WO2007024249A2 - Engineering fc antibody regions to confer effector function - Google Patents

Engineering fc antibody regions to confer effector function Download PDF

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Publication number
WO2007024249A2
WO2007024249A2 PCT/US2005/040962 US2005040962W WO2007024249A2 WO 2007024249 A2 WO2007024249 A2 WO 2007024249A2 US 2005040962 W US2005040962 W US 2005040962W WO 2007024249 A2 WO2007024249 A2 WO 2007024249A2
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region
antibody
variant
antigen
modified antibody
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PCT/US2005/040962
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English (en)
French (fr)
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WO2007024249A3 (en
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Jeffrey Stavenhagen
Scott Koenig
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Macrogenics, Inc.
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Priority to CA002587766A priority Critical patent/CA2587766A1/en
Priority to AU2005335714A priority patent/AU2005335714B2/en
Priority to EP05857976A priority patent/EP1810035A4/en
Publication of WO2007024249A2 publication Critical patent/WO2007024249A2/en
Priority to IL182974A priority patent/IL182974A/en
Publication of WO2007024249A3 publication Critical patent/WO2007024249A3/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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • C07K2317/52—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
    • C07K2317/72—Increased effector function due to an Fc-modification
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to molecules having a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region.
  • These modified molecules confer an effector function to a molecule, where the parent molecule does not detectably exhibit this effector function.
  • the molecules of the invention have an increased effector cell function mediated by a Fc ⁇ R, such as, but not limited to, ADCC.
  • the variant Fc region binds Fc ⁇ RIIIA and/or Fc ⁇ RIIA with a greater affinity, relative to a comparable molecule comprising the wild-type Fc region.
  • the molecules of the invention have particular utility in treatment, prevention or management of a disease or disorder, such as cancer, in a sub- population of patients, wherein the target antigen is expressed at low levels in the target cell population, in particular, in patients refractory fo treatment with an existing therapeutic antibody due to the low level of target antigen expression on the cancer or associated cells.
  • the Fc receptors members of the immunoglobulin gene superfamily of proteins, are surface glycoproteins that can bind the Fc portion of immunoglobulin molecules. Each member of the family recognizes immunoglobulins of one or more isotypes through a recognition domain on the ⁇ chain of the Fc receptor. Fc receptors are defined by their specificity for immunoglobulin subtypes. Fc receptors for IgG are referred to as Fc ⁇ R, for IgE as F ⁇ R, and for IgA as Fc ⁇ R.
  • Different accessory cells bear Fc receptors for antibodies of different isotype, and the isotype of the antibody determines which accessory cells will be engaged in a given response (reviewed by Ravetch J.V. et al.
  • Each member of this family is an integral membrane glycoprotein, possessing extracellular domains related to a C2-set of immunoglobulin-related domains, a single membrane spanning domain and an intracytoplasmic domain of variable length.
  • Fc ⁇ Rs There are three known Fc ⁇ Rs, designated Fc ⁇ RI(CD64), Fc ⁇ RII(CD32), and Fc ⁇ RIII(CD16).
  • the three receptors are encoded by distinct genes; however, the extensive homology between the three family members suggest they arose from a common progenitor perhaps by gene duplication.
  • Fc ⁇ RII proteins are 40KDa integral membrane glycoproteins which bind only the complexed IgG due to a low affinity for monomeric Ig (10 6 M "1 ). This receptor is the most widely expressed Fc ⁇ R, present on all hematopoietic cells, including monocytes, macrophages, B cells, NK cells, neutrophils, mast cells, and platelets. Fc ⁇ RII has only two immunoglobulin-like regions in its immunoglobulin binding chain and hence a much lower affinity for IgG than Fc ⁇ RI.
  • Fc ⁇ RII-A There are three human Fc ⁇ RII genes (Fc ⁇ RII- A, Fc ⁇ RII-B, Fc ⁇ RII-C), all of which bind IgG in aggregates or immune complexes. [0006] Distinct differences within the cytoplasmic domains of Fc ⁇ RII-A and
  • Fc ⁇ RII-B create two functionally heterogenous responses to receptor ligation.
  • the fundamental difference is that the A isoform initiates intracellular signaling leading to cell activation such as phagocytosis and respiratory burst, whereas the B isoform initiates inhibitory signals, e.g., inhibiting B-cell activation.
  • Both activating and inhibitory signals are transduced through the Fc ⁇ Rs following ligation. These diametrically opposing functions result from structural differences among the different receptor isoforms.
  • Two distinct domains within the cytoplasmic signaling domains of the receptor called imrnunoreceptor tyrosine based activation motifs (ITAMs) or immunoreceptor tyrosine based inhibitory motifs (ITIMS) account for the different responses.
  • ITAMs imrnunoreceptor tyrosine based activation motifs
  • ITIMS immunoreceptor tyrosine based inhibitory motifs
  • Fc ⁇ RIIA gene express the Fc ⁇ RIIA gene.
  • Fc ⁇ RIIA clustering via