g., Lizak et al,
  • an Fc-containing polypeptide of the invention can be made in a lower eukaryotic host cell or organism.
  • yeast and filamentous fungi such as Pichia pastoris, Gerngross et al, US Patent 7,029,872 and US Patent No. 7,449,308, the disclosures of which are hereby incorporated by reference. See also Jacobs et al, Nature
  • an Fc-containing polypeptide of the invention is made in a lower eukaryotic host cell, more preferably a yeast or filamentous fungal host cell, that has been engineered to produce glycoproteins comprising human-like N-glycans.
  • an Fc-containing polypeptide of the invention is made in a lower eukaryotic host cell, more preferably a yeast or filamentous fungal host cell, that has been engineered to produce glycoproteins having a predominant N-glycan comprising a terminal sialic acid.
  • the predominant N-glycan is the a-2,6 linked form of
  • SA2Gal2GlcNAc2Man3GlcNAc2 produced in strains glycoengineered with a-2,6 sialyl transferase which do not produce any a-2,3 linked sialic acid.
  • yeast In general, lower eukaryotes such as yeast are used for expression of the proteins, particularly glycoproteins because they can be economically cultured, give high yields, and when appropriately modified are capable of suitable glycosylation.
  • Yeast particularly offers established genetics allowing for rapid transformations, tested protein localization strategies and facile gene knock-out techniques.
  • Suitable vectors have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.
  • Pichia pastoris While the invention has been demonstrated herein using the methylotrophic yeast Pichia pastoris, other useful lower eukaryote host cells include Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, 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, Asperg
  • Trichoderma reesei Chrysosporiumi lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum Yarrowia lipolytica and Neurospora crassa.
  • yeasts such as K. lactis, Pichia pastoris, Pichia methanolica, and Hansenula polymorpha are particularly suitable for cell culture because they are able to grow to high cell densities and secrete large quantities of recombinant protein.
  • filamentous fungi such as Aspergillus niger, Fusarium sp, Neurospora crassa and others can be used to produce glycoproteins of the invention at an industrial scale.
  • Lower eukaryotes particularly yeast and filamentous fungi, can be genetically modified so that they express glycoproteins in which the glycosylation pattern is human-like or humanized.
  • the term "human-like N-glycan”, as used herein refers, to the N-linked oligosaccharides which closely resemble the oligosaccharides produced by non- engineered, wild-type human cells.
  • 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 predominantly present on glycoproteins produced from the host cells of the invention will depend upon the specific engineering steps that are performed.
  • glycoprotein compositions can be produced in which a specific desired glycoform is predominant in the composition. Such can be achieved by eliminating selected endogenous glycosylation enzymes and/or genetically engineering the host cells and/or supplying exogenous enzymes to mimic all or part of the mammalian glycosylation pathway as described in US Patent No. 7,449,308.
  • glycosylation can be performed, such that the glycoprotein can be produced with or without core fucosylation.
  • Use of lower eukaryotic host cells is further advantageous in that these cells are able to produce highly homogenous compositions of glycoprotein, such that the predominant glycoform of the glycoprotein may be present as greater than thirty mole percent of the glycoprotein in the composition.
  • 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.
  • Lower eukaryotes, particularly yeast, can be genetically modified so that they express glycoproteins in which the glycosylation pattern is human-like or humanized. Such can be achieved by eliminating selected endogenous glycosylation enzymes and/or supplying exogenous enzymes as described by Gerngross et al, US Patent No. 7,449,308.
  • a host cell can be selected or engineered to be depleted in l,6-mannosyl transferase activities, which would otherwise add mannose residues onto the N-glycan on a glycoprotein.
  • the host cell further includes an l,2-mannosidase catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target the l,2-mannosidase activity to the ER or Golgi apparatus of the host cell.
  • Passage of a recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a Man5GlcNAc2 glycoform, for example, a recombinant glycoprotein composition comprising predominantly a Man5GlcNAc2 glycoform.
  • U.S. Patent Nos. 7,029,872 and 7,449,308 and U.S. Published Patent Application No. 2005/0170452 disclose lower eukaryote host cells capable of producing a glycoprotein comprising a Man5GlcNAc2 glycoform.
  • the immediately preceding host cell further includes a GlcNAc transferase I (GnT I) catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target GlcNAc transferase I activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a GlcNAc transferase I (GnT I) catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target GlcNAc transferase I activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a GlcNAc transferase I (GnT I) catalytic domain fused to
  • GlcNAcMan5GlcNAc2 glycoform for example a recombinant glycoprotein composition comprising predominantly a GlcNAcMan5GlcNAc2 glycoform.
  • U.S. Patent Nos. 7,029,872 and 7,449,308 and U.S. Published Patent Application No. 2005/0170452 disclose lower eukaryote host cells capable of producing a glycoprotein comprising a GlcNAcMan5GlcNAc2 glycoform.
  • the glycoprotein produced in the above cells can be treated in vitro with a hexosaminidase to produce a recombinant glycoprotein comprising a Man5GlcNAc2 glycoform.
  • the immediately preceding host cell further includes a mannosidase II catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target mannosidase II activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a
  • GlcNAcMan3GlcNAc2 glycoform for example a recombinant glycoprotein composition comprising predominantly a GlcNAcMan3GlcNAc2 glycoform.
  • U.S. Patent No, 7,029,872 and U.S. Published Patent Application No. 2004/0230042 discloses lower eukaryote host cells that express mannosidase II enzymes and are capable of producing glycoproteins having
  • the glycoprotein produced in the above cells can be treated in vitro with a hexosaminidase to produce a recombinant glycoprotein comprising a Man3GlcNAc2 glycoform.
  • the immediately preceding host cell further includes GlcNAc transferase II (GnT II) catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target GlcNAc transferase II activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a GlcNAcMan3GlcNAc2 glycoform.
  • GnT II GlcNAc transferase II
  • GlcNAc2Man3GlcNAc2 glycoform for example a recombinant glycoprotein composition comprising predominantly a GlcNAc2Man3GlcNAc2 glycoform.
  • U.S. Patent Nos. 7,029,872 and 7,449,308 and U.S. Published Patent Application No. 2005/0170452 disclose lower eukaryote host cells capable of producing a glycoprotein comprising a GlcNAc2Man3GlcNAc2 glycoform.
  • the glycoprotein produced in the above cells can be treated in vitro with a hexosaminidase to produce a recombinant glycoprotein comprising a Man3GlcNAc2 glycoform.
  • the immediately preceding host cell further includes a galactosyltransferase catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target galactosyltransferase activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a
  • U.S. Patent No, 7,029,872 and U.S. Published Patent Application No. 2006/0040353 discloses lower eukaryote host cells capable of producing a glycoprotein comprising a Gal2GlcNAc2
  • Man3GlcNAc2 glycoform The glycoprotein produced in the above cells can be treated in vitro with a galactosidase to produce a recombinant glycoprotein comprising a GlcNAc2Man3 GlcNAc2 glycoform, for example a recombinant glycoprotein composition comprising predominantly a GlcNAc2Man3GlcNAc2 glycoform.
  • the immediately preceding host cell further includes a sialyltransferase catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target sialyltransferase activity to the ER or Golgi apparatus of the host cell.
  • the sialyltransferase is an a-2,6- sialyltransferase. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising predominantly a
  • the host cell further include a means for providing CMP-sialic acid for transfer to the N- glycan.
  • U.S. Published Patent Application No. 2005/0260729 discloses a method for genetically engineering lower eukaryotes to have a CMP-sialic acid synthesis pathway and U.S. Published Patent Application No. 2006/0286637 discloses a method for genetically engineering lower eukaryotes to produce sialylated glycoproteins.
  • the host cell can include two or more copies of the CMP-sialic acid synthesis pathway or two or more copies of the sialylatransferase.
  • 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
  • Any one of the preceding host cells can further include one or more GlcNAc transferase selected from the group consisting of GnT III, GnT IV, GnT V, GnT VI, and GnT IX to produce glycoproteins having bisected (GnT III) and/or multiantennary (GnT IV, V, VI, and IX) N- glycan structures such as disclosed in U.S. Published Patent Application Nos. 2005/0208617 and 2007/0037248.
  • the proceeding host cells can produce recombinant glycoproteins (for example, antibodies) comprising SA(l-4)Gal(l-4)GlcNAc(2-4) Man3GlcNAc2, including antibodies comprising NANA (l-4)Gal(l-4)GlcNAc(2-4) Man3GlcNAc2, NGNA(l-4)Gal(l- 4)GlcNAc(2-4)Man3GlcNAc2 or a combination of NANA (l-4)Gal(l-4)GlcNAc(2-4)
  • the recombinant glycoprotein will comprise N-glycans comprising a structure selected from the group consisting of SA(l-4)Gal(l-4)GlcNAc(2-4) Man3GlcNAc2 and devoid of any a2-3 linked SA.
  • GlcNAcMan5GlcNAc2 N-glycans further includes a galactosyltransferase catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target the galactosyltransferase activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising predominantly the GalGlcNAcMan5GlcNAc2 glycoform.
  • the immediately preceding host cell that produced glycoproteins that have predominantly the GalGlcNAcMan5GlcNAc2 N-glycans further includes a
  • sialyltransferase catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target sialyltransferase activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a
  • SAGalGlcNAcMan5GlcNAc2 glycoform for example NANAGalGlcNAcMan5GlcNAc2 or NGNAGalGlcNAcMan5GlcNAc2 or a mixture thereof.
  • any of the preceding host cells can further include one or more sugar transporters such as UDP-GlcNAc transporters (for example, Kluyveromyces lactis and Mus musculus UDP-GlcNAc transporters), UDP-galactose transporters (for example, Drosophila melanogaster UDP- galactose transporter), and CMP-sialic acid transporter (for example, human sialic acid transporter).
  • UDP-GlcNAc transporters for example, Kluyveromyces lactis and Mus musculus UDP-GlcNAc transporters
  • UDP-galactose transporters for example, Drosophila melanogaster UDP- galactose transporter
  • CMP-sialic acid transporter for example, human sialic acid transporter
  • any of the preceding host cells can be further manipulated to increase N-glycan occupancy. See e, g., Gaulitzek et al, Biotechnol. Bioengin. 103:1164-1175 (2009); Jones et al, Biochim. Biospyhs. Acta 1726: 121-137 (2005); WO2006/107990.
  • any of the preceding host cells can be further engineered to comprise at least one nucleic acid molecule encoding a heterologous single-subunit oligosaccharyltransferase (for example, Leishmania sp.
  • any of the preceding host cells can be further 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 host cell genes encoding the proteins comprising the endogenous OTase complex.
  • Host cells further include lower eukaryote cells (e.g., yeast such as Pichia pastoris) that are genetically engineered to produce glycoproteins that do not have a-mannosidase-resistant N- glycans.
  • yeast such as Pichia pastoris
  • This can be achieved by deleting or disrupting one or more of the ⁇ - mannosyltransferase genes (e.g., BMT1, BMT2, BMT3, and BMT4) (See, U.S. Published Patent Application No. 2006/0211085) and glycoproteins having phosphomannose residues by deleting or disrupting one or both of the phosphomannosyl transferase genes PNOl and MNN4B (See for example, U.S. Patent Nos.
  • ⁇ - mannosyltransferase genes e.g., BMT1, BMT2, BMT3, and BMT4
  • PNOl and MNN4B See for example, U.S. Patent Nos.
  • Disruption includes disrupting the open reading frame encoding the particular enzymes or disrupting expression of the open reading frame or abrogating translation of RNAs encoding one or more of the ⁇ -mannosyltransferases and/or phosphomannosyltransferases using interfering RNA, antisense RNA, or the like.
  • cells can produce glycoproteins with a-mannosidase-resistant N-glycans through the addition of chemical hinhibios or through modification of the cell culture condition. These host cells can be further modified as described above to produce particular N-glycan structures.
  • Host cells further include lower eukaryote cells (e.g., yeast such as Pichia pastoris) that are genetically modified to control O-glycosylation of the glycoprotein by deleting or disrupting one or more of the protein O-mannosyltransferase (Dol-P-Man:Protein (Ser/Thr) Mannosyl Transferase genes) (PMTs) (See U.S. Patent No. 5,714,377) or grown in the presence of Pmtp inhibitors and/or an a -mannosidase as disclosed in Published International Application No. WO 2007/061631, or both.
  • yeast eukaryote cells
  • 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 interfering RNA, antisense RNA, or the like.
  • the host cells can further include any one of the aforementioned host cells modified to produce particular N-glycan structures.
  • Pmtp inhibitors include but are not limited to a benzylidene thiazolidinediones.
  • Examples of benzylidene thiazolidinediones that can be used are 5-[[3,4-bis(phenylmethoxy) phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic Acid; 5-[[3-(l-Phenylethoxy)-4-(2- phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic Acid; and 5-[[3-(l- Phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3- thiazolidineacetic acid.
  • the function or expression of at least one endogenous PMT gene is reduced, disrupted, or deleted.
  • the function or expression of at least one endogenous PMT gene selected from the group consisting of the PMT I, PMT2, PMT3, and PMT 4 genes is reduced, disrupted, or deleted; or the host cells are cultivated in the presence of one or more PMT inhibitors.
  • the host cells include one or more PMT gene deletions or disruptions and the host cells are cultivated in the presence of one or more Pmtp inhibitors.
  • the host cells also express a secreted a -1,2-mannosidase.
  • PMT deletions or disruptions and/or Pmtp inhibitors control O-glycosylation by reducing O-glycosylation occupancy, that is, by reducing the total number of O-glycosylation sites on the glycoprotein that are glycosylated.
  • the further addition of an a -1,2-mannsodase that is secreted by the cell controls O-glycosylation by reducing the mannose chain length of the O-glycans that are on the glycoprotein.
  • the particular combination of PMT deletions or disruptions, Pmtp inhibitors, and a -1,2-mannosidase is determined empirically as particular heterologous glycoproteins (Fabs and antibodies, for example) may be expressed and transported through the Golgi apparatus with different degrees of efficiency and thus may require a particular combination of PMT deletions or disruptions, Pmtp inhibitors, and a -1,2- mannosidase.
  • genes encoding one or more endogenous mannosyltransferase enzymes are deleted. This deletion(s) can be in combination with providing the secreted a- 1,2- mannosidase and/or PMT inhibitors or can be in lieu of providing the secreted a- 1,2- mannosidase and/or PMT inhibitors.
  • control of O-glycosylation can be useful for producing particular glycoproteins in the host cells disclosed herein in better total yield or in yield of properly assembled
  • glycoprotein The reduction or elimination of O-glycosylation appears to have a beneficial effect on the assembly and transport of whole antibodies and Fab fragments as they traverse the secretory pathway and are transported to the cell surface.
  • O- glycosylation the yield of properly assembled antibodies or Fab fragments is increased over the yield obtained in host cells in which O-glycosylation is not controlled.
  • the recombinant glycoengineered Pichia pastoris host cells are genetically engineered to eliminate glycoproteins having a-mannosidase- resistant N-glycans by deleting or disrupting one or more of the ⁇ -mannosyltransferase genes (e.g., BMT1, BMT2, BMT3, and BMT4) (See, U.S. Patent No. 7,465,577 and U.S. Patent No. 7,713,719).
  • the deletion or disruption ⁇ 2 and one or more ⁇ , BMT3, and BMT4 also reduces or eliminates detectable cross reactivity to antibodies against host cell protein.
  • Yield of glycoprotein can in some situations be improved by overexpressing nucleic acid molecules encoding mammalian or human chaperone proteins or replacing the genes encoding one or more endogenous chaperone proteins with nucleic acid molecules encoding one or more mammalian or human chaperone proteins.
  • the expression of mammalian or human chaperone proteins in the host cell also appears to control O-glycosylation in the cell.
  • the host cells herein wherein 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 homolog of the chaperone protein is expressed in the host cell.
  • the lower eukaryotic host cell is a yeast or filamentous fungi host cell.
  • Examples of the use of chaperones of host cells in which human chaperone proteins are introduced to improve the yield and reduce or control O- glycosylation of recombinant proteins has been disclosed in Published International Application No. WO 2009105357 and WO2010019487 (the disclosures of which are incorporated herein by reference).
  • lower eukaryotic host cells wherein, 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
  • the function or expression of at least one endogenous gene encoding a protein O- mannosyltransferase (PMT) protein is reduced, disrupted, or deleted.
  • the function of at least one endogenous PMT gene selected from the group consisting of the PMT1, PMT2, PMT3, and PMT 4 genes is reduced, disrupted, or deleted.
  • O-glycosylation may have an effect on an antibody or Fab fragment's affinity and/or avidity for an antigen. This can be particularly significant when the ultimate host cell for production of the antibody or Fab is not the same as the host cell that was used for selecting the antibody.
  • O-glycosylation might interfere with an antibody's or Fab fragment's affinity for an antigen, thus an antibody or Fab fragment that might otherwise have high affinity for an antigen might not be identified because O-glycosylation may interfere with the ability of the antibody or Fab fragment to bind the antigen.
  • an antibody or Fab fragment that has high avidity for an antigen might not be identified because O-glycosylation interferes with the antibody's or Fab fragment's avidity for the antigen.
  • an antibody or Fab fragment that might be particularly effective when produced in a mammalian cell line might not be identified because the host cells for identifying and selecting the antibody or Fab fragment was of another cell type, for example, a yeast or fungal cell (e.g., a Pichia pastoris host cell).
  • a yeast or fungal cell e.g., a Pichia pastoris host cell.
  • 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 mucin-type or dystroglycan type O-glycosylation in mammals.
  • O-glycosylation might enhance the antibody or Fab fragments affinity or avidity for an antigen instead of interfere with antigen binding.
  • This effect is undesirable when the production host cell is to be different from the host cell used to identify and select the antibody or Fab fragment (for example, identification and selection is done in yeast and the production host is a mammalian cell) because in the production host the O-glycosylation will no longer be of the type that caused the enhanced affinity or avidity for the antigen. Therefore, controlling O-glycosylation can enable use of the materials and methods herein to identify and select antibodies or Fab fragments with specificity for a particular antigen based upon affinity or avidity of the antibody or Fab fragment for the antigen without identification and selection of the antibody or Fab fragment being influenced by the O-glycosylation system of the host cell.
  • controlling O-glycosylation further enhances the usefulness of yeast or fungal host cells to identify and select antibodies or Fab fragments that will ultimately be produced in a mammalian cell line.
  • Those of ordinary skill in the art would 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.
  • an increase or decrease in anti-inflammatory activity can be detected using any method known in the art.
  • 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- ⁇ , IL-6, RANKL, TRAP, ATP6v0d2, MDL-1, DAP 12, CDl lb, TIMP-1, MMP9, CTSK, PU-1, MCP1, ⁇ , Cxcll- Groa, CXcl2-Grob, CD 18, TNF, FcyRI, FcyRIIb, FcyRIII and FcyRIV.
  • a decrease in anti-inflammatory activity is detected by measuring a change in the expression of a gene selected from the group consisting of: IL- ⁇ , IL-6, RANKL, TRAP, ATP6v0d2, MDL-1, DAP 12, CDl lb, TIMP-1, MMP9, CTSK, PU-1, MCP1, ⁇ , Cxcll-Groa, CXcl2-Grob, CD 18, TNF, FcyRI, FcyRIIb, FcyRIII and FcyRIV.
  • a gene selected from the group consisting of: IL- ⁇ , IL-6, RANKL, TRAP, ATP6v0d2, MDL-1, DAP 12, CDl lb, TIMP-1, MMP9, CTSK, PU-1, MCP1, ⁇ , Cxcll-Groa, CXcl2-Grob, CD 18, TNF, FcyRI, FcyRIIb, FcyRIII and FcyRIV
  • the Fc-containing polypeptides of the invention can be made according to any method known in the art suitable for generating polypeptides.
  • 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).
  • the antibody or antibody fragment is of the IgG type.
  • the antibody or antibody fragment is of the IgGl subtype.
  • the antibody or antibody fragment is of the IgG2 subtype.
  • the antibody or antibody fragment is of the IgG3 subtype.
  • the antibody or antibody fragment is of the IgG4 subtype.
  • the antibody or antibody fragment is of the IgA type.
  • the antibody or antibody fragment is of the IgD type. In one embodiment, the antibody or antibody fragment is of the IgE type. In one embodiment, the antibody or antibody fragment is of the IgM type. In another embodiment, the Fc-containing polypeptide is an immunoadhesin. Methods of preparing antibody and antibody fragments are well known in the art. Methods of introducing point mutations into a polypeptide, for example site directed mutagenesis, are also well known in the art.
  • the Fc-containing polypeptide comprises N-glycans. In one embodiment, the Fc-containing polypeptide of the invention comprises N-glycans, wherein at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc-containing
  • polypeptide comprise an N-linked oligosaccharide structure selected from the group consisting of SA(i-4)Gal(i-4)GlcNAc(2-4)Man3GlcNAc2. In one embodiment, at least 30%, 40%, 50%, 60%), 70%), 80%) or 90%> of the N-glycans on the Fc-containing polypeptide comprise a
  • At least 30%, 40%, 50%, 60%, 70%), 80%) or 90%) of the N-glycans on the Fc-containing polypeptide comprise a
  • N-glycosylation is important for biological properties.
  • sialylation of the terminal N-linked glycan of an IgG Fc region is important for producing glycoproteins and antibodies that have the correct
  • sialylation requires the presence of a penultimate galactose, upon which the sialyl transferase acts to form the sialylated glycan.
  • the N-glycan composition of the antibodies can be analyzed by matrix-assisted laser desorption ionization/time-of- flight (MALDI-TOF) mass spectrometry after release from the antibody with peptide-N-glycosidase F. Released carbohydrate composition can be quantitated by HPLC on an Allentech Prevail carbo (Alltech Associates, Deerfield IL) column.
  • MALDI-TOF matrix-assisted laser desorption ionization/time-of- flight
  • the DC-SIGN, FcyR and FcRn binding of Fc muteins can be determined using any method known in the art. See, e.g., Shields et al, J. Biol. Chem. 276: 6591-6604 (2001);
  • the Fc region of the Fc-containing polypeptide could 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 IgGl, IgG2, IgG3 or IgG4. In one embodiment, Fc region of the Fc- containing polypeptide is from an IgGl .
  • the antibodies or antibody fragments produced by the materials and methods herein can be chimeric, humanized or human antibodies.
  • the Fc-containing polypeptides of the invention will bind to a biological target that is involved in inflammation.
  • the Fc-containing polypeptide of the invention will bind to a proinflammatory cytokine. In some embodiments, the Fc-containing polypeptide of the invention will bind to a molecule selected from the group consisting of: TNF-a, 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, a4p7-integrin, E-selectin, Fact II, ICAM-3,
  • the Fc-containing polypeptide of the invention will bind to TNF-a. 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. In another embodiment, the Fc-containing polypeptide of the invention will bind to aBLys.
  • 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, e.g., the antigens listed in Table 1 of WO2010/10910, which is incorporated herein by reference.
  • 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 useful in treating an inflammatory condition (for example, an antibody or immunoadhesin that binds to an antigen that is involved in inflammation) and introducing one or more mutations in the Fc region, wherein the mutations are selected from the group consisting of:
  • L351S/N384D/S426P/K447E F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274R/K360E/Y373H/P387S,
  • K246E/M252T/E272G/S375P D249G, F241S/V263A/T335A/N389S, E269G/N389S/S400P, N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G and
  • the Fc-containing polypeptide comprises N-glycans. In one embodiment, the Fc-containing polypeptide comprises sialylated N-glycans. In one embodiment, the parent Fc-containing polypeptide is an antibody, antibody fragment or immunoadhesin that binds to an antigen that is involved in inflammation.
  • 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 useful in treating an inflammatory condition (for example, an antibody or immunoadhesin that binds to an antigen that is involved in inflammation), wherein the parent Fc- containing polypeptide is a human IgGl or a fragment of human IgGl, and introducing one or more mutations in the Fc region, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R, M252V/V303A/N315S, S239P/F241L/I377V,
  • L351S/N384D/S426P/K447E F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274R/K360E/Y373H/P387S,
  • K246E/M252T/E272G/S375P D249G, F241S/V263A/T335A/N389S, E269G/N389S/S400P, N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G and
  • the Fc-containing polypeptide comprises N-glycans. In one embodiment, the Fc-containing polypeptide comprises sialylated N-glycans. In one embodiment, the parent Fc-containing polypeptide is an antibody, antibody fragment or immunoadhesin that binds to an antigen that is involved in inflammation.
  • the parent Fc-containing polypeptide is an antibody, antibody fragment or immunoadhesin that is already marketed or under development for the treatment of an inflammatory conditions.
  • 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-TNFa antibody), Trastuzumab (anti-Her2 antibody),
  • Ibritumomab tiuxeten (anti-CD20 antibody), Adalimumab (anti-TNFa antibody), Omalizumab (anti-IgE antibody), Tositumomab-1311 (iodinated derivative of an anti-CD20 antibody), Efalizumab (anti-CDl la antibody), Cetuximab (anti-EGF receptor antibody), Golimumab (anti- TNFa antibody), Bevacizumab (anti VEGF-A antibody), Natalizumab (anti a4 integrin), Efalizumab (anti CD 11a), Cetolizumab (anti-TNFa antibody) , Tocilizumab (anti-IL-6R), Ustenkinumab (anti IL-12/23), alemtuzumab (anti CD52), and natalizumab (anti a4 integrin), and variants thereof.
  • the parent Fc-contanining polypeptide is an Fc- fusion protein selected from the group consisting of: Arcalyst/ rilonacept (ILIR-Fc fusion), Orencia/ abatacept (CTLA-4-Fc fusion), Amevive/ alefacept (LFA-3-Fc fusion), Anakinra-Fc fusion (IL-lRa-Fc fusion protein), etanercept (TNFR-Fc fusion protein), FGF-21-Fc fusion protein, GLP-l-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
  • the invention comprises a method of treating a subject in need thereof comprising: administering to the subject a therapeutically effective amount of an Fc-containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R, M252V/V303A/N315S,
  • F241S/V263A/T335A/N389S E269G/N389S/S400P, N297D/S354P/K409R/S424P,
  • the parent Fc- containing polypeptide is human IgGl or a fragment of human IgGl .
  • the invention also comprises a method of treating a subject in need thereof comprising: administering to the subject a therapeutically effective amount of an Fc-containing polypeptide, wherein the Fc-containing polypeptide comprises any one of SEQ ID NOs: 11-33.
  • the Fc-containing polypeptide does not comprise N-glycans and the Fc-containing polypeptide is a human IgGl or a fragment of human IgGl comprising one or more of the mutations selected from the group consisting of: N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G and V259A/N297S/N389S/G446D.
  • the Fc-containing polypeptide does not comprise N-glycans and the Fc-containing polypeptide comprises any one of SEQ ID NOs:30-33.
  • the Fc-containing polypeptide comprises N-glycans and the Fc- containing polypeptide is a human IgGl or a fragment of human IgGl comprising one or more of the mutations selected from the group consisting of: Q295R/V302A/S344P/K414R ,
  • the Fc-containing polypeptide comprises sialylated N-glycans.
  • the Fc-containing polypeptide comprises N-glycans and the Fc- containing polypeptide comprises any one of SEQ ID NOs: 11-29. In one embodiment, the Fc- containing polypeptide comprises sialylated N-glycans.
  • the Fc-containing polypeptide of the invention can 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.
  • the subject is in need of treatment with a vaccine
  • the method further comprises the administration of a vaccine.
  • the invention comprises a method of increasing the efficacy of a vaccine comprising administering to a subject in need thereof an Fc- containing polypeptide of the invention.
  • the subject in need of treatment has an inflammatory condition.
  • the method further comprises the administration of another anti-inflammatory compound.
  • the method further comprises the administration of an agent that increases expression of FcyRIIB.
  • the method further comprises the administration of an agent that binds DC-SIGN.
  • inflammatory condition refers to a condition that is characterized by abnormal or unwanted inflammation, such as autoimmune disease.
  • Autoimmune diseases are disorders characterized by the chronic activation of immune cells under non-activating conditions. Examples include: psoriasis, inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis), rheumatoid arthritis, psoriatic arthritis, myasthenia gravis, multiple sclerosis, systemic lupus erythematosus, type I diabetes, primary biliary cirrhosis, and transplant.
  • psoriasis e.g., Crohn's disease and ulcerative colitis
  • rheumatoid arthritis e.g., Crohn's disease and ulcerative colitis
  • rheumatoid arthritis e.g., Crohn's disease and ulcerative colitis
  • rheumatoid arthritis e.g., Crohn's disease and ulcerative colitis
  • 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.
  • the inflammatory condition is a primary immunodeficiency syndrome, including congential agammaglobulinemia and hypogammaglobulinemia, common variable immunodeficiency, severed combined immunodeficiency, or Wiskott Aldrich syndrome.
  • the inflammatory condition is a secondary immunodeficiency syndrome, including B-cell lymphocytic leukemia, HIV infection or an allogeneic bone marrow transplantation.
  • the inflammatory condition is idiopathic thrombocytopenic purpura
  • the inflammatory condition is multiple myeloma.
  • the inflammatory condition is Guillain-Barre syndrome.
  • the inflammatory condition is Kawasaki disease.
  • the inflammatory condition is chronic inflammatory demyelinating polyneropathy (CIDP).
  • CIDP chronic inflammatory demyelinating polyneropathy
  • the inflammatory condition is autoimmune neutropenia.
  • the inflammatory condition is hemolytic anemia.
  • the inflammatory condition is anti-Factor VIII autoimmune disease.
  • the inflammatory condition is multifocal neuropathy.
  • the inflammatory condition is myasthenia gravis. In one embodiment, the inflammatory condition is systemic vasculitis (ANCA positive).
  • the inflammatory condition is polymyositis.
  • the inflammatory condition is dermatomyositis.
  • the inflammatory condition is antiphospholipid syndrome.
  • the inflammatory condition is sepsis syndrome.
  • the inflammatory condition is graft-v-host disease.
  • the inflammatory condition is allergy.
  • the inflammatory condition is an anti-Rhesus factor D reaction.
  • the inflammatory condition is an inflammatory condition of the cardiovascular system.
  • the Fc-containing polypeptides of the invention may be used to treat atherosclerosis, atherothrombosis, coronary artery hypertension, acute coronary syndrome and heart failure, all of which are associated with inflammation.
  • the inflammatory condition is an inflammatory condition of the central nervous system.
  • the inflammatory condition will be an inflammatory condition of the peripheral nervous system.
  • the Fc-containing polypeptides of the invention may be used for the treatment of, e.g., Alzheimer's disease, amyotrophic lateral sclerosis (a.k.a. ALS; Lou Gehrig's disease), ischemic brain injury, prion diseases, and HIV-associated dementia.
  • the inflammatory condition is an inflammatory condition of the gastrointestinal tract.
  • the Fc-containing polypeptides of the invention may be used for treating inflammatory bowel disorders, e.g., Crohn's disease, ulcerative colitis, celiac disease, and irritable bowel syndrome.
  • the inflammatory condition is psoriasis, atopic dermatitis, arthritis, including rheumatoid arthritis, osteoarthritis, and psoriatic arthritis.
  • the inflammatory condition is steroid-dependent atopic dermatitis.
  • the inflammatory condition is cachexia.
  • Examples of other inflammatory disorders that can 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.
  • a subject to be treated for an inflammatory condition can be identified by standard diagnosing techniques for the disorder.
  • the subject can be examined for the level or percentage of one or more of cytokines or cells a test sample obtained from the subject by methods known in the art.
  • the subject is a candidate for treatment described herein.
  • a threshold value which can be obtained from a normal subject
  • the subject is a candidate for treatment described herein.
  • the subject in need of treatment has infectious disease.
  • the subject in need of treatment has cancer.
  • Treating refers to administration of a compound or agent to a subject who has a disorder with the purpose to cure, alleviate, relieve, remedy, delay the onset of, prevent, or ameliorate the disorder, the symptom of the disorder, the disease state secondary to the disorder, or the predisposition toward the disorder.
  • terapéuticaally effective amount refers 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 to cause a measurable improvement in one or more symptoms of a disease or condition or the progression of such disease or condition.
  • therapeutically effective dose further refers to that amount of the Fc-containing polypeptide sufficient to result in at least partial amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to 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 will result in an improvement of a diagnostic measure or parameter by at least 10%; usually by at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%.
  • an effective amount can also result in an improvement in a subjective measure in cases where subjective measures are used to assess disease severity.
  • the agent can be administered in vivo or ex vivo, alone or co-administered in conjunction with other drugs or therapy, i.e., a cocktail therapy.
  • co-administered refers to the administration of at least two agents or therapies to a subject. In some embodiments, the co-administration of two or more
  • agents/therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy.
  • a first agent/therapy is administered prior to a second agent/therapy.
  • a compound or agent is administered to a subject.
  • the compound or agent is suspended in a pharmaceutically- acceptable carrier (such as, for example, but not limited to, physiological saline) and administered orally or by intravenous infusion, or injected or implanted subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily.
  • a pharmaceutically- acceptable carrier such as, for example, but not limited to, physiological saline
  • the dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the patient's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.001-100 mg/kg.
  • the Fc-containing polypeptide of the invention will be administered a dose of between 1 to 100 milligrams per kilograms of body weight. In one embodiment, the Fc- containing polypeptide of the invention will be administered a dose of between 0.001 to 10 milligrams per kilograms of body weight. In one embodiment, the Fc-containing polypeptide of the invention will be administered a dose of between 0.001 to 0.1 milligrams per kilograms of body weight. In one embodiment, the Fc-containing polypeptide of the invention will be administered a dose of between 0.001 to 0.01 milligrams per kilograms of body weight.
  • Fc-containing polypeptides can be provided by continuous infusion, or by doses administered, e.g., daily, 1-7 times per week, weekly, bi-weekly, monthly, bimonthly, quarterly, semiannually, annually etc.
  • the invention also comprises pharmaceutical formulations comprising an Fc-containing polypeptide of the invention and a pharmaceutically acceptable carrier.
  • the Fc-containing polypeptide of the invention is an IgG antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide of the invention is an IgGl antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide of the invention is a human IgGl antibody or antibody fragment.
  • the Fc-containing polypeptide of the invention comprises or consists essentially of any one of SEQ ID NOs: 11-33.
  • the Fc-containing polypeptide comprises N-glycans and the Fc- containing polypeptide comprises or consists essentially of any one of SEQ ID NOs: 11-29.
  • the Fc-containing polypeptide is a human IgGl or a fragment of human IgGl that comprises N-glycans and the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of: Q295R/V302A/S344P/K414R ,
  • the Fc- containing polypeptide comprises sialylated N-glycans.
  • the Fc-containing polypeptide is a human IgGl or a fragment of human IgGl that does not comprise N-glycans and the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of: N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G, and V259A/N297S/N389S/G446D.
  • the Fc-containing polypeptide is a human IgGl or a fragment of human IgGl that does not comprise N-glycans and the Fc-containing polypeptide comprises or consists essentially of any one of SEQ ID NOs:30-33.
  • the pharmaceutical formulation comprises and Fc-containing polypeptide comprising N-glycans.
  • the pharmaceutical formulation comprises and Fc-containing polypeptide comprising sialylated N-glycans.
  • the term "pharmaceutically acceptable” means a non-toxic 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 or a state government or listed in the U.S.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered and includes, but is not limited to such sterile liquids as water and oils. The characteristics of the carrier will depend on the route of administration.
  • compositions of therapeutic and diagnostic agents may be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions (see, e.g., Hardman et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY;
  • the mode of administration can vary. Suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal,
  • the Fc-containing polypeptides of the invention can be administered by an invasive route such as by injection (see above).
  • the Fc-containing polypeptides of the invention, or pharmaceutical composition thereof is administered intravenously, subcutaneously, intramuscularly, intraarterially, intra- articularly (e.g. in arthritis joints), intratumorally, or by inhalation, aerosol delivery.
  • Administration by non-invasive routes e.g., orally; for example, in a pill, capsule or tablet
  • non-invasive routes e.g., orally; for example, in a pill, capsule or tablet
  • the Fc-containing polypeptides of the invention can be administered by an invasive route such as by injection (see above).
  • the Fc-containing polypeptides of the invention, or pharmaceutical composition thereof is administered intravenously, subcutaneously, intramuscularly, intraarterially, intra- articularly (e.g. in arthritis joints), intratumorally, or by inhalation, aerosol delivery.
  • non-invasive routes e.g., orally; for example, in a pill, capsule or tablet
  • administration by non-invasive routes is also within the scope of the present invention.
  • compositions can be administered with medical devices known in the art.
  • a pharmaceutical composition of the invention can be administered by injection with a hypodermic needle, including, e.g., a prefilled syringe or autoinjector.
  • compositions of the invention may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Patent Nos.
  • compositions of the invention may also be administered by infusion.
  • implants and modules form administering pharmaceutical compositions include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent. No. 4,439, 196, which discloses an osmotic drug delivery system having multi-chamber compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art.
  • one may administer the antibody in a targeted drug delivery system for example, in a liposome coated with a tissue-specific antibody, targeting, for example, arthritic joint or pathogen-induced lesion characterized by immunopathology.
  • the liposomes will be targeted to and taken up selectively by the afflicted tissue.
  • 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 the target cells in the biological matrix.
  • the administration regimen delivers sufficient therapeutic antibody to effect improvement in the target disease state, while simultaneously minimizing undesired side effects.
  • the amount of biologic delivered depends in part on the particular therapeutic antibody and the severity of the condition being treated. Guidance in selecting appropriate doses of therapeutic antibodies is available (see, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub.
  • a library of Fc variants was constructed using error prone PCR to introduce random mutations in a region encompassing the CH2-CH3 domains of a human immunoglobulin G 1 (IgGl) gene. These PCR fragments encoding variant Fc molecules were cloned in frame to VH1-CH1 region of an anti-Her2 monoclonal antibody (mAb) under the expression of Pichia AOX1 promoter in pGLYl 1576 ( Figure 3). The complete amino acid sequence of the heavy chain of the Anti-Her2 antibody used in this experiment shown in SEQ ID NO:3. The VH1-CH1 sequence corresponds to amino acids 1-220 of SEQ ID NO: 3.
  • DNA isolated as described above was incubated for 2 hr with Spel restriction enzyme at 37°C.
  • Linearized plasmid DNA was precipitated with 3M sodium acetate pH 5.2 and isopropanol.
  • Digested DNA was reconstituted in deionized water at concentration of 1 ⁇ g/ ⁇ L. 1 mg of this DNA was introduced in a Pichia pastoris strain that is capable of producing sialylated N-glycans and which contained plasmid pGLYl 1714 ( Figure 1, SEQ ID NO:4), generating a library of 10 6 clones. These clones were scraped off plates and pooled in liquid media.
  • the plasmid designated as pGLYl 1714 contains a cassette expressing IgG Light chain fused to surface anchor Sedl (Lc-Sedl) on the cell surface.
  • Lc-Sedl surface anchor Sedl
  • a random set of 80 yeast clones were cultured in a 96-well plate, induced and analyzed for secreting full length IgGs by SDS-PAGE. 65% of the clones were shown to produce full-length IgGs.
  • ⁇ Pichia pastoris strains capable of producing sialylated N-glycans are described in the art, for example, in WO2011/149999.)
  • Figure 1 contains a plasmid map of pGLYl 1714.
  • the plasmid pGLYl 1714 containing anti-PC SK9 Lc bait cassette was constructed using a codon optimized sequence of human IgG2 Lc (VL+CL) fragment, which was synthesized and fused in frame to the 3' end of the nucleic acid sequence of S. cerevisiae a-mating factor signal sequence.
  • the nucleic acid sequence encoding three repeats of the amino acids (GGGS) linker was used to link the nucleic acid sequence of Lc 3' end to the 5'end of S. cerevisiae Sedlp.
  • the construct was subcloned into pGLY9008 at EcoRI -Sail (replacing the Fc) by the contracting research organization (CRO) Genewiz (South Plainfield, NJ).
  • CRO contracting research organization
  • SEQ ID NO: 4 provides the sequence of plasmid pGLYl 1714.
  • SEQ ID NO:5 provides the sequence of the light chain of the anti-PCSK9 antibody.
  • SEQ ID NO:6 provides the amino acid sequence alpha mating factor-antipPCSK9 Lc-(GGGS) linker- ⁇ * .
  • Strains that comprise plasmid pGLYl 1714 are able to display an antibody light chain and utilize the covalent interaction of this light chain with the heavy chain of an antibody molecule that is co-expressed in the same host. This interaction tethers the IgG molecule on the cell surface (See Figure 2). This surface display allows for the discovery of novel Fc variants that possess specific desired biological properties, such as Fc receptor binding affinities.
  • YGLY32444 also allows co-secretion of the displayed molecule allowing further in vitro analysis.
  • FIG. 5 is a schematic representation of the method used to isolate the novel IgGl Fc variants of the invention having improved binding to human DC-SIGN.
  • DM refers to cells displaying an anti-Her2 IgGl antibody having mutations at F243A/V264A in the Fc region. These cells reacted with anti-Fc fluorescent antibodies but did not exhibit detectable binding to biotinylated DC-SIGN.
  • Unsorted library refers to the cells displaying the IgGl Fc variants from the library described in Example 1. This library was labeled and gated to isolate clones with higher fluorescence intensity.
  • Olince-sorted library refers to cells that were labeled and isolated from the unsorted library and displayed a shift in fluorescence intensity as expected. A second round of sorting was performed to isolate highly purified IgGl Fc variants having improved binding to human DC-SIGN.
  • Pichia clones from the library described in Example 1 were cultured in a shake flask in growth medium BMGY for 24 hours and expression and surface display was initiated by re- suspending and incubating for 24 hour in 2% methanol containing medium for at room temperature.
  • 2 OD 6 oo of cells ( ⁇ 10 7 cells) were collected into a 1.5-ml microfuge tube and washed twice with phosphate-buffered saline (PBS) and then re- suspended in 100 ⁇ of the Lectin Buffer (100 mM HEPES, 1.5 M NaCl, pH 7.4, 1 mM CaC12, 1 mM MgC12, 0.01% Tween 20) containing 1 ⁇ (2 ⁇ g) of fluorescently-labeled goat anti-human Fc DyeLight 647 (Jackson Immunoresearch Labs, West Grove, PA), and 1 ⁇ g of biotinylated human DC-SIGN (Elicityl, Grenoble, France).
  • PBS phosphate-buffered saline
  • the labeling cells/receptors mixtures were incubated at room temperature for 30 min. Biotinylated DC-SIGN was detected with fluorescently-labeled (DyeLight 488) streptavdin (purchased from R&D Systems, Minneapolis, MN). Cells were washed once with the Lectin Buffer and suspended in 300 ⁇ of PBS for flow cytometric analysis and sorting. Clones with higher fluorescence intensity indicative of receptor labeling were isolated and cultured in liquid medium.
  • the sequences of novel IgGl Fc variants isolated as described herein were determined by DNA sequencing (Genewiz, South Plainfield, NJ).
  • Table 1 describes the mutations identified in the Fc region of the identified IgGl Fc variants. The mutations are identified by reference to the Kabat numbering system using wild type human IgGl Fc as a reference and the single letter amino acid code.
  • Figure 6 shows an alignment of the Fc regions of the novel binders isolated as described in Examples 2 and of the consensus sequence for the Fc region of human IgGl . (Residue number 1 in the alignment corresponds to amino acid position 215 according to EU numbering system as in Kabat).
  • DNA regions encoding Fc fragments of SEQ ID NOs: 11-33 were synthesized by Genewiz (South Plainfield, NJ) and cloned in Pichia expression vector pGLY4464 ( Figure 4) under the AOXI promoter.
  • the resulting plasmids contain Pichia pastoris TRP2 gene which is used to integrate expression cassettes into the yeast TRP2 locus.
  • Yeast strains producing each of these variant Fc fragments were generated by introducing Spel linearized plasmids into humanized Pichia pastoris strain that have been engineered to produce sialylated N-glycans (of the type NANA ( i_4 ) Gal ( i_ 4) GlcNAc ( i_4 ) Man3GlcNAc 2 ). Resulting clones were fermented in 1 L bioreactor vessels to produce each of the described variants.
  • N-glycan composition of the purified IgG variants produced herein were analyzed by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry after release from the antibody with peptide -N-glycosidase F. Released carbohydrate compositions were also quantitated by HPLC on an Allentech Prevail carbo (Alltech Associates, Deerfield IL) column.
  • MALDI-TOF matrix-assisted laser desorption ionization/time-of-flight
  • Herceptin - refers to an anti-Her 2 IgGl antibody comprising a wild type human Fc region expressed in CHO cells and purchased from Genentech (San Francisco CA, Lot#724370).
  • the heavy chain amino acid sequence is provided in SEQ ID NO: 3 and the light chain amino acid sequence is provided in SEQ ID NO:9.
  • WT hFc - refers to human Fc fragment (wild-type) comprising the amino acid of SEQ ID NO: 2 (obtained by expression in a Pichia pastoris strain that has been engineered to produce sialylated N-glycans (of the type NANA(i_ 4) Gal(i_
  • shFc - - refers to a sialylated human Fc fragment comprising mutations (F243A/V264A) (SEQ ID NO: 7) (obtained expression in a Pichia pastoris strain that has been engineered to produce sialylated N-glycans (of the type NANA(i_ 4 ) Gal(i_ 4 GlcNAc(i_ 4) Man 3 GlcNAc 2 )).
  • shFc F241- - refers to a sialylated human Fc fragment comprising mutation
  • F241A (SEQ ID NO: 8) (obtained by expression in a Pichia pastoris strain that has been engineered to produce sialylated N-glycans (of the type NANA(i_ 4) Gal(i_ 4 ) GlcNAc(i_ 4) Man 3 GlcNAc 2 )).
  • mFc IgGl - - refers to wild type murine Fc fragment comprising the amino acid sequence of SEQ ID NO: 10 obtained by expression in a Pichia pastoris strain that has been engineered to produce sialylated N-glycans (of the type NANA(i_ 4) Gal(i_ 4 ) GlcNAc(i_ 4) Man 3 GlcNAc 2 )).
  • the running buffer comprised of 10 mM HEPES, 150 mM NaCl (l HBS-N) (GE Healthcare, cat. no. BR-1006-70), 1 mM CaC12 (Sigma, cat. no. 21115 - 250 mL), 1 mM MgC12 (Sigma, cat. no.
  • CM5 carboxymethylated dextran chip
  • l HBS-EP+ 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% Surfactant P20
  • the results are also shown in Figure 7.
  • the CM5 chip (GE Healthcare, Wauwatusa, WI) was immobilized on all flow cells with mouse anti-human IgG (Fc specific) (obtained from GE Healthcare, Wauwatusa, WI) according to the Biacore Human Antibody Capture Kit to ⁇ 7000 RU.
  • IgGl variants containing mutations in Fc region were captured on the chip through immobilized mouse anti-human IgG (obtained from GE Healthcare, Wauwatusa, WI) followed by injection of human or mouse FcyRs.
  • Human receptors FcyRIIb and FcyRIIIA-LF
  • mFcYRIIb and mFcyRIV mouse receptors

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Abstract

The present invention is directed to methods and compositions for the production of Fc-containing polypeptides which are useful as human or animal therapeutic agents, and which comprise increased anti-inflammatory properties and improved CD209 (DC-SIGN) binding.

Description

TITLE OF THE INVENTION
Fc CONTAINING POLYPEPTIDES HAVING INCREASED BINDING TO HUMAN DC- SIGN
FIELD OF THE INVENTION
The present invention is directed to methods and compositions for the production of Fc- containing polypeptides which are useful as human or animal therapeutic agents, and which comprise increased binding to human DC-SIGN.
BACKGROUND OF THE INVENTION
IgG and IgE mediate their pro-inflammatory properties through the crosslinking of the Fc receptor (FcR) with the Fc portion of the antibodies. IgG and IgE can also engage a second class of receptors, the evolutionarily related, C-type lectins Dendritic Cell-specified Intercellular adhesion molecule-3 -Grabbing Nonintegrin (DC-SIGN or CD209) and CD23 resulting in antiinflammatory and immunosuppressive responses. See, e.g., Sondermann et al, PNAS
110(24)9868-9872 (2013). DC-SIGN is a structurally homologous, calcium-dependent, carbohydrate-binding, type II lectin, displaying ligand specificity for mannose-containing glycoconjugates and fucose-containing Lewis antigens.
It has been reported that the anti-inflammatory activity of intravenous immunoglobulin (IVIG) results from a the population of the pooled IgG molecules that contains terminal alpha2,6-sialic acid linkages on their Fc-linked glycans (Kaneko et al., Science 313(5787):670- 3(2006) and Anthony et al, PNAS 105: 19571-19578 (2008). It has also been reported that the anti-inflammatory properties of IVIG can be seen using a preparation of sialylated IgG Fc fragments (See, e.g., WO2011/149999). It appears that the anti-inflammatory properties of IVIG and sialylated Fc preparation require binding to DC-SIGN (Anthony et al., PNAS 105: 19571- 19578 (2008) and Anthony et al, Nature 475, 110-113 (2011)). It has been postulated that sialylation of the Fc region induces a conformation that will bind DC-SIGN and CD23, thereby providing a mechanism for suppressing follicular B-cell activation. See, e.g., Sondermann et al, PNAS 110(24)9868-9872 (2013). Given the importance of monoclonal antibodies as therapeutics, it would be desirable to engineer antibodies or other Fc-containing polypeptides to have enhanced binding to DC-SIGN.
SUMMARY OF THE INVENTION
Fc-containing Polypeptides
The invention comprises an Fc-containing polypeptide (such as antibody or an antibody fragment or immunoadhesin) comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R,
M252V/V303A/N315S, S239P/F241L/I377V, L351S/N384D/S426P/K447E,
F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274P K360E/Y373H/P387S, T289A/E345G/E357V/Q386R/N434S,
A231V/P232S/S267F/S354P, R301G/K360E, K246E/M252T/E272G/S375P, D249G,
F241S/V263A/T335A/N389S, E269G/N389S/S400P, N297D/S354P/K409R/S424P,
N297S/N390D/P395L, N297S/R301G and V259A/N297S/N389S/G446D; wherein the numbering is according to the EU index as in Kabat.
The invention also comprises an Fc-containing polypeptide (such as antibody or an antibody fragment or immunoadhesin), wherein the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of: Q295R/V302A/S344P/K414R , M252V/V303A/N315S, S239P/F241L/I377V, L351S/N384D/S426P/K447E,
F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274R/K360E/Y373H/P387S, T289A/E345G/E357V/Q386R/N434S,
A231V/P232S/S267F/S354P, R301G/K360E, K246E/M252T/E272G/S375P, D249G,
F241S/V263A/T335A/N389S and E269G/N389S/S400P, wherein the numbering is according to the EU index as in Kabat, and wherein the Fc-containing polypeptide comprises N-glycans.
The invention also comprises an Fc-containing polypeptide (such as antibody or an antibody fragment or immunoadhesin) comprising a mutation in the Fc region selected from the group consisting of: 246, 276, 360, 389, 395, and 414, wherein the numbering is according to the EU index as in Kabat. The invention also comprises an Fc-containing polypeptide comprising a mutation in the Fc region selected from the group consisting of: F241L, F241S, K246E, E272G, N276D, N276S, R301G, S354P, K360E, N389S, N389D, P395L, P395S and K414R, wherein the numbering is according to the EU index as in Kabat, and wherein the Fc-containing polypeptide has enhanced binding to human DC-SIGN when compared to a parent Fc-containing
polypeptide.
Any of the Fc-containing polypeptides of the invention can further comprise one or more mutations at positions selected from the group consisting of: 241, 243, 246, 252, 254, 256, 264, 267, 272, 276, 301, 328, 339, 342, 354, 360, 389, 395, 414, 433 and 434 of the Fc region, wherein the numbering is according to the EU index as in Kabat.
Any of the Fc-containing polypeptides of the invention can further comprise one or more mutations at positions selected from the group consisting of: F241L, F241S, K246E, E272G, N276D, N276S, R301G, S354P, K360E, N389S, N389D, P395L, P395S and K414R, wherein the numbering is according to the EU index as in Kabat.
Any of the Fc-containing polypeptides of the invention can comprise N-glycans. In one embodiment, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc- containing polypeptide comprise an N-linked oligosaccharide structure selected from the group consisting of SA(i_4)Gal(i_4)GlcNAc(2-4)Man3GlcNAc2. In one embodiment, the sialic acid residues in the sialylated N-glycans are attached via a-2,6 linkages. In one embodiment, the N- linked oligosaccharides recited above further comprise a core fucose.
The invention also comprises an Fc-containing polypeptide (such as antibody or an antibody fragment or immunoadhesin), wherein the Fc-containing polypeptide does not comprise N-glycans and wherein the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of: N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G, and V259A/N297S/N389S/G446D, wherein the numbering is according to the EU index as in Kabat.
In some embodiments, the Fc-containing polypeptide of the invention is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide of the invention is an IgGl antibody or an antibody fragment.
In one embodiment, the Fc-containing polypeptide of the invention is a human IgGl or a fragment of human IgGl that comprises or consists of any one of SEQ ID NOs: 11-33. In one embodiment, the Fc-containing polypeptide comprises N-glycans and comprises or consists essentially of any one of SEQ ID NOs: 11-29. In one embodiment, the Fc-containing polypeptide does not comprise N-glycans and comprises or consists essentially of any one of SEQ ID
NOs:30-33.
In some embodiments, the Fc-containing polypeptide of the invention has enhanced binding to human DC-SIGN when compared to a parent Fc-containing polypeptide.
In some embodiments, the Fc-containing polypeptide of the invention has altered inflammatory properties when compared to a parent Fc-containing polypeptide.
In some embodiments, the Fc-containing polypeptide of the invention has enhanced inflammatory properties when compared to a parent Fc-containing polypeptide.
Methods for Producing Fc-containing Polypeptides
The invention also comprises a method for producing an Fc-containing polypeptide in a host cell comprising: (a) providing a host cell comprising a nucleic acid encoding an Fc- containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R,
M252V/V303A/N315S, S239P/F241L/I377V, L351S/N384D/S426P/K447E,
F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274P K360E/Y373H/P387S, T289A/E345G/E357V/Q386R/N434S,
A231V/P232S/S267F/S354P, R301G/K360E, K246E/M252T/E272G/S375P, D249G,
F241S/V263A/T335A/N389S, E269G/N389S/S400P, N297D/S354P/K409R/S424P,
N297S/N390D/P395L, N297S/R301G and V259A/N297S/N389S/G446D; wherein the numbering is according to the EU index as in Kabat, (b) culturing the host cell under conditions which cause expression of the Fc-containing polypeptide; and (c) isolating the Fc-containing polypeptide from the host cell. In one embodiment, the host cell is capable of producing an Fc- containing polypeptide comprising N-glycans. In one embodiment, the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof. In one embodiment, the host cell is a lower eukaryotic host cell. In one embodiment, the host cell is Pichia pastoris. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce hybrid or complex type N-glycans. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce sialylated N-glycans.
The invention also comprises a method for producing an Fc-containing polypeptide in a host cell comprising: (a) providing a host cell comprising a nucleic acid encoding an Fc- containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R ,
M252V/V303A/N315S, S239P/F241L/I377V, L351S/N384D/S426P/K447E,
F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274R/K360E/Y373H/P387S, T289A/E345G/E357V/Q386R/N434S,
A231V/P232S/S267F/S354P, R301G/K360E, K246E/M252T/E272G/S375P, D249G,
F241S/V263A/T335A/N389S and E269G/N389S/S400P; wherein the numbering is according to the EU index as in Kabat, (b) culturing the host cell under conditions which cause expression of the Fc-containing polypeptide; and (c) isolating the Fc-containing polypeptide from the host cell. In one embodiment, the host cell is capable of producing an Fc-containing polypeptide comprising N-glycans. In one embodiment, the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof. In one embodiment, the host cell is a lower eukaryotic host cell. In one embodiment, the host cell is Pichia pastoris. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce hybrid or complex type N-glycans. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce sialylated N-glycans.
The invention also comprises a method for producing an Fc-containing polypeptide in a host cell comprising: (a) providing a host cell comprising a nucleic acid encoding an Fc- containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: N297D/S354P/K409R/S424P,
N297S/N390D/P395L, N297S/R301G and V259A/N297S/N389S/G446D; wherein the numbering is according to the EU index as in Kabat, (b) culturing the host cell under conditions which cause expression of the Fc-containing polypeptide; and (c) isolating the Fc-containing polypeptide from the host cell. In one embodiment, the method results in the production of an Fc-containing polypeptide that does not comprise N-glycans. In one embodiment, the Fc- containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc- containing polypeptide is a human IgGl antibody or a fragment thereof. In one embodiment, the host cell is a lower eukaryotic host cell. In one embodiment, the host cell is Pichia pastoris. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce hybrid or complex type N-glycans. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce sialylated N-glycans.
The invention also comprises a method for producing an Fc-containing polypeptide in a host cell comprising: (a) providing a host cell comprising a nucleic acid encoding an Fc- containing polypeptide comprising the amino acid sequence of any of SEQ ID NO: 11-33, (b) culturing the host cell under conditions which cause expression of the Fc-containing polypeptide; and (c) isolating the Fc-containing polypeptide from the host cell. In one embodiment, the host cell is capable of producing an Fc-containing polypeptide comprising N-glycans. In one embodiment, the host cell is a lower eukaryotic host cell. In one embodiment, the host cell is Pichia pastoris. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce hybrid or complex type N-glycans. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce sialylated N-glycans.
The invention also comprises a method for producing an Fc-containing polypeptide in a host cell comprising: (a) providing a host cell comprising a nucleic acid encoding an Fc- containing polypeptide comprising one or more of mutations in the Fc region, at positions selected from the group consisting of: 241, 243, 246, 252, 254, 256, 264, 267, 272, 276, 301, 328, 339, 342, 354, 360, 389, 395, 414, 433 and 434 of the Fc region, wherein the numbering is according to the EU index as in Kabat (b) culturing the host cell under conditions which cause expression of the Fc-containing polypeptide; and (c) isolating the Fc-containing polypeptide from the host cell. In one embodiment, the host cell is capable of producing an Fc-containing polypeptide comprising N-glycans. In one embodiment, the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof. In one embodiment, the host cell is a lower eukaryotic host cell. In one embodiment, the host cell is Pichia pastoris. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce hybrid or complex type N-glycans. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce sialylated N-glycans.
The invention also comprises a method for producing an Fc-containing polypeptide in a host cell comprising: (a) providing a host cell comprising a nucleic acid encoding an Fc- containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: F241L, F241S, K246E, E272G, N276D, N276S, R301G, S354P, K360E, N389S, N389D, P395L, P395S and K414R, wherein the numbering is according to the EU index as in Kabat (b) culturing the host cell under conditions which cause expression of the Fc-containing polypeptide; and (c) isolating the Fc-containing polypeptide from the host cell. In one embodiment, the host cell is capable of producing an Fc- containing polypeptide comprising N-glycans. In one embodiment, the Fc-containing
polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof. In one embodiment, the host cell is a lower eukaryotic host cell. In one embodiment, the host cell is Pichia pastoris. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce hybrid or complex type N-glycans. In one embodiment, the host cell is a Pichia pastoris host cell that has been engineered to produce sialylated N-glycans.
Methods for Altering the Inflammatory Properties of An Fc-containing Polypeptide
The invention also comprises a method of altering the inflammatory properties of an Fc- containing polypeptide comprising: introducing one or more of mutations in the Fc region of the Fc-containing polypeptide, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R, M252V/V303A/N315S, S239P/F241L/I377V,
L351S/N384D/S426P/K447E, F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274R/K360E/Y373H/P387S,
T289A/E345G/E357V/Q386R/N434S, A231V/P232S/S267F/S354P, R301G/K360E, K246E/M252T/E272G/S375P, D249G, F241S/V263A/T335A/N389S, E269G/N389S/S400P, N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G and
V259A/N297S/N389S/G446D, wherein the numbering is according to the EU index as in Kabat, and wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or an antibody fragment thereof. In one embodiment, the Fc-containing polypeptide comprises or consists essentially of the amino acid sequence of any of SEQ ID NOs: l l-33.
The invention also comprises a method of altering the inflammatory properties of an Fc- containing polypeptide comprising: introducing one or more of mutations in the Fc region of the Fc-containing polypeptide, at positions selected from the group consisting of: 241, 243, 246, 252, 254, 256, 264, 267, 272, 276, 301, 328, 339, 342, 354, 360, 389, 395, 414, 433 and 434 of the Fc region, wherein the numbering is according to the EU index as in Kabat, and wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc- containing polypeptide. In one embodiment, the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof.
The invention also comprises a method of altering the inflammatory properties of an Fc- containing polypeptide comprising: introducing one or more of mutations in the Fc region of the Fc-containing polypeptide, wherein the mutations are selected from the group consisting of: F241L, F241S, K246E, E272G, N276D, N276S, R301G, S354P, K360E, N389S, N389D, P395L, P395S and K414R, wherein the numbering is according to the EU index as in Kabat, and wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof.
The invention also comprises a method of increasing the anti-inflammatory properties of an Fc-containing polypeptide comprising: introducing one or more of mutations in the Fc region of the Fc-containing polypeptide, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R, M252V/V303A/N315S, S239P/F241L/I377V, L351S/N384D/S426P/K447E, F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274R/K360E/Y373H/P387S,
T289A/E345G/E357V/Q386R/N434S, A231V/P232S/S267F/S354P, R301G/K360E,
K246E/M252T/E272G/S375P, D249G, F241S/V263A/T335A/N389S, E269G/N389S/S400P, N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G and
V259A/N297S/N389S/G446D, wherein the numbering is according to the EU index as in Kabat, and wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof. In one embodiment, the Fc-containing polypeptide comprises or consists essentially of the amino acid sequence of any of SEQ ID NOs: 11-33.
The invention also comprises a method of increasing the anti-inflammatory properties of an Fc-containing polypeptide, wherein the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of: Q295R/V302A/S344P/K414R ,
M252V/V303A/N315S, S239P/F241L/I377V, L351S/N384D/S426P/K447E,
F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274R/K360E/Y373H/P387S, T289A/E345G/E357V/Q386R/N434S,
A231V/P232S/S267F/S354P, R301G/K360E, K246E/M252T/E272G/S375P, D249G,
F241S/V263A/T335A/N389S and E269G/N389S/S400P, wherein the numbering is according to the EU index as in Kabat, wherein the Fc-containing polypeptide comprises N-glycans, and wherein the Fc-containing polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof. In one embodiment, the Fc- containing polypeptide comprises or consists essentially of the amino acid sequence of any of SEQ ID NOs: 11-29. The invention also comprises a method of increasing the anti-inflammatory properties of an Fc-containing polypeptide, wherein the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of: N297D/S354P/K409R/S424P,
N297S/N390D/P395L, N297S/R301G and V259A/N297S/N389S/G446D, wherein the numbering is according to the EU index as in Kabat, wherein the Fc-containing polypeptide is does not comprise N-glycans, and wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc- containing polypeptide is a human IgGl antibody or a fragment thereof. In one embodiment, the Fc-containing polypeptide comprises or consists essentially of the amino acid sequence of any of SEQ ID NOs:30-33.
The invention also comprises a method of increasing the anti-inflammatory properties of an Fc-containing polypeptide comprising: introducing one or more of mutations in the Fc region of the Fc-containing polypeptide at positions selected from the group consisting of: 241, 243, 246, 252, 254, 256, 264, 267, 272, 276, 301, 328, 339, 342, 354, 360, 389, 395, 414, 433 and 434 of the Fc region, wherein the numbering is according to the EU index as in Kabat, and wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc- containing polypeptide. In one embodiment, the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof.
The invention also comprises a method of increasing the anti-inflammatory properties of an Fc-containing polypeptide comprising: introducing one or more of mutations in the Fc region of the Fc-containing polypeptide, wherein the mutations are selected from the group consisting of: F241L, F241S, K246E, E272G, N276D, N276S, R301G, S354P, K360E, N389S, N389D, P395L, P395S and K414R, wherein the numbering is according to the EU index as in Kabat, and wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof. Methods of Treatment
The invention also comprises a method of treating a subject in need thereof comprising: administering to the subject a therapeutically effective amount of an Fc-containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R, M252V/V303A/N315S,
S239P/F241L/I377V, L351S/N384D/S426P/K447E, F241S/N276D/N361S, V266A,
K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S,
S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274R/K360E/Y373H/P387S, T289A/E345G/E357V/Q386R/N434S,
A231V/P232S/S267F/S354P, R301G/K360E, K246E/M252T/E272G/S375P, D249G,
F241S/V263A/T335A/N389S, E269G/N389S/S400P, N297D/S354P/K409R/S424P,
N297S/N390D/P395L, N297S/R301G and V259A/N297S/N389S/G446D, wherein the numbering is according to the EU index as in Kabat, and wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide of is a human IgGl antibody or a fragment thereof. In one embodiment, the Fc-containing polypeptide comprises or consists essentially of the amino acid sequence of any of SEQ ID NOs: 11-33. In one embodiment, the method further comprises the administration of a vaccine. In one embodiment, the method further comprises the administration of an anti-inflammatory compound. In one embodiment, the method further comprises the administration of an agent that binds to FcyRIIB. In one embodiment, the subject has an inflammatory condition.
The invention also comprises a method of treating a subject in need thereof comprising: administering to the subject a therapeutically effective amount of an Fc-containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R , M252V/V303A/N315S,
S239P/F241L/I377V, L351S/N384D/S426P/K447E, F241S/N276D/N361S, V266A,
K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S,
S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274R/K360E/Y373H/P387S, T289A/E345G/E357V/Q386R/N434S, A231V/P232S/S267F/S354P, R301G/K360E, K246E/M252T/E272G/S375P, D249G,
F241S/V263A/T335A/N389S and E269G/N389S/S400P, wherein the numbering is according to the EU index as in Kabat, wherein the polypeptide comprises N-glycans, and wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc- containing polypeptide. In one embodiment, the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof. In one embodiment, the Fc-containing polypeptide comprises or consists essentially of the amino acid sequence of any of SEQ ID NOs: 11-29. In one embodiment, the method further comprises the administration of a vaccine. In one embodiment, the method further comprises the administration of an anti-inflammatory compound. In one embodiment, the method further comprises the administration of an agent that binds to FcyRIIB. In one embodiment, the subject has an inflammatory condition.
The invention also comprises a method of treating a subject in need thereof comprising: administering to the subject a therapeutically effective amount of an Fc-containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G and V259A/N297S/N389S/G446D, wherein the numbering is according to the EU index as in Kabat, wherein the polypeptide does not comprise N-glycans, and wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc-containing polypeptide. In one embodiment, the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof. In one embodiment, the Fc-containing polypeptide comprises or consists essentially of the amino acid sequence of any of SEQ ID NOs:30-33. In one embodiment, the method further comprises the administration of a vaccine. In one embodiment, the method further comprises the administration of an anti-inflammatory compound. In one embodiment, the method further comprises the administration of an agent that binds to FcyRIIB. In one embodiment, the subject has an inflammatory condition.
The invention also comprises a method of treating a subject in need thereof comprising: administering to the subject a therapeutically effective amount of an Fc-containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are at positions selected from the group consisting of: 241, 243, 246, 252, 254, 256, 264, 267, 272, 276, 301, 328, 339, 342, 354, 360, 389, 395, 414, 433 and 434, wherein the numbering is according to the EU index as in Kabat and wherein the polypeptide comprises enhanced binding to human DC- SIGN when compared to a parent Fc-containing polypeptide. In one embodiment, the Fc- containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc- containing polypeptide is a human IgGl antibody or a fragment thereof. In one embodiment, the method further comprises the administration of a vaccine. In one embodiment, the method further comprises the administration of an anti-inflammatory compound. In one embodiment, the method further comprises the administration of an agent that binds to FcyRIIB. In one embodiment, the subject has an inflammatory condition.
The invention also comprises a method of treating a subject in need thereof comprising: administering to the subject a therapeutically effective amount of an Fc-containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: F241L, F241S, K246E, E272G, N276D, N276S, R301G, S354P, K360E, N389S, N389D, P395L, P395S and K414R, wherein the numbering is according to the EU index as in Kabat, and wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc-containing polypeptide. In one embodiment, the method further comprises the administration of a vaccine. In one embodiment, the method further comprises the administration of an anti-inflammatory compound. In one embodiment, the method further comprises the administration of an agent that binds to FcyRIIB. In one embodiment, the subject has an inflammatory condition.
Pharmaceutical Compositions
The invention also comprises a pharmaceutical formulation comprising: an Fc-containing polypeptide as described above and a pharmaceutically acceptable carrier.
The invention comprises a pharmaceutical formulation comprising: (a) an Fc-containing polypeptide (such as antibody or an antibody fragment or immunoadhesin) comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R, M252V/V303A/N315S, S239P/F241L/I377V,
L351S/N384D/S426P/K447E, F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274R/K360E/Y373H/P387S,
T289A/E345G/E357V/Q386R/N434S, A231V/P232S/S267F/S354P, R301G/K360E,
K246E/M252T/E272G/S375P, D249G, F241S/V263A/T335A/N389S, E269G/N389S/S400P, N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G and
V259A/N297S/N389S/G446D, wherein the numbering is according to the EU index as in Kabat, and (b) a pharmaceutically acceptable carrier. In one embodiment, the Fc-containing
polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or a fragment thereof. In one embodiment, the Fc- containing polypeptide is a human IgGl or a fragment of human IgGl that comprises or consists of any one of SEQ ID NOs: 11-33.
The invention also comprises a pharmaceutical formulation comprising: (a) an Fc- containing polypeptide (such as antibody or an antibody fragment or immunoadhesin) comprising N-glycans, wherein the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of: Q295R/V302A/S344P/K414R ,
M252V/V303A/N315S, S239P/F241L/I377V, L351S/N384D/S426P/K447E,
F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274P K360E/Y373H/P387S, T289A/E345G/E357V/Q386R/N434S,
A231V/P232S/S267F/S354P, R301G/K360E, K246E/M252T/E272G/S375P, D249G,
F241S/V263A/T335A/N389S, and E269G/N389S/S400P, wherein the numbering is according to the EU index as in Kabat, and (b) a pharmaceutically acceptable carrier. In one embodiment, the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc- containing polypeptide is a human IgGl antibody or a fragment thereof. In one embodiment, the Fc-containing polypeptide is a human IgGl or a fragment of human IgGl that comprises or consists of any one of SEQ ID NOs: 11 -29. In one embodiment, at least 30%, 40%, 50%, 60%, 70%), 80%) or 90%) of the N-glycans on the Fc-containing polypeptide comprise an N-linked oligosaccharide structure selected from the group consisting of SA(i_4)Gal(i_4)GlcNAc(2- 4)Man3GlcNAc2- In one embodiment, the sialic acid residues in the sialylated N-glycans are attached via a-2,6 linkages. The invention also comprises a pharmaceutical formulation comprising: (a) an Fc- containing polypeptide (such as antibody or an antibody fragment or immunoadhesin) comprising a mutation in the Fc region selected from the group consisting of: 246, 276, 360, 389, 395, and 414, wherein the numbering is according to the EU index as in Kabat, and (b) a pharmaceutically acceptable carrier. In one embodiment, the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or an antibody fragment thereof.
The invention also comprises a pharmaceutical formulation comprising: (a) an Fc- containing polypeptide (such as antibody or an antibody fragment or immunoadhesin) comprising a mutation in the Fc region selected from the group consisting of: F241L, F241S, K246E, E272G, N276D, N276S, R301G, S354P, K360E, N389S, N389D, P395L, P395S; and K414R, wherein the numbering is according to the EU index as in Kabat, and (b) a
pharmaceutically acceptable carrier. In one embodiment, the Fc-containing polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or an fragment thereof.
The invention also comprises a pharmaceutical formulation comprising: (a) an Fc- containing polypeptide (such as antibody or an antibody fragment or immunoadhesin), wherein the Fc-containing polypeptide does not comprise N-glycans and wherein the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of:
N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G, and
V259A/N297S/N389S/G446D, wherein the numbering is according to the EU index as in Kabat, and (b) a pharmaceutically acceptable carrier. In one embodiment, the Fc-containing
polypeptide is an antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide is a human IgGl antibody or an fragment thereof. In one embodiment, the Fc- containing polypeptide is a human IgGl or a fragment of human IgGl that comprises or consists of any one of SEQ ID NOs:30-33.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 illustrates the plasmid designated pGLYl 1714 containing anti-PC SK9 Lc bait cassette. FIGURE 2 is a schematic representation of the Lc-Sedlp antibody display system described in Example 1. The DNA sequence comprising the IgG light chain (Lc) is fused through a flexible linker to Sedlp. When co-expressed in the same host with a secretable full length IgG molecule, the Lc portion of the anchored fusion (the bait) heterodimerizes in the ER with the heavy chain (He) region of the IgG molecule, forming disulfide bridges. Since the surface displayed half IgG molecule can still pair with secreted heavy and light chains (H+L), this complex results in surface display of the full length IgG molecule (two heavy chains paired with two light chains, i.e., H2+L2). Meanwhile the assembly of soluble full length IgG occurs with equal probability resulting in secretion of the bivalent (H2+L2) in the culture medium.
FIGURE 3 illustrates the plasmid designated pGLYl 1576.
FIGURE 4 illustrates the plasmid designated pGLY4464.
FIGURE 5 is a schematic representation of the method used to isolate the novel IgGl Fc variants of the invention having improved binding to human DC-SIGN.
FIGURE 6 is an alignment of the IgGl Fc variants isolated as described in Example 2 and of the consensus sequence for the Fc region of human IgGl . (Residue number 1 in the alignment corresponds to amino acid position 215 according to EU numbering system as in Kabat).
FIGURE 7 shows the binding affinity of the IgGl Fc variants of the invention towards DC-SIGN and various FcyRs.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
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 encompasses not only intact polyclonal or monoclonal antibodies, consisting of four polypeptide chains, i.e. two identical pairs of polypeptide chains, each pair having one "light" chain (LC) (about 25 kDa) and one "heavy" chain (HC) (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 that comprises an antigen recognition site and at least the portion of the Cpf2 domain of the heavy chain immunoglobulin constant region which comprises an N-linked glycosylation site of the Cpf2 domain, or a variant thereof. As used herein the term includes an antibody of any class, such as IgG (for example, IgGl, IgG2, IgG3 or IgG4), IgM, IgA, IgD and IgE, respectively.
The term "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch 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 can optionally comprise a hinge region. In one embodiment, the Fc region comprises or consists essentially of the amino acid sequence of SEQ ID NO: 1 (or a variant thereof comprising point mutations). In one embodiment, the Fc region comprises or consists essentially of the amino acid sequence of SEQ ID NO:2 (or a variant thereof comprising point mutations). In another embodiment, the Fc region comprises or consists essentially of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 with the addition of a lysine (K) residue at the 3' end. The Fc region contains a single N-linked glycosylation site in the CH2 domain that corresponds to the Asn297 site of a full-length heavy chain of an antibody, wherein the numbering is according to the EU index as in Kabat.
The term "Fc-containing polypeptide" refers to a polypeptide, such as an antibody or immunoadhesin, which comprises an Fc region or fragment of an Fc region which retains the N- linked glycosylation site in the CH2 domain and retains the ability to recruit immune cells. This term encompasses polypeptides comprising or consisting of (or consisting essentially of) an Fc region either as a monomore or dimeric species. Polypeptides comprising an Fc region can be generated by papain digestion of antibodies or by recombinant DNA technology.
The term "parent antibody", "parent immunoglobulin" or "parent Fc-containing polypeptide" when used herein refers to an antibody or Fc-containing polypeptide which lacks the Fc region mutations disclosed herein. A parent Fc-containing polypeptide may comprise a native sequence Fc region or an Fc region with pre-existing amino acid sequence modifications.
A native sequence Fc region comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence Fc regions include the native sequence human IgGl Fc region, the native sequence human IgG2 Fc region, the native sequence human IgG3 Fc region and the native sequence human IgG4 Fc region as well as naturally occurring variants thereof. When used as a comparator, a parent antibody or a parent Fc- containing polypeptide can be expressed in any cell. In one embodiment, the parent antibody or a parent Fc-containing polypeptide is expressed in the same cell as the Fc-containing polypeptide of the invention.
As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the "binding domain" of a heterologous "adhesin" protein (e.g. a receptor, ligand or enzyme) with an immunoglobulin constant domain. Structurally, the 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 native cell-surface receptor or any region or derivative thereof retaining at least a qualitative ligand binding ability of a corresponding native receptor. In a specific embodiment, the receptor is from a cell-surface polypeptide having an extracellular domain that is homologous to a member of the immunoglobulin supergenefamily. Other receptors, which are not members of the immunoglobulin supergenefamily but are nonetheless specifically covered by this definition, are receptors for cytokines, and in particular receptors with tyrosine kinase activity (receptor tyrosine kinases), members of the hematopoietin and nerve growth factor which predispose the mammal to the disorder in question. In one
embodiment, the disorder is cancer. Methods of making immunoadhesins are well known in the art. See, e.g., WO00/42072.
The term "Fc mutein" or "Fc mutein antibody" when used herein refers to an Fc- containing polypeptide in which one or more point mutations have been made to the Fc region.
The term "Fc mutation" when used herein refers to a mutation made to the Fc region of an Fc-containing polypeptide.
Throughout the present specification and claims, the numbering of the 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 IgGl EU antibody.
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 Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); Clq binding; complement dependent cytotoxicity (CDC) phagocytosis; down regulation of cell surface receptors (e. g. B cell receptor; BCR), etc. Such effector functions can be assessed using various assays known in the art.
The terms "Fc receptor" or "FcR" refer to a receptor that binds to the Fc region of an antibody. The preferred Fc receptor is a native sequence of human FcR. Moreover, a preferred FcR is one that binds an IgG antibody - an FcyR - and includes receptors FcyRI, FcyRII, and FcyRIII, including allelic variants and alternatively spliced forms of these receptors. The term also includes the neonatal receptor, FcRn, that is responsible for the transfer of maternal IgGs to the fetus.
The term FcyRIIB refers to a polyeptide en coded by the human FcRIIB gene, and includes, but is not limited to FcyRIIBl (GenBank Accession No. NP 003992), FcyRIIB2 (GenBank Accession No. NP 001002273), FcyRIIB3 GenBank Accession No.
(NP 001002274), and allelic variants thereof.
DC-SIGN refers to a Dendritic Cell-Specific Adhesion Molecule-3 -Grabbing
Nonintegrin [CD209]. The human DC-SIGN receptor, identified by Geijtenbeek, et al, Cell 100:575-585 (2000); US 6,391,507) is a C-type lectin (calcium-dependent) receptor which is expressed at least on dendritic cells, macrophages and activated B cells. A polynucleotide sequence encoding human DC-SIGN can be found using GenBank Accession No. NM 021155. A mouse ortholog to human DC-SIGN is SIGN-R1. Method of detecting binding between a compound and DC-SIGN are well known in the art. See, e.g. WO2011/031736 and
WO2013/095966.
The term "glycoengineered Pichia pastoris" when used herein refers to a strain of Pichia pastoris that has been genetically altered to express human- like N-glycans. For example, the GFI 5.0, GFI 5.5 and GFI 6.0 strains. The term "GFI 5.0" when used herein refers to glycoengineered Pichia pastoris strains that produce glycoproteins having predominantly Gal2GlcNAc2Man3GlcNAc2 N-glycans.
The term "GFI 6.0" when used herein refers to glycoengineered Pichia pastoris strains that produce glycoproteins having predominantly NANA2Gal2GlcNAc2Man3GlcNAc2 N- glycans.
The term "GS5.0", when used herein refers to the N-glycosylation structure
Gal2GlcN Ac2Man3 GlcN Ac2.
The term "GS5.5", when used herein refers to the N-glycosylation structure
NANAGal2GlcNAc2Man3GlcNAc2, which when produced in Pichia pastoris strains to which a 2,6 sialyl transferase has been glycoengineered result in a-2,6-linked sialic acid and which when produced in Pichia pastoris strains to which a-2,3 sialyl transferase has been glycoengineered result in a-2,3-linked sialic acid.
The term "GS6.0", when used herein refers to the N-glycosylation structure
NANA2Gal2GlcNAc2Man3GlcNAc2, which when produced in Pichia pastoris strains to which a-2,6 sialyltransferase has been glycoengineered result in a-2,6-linked sialic acid and which when produced in Pichia pastoris strains to which a-2,3 sialyl transferase has been
glycoengineered result in a-2,3-linked sialic acid.
The term "wild type" or "wt" when used herein in connection to a Pichia pastoris strain refers to a native Pichia pastoris strain that has not been subjected to genetic modification to control glycosylation.
The term "wild type" or "wt" when used herein in connection to an FcR refers to a an FcR polypeptode or amino acid that comprises the a native nucleotide or amino acid sequence - a sequence that has not been subject to any genetic modification.
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 linkage to an asparagine residue of a polypeptide. Predominant sugars found on glycoproteins are glucose, galactose, mannose, fucose, ^-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialic acid (SA, including NANA, NGNA and derivatives and analogs thereof, including acetylated NANA or acetylated NGNA). In glycoengineered Pichia pastoris, sialic acid is exclusively N-acetyl-neuraminic acid (NANA) (Hamilton et al., Science 313 (5792): 1441-1443 (2006)). N-glycans have a common pentasaccharide core of Man3GlcNAc2, wherein "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 (antennae) comprising peripheral sugars (e.g., GlcNAc, galactose, fucose and sialic acid) that are added to the Man3GlcNAc2 ("Man3") core structure which is also referred to as the "trimannose core", the "pentasaccharide core" or the "paucimannose core". N-glycans are classified according to their branched constituents (e.g., high mannose, complex or hybrid).
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 analog or derivative thereof (including those arising from acetylation at any position on 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 usually the terminal residue of the oligosaccharides. N-acetylneuraminic acid (NANA) is the most common sialic acid form and N-glycolylneuraminic acid (NGNA) is the second most common form. Schauer, Glycobiology, 1, 449-452 (1991). NGNA is widespread throughout the animal kingdom and, according to species and tissue, often constitutes a significant proportion of the glycoconjugate-bound sialic acid. Certain species such as chicken and human are exceptional, since they lack 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).
The term "human-like N-glycan", as used herein, refers to the N-linked oligosaccharides which closely resemble the oligosaccharides produced by non-engineered, wild-type human cells. For example, wild-type Pichia pastoris and other lower eukaryotic cells typically produce hypermannosylated proteins at N-glycosylation sites. The host cells described herein produce glycoproteins (for example, antibodies) comprising human-like N-glycans that are not hypermannosylated. In some embodiments, the host cells of the present invention are capable of producing human-like N-glycans with hybrid and/or complex N-glycans. The specific type of "human-like" glycans present on a specific glycoprotein produced from a host cell of the invention will depend upon the specific glycoengineering steps that are performed in the host cell.
The term "high mannose" type N-glycan when used herein refers to an N-glycan having five or more mannose residues.
The term "complex" type N-glycan when used herein refers to an N-glycan having 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 have galactose ("Gal") or N- acetylgalactosamine ("GalNAc") residues that 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 have intrachain substitutions comprising "bisecting" GlcNAc and core fucose ("Fuc"). As an example, when a N-glycan comprises a bisecting GlcNAc on the trimannose core, the structure can be represented as Man3GlcNAc2(GlcNAc) or Man3GlcNAc3. When an N-glycan comprises a core fucose attached to the trimannose core, the structure may be represented as Man3GlcNAc2(Fuc). Complex N-glycans may also have multiple antennae on the "trimannose core," often referred to as "multiple antennary glycans."
The term "hybrid" N-glycan when used herein refers to an N-glycan having at least one GlcNAc on the terminal 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.
When referring to "mole percent" of a glycan present in a preparation of a glycoprotein, the term means the molar percent of a particular glycan present in the pool of N-linked oligosaccharides released when the protein preparation is treated with PNGase and then quantified by a method that is not affected by glycoform composition, (for instance, labeling a PNGase released glycan pool with a fluorescent tag such as 2-aminobenzamide and then separating by high performance liquid chromatography or capillary electrophoresis and then quantifying 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. Thus, in this application, the terms "mole percent" and "percent" are used interchangeably.
The term "anti-inflammatory antibody" as used herein, refers to an antibody intended to be used 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), WO201 1/149999 and WO2013/095966. In another embodiment, the antiinflammatory properties of an Fc-containing polypeptide are measured by determining the level of a biomarker related to inflammation (including without limitation: CRP, pro-inflammatory cytokines such as tumor necrosis factors (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 polypetpide is 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 has anti-inflammatory properties.
"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 frequently be made without altering the biological activity of the protein. Those of skill in this 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.)). In addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are listed below:
Figure imgf000024_0001
Figure imgf000025_0001
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, glycosylation engineering in the constant region of IgG has become an area of active research for the development of therapeutic monoclonal antibodies (mAbs). It has been established that the presence of N-linked glycosylation at Asn297 is critical for mAb activity in immune effector function assays 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 lacking when glycosylation is absent. More specifically, IgG which lacks glycosylation in the Fc Cpf2 domain does not bind to FcyR, including FcyRI, FcyRII, and FcyRIII, Rothman (1989). 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 in vitro FcyRIIIa binding and in vitro ADCC, 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 predominately 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 over a lectin column to enrich for the sialylated form showed a distinct anti-inflammatory effect limited to the sialylated Fc fragment and was linked to an increase in expression of the inhibitory receptor FcyRIIb, Nimmerjahn and Ravetch., J. Exp. Med. 204:11-15 (2007).
Glycosylation in the Fc region of an antibody derived from mammalian cell lines typically consists of a heterogeneous mix of glycoforms, with the predominant forms typically being comprised of the complex fucosylated glycoforms: G0F, GIF, and, to a lesser extent, G2F. Possible conditions resulting in incomplete galactose transfer to the G0F structure include, but are not limited to, non-optimized galactose transfer machinery, such as β-1,4 galactosyl transferase, and poor UDP-galactose transport into the Golgi apparatus, suboptimal cell culture and protein expression conditions, and steric hindrance by amino acid residues neighboring the oligosaccharide. While each of these conditions may modulate the ultimate degree of terminal galactose, it is thought that subsequent sialic acid transfer to the Fc oligosaccharide is inhibited by the closed pocket configuration of the CH2 domain. See, for example, Fig. 1, Jefferis, R., Nature Biotech., 24 (10): 1230-1231, 2006. Without the correct terminal monosaccharide, specifically galactose, or with insufficient terminal galactosylated forms, there is little possibility of producing a sialylated form, capable of acting as a therapeutic protein, even when produced in the presence of sialyl transferase. Protein engineering and structural analysis of human IgG-Fc glycoforms has shown that glycosylation profiles are affected by Fc conformation, such as the finding that increased levels of galactose and sialic acid on oligosaccharides derived from CHO- produced IgG3 could be achieved when specific amino acids from the Fc pocket were mutated, to an alanine including F241, F243, V264, D265, Y296 and R301. Lund et al., J. Immunol. 157(11); 4963-4969 (1996). It was further shown 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 Clq binding, respectively.
Yeast 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) describes the use of libraries of a 1,2 mannosidase catalytic domains and N- acetylglucosaminyltransferase I catalytic domains in combination with a library of fungal type II membrane protein leader sequences to localize the catalytic domains to the secretory pathway. In this way, strains were isolated that produced in vivo glycoproteins 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 having a glycan composition that consisted predominantly of a bisialylated glycan structure, GS6.0, NANA2Gal2GlcNAc2Man3GlcNAc2 (90.5%) and monosialylated, GS5.5, NANAGal2GlcNAc2 Man3GlcNAc2 (7.9%). WO201//149999 describes the production of sialyulated antibodies in Pichia pastoris.
Host organisms and cell lines
The Fc-containing polypeptides of this invention can be made in any host organism or cell line.
In one embodiment, an Fc-containing polypeptide of the invention is made in a host cell which is capable of producing proteins comprising N-glycans.
In one embodiment, an Fc-containing polypeptide of the invention is made in a host cell which is capable of producing proteins comprising sialylated N-glycans.
In one embodiment, an Fc-containing polypeptide of the invention is made in a mammalian cell where the cell either endogenously or through genetic or process manipulation produces glycoproteins containing either a mixture of terminal a2-6 and a2-3 sialic acid, or only terminal a2-6 sialic acid. The propagation of mammalian cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney
CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby 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 3 A, 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 line (Hep G2).
In one embodiment, an Fc-containing polypeptide of the invention can be made in a plant cell which is engineered 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).
In one embodiment, an Fc-containing polypeptide of the invention can be made in an insect cell which is engineered to produce sialylated N-glycans. See, e.g., Harrison and Jarvis, Adv. Virus Res. 68: 159-91 (2006).
In one embodiment, an Fc-containing polypeptide of the invention can be made in a bacterial cell which is engineered to produce sialylated N-glycans. See, e.g., Lizak et al,
Bioconiugate Chem. 22:488-496 (2011).
Lower Eukaryotic Organisms as Host Cells
In one embodiment, an Fc-containing polypeptide of the invention can be made in a lower eukaryotic host cell or organism. Recent developments allow the production of fully humanized therapeutics in lower eukaryotic host organisms, yeast and filamentous fungi, such as Pichia pastoris, Gerngross et al, US Patent 7,029,872 and US Patent No. 7,449,308, the disclosures of which are hereby incorporated by reference. See also Jacobs et al, Nature
Protocols 4(l):58-70 (2009).
In one embodiment, an Fc-containing polypeptide of the invention is made in a lower eukaryotic host cell, more preferably a yeast or filamentous fungal host cell, that has been engineered to produce glycoproteins comprising human-like N-glycans. In another embodiment, an Fc-containing polypeptide of the invention is made in a lower eukaryotic host cell, more preferably a yeast or filamentous fungal host cell, that has been engineered to produce glycoproteins having a predominant N-glycan comprising a terminal sialic acid. In one embodiment of the invention, the predominant N-glycan is the a-2,6 linked form of
SA2Gal2GlcNAc2Man3GlcNAc2, produced in strains glycoengineered with a-2,6 sialyl transferase which do not produce any a-2,3 linked sialic acid.
Those of ordinary skill in the art would recognize and appreciate that the materials and methods described herein are not limited to the specific strain of Pichia pastoris provided as an example herein, but could include any Pichia pastoris strain or other yeast or filamentous fungal strains capable of producing human-like N-glycans.
In general, lower eukaryotes such as yeast are used for expression of the proteins, particularly glycoproteins because they can be economically cultured, give high yields, and when appropriately modified are capable of suitable glycosylation. Yeast particularly offers established genetics allowing for rapid transformations, tested protein localization strategies and facile gene knock-out techniques. Suitable vectors have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.
While the invention has been demonstrated herein using the methylotrophic yeast Pichia pastoris, other useful lower eukaryote host cells include Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, 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 Yarrowia lipolytica and Neurospora crassa. Various yeasts, such as K. lactis, Pichia pastoris, Pichia methanolica, and Hansenula polymorpha are particularly suitable for cell culture because they are able to grow to high cell densities and secrete large quantities 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 at an industrial scale. Lower eukaryotes, particularly yeast and filamentous fungi, can be genetically modified so that they 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 the N-linked oligosaccharides which closely resemble the oligosaccharides produced by non- engineered, wild-type 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 predominantly present on glycoproteins produced from the host cells of the invention will depend upon the specific engineering steps that are performed. In this manner, glycoprotein compositions can be produced in which a specific desired glycoform is predominant in the composition. Such can be achieved by eliminating selected endogenous glycosylation enzymes and/or genetically engineering the host cells and/or supplying exogenous enzymes to mimic all or part of the mammalian glycosylation pathway as described in US Patent No. 7,449,308. If desired, additional genetic engineering of the glycosylation can be performed, such that the glycoprotein can be produced with or without core fucosylation. Use of lower eukaryotic host cells is further advantageous in that these cells are able to produce highly homogenous compositions of glycoprotein, 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.
Lower eukaryotes, particularly yeast, can be genetically modified so that they express glycoproteins in which the glycosylation pattern is human-like or humanized. Such can be achieved by eliminating selected endogenous glycosylation enzymes and/or supplying exogenous enzymes as described by Gerngross et al, US Patent No. 7,449,308. For example, a host cell can be selected or engineered to be depleted in l,6-mannosyl transferase activities, which would otherwise add mannose residues onto the N-glycan on a glycoprotein.
In one embodiment, the host cell further includes an l,2-mannosidase catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target the l,2-mannosidase activity to the ER or Golgi apparatus of the host cell. Passage of a recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a Man5GlcNAc2 glycoform, for example, a recombinant glycoprotein composition comprising predominantly a Man5GlcNAc2 glycoform. For example, U.S. Patent Nos. 7,029,872 and 7,449,308 and U.S. Published Patent Application No. 2005/0170452 disclose lower eukaryote host cells capable of producing a glycoprotein comprising a Man5GlcNAc2 glycoform.
In a further embodiment, the immediately preceding host cell further includes a GlcNAc transferase I (GnT I) catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target GlcNAc transferase I activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a
GlcNAcMan5GlcNAc2 glycoform, for example a recombinant glycoprotein composition comprising predominantly a GlcNAcMan5GlcNAc2 glycoform. U.S. Patent Nos. 7,029,872 and 7,449,308 and U.S. Published Patent Application No. 2005/0170452 disclose lower eukaryote host cells capable of producing a glycoprotein comprising a GlcNAcMan5GlcNAc2 glycoform. The glycoprotein produced in the above cells can be treated in vitro with a hexosaminidase to produce a recombinant glycoprotein comprising a Man5GlcNAc2 glycoform.
In a further embodiment, the immediately preceding host cell further includes a mannosidase II catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target mannosidase II activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a
GlcNAcMan3GlcNAc2 glycoform, for example a recombinant glycoprotein composition comprising predominantly a GlcNAcMan3GlcNAc2 glycoform. U.S. Patent No, 7,029,872 and U.S. Published Patent Application No. 2004/0230042 discloses lower eukaryote host cells that express mannosidase II enzymes and are capable of producing glycoproteins having
predominantly a GlcNAcMan3GlcNAc2 glycoform. The glycoprotein produced in the above cells can be treated in vitro with a hexosaminidase to produce a recombinant glycoprotein comprising a Man3GlcNAc2 glycoform. In a further embodiment, the immediately preceding host cell further includes GlcNAc transferase II (GnT II) catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target GlcNAc transferase II activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a
GlcNAc2Man3GlcNAc2 glycoform, for example a recombinant glycoprotein composition comprising predominantly a GlcNAc2Man3GlcNAc2 glycoform. U.S. Patent Nos. 7,029,872 and 7,449,308 and U.S. Published Patent Application No. 2005/0170452 disclose lower eukaryote host cells capable of producing a glycoprotein comprising a GlcNAc2Man3GlcNAc2 glycoform. The glycoprotein produced in the above cells can be treated in vitro with a hexosaminidase to produce a recombinant glycoprotein comprising a Man3GlcNAc2 glycoform.
In a further embodiment, the immediately preceding host cell further includes a galactosyltransferase catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target galactosyltransferase activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a
GalGlcNAc2 Man3GlcNAc2 or Gal2GlcNAc2Man3GlcNAc2 glycoform, or mixture thereof for example a recombinant glycoprotein composition comprising predominantly a GalGlcNAc2 Man3GlcNAc2 glycoform or Gal2GlcNAc2Man3GlcNAc2 glycoform or mixture thereof. U.S. Patent No, 7,029,872 and U.S. Published Patent Application No. 2006/0040353 discloses lower eukaryote host cells capable of producing a glycoprotein comprising a Gal2GlcNAc2
Man3GlcNAc2 glycoform. The glycoprotein produced in the above cells can be treated in vitro with a galactosidase to produce a recombinant glycoprotein comprising a GlcNAc2Man3 GlcNAc2 glycoform, for example a recombinant glycoprotein composition comprising predominantly a GlcNAc2Man3GlcNAc2 glycoform.
In a further embodiment, the immediately preceding host cell further includes a sialyltransferase catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target sialyltransferase activity to the ER or Golgi apparatus of the host cell. In a preferred embodiment, the sialyltransferase is an a-2,6- sialyltransferase. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising predominantly a
SA2Gal2GlcNAc2Man3GlcNAc2 glycoform or SAGal2GlcNAc2Man3GlcNAc2 glycoform or mixture thereof. For lower eukaryote host cells such as yeast and filamentous fungi, it is useful that the host cell further include a means for providing CMP-sialic acid for transfer to the N- glycan. U.S. Published Patent Application No. 2005/0260729 discloses a method for genetically engineering lower eukaryotes to have a CMP-sialic acid synthesis pathway and U.S. Published Patent Application No. 2006/0286637 discloses a method for genetically engineering lower eukaryotes to produce sialylated glycoproteins. To enhance the amount of sialylation, it can be advantageous to construct the host cell to include two or more copies of the CMP-sialic acid synthesis pathway or two or more copies of the sialylatransferase. 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.
Any one of the preceding host cells can further include one or more GlcNAc transferase selected from the group consisting of GnT III, GnT IV, GnT V, GnT VI, and GnT IX to produce glycoproteins having bisected (GnT III) and/or multiantennary (GnT IV, V, VI, and IX) N- glycan structures such as disclosed in U.S. Published Patent Application Nos. 2005/0208617 and 2007/0037248. Further, the proceeding host cells can produce recombinant glycoproteins (for example, antibodies) comprising SA(l-4)Gal(l-4)GlcNAc(2-4) Man3GlcNAc2, including antibodies comprising NANA (l-4)Gal(l-4)GlcNAc(2-4) Man3GlcNAc2, NGNA(l-4)Gal(l- 4)GlcNAc(2-4)Man3GlcNAc2 or a combination of NANA (l-4)Gal(l-4)GlcNAc(2-4)
Man3GlcNAc2 and NGNA(l-4)Gal(l-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(l-4)Gal(l-4)GlcNAc(2-4) Man3GlcNAc2 and devoid of any a2-3 linked SA.
In further embodiments, the host cell that produces glycoproteins that have
predominantly GlcNAcMan5GlcNAc2 N-glycans further includes a galactosyltransferase catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target the galactosyltransferase activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising predominantly the GalGlcNAcMan5GlcNAc2 glycoform.
In a further embodiment, the immediately preceding host cell that produced glycoproteins that have predominantly the GalGlcNAcMan5GlcNAc2 N-glycans further includes a
sialyltransferase catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target sialyltransferase activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a
SAGalGlcNAcMan5GlcNAc2 glycoform (for example NANAGalGlcNAcMan5GlcNAc2 or NGNAGalGlcNAcMan5GlcNAc2 or a mixture thereof).
Any of the preceding host cells can further include one or more sugar transporters such as UDP-GlcNAc transporters (for example, Kluyveromyces lactis and Mus musculus UDP-GlcNAc transporters), UDP-galactose transporters (for example, Drosophila melanogaster UDP- galactose transporter), and CMP-sialic acid transporter (for example, human sialic acid transporter). Because lower eukaryote host cells such as yeast and filamentous fungi lack the above transporters, it is preferable that lower eukaryote host cells such as yeast and filamentous fungi be genetically engineered to include the above transporters.
Further, any of the preceding host cells can be further manipulated to increase N-glycan occupancy. See e, g., Gaulitzek et al, Biotechnol. Bioengin. 103:1164-1175 (2009); Jones et al, Biochim. Biospyhs. Acta 1726: 121-137 (2005); WO2006/107990. In one embodiment, any of the preceding host cells can be further engineered to comprise at least one nucleic acid molecule encoding a heterologous single-subunit oligosaccharyltransferase (for example, 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 host cell genes encoding the proteins comprising the endogenous OTase complex. In one embodiment, any of the preceding host cells can be further 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 host cell genes encoding the proteins comprising the endogenous OTase complex.
Host cells further include lower eukaryote cells (e.g., yeast such as Pichia pastoris) that are genetically engineered to produce glycoproteins that do not have a-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, U.S. Published Patent Application No. 2006/0211085) and glycoproteins having phosphomannose residues by deleting or disrupting one or both of the phosphomannosyl transferase genes PNOl and MNN4B (See for example, U.S. Patent Nos. 7,198,921 and 7,259,007), which in further aspects can also include deleting or disrupting the MNN4A gene. Disruption includes disrupting the open reading frame encoding the 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 interfering RNA, antisense RNA, or the like. Further, cells can produce glycoproteins with a-mannosidase-resistant N-glycans through the addition of chemical hinhibios or through modification of the cell culture condition. These host cells can be further modified as described above to produce particular N-glycan structures.
Host cells further include lower eukaryote cells (e.g., yeast such as Pichia pastoris) that are genetically modified to control O-glycosylation of the glycoprotein by deleting or disrupting one or more of the protein O-mannosyltransferase (Dol-P-Man:Protein (Ser/Thr) Mannosyl Transferase genes) (PMTs) (See U.S. Patent No. 5,714,377) or grown in the presence of Pmtp inhibitors and/or an a -mannosidase as disclosed in Published International Application No. 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 interfering RNA, antisense RNA, or the like. The host cells can further include any one of the aforementioned host cells modified to produce particular N-glycan structures.
Pmtp inhibitors include but are not limited to a benzylidene thiazolidinediones.
Examples of benzylidene thiazolidinediones that can be used are 5-[[3,4-bis(phenylmethoxy) phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic Acid; 5-[[3-(l-Phenylethoxy)-4-(2- phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic Acid; and 5-[[3-(l- Phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3- thiazolidineacetic acid.
In particular embodiments, the function or expression of at least one endogenous PMT gene is reduced, disrupted, or deleted. For example, in particular embodiments the function or expression of at least one endogenous PMT gene selected from the group consisting of the PMT I, PMT2, PMT3, and PMT 4 genes is reduced, disrupted, or deleted; or the host cells are cultivated in the presence of one or more PMT inhibitors. In further embodiments, the host cells include one or more PMT gene deletions or disruptions and the host cells are cultivated in the presence of one or more Pmtp inhibitors. In particular aspects of these embodiments, the host cells also express a secreted a -1,2-mannosidase.
PMT deletions or disruptions and/or Pmtp inhibitors control O-glycosylation by reducing O-glycosylation occupancy, that is, by reducing the total number of O-glycosylation sites on the glycoprotein that are glycosylated. The further addition of an a -1,2-mannsodase that is secreted by the cell controls O-glycosylation by reducing the mannose chain length of the O-glycans that are on the glycoprotein. Thus, combining PMT deletions or disruptions and/or Pmtp inhibitors with expression of a secreted a -1,2-mannosidase controls O-glycosylation by reducing occupancy and chain length. In particular circumstances, the particular combination of PMT deletions or disruptions, Pmtp inhibitors, and a -1,2-mannosidase is determined empirically as particular heterologous glycoproteins (Fabs and antibodies, for example) may be expressed and transported through the Golgi apparatus with different degrees of efficiency and thus may require a particular combination of PMT deletions or disruptions, Pmtp inhibitors, and a -1,2- mannosidase. In another aspect, genes encoding one or more endogenous mannosyltransferase enzymes are deleted. This deletion(s) can be in combination with providing the secreted a- 1,2- mannosidase and/or PMT inhibitors or can be in lieu of providing the secreted a- 1,2- mannosidase and/or PMT inhibitors.
Thus, the control of O-glycosylation can be useful for producing particular glycoproteins in the host cells disclosed herein in better total yield or in yield of properly assembled
glycoprotein. The reduction or elimination of O-glycosylation appears to have a beneficial effect on the assembly and transport of whole 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 assembled antibodies or Fab fragments is increased over the yield obtained in host cells in which O-glycosylation is not controlled.
To reduce or eliminate the likelihood of N-glycans and O-glycans with β-linked mannose residues, which are resistant to a-mannosidases, the recombinant glycoengineered Pichia pastoris host cells are genetically engineered to eliminate glycoproteins having a-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 No. 7,465,577 and U.S. Patent No. 7,713,719). The deletion or disruption ΟΪΒΜΤ2 and one or more οΐΒΜΤΙ, BMT3, and BMT4 also reduces or eliminates detectable cross reactivity to antibodies against host cell protein.
Yield of glycoprotein can in some situations be improved by overexpressing nucleic acid molecules encoding mammalian or human chaperone proteins or replacing the genes encoding one or more endogenous chaperone proteins with nucleic acid molecules encoding one or more mammalian or human chaperone proteins. In addition, the expression of mammalian or human chaperone proteins in the host cell also appears to control O-glycosylation in the cell. Thus, further included are the host cells herein wherein 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 homolog of the chaperone protein is expressed in the host cell. Also included are host cells in which the endogenous host cell chaperones and the mammalian or human chaperone proteins are expressed. In further aspects, the lower eukaryotic host cell is a yeast or filamentous fungi host cell. Examples of the use of chaperones of host cells in which human chaperone proteins are introduced to improve the yield and reduce or control O- glycosylation of recombinant proteins has been disclosed in Published International Application No. WO 2009105357 and WO2010019487 (the disclosures of which are incorporated herein by reference). Like above, further included are lower eukaryotic host cells wherein, 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 as described above, the function or expression of at least one endogenous gene encoding a protein O- mannosyltransferase (PMT) protein is reduced, disrupted, or deleted. In particular embodiments, the function of at least one endogenous PMT gene selected from the group consisting of the PMT1, PMT2, PMT3, and PMT 4 genes is reduced, disrupted, or deleted.
In addition, O-glycosylation may have an effect on an antibody or Fab fragment's affinity and/or avidity for an antigen. This can be particularly significant when the ultimate host cell for production of the antibody or Fab is not the same as the host cell that was used for selecting the antibody. For example, O-glycosylation might interfere with an antibody's or Fab fragment's affinity for an antigen, thus an antibody or Fab fragment that might otherwise have high affinity for an antigen might not be identified because O-glycosylation may interfere with the ability of the antibody or Fab fragment to bind the antigen. In other cases, an antibody or Fab fragment that has high avidity for an antigen might not be identified because O-glycosylation interferes with the antibody's or Fab fragment's avidity for the antigen. In the preceding two cases, an antibody or Fab fragment that might be particularly effective when produced in a mammalian cell line might not be identified because the host cells for identifying and selecting the antibody or Fab fragment was of another cell type, for example, 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 mucin-type or dystroglycan type O-glycosylation in mammals. In particular cases, O-glycosylation might enhance the antibody or Fab fragments affinity or avidity for an antigen instead of interfere with antigen binding. This effect is undesirable when the production host cell is to be different from the host cell used to identify and select the antibody or Fab fragment (for example, identification and selection is done in yeast and the production host is a mammalian cell) because in the production host the O-glycosylation will no longer be of the type that caused the enhanced affinity or avidity for the antigen. Therefore, controlling O-glycosylation can enable use of the materials and methods herein to identify and select antibodies or Fab fragments with specificity for a particular antigen based upon affinity or avidity of the antibody or Fab fragment for the antigen without identification and selection of the antibody or Fab fragment being influenced by the O-glycosylation system of the host cell. Thus, controlling O-glycosylation further enhances the usefulness of yeast or fungal host cells to identify and select antibodies or Fab fragments that will ultimately be produced in a mammalian cell line. Those of ordinary skill in the art would 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, for example, US 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 modified, the description of which is incorporated herein as if set forth at length. See also WO2011/149999.
Biological Properties of Fc muteins
For many Fc-containing polypeptides altered anti-inflammatory properties would be desirable characteristics. In any of the embodiments of the invention, an increase or decrease 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-Ιβ, IL-6, RANKL, TRAP, ATP6v0d2, MDL-1, DAP 12, CDl lb, TIMP-1, MMP9, CTSK, PU-1, MCP1, ΜΙΡΙα, Cxcll- Groa, CXcl2-Grob, CD 18, TNF, FcyRI, FcyRIIb, FcyRIII and FcyRIV. In one embodiment, a decrease in anti-inflammatory activity is detected by measuring a change in the expression of a gene selected from the group consisting of: IL-Ιβ, IL-6, RANKL, TRAP, ATP6v0d2, MDL-1, DAP 12, CDl lb, TIMP-1, MMP9, CTSK, PU-1, MCP1, ΜΙΡΙα, Cxcll-Groa, CXcl2-Grob, CD 18, TNF, FcyRI, FcyRIIb, FcyRIII and FcyRIV.
Production of Fc-containing polypeptides
The Fc-containing polypeptides of the invention can be made according to any method known in the art suitable for generating polypeptides. 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 one embodiment, the antibody or antibody fragment is of the IgG type. In one embodiment, the antibody or antibody fragment is of the IgGl subtype. In one embodiment, the antibody or antibody fragment is of the IgG2 subtype. In one embodiment, the antibody or antibody fragment is of the IgG3 subtype. In one embodiment, the antibody or antibody fragment is of the IgG4 subtype. In one embodiment, the antibody or antibody fragment is of the IgA type. In one embodiment, the antibody or antibody fragment is of the IgD type. In one embodiment, the antibody or antibody fragment is of the IgE type. In one embodiment, the antibody or antibody fragment is of the IgM type. In another embodiment, the Fc-containing polypeptide is an immunoadhesin. Methods of preparing antibody and antibody fragments are well known in the art. Methods of introducing point mutations into a polypeptide, for example site directed mutagenesis, are also well known in the art.
In one embodiment, the Fc-containing polypeptide comprises N-glycans. In one embodiment, the Fc-containing polypeptide of the invention comprises N-glycans, wherein at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc-containing
polypeptide comprise an N-linked oligosaccharide structure selected from the group consisting of SA(i-4)Gal(i-4)GlcNAc(2-4)Man3GlcNAc2. In one embodiment, at least 30%, 40%, 50%, 60%), 70%), 80%) or 90%> of the N-glycans on the Fc-containing polypeptide comprise a
SA2Gal2GlcNAc2Man3GlcNAc structure. In one embodiment, at least 30%, 40%, 50%, 60%, 70%), 80%) or 90%) of the N-glycans on the Fc-containing polypeptide comprise a
NANA2Gal2GlcNAc2Man3GlcNAc structure.
N-Glycan analysis of Fc muteins
For many glycoproteins, including certain antibodies, N-glycosylation is important for biological properties. For example, sialylation of the terminal N-linked glycan of an IgG Fc region is important for producing glycoproteins and antibodies that have the correct
conformation to impart therapeutic activity. See, for example, Anthony et al., Science, 320: 373- 376 (2008), where terminal sialylation was correlated to anti-inflammatory activity for an IVIG preparation. Sialylation requires the presence of a penultimate galactose, upon which the sialyl transferase acts to form the sialylated glycan.
The N-glycan composition of the antibodies can be analyzed by matrix-assisted laser desorption ionization/time-of- flight (MALDI-TOF) mass spectrometry after release from the antibody with peptide-N-glycosidase F. Released carbohydrate composition can be quantitated by HPLC on an Allentech Prevail carbo (Alltech Associates, Deerfield IL) column.
DC-SIGN, FcyR and FcRn binding of Fc muteins
The DC-SIGN, FcyR and FcRn binding of Fc muteins can be determined using any method known in the art. See, e.g., Shields et al, J. Biol. Chem. 276: 6591-6604 (2001);
Vaccaro et al, Nat Biotechnol 23(10): 1283-8 (2005); Anthony et al, PNAS 105(50): 19571-8 (2008).
Biological Targets
Those of ordinary skill in the art would recognize and appreciate that the materials and methods herein could be used to produce any Fc-containing polypeptide for which the characteristics of altered anti-inflammatory activity would be desirable. It should further be noted that there is no restriction as to the type of Fc-containing polypeptide or antibody so produced by the invention. The Fc region of the Fc-containing polypeptide could 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 IgGl, IgG2, IgG3 or IgG4. In one embodiment, Fc region of the Fc- containing polypeptide is from an IgGl . In specific embodiments the antibodies or antibody fragments produced by the materials and methods herein can be chimeric, humanized or human antibodies.
In some embodiments, the Fc-containing polypeptides of the invention will bind to a biological target that is involved in inflammation.
In some embodiments, the Fc-containing polypeptide of the invention will bind to a proinflammatory cytokine. In some embodiments, the Fc-containing polypeptide of the invention will bind to a molecule selected from the group consisting of: TNF-a, 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, a4p7-integrin, E-selectin, Fact II, ICAM-3, beta2-integrin, IFNy, C5, CBL, LCAT, CR3, MDL-1, GITR, ADDL, CGRP, TRKA, IGF1R, RANKL, GTC, aBLys, or the receptor for any of the above mentioned molecules. In one embodiment, the Fc-containing polypeptide of the invention will bind to TNF-a. 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. In another embodiment, the Fc-containing polypeptide of the invention will bind to aBLys.
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, e.g., the antigens listed in Table 1 of WO2010/10910, which is incorporated herein by reference.
Methods of Increasing Anti-Inflammatory Properties
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 useful in treating an inflammatory condition (for example, an antibody or immunoadhesin that binds to an antigen that is involved in inflammation) and introducing one or more mutations in the Fc region, wherein the mutations are selected from the group consisting of:
Q295R/V302A/S344P/K414R, M252V/V303A/N315S, S239P/F241L/I377V,
L351S/N384D/S426P/K447E, F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274R/K360E/Y373H/P387S,
T289A/E345G/E357V/Q386R/N434S, A231V/P232S/S267F/S354P, R301G/K360E,
K246E/M252T/E272G/S375P, D249G, F241S/V263A/T335A/N389S, E269G/N389S/S400P, N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G and
V259A/N297S/N389S/G446D, wherein the numbering is according to the EU index as in Kabat. In one embodiment, the Fc-containing polypeptide comprises N-glycans. In one embodiment, the Fc-containing polypeptide comprises sialylated N-glycans. In one embodiment, the parent Fc-containing polypeptide is an antibody, antibody fragment or immunoadhesin that binds to an antigen that is involved in inflammation.
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 useful in treating an inflammatory condition (for example, an antibody or immunoadhesin that binds to an antigen that is involved in inflammation), wherein the parent Fc- containing polypeptide is a human IgGl or a fragment of human IgGl, and introducing one or more mutations in the Fc region, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R, M252V/V303A/N315S, S239P/F241L/I377V,
L351S/N384D/S426P/K447E, F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274R/K360E/Y373H/P387S,
T289A/E345G/E357V/Q386R/N434S, A231V/P232S/S267F/S354P, R301G/K360E,
K246E/M252T/E272G/S375P, D249G, F241S/V263A/T335A/N389S, E269G/N389S/S400P, N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G and
V259A/N297S/N389S/G446D, wherein the numbering is according to the EU index as in Kabat. In one embodiment, the Fc-containing polypeptide comprises N-glycans. In one embodiment, the Fc-containing polypeptide comprises sialylated N-glycans. In one embodiment, the parent Fc-containing polypeptide is an antibody, antibody fragment or immunoadhesin that binds to an antigen that is involved in inflammation.
In one embodiment, the parent Fc-containing polypeptide is an antibody, antibody fragment or immunoadhesin that is already marketed or under development for the treatment of an 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-TNFa antibody), Trastuzumab (anti-Her2 antibody),
Gemtuzumab ozogamicin (anti-CD33 antibody), Alemtuzumab (anti-CD52 antibody),
Ibritumomab tiuxeten (anti-CD20 antibody), Adalimumab (anti-TNFa antibody), Omalizumab (anti-IgE antibody), Tositumomab-1311 (iodinated derivative of an anti-CD20 antibody), Efalizumab (anti-CDl la antibody), Cetuximab (anti-EGF receptor antibody), Golimumab (anti- TNFa antibody), Bevacizumab (anti VEGF-A antibody), Natalizumab (anti a4 integrin), Efalizumab (anti CD 11a), Cetolizumab (anti-TNFa antibody) , Tocilizumab (anti-IL-6R), Ustenkinumab (anti IL-12/23), alemtuzumab (anti CD52), and natalizumab (anti a4 integrin), and variants thereof. In another embodiment, the parent Fc-contanining polypeptide is an Fc- fusion protein selected from the group consisting of: Arcalyst/ rilonacept (ILIR-Fc fusion), Orencia/ abatacept (CTLA-4-Fc fusion), Amevive/ alefacept (LFA-3-Fc fusion), Anakinra-Fc fusion (IL-lRa-Fc fusion protein), etanercept (TNFR-Fc fusion protein), FGF-21-Fc fusion protein, GLP-l-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 and variants thereof.
Methods of Treatment
The invention comprises a method of treating a subject in need thereof comprising: administering to the subject a therapeutically effective amount of an Fc-containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of: Q295R/V302A/S344P/K414R, M252V/V303A/N315S,
S239P/F241L/I377V, L351S/N384D/S426P/K447E, F241S/N276D/N361S, V266A,
K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S,
S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274R/K360E/Y373H/P387S, T289A/E345G/E357V/Q386R/N434S,
A231V/P232S/S267F/S354P, R301G/K360E, K246E/M252T/E272G/S375P, D249G,
F241S/V263A/T335A/N389S, E269G/N389S/S400P, N297D/S354P/K409R/S424P,
N297S/N390D/P395L, N297S/R301G and V259A/N297S/N389S/G446D, wherein the numbering is according to the EU index as in Kabat. In one embodiment, the parent Fc- containing polypeptide is human IgGl or a fragment of human IgGl .
The invention also comprises a method of treating a subject in need thereof comprising: administering to the subject a therapeutically effective amount of an Fc-containing polypeptide, wherein the Fc-containing polypeptide comprises any one of SEQ ID NOs: 11-33.
In one embodiment, the Fc-containing polypeptide does not comprise N-glycans and the Fc-containing polypeptide is a human IgGl or a fragment of human IgGl comprising one or more of the mutations selected from the group consisting of: N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G and V259A/N297S/N389S/G446D.
In one embodiment, the Fc-containing polypeptide does not comprise N-glycans and the Fc-containing polypeptide comprises any one of SEQ ID NOs:30-33.
In one embodiment, the Fc-containing polypeptide comprises N-glycans and the Fc- containing polypeptide is a human IgGl or a fragment of human IgGl comprising one or more of the mutations selected from the group consisting of: Q295R/V302A/S344P/K414R ,
M252V/V303A/N315S, S239P/F241L/I377V, L351S/N384D/S426P/K447E,
F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274R/K360E/Y373H/P387S, T289A/E345G/E357V/Q386R/N434S,
A231V/P232S/S267F/S354P, R301G/K360E, K246E/M252T/E272G/S375P, D249G,
F241S/V263A/T335A/N389S and E269G/N389S/S400P. In one embodiment, the Fc-containing polypeptide comprises sialylated N-glycans.
In one embodiment, the Fc-containing polypeptide comprises N-glycans and the Fc- containing polypeptide comprises any one of SEQ ID NOs: 11-29. In one embodiment, the Fc- containing polypeptide comprises sialylated N-glycans.
The Fc-containing polypeptide of the invention can 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.
In one embodiment, the subject is in need of treatment with a vaccine, and the method further comprises the administration of a vaccine. Thus, the invention comprises a method of increasing the efficacy of a vaccine comprising administering to a subject in need thereof an Fc- containing polypeptide of the invention.
In one embodiment, the subject in need of treatment has an inflammatory condition. In one embodiment, the method further comprises the administration of another anti-inflammatory compound. In another embodiment, the method further comprises the administration of an agent that increases expression of FcyRIIB. In another embodiment, the method further comprises the administration of an agent that binds DC-SIGN.
The term "inflammatory condition" refers to a condition that is characterized by abnormal or unwanted inflammation, such as autoimmune disease. Autoimmune diseases are disorders characterized by the chronic activation of immune cells under non-activating conditions. Examples include: psoriasis, inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis), rheumatoid arthritis, psoriatic arthritis, myasthenia gravis, multiple sclerosis, systemic lupus erythematosus, type I diabetes, primary biliary cirrhosis, and transplant.
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.
In one embodiment, the inflammatory condition is a primary immunodeficiency syndrome, including congential agammaglobulinemia and hypogammaglobulinemia, common variable immunodeficiency, severed combined immunodeficiency, or Wiskott Aldrich syndrome.
In one embodiment, the inflammatory condition is a secondary immunodeficiency syndrome, including B-cell lymphocytic leukemia, HIV infection or an allogeneic bone marrow transplantation.
In one embodiment, the inflammatory condition is idiopathic thrombocytopenic purpura
(ITP).
In one embodiment, the inflammatory condition is multiple myeloma.
In one embodiment, the inflammatory condition is Guillain-Barre syndrome.
In one embodiment, the inflammatory condition is Kawasaki disease.
In one embodiment, the inflammatory condition is chronic inflammatory demyelinating polyneropathy (CIDP).
In one embodiment, the inflammatory condition is autoimmune neutropenia.
In one embodiment, the inflammatory condition is hemolytic anemia.
In one embodiment, the inflammatory condition is anti-Factor VIII autoimmune disease.
In one embodiment, the inflammatory condition is multifocal neuropathy.
In one embodiment, the inflammatory condition is myasthenia gravis. In one embodiment, the inflammatory condition is systemic vasculitis (ANCA positive).
In one embodiment, the inflammatory condition is polymyositis.
In one embodiment, the inflammatory condition is dermatomyositis.
In one embodiment, the inflammatory condition is antiphospholipid syndrome.
In one embodiment, the inflammatory condition is sepsis syndrome.
In one embodiment, the inflammatory condition is graft-v-host disease.
In one embodiment, the inflammatory condition is allergy.
In one embodiment, the inflammatory condition is an anti-Rhesus factor D reaction.
In one embodiment, the inflammatory condition is an inflammatory condition of the cardiovascular system. The Fc-containing polypeptides of the invention may be used to treat atherosclerosis, atherothrombosis, coronary artery hypertension, acute coronary syndrome and heart failure, all of which are associated with inflammation.
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 may be used for the treatment of, e.g., Alzheimer's disease, amyotrophic lateral sclerosis (a.k.a. ALS; Lou Gehrig's disease), ischemic brain injury, prion diseases, and HIV-associated dementia.
In one embodiment, the inflammatory condition is an inflammatory condition of the gastrointestinal tract. For example, the Fc-containing polypeptides of the invention may be used for treating inflammatory bowel disorders, e.g., Crohn's disease, ulcerative colitis, celiac disease, and irritable bowel syndrome.
In one embodiment, the inflammatory condition is psoriasis, atopic dermatitis, arthritis, including rheumatoid arthritis, osteoarthritis, and psoriatic arthritis.
In one embodiment, the inflammatory condition is steroid-dependent atopic dermatitis.
In one embodiment, the inflammatory condition is cachexia.
Examples of other inflammatory disorders that can 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. A subject to be treated for an inflammatory condition can be identified by standard diagnosing techniques for the disorder. Optionally, the subject can be examined for the level or percentage of one or more of cytokines or cells a test sample obtained from the subject by methods known in the art. If the level or percentage is at or below a threshold value (which can be obtained from a normal subject), the subject is a candidate for treatment described herein. To confirm the inhibition or treatment, one can evaluate and/or verify the level or percentage of one or more of the above-mentioned cytokines or cells in the subject after treatment.
In another embodiment, the subject in need of treatment has infectious disease.
In another embodiment, the subject in need of treatment has cancer.
"Treating" or "treatment" refers to administration of a compound or agent to a subject who has a disorder with the purpose to cure, alleviate, relieve, remedy, delay the onset of, prevent, or ameliorate the disorder, the symptom of the disorder, the disease state secondary to the disorder, or the predisposition toward the disorder.
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 to cause a measurable improvement in one or more symptoms of a disease or condition or the progression of such disease or condition. A
therapeutically effective dose further refers to that amount of the Fc-containing polypeptide sufficient to result in at least partial amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in 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 to a combination, a therapeutically effective dose refers to 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 will result in an improvement of a diagnostic measure or parameter by at least 10%; usually by at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%. An effective amount can also result in an improvement in a subjective measure in cases where subjective measures are used to assess disease severity. The agent can be administered in vivo or ex vivo, alone or co-administered in conjunction with other drugs or therapy, i.e., a cocktail therapy. As used herein, the term "coadministration" or "co-administered" refers to the administration of at least two agents or therapies to a subject. In some embodiments, the co-administration of two or more
agents/therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents/therapies used may vary.
In an in vivo approach, a compound or agent is administered to a subject. Generally, the compound or agent is suspended in a pharmaceutically- acceptable carrier (such as, for example, but not limited to, physiological saline) and administered orally or by intravenous infusion, or injected or implanted subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily.
The dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the patient's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.001-100 mg/kg.
In one embodiment, the Fc-containing polypeptide of the invention will be administered a dose of between 1 to 100 milligrams per kilograms of body weight. In one embodiment, the Fc- containing polypeptide of the invention will be administered a dose of between 0.001 to 10 milligrams per kilograms of body weight. In one embodiment, the Fc-containing polypeptide of the invention will be administered a dose of between 0.001 to 0.1 milligrams per kilograms of body weight. In one embodiment, the Fc-containing polypeptide of the invention will be administered a dose of between 0.001 to 0.01 milligrams per kilograms of body weight.
Fc-containing polypeptides can be provided by continuous infusion, or by doses administered, e.g., daily, 1-7 times per week, weekly, bi-weekly, monthly, bimonthly, quarterly, semiannually, annually etc. Pharmaceutical Formulations
The invention also comprises pharmaceutical formulations comprising an Fc-containing polypeptide of the invention and a pharmaceutically acceptable carrier.
In one embodiment, the Fc-containing polypeptide of the invention is an IgG antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide of the invention is an IgGl antibody or antibody fragment. In one embodiment, the Fc-containing polypeptide of the invention is a human IgGl antibody or antibody fragment.
In one embodiment, the Fc-containing polypeptide of the invention comprises or consists essentially of any one of SEQ ID NOs: 11-33.
In one embodiment, the Fc-containing polypeptide comprises N-glycans and the Fc- containing polypeptide comprises or consists essentially of any one of SEQ ID NOs: 11-29.
In one embodiment, the Fc-containing polypeptide is a human IgGl or a fragment of human IgGl that comprises N-glycans and the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of: Q295R/V302A/S344P/K414R ,
M252V/V303A/N315S, S239P/F241L/I377V, L351S/N384D/S426P/K447E,
F241S/N276D/N361S, V266A, K246E/E272G/N286D/Y300C, H285R/V412A/S442P/L443S, S254F/N276S/P395S/F404L/P445S, V420I/I336V/K414R, D270G/K317R/E333G/N389D, K248E/K274P K360E/Y373H/P387S, T289A/E345G/E357V/Q386R/N434S,
A231V/P232S/S267F/S354P, R301G/K360E, K246E/M252T/E272G/S375P, D249G,
F241S/V263A/T335A/N389S, and E269G/N389S/S400P. In one embodiment, the Fc- containing polypeptide comprises sialylated N-glycans.
In one embodiment, the Fc-containing polypeptide is a human IgGl or a fragment of human IgGl that does not comprise N-glycans and the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of: N297D/S354P/K409R/S424P, N297S/N390D/P395L, N297S/R301G, and V259A/N297S/N389S/G446D.
In one embodiment, the Fc-containing polypeptide is a human IgGl or a fragment of human IgGl that does not comprise N-glycans and the Fc-containing polypeptide comprises or consists essentially of any one of SEQ ID NOs:30-33. In one embodiment, the pharmaceutical formulation comprises and Fc-containing polypeptide comprising N-glycans. In one embodiment, the pharmaceutical formulation comprises and Fc-containing polypeptide comprising sialylated N-glycans.
As utilized herein, the term "pharmaceutically acceptable" means a non-toxic 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 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 is administered and includes, but is not limited to such sterile liquids as water and oils. The characteristics of the carrier will depend on the route of administration.
Pharmaceutical Formulations of therapeutic and diagnostic agents may be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions (see, e.g., 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).
The mode of administration can 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 intra-arterial.
In certain embodiments, the Fc-containing polypeptides of the invention can be administered by an invasive route such as by injection (see above). In some embodiments of the invention, the Fc-containing polypeptides of the invention, or pharmaceutical composition thereof, is administered intravenously, subcutaneously, intramuscularly, intraarterially, intra- articularly (e.g. in arthritis joints), intratumorally, or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., orally; for example, in a pill, capsule or tablet) is also within the scope of the present invention.
In certain embodiments, the Fc-containing polypeptides of the invention can be administered by an invasive route such as by injection (see above). In some embodiments of the invention, the Fc-containing polypeptides of the invention, or pharmaceutical composition thereof, is administered intravenously, subcutaneously, intramuscularly, intraarterially, intra- articularly (e.g. in arthritis joints), intratumorally, or by inhalation, aerosol delivery.
Administration by non-invasive routes (e.g., orally; for example, in a pill, capsule or tablet) is also within the scope of the present invention.
Compositions can be administered with medical devices known in the art. For example, a pharmaceutical composition of the invention can be administered by injection with a hypodermic needle, including, e.g., a prefilled syringe or autoinjector.
The pharmaceutical compositions of the invention may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Patent Nos.
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.
The pharmaceutical compositions of the invention may also be administered by infusion. Examples of well-known implants and modules form administering pharmaceutical compositions include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent. No. 4,439, 196, which discloses an osmotic drug delivery system having multi-chamber compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art.
Alternately, one may administer the antibody in a local rather than systemic manner, for example, via injection of the antibody directly into an arthritic joint, often in a depot or sustained release formulation. Furthermore, one may administer the antibody in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody, targeting, for example, arthritic joint or pathogen-induced lesion characterized by immunopathology. The liposomes will be targeted to and taken up selectively by the afflicted tissue.
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 the target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic antibody to effect improvement in the target disease state, while simultaneously minimizing undesired side effects. Accordingly, the amount of biologic delivered depends in part on the particular therapeutic antibody and the severity of the condition being treated. Guidance in selecting appropriate doses of therapeutic antibodies is available (see, e.g., 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).
EXAMPLE 1:
Construction of Library of Full-Length IgGl Fc Variants in Pichia pastoris
A library of Fc variants was constructed using error prone PCR to introduce random mutations in a region encompassing the CH2-CH3 domains of a human immunoglobulin G 1 (IgGl) gene. These PCR fragments encoding variant Fc molecules were cloned in frame to VH1-CH1 region of an anti-Her2 monoclonal antibody (mAb) under the expression of Pichia AOX1 promoter in pGLYl 1576 (Figure 3). The complete amino acid sequence of the heavy chain of the Anti-Her2 antibody used in this experiment shown in SEQ ID NO:3. The VH1-CH1 sequence corresponds to amino acids 1-220 of SEQ ID NO: 3. Briefly, 1 microgram of ligation mixture was used to transform MegaX DH10B electro-competent E. coli cells (obtained from Life Technologies, Carlsbad, CA) by electroporation. Plated transformations resulted in 107 clones carrying unique sequences encoding full-length IgGl Fc variants. Clones were pooled cultured and lysed to isolate plasmid DNA using a Qiagen Maxi Plasmid Preparation kit (obtained from Qiagen, Germantown, MD).
DNA isolated as described above (encoding full-length IgGl Fc variants) was incubated for 2 hr with Spel restriction enzyme at 37°C. Linearized plasmid DNA was precipitated with 3M sodium acetate pH 5.2 and isopropanol. Digested DNA was reconstituted in deionized water at concentration of 1 μg/μL. 1 mg of this DNA was introduced in a Pichia pastoris strain that is capable of producing sialylated N-glycans and which contained plasmid pGLYl 1714 (Figure 1, SEQ ID NO:4), generating a library of 106 clones. These clones were scraped off plates and pooled in liquid media. The plasmid designated as pGLYl 1714 contains a cassette expressing IgG Light chain fused to surface anchor Sedl (Lc-Sedl) on the cell surface. A random set of 80 yeast clones were cultured in a 96-well plate, induced and analyzed for secreting full length IgGs by SDS-PAGE. 65% of the clones were shown to produce full-length IgGs. {Pichia pastoris strains capable of producing sialylated N-glycans are described in the art, for example, in WO2011/149999.)
Construction of pGLY11714: Figure 1 contains a plasmid map of pGLYl 1714.
Briefly, a polynucleotide encoding the N-terminus of a cell surface anchoring protein S.
cerevisiae Sedlp that inherently contains an attached glycophosphotidylinositol (GPI) post- translational modification that anchors the protein on the yeast cell wall was linked to a nucleic acid sequence that encodes the human IgG2 anti PCSK9 (1F11) Light Chain (Lc). The plasmid pGLYl 1714 containing anti-PC SK9 Lc bait cassette was constructed using a codon optimized sequence of human IgG2 Lc (VL+CL) fragment, which was synthesized and fused in frame to the 3' end of the nucleic acid sequence of S. cerevisiae a-mating factor signal sequence. The nucleic acid sequence encoding three repeats of the amino acids (GGGS) linker was used to link the nucleic acid sequence of Lc 3' end to the 5'end of S. cerevisiae Sedlp. The construct was subcloned into pGLY9008 at EcoRI -Sail (replacing the Fc) by the contracting research organization (CRO) Genewiz (South Plainfield, NJ). The resulting plasmid enables delivery of the Lc-SEDl cassette under the control of the Pichia pastoris AOXl promoter at the URA6 locus in Pichia pastoris. SEQ ID NO: 4 provides the sequence of plasmid pGLYl 1714. SEQ ID NO:5 provides the sequence of the light chain of the anti-PCSK9 antibody. SEQ ID NO:6 provides the amino acid sequence alpha mating factor-antipPCSK9 Lc-(GGGS) linker-^*.
cerevisiae Sedlp.
Strains that comprise plasmid pGLYl 1714 are able to display an antibody light chain and utilize the covalent interaction of this light chain with the heavy chain of an antibody molecule that is co-expressed in the same host. This interaction tethers the IgG molecule on the cell surface (See Figure 2). This surface display allows for the discovery of novel Fc variants that possess specific desired biological properties, such as Fc receptor binding affinities.
YGLY32444 also allows co-secretion of the displayed molecule allowing further in vitro analysis.
EXAMPLE 2:
Screening for Clones with Improved Binding to Human DC-SIGN using Surface Display
Figure 5 is a schematic representation of the method used to isolate the novel IgGl Fc variants of the invention having improved binding to human DC-SIGN. "DM" refers to cells displaying an anti-Her2 IgGl antibody having mutations at F243A/V264A in the Fc region. These cells reacted with anti-Fc fluorescent antibodies but did not exhibit detectable binding to biotinylated DC-SIGN. "Unsorted library" refers to the cells displaying the IgGl Fc variants from the library described in Example 1. This library was labeled and gated to isolate clones with higher fluorescence intensity. "Once-sorted library" refers to cells that were labeled and isolated from the unsorted library and displayed a shift in fluorescence intensity as expected. A second round of sorting was performed to isolate highly purified IgGl Fc variants having improved binding to human DC-SIGN.
Pichia clones from the library described in Example 1 were cultured in a shake flask in growth medium BMGY for 24 hours and expression and surface display was initiated by re- suspending and incubating for 24 hour in 2% methanol containing medium for at room temperature. After induction on methanol, 2 OD6oo of cells (~ 107 cells) were collected into a 1.5-ml microfuge tube and washed twice with phosphate-buffered saline (PBS) and then re- suspended in 100 μΐ of the Lectin Buffer (100 mM HEPES, 1.5 M NaCl, pH 7.4, 1 mM CaC12, 1 mM MgC12, 0.01% Tween 20) containing 1 μΐ (2 μg) of fluorescently-labeled goat anti-human Fc DyeLight 647 (Jackson Immunoresearch Labs, West Grove, PA), and 1 μg of biotinylated human DC-SIGN (Elicityl, Grenoble, France). The labeling cells/receptors mixtures were incubated at room temperature for 30 min. Biotinylated DC-SIGN was detected with fluorescently-labeled (DyeLight 488) streptavdin (purchased from R&D Systems, Minneapolis, MN). Cells were washed once with the Lectin Buffer and suspended in 300 μΐ of PBS for flow cytometric analysis and sorting. Clones with higher fluorescence intensity indicative of receptor labeling were isolated and cultured in liquid medium.
The sequences of novel IgGl Fc variants isolated as described herein were determined by DNA sequencing (Genewiz, South Plainfield, NJ). Table 1 describes the mutations identified in the Fc region of the identified IgGl Fc variants. The mutations are identified by reference to the Kabat numbering system using wild type human IgGl Fc as a reference and the single letter amino acid code.
Table 1.
SEQ ID NO: (Fc region, starting at amino acid residues 215 according to
Variant ID Mutations the EU index as in Kabat)
D 132443 Q295R/V302A/S344P/K414R 11
D 132444 M252V/V303A/N315S 12
D 132445 S239P/F241L/I377V 13
D 132446 L351S/N384D/S426P/K447E 14
D 132447 F241S/N276D/N361S 15
D 132448 V266A 16
D132733 K246E/E272G/N286D/Y300C 17
D132734 H285R/V412A/S442P/L443S 18
D132735 S254F/N276S/P395S/F404L/P445S 19
D132736 V240I/I336V/K414R 20
D132737 D270G/K317R/E333G/N389D 21
D132738 K248E/K274R/K360E/Y373H/P387S 22
D132739 T289A/E345G/E357V/Q386R/N434S 23
D 132740 A231 V/P232S/S267F/S354P 24
D 132921 R301G/K360E 25
D 132922 K246E/M252T/E272G/S375P 26
D 132923 D249G 27
D 132924 F241S/V263A/T335A/N389S 28
D 132925 E269G/N389S/S400P 29
D 132926 N297D/S354P/K409R/S424P 30 SEQ ID NO: (Fc region, starting at amino acid residues 215 according to
Variant ID Mutations the EU index as in Kabat)
D 132927 N297S/N390D/P395L 31
D132928 N297S/R301G 32
D 132929 V259A/N297S/N389S/G446D 33
Figure 6 shows an alignment of the Fc regions of the novel binders isolated as described in Examples 2 and of the consensus sequence for the Fc region of human IgGl . (Residue number 1 in the alignment corresponds to amino acid position 215 according to EU numbering system as in Kabat).
EXAMPLE 3:
Generation of Fc fragments of novel IgGls with enhanced binding to human DC-SIGN
DNA regions encoding Fc fragments of SEQ ID NOs: 11-33 were synthesized by Genewiz (South Plainfield, NJ) and cloned in Pichia expression vector pGLY4464 (Figure 4) under the AOXI promoter. The resulting plasmids contain Pichia pastoris TRP2 gene which is used to integrate expression cassettes into the yeast TRP2 locus.
Yeast strains producing each of these variant Fc fragments were generated by introducing Spel linearized plasmids into humanized Pichia pastoris strain that have been engineered to produce sialylated N-glycans (of the type NANA(i_4)Gal(i_4)GlcNAc(i_4)Man3GlcNAc2). Resulting clones were fermented in 1 L bioreactor vessels to produce each of the described variants.
Culture supematants were passed over protein A coated beads to capture secreted Fc fragments. Purified Fc fragments were eluted at pH 3.0 into tubes containing 1 M Tris (pH 8.0).
The N-glycan composition of the purified IgG variants produced herein were analyzed by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry after release from the antibody with peptide -N-glycosidase F. Released carbohydrate compositions were also quantitated by HPLC on an Allentech Prevail carbo (Alltech Associates, Deerfield IL) column.
The binding of the purified Fc fragments to various receptors were analyzed by surface plasmon resonance (SPR), and compared to the binding of: • Herceptin - refers to an anti-Her 2 IgGl antibody comprising a wild type human Fc region expressed in CHO cells and purchased from Genentech (San Francisco CA, Lot#724370). The heavy chain amino acid sequence is provided in SEQ ID NO: 3 and the light chain amino acid sequence is provided in SEQ ID NO:9.
• WT hFc - refers to human Fc fragment (wild-type) comprising the amino acid of SEQ ID NO: 2 (obtained by expression in a Pichia pastoris strain that has been engineered to produce sialylated N-glycans (of the type NANA(i_4)Gal(i_
4)GlcNAc(i_4)Man3GlcNAc2)).
• shFc - - refers to a sialylated human Fc fragment comprising mutations (F243A/V264A) (SEQ ID NO: 7) (obtained expression in a Pichia pastoris strain that has been engineered to produce sialylated N-glycans (of the type NANA(i_ 4)Gal(i_4GlcNAc(i_4)Man3GlcNAc2)).
• shFc F241- - refers to a sialylated human Fc fragment comprising mutation
F241A (SEQ ID NO: 8) (obtained by expression in a Pichia pastoris strain that has been engineered to produce sialylated N-glycans (of the type NANA(i_4)Gal(i_ 4)GlcNAc(i_4)Man3GlcNAc2)).
• mFc IgGl - - refers to wild type murine Fc fragment comprising the amino acid sequence of SEQ ID NO: 10 obtained by expression in a Pichia pastoris strain that has been engineered to produce sialylated N-glycans (of the type NANA(i_4)Gal(i_ 4)GlcNAc(i_4)Man3GlcNAc2)).
The binding of the purified Fc fragments to recombinant human DC-SIGN (rhDC-SIGN) was measured by surface plasmon resonance technology using a Biacore T100 (GE Healthcare,
Piscataway, NJ). The rhDC-SIGN at densities between 2500 to 10000 RU was immobilized to a
Series S CM5 chip (GE Healthcare, cat. no. BR- 1005-30) via amine coupling chemistry. The running buffer comprised of 10 mM HEPES, 150 mM NaCl (l HBS-N) (GE Healthcare, cat. no. BR-1006-70), 1 mM CaC12 (Sigma, cat. no. 21115 - 250 mL), 1 mM MgC12 (Sigma, cat. no.
68475 - 100 mL - F), and 0.01% Tween 20 (Sigma, cat. no. PI 379 - 100 mL) and was applied at a flow rate of 30 μΕ/ηώηιίε. IgGs were injected over the immobilized rhDC SIGN surface at a concentration of either 2 μΜ for 600 seconds. The rhDC-SIGN sensor surface was regenerated after each injection with 20 mM HEPES, 300 mM NaCl, and 25 mM EDTA at a flow rate of 10 μΕ/ηώηιίε for 60 seconds. Sensogram data was double referenced with both an empty reference flow cell and 0 μΜ concentration injection using Biacore T100 Evaluation Software (version 2.0.3). The maximum Response Units (RU's) for each IgG variant were tabulated. The ratios of variant RU's versus Herceptin were calculated and reported in Figure 7.
The binding of the purified Fc fragments to human FcyRIIb and FcyRIIIA-LF and mouse FcyRIIb and FcyRIV were measured by SPR on a Biacore T100 instrument with a
carboxymethylated dextran chip (CM5) and l HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% Surfactant P20) as the running buffer. The results are also shown in Figure 7. The CM5 chip (GE Healthcare, Wauwatusa, WI) was immobilized on all flow cells with mouse anti-human IgG (Fc specific) (obtained from GE Healthcare, Wauwatusa, WI) according to the Biacore Human Antibody Capture Kit to ~ 7000 RU. Purified IgGl variants containing mutations in Fc region (described in Example 1) were captured on the chip through immobilized mouse anti-human IgG (obtained from GE Healthcare, Wauwatusa, WI) followed by injection of human or mouse FcyRs. Human receptors (FcyRIIb and FcyRIIIA-LF) were expressed in Chinese Hamster Ovary cells (Merck, Whitehouse Station, NJ) and mouse receptors (mFcYRIIb and mFcyRIV) were expressed in NS0 murine myeloma (R&D Systems,
Minneapolis, MN)). Each flow cell was regenerated between each analyte injection with 3 M MgCl for 40 seconds at 10 μΐ/min. Data was analyzed with Biacore T100 Evaluation Software using the 1/1 binding model for KD values, where reported. "RU" or "Relative Response" refers to the measured change in mass on the surface of the sensor chip that is coated with equal moles of capture samples and exposed to equal amounts of the said receptors. 1 RU = lpg/mm2, therefore an increase in response is related to an increase in mass on the surface due to binding of the receptor to the capture samples. Higher affinity capture samples display higher RUs.
As listed in Figure 7, IgGl variants isolated though FACS/surface display screening exhibited higher binding affinity to immobilized hDC-SIGN in the SPR screen. Sialylated human Fc variants F243A/V264A and F241A also showed increased binding to hDC-SIGN compared to WT IgGl, albeit at lower affinity than the novel variants obtained from our screen.
We also analyzed N297 IgG glycans on these variants (Figure 7) and concluded that we were able to obtain novel muteins with diverse glycan signatures. We were also able to isolate hDC-SIGN binders that did not contain any N-glycosylation due to mutations in N297 residues. SEQUENCE LISTING
SEQ ID DESCRIPTION SEQUENCE NO:
1 Human IgGl Fc T C P P C P A P E L L G G P S V F L F P P region K P K D T L M I S R T P E V T C V V V D V
(wildtype) S H E D P E V K F N W Y V D G V E V H N A
K T K P R E E Q Y N s T Y R V V S V L T V
L H Q D W L N G K E Y K C K V S N K A L P
A P I E K T I S K A K G Q P R E P Q V Y T
L P P S R D E L T K N Q V S L T C L V K G
F Y P S D I A V E W E S N G Q P E N N Y K
T T P P V L D S D G S F F L Y S K L T V D
K S R W Q Q G N V F S C S V M H E A L H N
H Y T Q K S L S L S P G
2 Human IgGl Fc A E P K S C D K T H T C P P C P A P E L L region G G P s V F L F P P K P K D T L M I S R T
(wildtype) P E V T C V V V D V S H E D P E V K F N W
Y V D G V E V H N A K T K P R E E Q Y N s
T Y R V V S V L T V L H Q D W L N G K E Y
K C K V s N K A L P A P I E K T I S K A K
G Q P R E P Q V Y T L P P S R D E L T K N
Q V S L T C L V K G F Y P S D I A V E w E
S N G Q P E N N Y K T T P P V L D S D G S
F F L Y S K L T V D K S R W Q Q G N V F S
C S V M H E A L H N H Y T Q K S L S L S P
G
3 Heavy chain E V Q L V E S G G G L V Q P G G S L R L S amino acid C A A S G F N I K D T Y I H W V R Q A P G sequence of Her2 K G L E W V A R I Y P T N G Y T R Y A D S
IgGl antibody V K G R F T I S A D T S K N T A Y L Q M N
S L R A E D T A V Y Y C S R w G G D G F Y
A M D Y W G Q G T L V T V S s A S T K G P
S V F P L A P S s K S T S G G T A A L G C
L V K D Y F P E P V T V S W N S G A L T S
G V H T F P A V L Q S s G L Y S L S S V V
T V P S S S L G T Q T Y I C N V N H K P S
N T K V D K K V E P K s C D K T H T C P P
C P A P E L L G G P S V F L F P P K P K D
T L M I S R T P E V T c V V V D V S H E D
P E V K F N W Y V D G V E V H N A K T K P
R E E Q Y N s T Y R V V S V L T V L H Q D
W L N G K E Y K C K V s N K A L P A P I E
K T I S K A K G Q P R E P Q V Y T L P P S
R E E M T K N Q V S L T C L V K G F Y P S
D I A V E W E S N G Q P E N N Y K T T P P
V L D S D G S F F L Y s K L T V D K S R W
Q Q G N V F S C S V M H E A L H N H Y T Q
K S L S L S P G
4 Nucleic Acid TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACA Sequence of GCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGG pGLY11714 CGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATA
TGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCA TTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCT GGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCAC GACGTTGTAAAACGACGGCCAGTGAATTGAGATCTAACATCCAAAGACGAAAGGTTGAATGA
AGGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACA
ATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGG CGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTC CAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACT GACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAAC TAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGGC ATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTA GCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAAC
ATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGC
TACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTTAACGAC AACTTGAGAAGATCAAAAAACAACTAATTATTCGAAACGGAATTCACGATGAGATTCCCATC CATCTTCACTGCTGTTTTGTTCGCTGCTTCCTCTGCTTTGGCTGACATTCAAATGACTCAGT CCCCATCTTCCTTGTCTGCTTCCGTTGGTGACAGAGTTACTATCACTTGTAAGGCTTCCCAG AACGTTGGAACTAACGTTGTTTGGTATCAGCAGAAGCCAGGTAAGGCTCCAAAGGCTTTGAT TCACTCCGCTTCATACAGATACTCCGGTGTTCCATCCAGATTCTCTGGTTCTGGTTCCGGTA CTGACTTTACTTTGACTATCTCCTCATTGCAGCCAGAGGACTTCGCTACTTACTACTGTCAG CAGTACAAGACTTACCCATACACTTTCGGTCAGGGTACCAAGGTTGAGATCAAGAGAACTGT TGCTGCTCCATCCGTTTTCATTTTCCCACCATCCGACGAACAGTTGAAGTCTGGTACAGCTT CCGTTGTTTGTTTGTTGAACAACTTCTACCCAAGAGAGGCTAAGGTTCAGTGGAAGGTTGAC AACGCTTTGCAATCCGGTAACTCCCAAGAATCCGTTACTGAGCAAGACTCTAAGGACTCCAC TTACTCCTTGTCCTCCACTTTGACTTTGTCCAAGGCTGATTACGAGAAGCACAAGGTTTACG CTTGTGAGGTTACACATCAGGGTTTGTCCTCCCCAGTTACTAAGTCCTTCAACAGAGGAGAG TGTGGTGGTGGTGGTTCCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGTCGACCAATTCTC TAACTCTACTTCCGCTTCCTCTACTGACGTTACTTCCTCCTCCTCTATTTCTACTTCCTCCG GTTCCGTTACTATTACTTCCTCTGAGGCTCCAGAATCTGACAACGGTACTTCTACTGCTGCT CCAACTGAAACTTCTACTGAGGCTCCTACTACTGCTATTCCAACTAACGGAACTTCCACAGA GGCTCCAACAACAGCTATCCCTACAAACGGTACATCCACTGAAGCTCCTACTGACACTACTA CAGAAGCTCCAACTACTGCTTTGCCTACTAATGGTACATCAACAGAGGCTCCTACAGATACA ACAACTGAAGCTCCAACAACTGGATTGCCAACAAACGGTACTACTTCTGCTTTCCCACCAAC TACTTCCTTGCCACCATCCAACACTACTACTACTCCACCATACAACCCATCCACTGACTACA CTACTGACTACACAGTTGTTACTGAGTACACTACTTACTGTCCAGAGCCAACTACTTTCACA ACAAACGGAAAGACTTACACTGTTACTGAGCCTACTACTTTGACTATCACTGACTGTCCATG TACTATCGAGAAGCCAACTACTACTTCCACTACAGAGTATACTGTTGTTACAGAATACACAA CATATTGTCCTGAGCCAACAACATTCACTACTAATGGAAAAACATACACAGTTACAGAACCA ACTACATTGACAATTACAGATTGTCCTTGTACAATTGAGAAGTCCGAGGCTCCTGAATCTTC TGTTCCAGTTACTGAATCCAAGGGTACTACTACTAAAGAAACTGGTGTTACTACTAAGCAGA CTACTGCTAACCCATCCTTGACTGTTTCCACTGTTGTTCCAGTTTCTTCCTCTGCTTCTTCC CACTCCGTTGTTATCAACTCCAACGGTGCTAACGTTGTTGTTCCTGGTGCTTTGGGATTGGC TGGTGTTGCTATGTTGTTCTTGTAATAGGGCCGGCCATTTAAATACAGGCCCCTTTTCCTTT GTCGATATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCTCCCACATCCGCTCT
TATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAAACGCG TGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCT TTAATTTGCAAGCTGGATCCGCGGCCGCTTACGCGCCGTTCTTCGCTTGGTCTTGTATCTCC TTACACTGTATCTTCCCATTTGCGTTTAGGTGGTTATCAAAAACTAAAAGGAAAAATTTCAG
ATTACGTAAGTTATTTTGGTCCGGTGGGTAAGTGGGTAAGAATAGAAAGCATGAAGGTTTAC AAAAACGCAGTCACGAATTATTGCTACTTCGAGCTTGGAACCACCCCAAAGATTATATTGTA CTGATGCACTACCTTCTCGATTTTGCTCCTCCAAGAACCTACGAAAAACATTTCTTGAGCCT TTTCAACCTAGACTACACATCAAGTTATTTAAGGTATGTTCCGTTAACATGTAAGAAAAGGA GAGGATAGATCGTTTATGGGGTACGTCGCCTGATTCAAGCGTGACCATTCGAAGAATAGGCC TTCGAAAGCTGAATAAAGCAAATGTCAGTTGCGATTGGTATGCTGACAAATTAGCATAAAAA GCAATAGACTTTCTAACCACCTGTTTTTTTCCTTTTACTTTATTTATATTTTGCCACCGTAC TAACAAGTTCAGACAAATTAATTAACACCATGTCAGAAGATCAAAAAAGTGAAAATTCCGTA CCTTCTAAGGTTAATATGGTGAATCGCACCGATATACTGACTACGATCAAGTCATTGTCATG GCTTGACTTGATGTTGCCATTTACTATAATTCTCTCCATAATCATTGCAGTAATAATTTCTG TCTATGTGCCTTCTTCCCGTCACACTTTTGACGCTGAAGGTCATCCCAATCTAATGGGAGTG TCCATTCCTTTGACTGTTGGTATGATTGTAATGATGATTCCCCCGATCTGCAAAGTTTCCTG GGAGTCTATTCACAAGTACTTCTACAGGAGCTATATAAGGAAGCAACTAGCCCTCTCGTTAT TTTTGAATTGGGTCATCGGTCCTTTGTTGATGACAGCATTGGCGTGGATGGCGCTATTCGAT TATAAGGAATACCGTCAAGGCATTATTATGATCGGAGTAGCTAGATGCATTGCCATGGTGCT AATTTGGAATCAGATTGCTGGAGGAGACAATGATCTCTGCGTCGTGCTTGTTATTACAAACT CGCTTTTACAGATGGTATTATATGCACCATTGCAGATATTTTACTGTTATGTTATTTCTCAT GACCACCTGAATACTTCAAATAGGGTATTATTCGAAGAGGTTGCAAAGTCTGTCGGAGTTTT TCTCGGCATACCACTGGGAATTGGCATTATCATACGTTTGGGAAGTCTTACCATAGCTGGTA AAAGTAATTATGAAAAATACATTTTGAGATTTATTTCTCCATGGGCAATGATCGGATTTCAT TACACTTTATTTGTTATTTTTATTAGTAGAGGTTATCAATTTATCCACGAAATTGGTTCTGC AATATTGTGCTTTGTCCCATTGGTGCTTTACTTCTTTATTGCATGGTTTTTGACCTTCGCAT TAATGAGGTACTTATCAATATCTAGGAGTGATACACAAAGAGAATGTAGCTGTGACCAAGAA CTACTTTTAAAGAGGGTCTGGGGAAGAAAGTCTTGTGAAGCTAGCTTTTCTATTACGATGAC GCAATGTTTCACTATGGCTTCAAATAATTTTGAACTATCCCTGGCAATTGCTATTTCCTTAT ATGGTAACAATAGCAAGCAAGCAATAGCTGCAACATTTGGGCCGTTGCTAGAAGTTCCAATT TTATTGATTTTGGCAATAGTCGCGAGAATCCTTAAACCATATTATATATGGAACAATAGAAA TTAATTAACAGGCCCCTTTTCCTTTGTCGATATCATGTAATTAGTTATGTCACGCTTACATT CACGCCCTCCTCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAG GTCCCTATTTATTTTTTTTAATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATT TTTCTTTTTTTTCTGTACAAACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGA GAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGCGGCCTAAGGCGCGCCAGGCCA TAATGGCCCAAATGCAAGAGGACATTAGAAATGTGTTTGGTAAGAACATGAAGCCGGAGGCA TACAAACGATTCACAGATTTGAAGGAGGAAAACAAACTGCATCCACCGGAAGTGCCAGCAGC CGTGTATGCCAACCTTGCTCTCAAAGGCATTCCTACGGATCTGAGTGGGAAATATCTGAGAT TCACAGACCCACTATTGGAACAGTACCAAACCTAGTTTGGCCGATCCATGATTATGTAATGC
CAAGCATGTTTACCAGGTCTGTTAGAAACTCCTTTGTGAGGGCAGGACCTATTCGTCTCGGT CCCGTTGTTTCTAAGAGACTGTACAGCCAAGCGCAGAATGGTGGCATTAACCATAAGAGGAT TCTGATCGGACTTGGTCTATTGGCTATTGGAACCACCCTTTACGGGACAACCAACCCTACCA AGACTCCTATTGCATTTGTGGAACCAGCCACGGAAAGAGCGTTTAAGGACGGAGACGTCTCT GTGATTTTTGTTCTCGGAGGTCCAGGAGCTGGAAAAGGTACCCAATGTGCCAAACTAGTGAG TAATTACGGATTTGTTCACCTGTCAGCTGGAGACTTGTTACGTGCAGAACAGAAGAGGGAGG GGTCTAAGTATGGAGAGATGATTTCCCAGTATATCAGAGATGGACTGATAGTACCTCAAGAG GTCACCATTGCGCTCTTGGAGCAGGCCATGAAGGAAAACTTCGAGAAAGGGAAGACACGGTT
AGTCCAAGGTGACACTTTTCTTTGATTGTCCCGAATCAGTGCTCCTTGAGAGATTACTTAAA AGAGGACAGACAAGCGGAAGAGAGGATGATAATGCGGAGAGTATCAAAAAAAGATTCAAAAC ATTCGTGGAAACTTCGATGCCTGTGGTGGACTATTTCGGGAAGCAAGGACGCGTTTTGAAGG TATCTTGTGACCACCCTGTGGATCAAGTGTATTCACAGGTTGTGTCGGTGCTAAAAGAGAAG GGGATCTTTGCCGATAACGAGACGGAGAATAAATAAACATTGTAATAAGATTTAGACTGTGA
TGCAAACTCATTGATTACTTGTGCAATGGGCAAGAAGGATAGCTCTAGAAAGAAGAAGAAAA AGGAGCCGCCTGAAGAGCTGGATCTTTCCGAGGTTGTTCCAACTTTTGGTTATGAGGAATTT CATGTTGAGCAAGAGGAGAATCCGGTCGATCAAGACGAACTTGACGGCCATAATGGCCTAGC TTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACA CAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCA CATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCAT TAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTC GCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGG CGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGC
CCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTAT AAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCG CTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACG CTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCC CCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGA CACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGG CGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTG
Figure imgf000062_0001
ACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTA AATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTA CCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTG CCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCT GCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGC CGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATT GTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATT GCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCA ACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTC CTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTG CATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAAC CAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGG ATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGG CGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACC
AAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTT TTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATG TATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACG TCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTT CGTC
Amino acid DIQMTQSPSSLSASVGDRVTITCKASQNVGTNWWYQQKPGKAPKALIHSASYRYSGVPSRF sequence of SGSGSGTDFTLTISSLQPEDFATYYCQQYKTYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQ anti-PCSK9 Light LKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY Chain EKHKVYACEVTHQGLSSPVTKSFNRGEC
Amino acid MRFPSIFTAVLFAASSALADIOMTOSPSSLSASVGDRVTITCKASOMVGTMWWYOOKPGKA sequence of PKALIHSASYRYSGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCOOYKTYPYTFGOGTKVE alpha mating IKRTVAAPSVFIFPPSDEOLKSGTASWCLLMMFYPREAKVOWKVDMALOSGMSOESVTEOD factor- SKDSTYSLSSTLTLSKADYEKHKVYACEVTHOGLSSPVTKSFNRGECGGGGSGGGGSGGGGS antipPCSK9 Lc- VDQFSNSTSASSTDVTSSSSISTSSGSVTITSSEAPESDNGTSTAAPTETSTEAPTTAIPTN (GGGS , SEQ ID GTSTEAPTTAIPTNGTSTEAPTDTTTEAPTTALPTNGTSTEAPTDTTTEAPTTGLPTNGTTS NO:3) linker-S. AFPPTTSLPPSNTTTTPPYNPSTDYTTDYTWTEYTTYCPEPTTFTTNGKTYTVTEPTTLTI cerevisiae Sedlp TDCPCTIEKPTTTSTTEYTWTEYTTYCPEPTTFTTNGKTYTVTEPTTLTITDCPCTIEKSE
APESSVPVTESKGTTTKETGVTTKQTTA PSLTVSTWPVSSSASSHSWINSNGA VWPG ALGLAGVAMLFL
Amino acid AEPKSCDKTHTCPPCPAPELLGGPSVFLAPPKPKDTLMISRTPEVTCWADVSHEDPEVKFN sequence of WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK human IgGl Fc AKGQPREPQVYTLPPSREEMTK QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS polypeptide DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
comprising
mutations at
positions F243A
and V264A
Amino acid AEPKSCDKTHTCPPCPAPELLGGPSVALFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN sequence of WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK human IgGl Fc AKGQPREPQVYTLPPSREEMTK QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS polypeptide DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
comprising a
mutation at
positions 241
(F241A)
Light chain D I Q M T Q S P S S L S A S V G D R V T I amino acid T C R A s Q D V N T A V A W Y Q Q K P G K sequence of Her2 A P K L L I Y S A S F L Y S G V P S R F S
IgGl antibody G S R S G T D F T L T I S S L Q P E D F A
T Y Y C Q Q H Y T T P P T F G Q G T K V E
I K R T V A A P S V F I F P P S D E Q L K
S G T A S V V C L L N N F Y P R E A K V Q
W K V D N A L Q S G N S Q E S V T E Q D S
K D S T Y S L S S T L T L S K A D Y E K H
K V Y A C E V T H Q G L S S P V T K S F N
R G E C
Mouse IgGl GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVWDISKDDPEVQFSWFVDDVEVHTA region QTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVY
TIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLN VQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG D132443 AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN WYVDGVEVHNAKTKPREERYNSTYRAVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVPLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDRSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
D132444 AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLVISR PEVTCVWDVSHEDPEVKFN
YVDGVEVHNAKTKPREEQYNS YRVASVL VLHQD LSGKEYKCKVSNKALPAPIEK ISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE ESNGQPENNYK PPVLDS DGSFFLYSKLTVDKSR QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
D132445 AEPKSCDKTHTCPPCPAPELLGGPPVLLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDVAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDRSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
D132446 AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTSPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESDGQPENNYKTTPPVLDS DGSFFLYSKLTVDRSRWQQGNVFSCPVMHEALHNHYTQKSLSLSPGE
D132447 AEPKSCDKTHTCPPCPAPELLGGPSVSLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFD
WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKSQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDRSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGR
D132448 AEPRSCDRTHTCPPCPAPELLGGPSVFLFPPRPRDTLMISRTPEVTCVWDASHEDPEVRFN
WYVDGVEVHNARTRPREEQYNSTYRWSVLTVLHQDWLNGREYRCRVSNRALPAPIERTISR ARGQPREPQVYTLPPSRDELTRNQVSLTCLVRGFYPSDIAVEWESNGQPENNYRTTPPVLDS DGSFFLYSRLTVDRSRWQQGNVFSCSVMHEALHNHYTQRSLSLSPGR
D132733 AEPRSCDRTHTCPPCPAPELLGGPSVFLFPPEPRDTLMISRTPEVTCVWDVSHEDPGVRFN
WYVDGVEVHDARTRPREEQYNSTCRWSVLTVLHQDWLNGREYRCRVSNRALPAPIERTISR ARGQPREPQVYTLPPSRDELTRNQVSLTCLVRGFYPSDIAVEWESNGQPENNYRTTPPVLDS DGSFFLYSRLTVDRSRWQQGNVFSCSVMHEALHNHYTQRSLSLSPGR
D132734 AEPRSCDRTHTCPPCPAPELLGGPSVFLFPPRPRDTLMISRTPEVTCVWDVSHEDPEVRFN
WYVDGVEVRNARTRPREEQYNSTYRWSVLTVLHQDWLNGREYRCRVSNRALPAPIERTISR ARGQPREPQVYTLPPSRDELTRNQVSLTCLVRGFYPSDIAVEWESNGQPENNYRTTPPVLDS DGSFFLYSRLTADRSRWQQGNVFSCSVMHEALHNHYTQRSLPSSPGR
D1327340 AEPRSCDRTHTCPPCPVSELLGGPSVFLFPPRPRDTLMISRTPEVTCVWDVFHEDPEVRFN
WYVDGVEVHNARTRPREEQYNSTYRWSVLTVLHQDWLNGREYRCRVSNRALPAPIERTISR ARGQPREPQVYTLPPPRDELTRNQVSLTCLVRGFYPSDIAVEWESNGQPENNYRTTPPVLDS DGSFFLYSRLTVDRSRWQQGNVFSCSVMHEALHNHYTQRSLSLSPGR
D132735 AEPRSCDRTHTCPPCPAPELLGGPSVFLFPPRPRDTLMIFRTPEVTCVWDVSHEDPEVRFS
WYVDGVEVHNARTRPREEQYNSTYRWSVLTVLHQDWLNGREYRCRVSNRALPAPIERTISR ARGQPREPQVYTLPPSRDELTRNQVSLTCLVRGFYPSDIAVEWESNGQPENNYRTTSPVLDS DGSLFLYSRLTVDRSRWQQGNVFSCSVMHEALHNHYTQRSLSLSSGR
D132736 AEPRSCDRTHTCPPCPAPELLGGPSIFLFPPRPRDTLMISRTPEVTCVWDVSHEDPEVRFN
WYVDGVEVHNARTRPREEQYNSTYRWSVLTVLHQDWLNGREYRCRVSNRALPAPIERTVSR ARGQPREPQVYTLPPSRDELTRNQVSLTCLVRGFYPSDIAVEWESNGQPENNYRTTPPVLDS DGSFFLYSRLTVDRSRWQQGNVFSCSVMHEALHNHYTQRSLSLSPGR
D132737 AEPRSCDRTHTCPPCPAPELLGGPSVFLFPPRPRDTLMISRTPEVTCVWDVSHEGPEVRFN
WYVDGVEVHNARTRPREEQYNSTYRWSVLTVLHQDWLNGREYRCRVSNRALPAPIGRTISR ARGQPREPQVYTLPPSRDELTRNQVSLTCLVRGFYPSDIAVEWESNGQPEDNYRTTPPVLDS DGSFFLYSRLTVDRSRWQQGNVFSCSVMHEALHNHYTQRSLSLSPGR
D132738 AEPRSCDRTHTCPPCPAPELLGGPSVFLFPPRPEDTLMISRTPEVTCVWDVSHEDPEVRFN
WYVDGVEVHNARTRPREEQYNSTYRWSVLTVLHQDWLNGREYRCRVSNRALPAPIERTISR ARGQPREPQVYTLPPSRDELTENQVSLTCLVRGFHPSDIAVEWESNGQSENNYRTTPPVLDS DGSFFLYSRLTVDRSRWQQGNVFSCSVMHEALHNHYTQRSLSLSPGR D132739 AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN WYVDGVEVHNAKAKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPRGPQVYTLPPSRDVLTKNQVSLTCLVKGFYPSDIAVEWESNGRPENNYKTTPPVLDS DGSFFLYS KLTVDKSRWQQGNVFS CS VMHEALHSHYTQKS LSLSPGK
D132921 AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYGWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYS KLTVDKSRWQQGNVFS CS VMHEALHNHYTQKS LSLSPGK
D132922 AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPEPKDTLTISRTPEVTCVWDVSHEDPGVKFN
WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPPDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYS KLTVDKSRWQQGNVFS CS VMHEALHNHYTQKS LSLSPGK
D132923 AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKGTLMISRTPEVTCVWDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYS KLTVDKSRWQQGNVFS CS VMHEALHNHYTQKS LSLSPGK
D132924 AEPKSCDKTHTCPPCPAPELLGGPSVSLFPPKPKDTLMISRTPEVTCVAVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKAI SK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPESNYKTTPPVLDS DGSFFLYS KLTVDKSRWQQGNVFS CS VMHEALHNHYTQKS LSLSPGK
D132925 AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHGDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPESNYKTTPPVLDP DGSFFLYS KLTVDKSRWQQGNVFS CS VMHEALHNHYTQKS LSLSPGK
D132926 AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYDSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPPRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQQGNVFPCS VMHEALHNHYTQKS LSLSPGK
D132927 AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYSSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENDYKTTLPVLDS DGSFFLYS KLTVDKSRWQQGNVFS CS VMHEALHNHYTQKS LSLSPGK
D132928 AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYSSTYGWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYS KLTVDKSRWQQGNVFS CS VMHEALHNHYTQKS LSLSPGK
D132929 AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEATCVWDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYSSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPESNYKTTPPVLDS DGSFFLYS KLTVDKSRWQQGNVFS CS VMHEALHNHYTQKS LS LS PDK

Claims

WHAT IS CLAIMED:
1) An Fc-containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of:
• Q295R/V302A/S344P/K414R,
• M252V/V303A/N315S,
• S239P/F241L/I377V,
• L351S/N384D/S426P/K447E,
• F241S/N276D/N361S,
• V266A,
• K246E/E272G/N286D/Y300C,
• H285R/V412A/S442P/L443S,
• S254F/N276S/P395S/F404L/P445S,
• V420I/I336V/K414R,
• D270G/K317R/E333G/N389D,
• K248E/K274R/K360E/Y373H/P387S,
• T289A/E345G/E357V/Q386R/N434S,
• A231V/P232S/S267F/S354P,
• R301G/K360E,
• K246E/M252T/E272G/S375P,
• D249G,
• F241S/V263A/T335A/N389S,
• E269G/N389S/S400P,
• N297D/S354P/K409R/S424P,
• N297S/N390D/P395L,
• N297S/R301G and
• V259A/N297S/N389S/G446D;
wherein the numbering is according to the EU index as in Kabat. The Fc-containing polypeptide of claim 1 , wherein the Fc-containing polypeptide does not comprise N-glycans and wherein the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of:
• N297D/S354P/K409R/S424P,
• N297S/N390D/P395L,
• N297S/R301G and
• V259A/N297S/N389S/G446D.
The Fc-containing polypeptide of claim 1 , wherein the Fc-containing polypeptide comprises N-glycans and wherein the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of:
• Q295R/V302A/S344P/K414R ,
• M252V/V303A/N315S,
• S239P/F241L/I377V,
• L351S/N384D/S426P/K447E,
• F241S/N276D/N361S,
• V266A,
• K246E/E272G/N286D/Y300C,
• H285R/V412A/S442P/L443S,
• S254F/N276S/P395S/F404L/P445S,
• V420I/I336V/K414R,
• D270G/K317R/E333G/N389D,
• K248E/K274R/K360E/Y373H/P387S,
• T289A/E345G/E357V/Q386R/N434S,
• A231V/P232S/S267F/S354P,
• R301G/K360E,
• K246E/M252T/E272G/S375P,
• D249G,
• F241S/V263A/T335A/N389S and • E269G/N389S/S400P.
4) An Fc-containing polypeptide comprising a mutation in the Fc region, wherein the mutation is at a position selected from the group consisting of: 246, 276, 360, 389, 395, and 414, wherein the numbering is according to the EU index as in Kabat.
5) An Fc-containing polypeptide comprising a mutation in the Fc region, wherein the mutation is selected from the group consisting of:
• F241L
• F241S
• K246E
• E272G
• N276D
• N276S
• R301G
• S354P
• K360E
• N389S
• N389D
• P395L,
• P395S and
• K414R.
6) The Fc-containing polypeptide of any one of claims 1, 3 or 4, wherein the Fc-containing polypeptide further comprises one or more mutations at positions selected from the group consisting of: 241, 243, 246, 252, 254, 256, 264, 267, 272, 276, 301, 328, 339, 342, 354, 360, 389, 395, 414, 433 and 434 of the Fc region, wherein the numbering is according to the EU index as in Kabat. 7) The Fc-containing polypeptide of any one of claim 6, wherein the Fc-containing polypeptide further comprises one or more mutations selected from the group consisting of:
• F241L,
• F241S ,
• K246E,
• E272G,
• N276D,
• N276S ,
• R301G ,
• S354P ,
• K360E ,
• N389S,
• N389D ,
• P395L,
• P395S and
• K414R.
8) The Fc-containing polypeptide of any one of claims 1 or 3-7, wherein the Fc-containing polypeptide comprises N-glycans, and wherein at least 30%, 40%>, 50%>, 60%>, 70%>, 80%> or 90% of the N-glycans on the Fc-containing polypeptide comprise an N-linked
oligosaccharide structure selected from the group consisting of SA(i_4)Gal(i_4)GlcNAc(2- 4)Man3GlcNAc2-
9) The Fc-containing polypeptide any one of claims 1 or 3-8, wherein the sialic acid residues in the sialylated N-glycans are attached via a-2,6 linkages.
10) The Fc-containing polypeptide of any one of claims 1-9, wherein the Fc-containing
polypeptide is an antibody or an antibody fragment. 11) The Fc-containing polypeptide of any one of claims 1-10, wherein the Fc-containing polypeptide is an IgGl antibody or an antibody fragment.
12) The Fc polypeptide of claim 1-11 , wherein the isolated polypeptide has one or more of the following properties when compared to a parent Fc-containing polypeptide:
a) enhanced binding to human DC-SIGN;
b) altered inflammatory properties.
13) A method for producing an Fc-containing polypeptide in a host cell comprising:
a) providing a host cell comprising a nucleic acid encoding an Fc-containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of:
• Q295R/V302A/S344P/K414R,
• M252V/V303A/N315S,
• S239P/F241L/I377V,
• L351S/N384D/S426P/K447E,
• F241S/N276D/N361S,
• V266A,
• K246E/E272G/N286D/Y300C,
• H285R/V412A/S442P/L443S,
• S254F/N276S/P395S/F404L/P445S,
• V420I/I336V/K414R,
• D270G/K317R/E333G/N389D,
• K248E/K274R/K360E/Y373H/P387S,
• T289A/E345G/E357V/Q386R/N434S,
• A231V/P232S/S267F/S354P,
• R301G/K360E,
• K246E/M252T/E272G/S375P,
• D249G, • F241S/V263A/T335A/N389S,
• E269G/N389S/S400P,
• N297D/S354P/K409R/S424P,
• N297S/N390D/P395L,
• N297S/R301G and
• V259A/N297S/N389S/G446D;
wherein the numbering is according to the EU index as in Kabat;
b) culturing the host cell under conditions which cause expression of the Fc-containing polypeptide; and
c) isolating the Fc-containing polypeptide from the host cell.
14) The method of claim 13, wherein the host cell is capable of producing an Fc-containing polypeptide comprising N-glycans.
15) The method of claim 13 or 14, wherein the host cell is a lower eukaryotic host cell.
16) A method of increasing the anti-inflammatory properties of an Fc-containing polypeptide comprising introducing one or more of mutations in the Fc region of the Fc-containing polypeptide, wherein the mutations are selected from the group consisting of:
• Q295R/V302A/S344P/K414R,
• M252V/V303A/N315S,
• S239P/F241L/I377V,
• L351S/N384D/S426P/K447E,
• F241S/N276D/N361S,
• V266A,
• K246E/E272G/N286D/Y300C,
• H285R/V412A/S442P/L443S,
• S254F/N276S/P395S/F404L/P445S,
• V420I/I336V/K414R, • D270G/K317R/E333G/N389D,
• K248E/K274R/K360E/Y373H/P387S,
• T289A/E345G/E357V/Q386R/N434S,
• A231V/P232S/S267F/S354P,
• R301G/K360E,
• K246E/M252T/E272G/S375P,
• D249G,
• F241S/V263A/T335A/N389S,
• E269G/N389S/S400P,
• N297D/S354P/K409R/S424P,
• N297S/N390D/P395L,
• N297S/R301G, and
• V259A/N297S/N389S/G446D;
wherein the numbering is according to the EU index as in Kabat, wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc- containing polypeptide.
17) The method of claim 16, wherein the Fc-containing polypeptide is an antibody or an antibody fragment that does not comprise N-glycans and wherein the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of:
• N297D/S354P/K409R/S424P,
• N297S/N390D/P395L,
• N297S/R301G, and
• V259A/N297S/N389S/G446D.
18) The method of claim 16, wherein the Fc-containing polypeptide is an antibody or an antibody fragment comprising N-glycans and wherein the Fc-containing polypeptide comprises one or more of the mutations selected from the group consisting of:
• Q295R/V302A/S344P/K414R , M252V/V303A/N315S,
S239P/F241L/I377V,
L351S/N384D/S426P/K447E,
F241S/N276D/N361S,
V266A,
K246E/E272G/N286D/Y300C,
H285R/V412A/S442P/L443S,
S254F/N276S/P395S/F404L/P445S,
V420I/I336V/K414R,
D270G/K317R/E333G/N389D,
K248E/K274R/K360E/Y373H/P387S,
T289A/E345G/E357V/Q386R/N434S,
A231 V/P232S/S267F/S354P,
R301G/K360E,
K246E/M252T/E272G/S375P,
D249G,
F241S/V263A/T335A/N389S, and
E269G/N389S/S400P.
19) A method of treating a subject in need thereof comprising: administering to the subject a therapeutically effective amount of an Fc-containing polypeptide comprising one or more of mutations in the Fc region, wherein the mutations are selected from the group consisting of:
Q295R/V302A/S344P/K414R,
M252V/V303A/N315S,
S239P/F241L/I377V,
L351S/N384D/S426P/K447E,
F241S/N276D/N361S,
V266A,
K246E/E272G/N286D/Y300C, • H285R/V412A/S442P/L443S,
• S254F/N276S/P395S/F404L/P445S,
• V420I/I336V/K414R,
• D270G/K317R/E333G/N389D,
• K248E/K274R/K360E/Y373H/P387S,
• T289A/E345G/E357V/Q386R/N434S,
• A231V/P232S/S267F/S354P,
• R301G/K360E,
• K246E/M252T/E272G/S375P,
• D249G,
• F241S/V263A/T335A/N389S,
• E269G/N389S/S400P,
• N297D/S354P/K409R/S424P,
• N297S/N390D/P395L,
• N297S/R301G, and
• V259A/N297S/N389S/G446D;
wherein the numbering is according to the EU index as in Kabat, wherein the polypeptide comprises enhanced binding to human DC-SIGN when compared to a parent Fc- containing polypeptide.
20) The method of claim 19, wherein the subject has an inflammatory condition.
21) The method of claim 19 , method further comprises the administration of an agent selected from the group consisting of:
a) another anti-inflammatory compound,
b) an agent that binds FcyRIIB.
22) A pharmaceutical formulation comprising:
a) the Fc-containing polypeptide of any one of claims 1-12, and
b) a pharmaceutically acceptable carrier.
PCT/US2014/064458 2013-11-13 2014-11-07 Fc CONTAINING POLYPEPTIDES HAVING INCREASED BINDING TO HUMAN DC-SIGN WO2015073307A2 (en)

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Cited By (4)

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US10513724B2 (en) 2014-07-21 2019-12-24 Glykos Finland Oy Production of glycoproteins with mammalian-like N-glycans in filamentous fungi
WO2020089811A1 (en) 2018-10-31 2020-05-07 Novartis Ag Dc-sign antibody drug conjugates
US10669344B2 (en) 2016-08-12 2020-06-02 Janssen Biotech, Inc. Engineered antibodies and other Fc-domain containing molecules with enhanced agonism and effector functions
US10724013B2 (en) 2013-07-04 2020-07-28 Glykos Finland Oy O-mannosyltransferase deficient filamentous fungal cells and methods of use thereof

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EP1888649A2 (en) * 2005-05-09 2008-02-20 GlycArt Biotechnology AG Antigen binding molecules having modified fc regions and altered binding to fc receptors
EP2233500A1 (en) * 2009-03-20 2010-09-29 LFB Biotechnologies Optimized Fc variants

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US10724013B2 (en) 2013-07-04 2020-07-28 Glykos Finland Oy O-mannosyltransferase deficient filamentous fungal cells and methods of use thereof
US10513724B2 (en) 2014-07-21 2019-12-24 Glykos Finland Oy Production of glycoproteins with mammalian-like N-glycans in filamentous fungi
US10669344B2 (en) 2016-08-12 2020-06-02 Janssen Biotech, Inc. Engineered antibodies and other Fc-domain containing molecules with enhanced agonism and effector functions
WO2020089811A1 (en) 2018-10-31 2020-05-07 Novartis Ag Dc-sign antibody drug conjugates

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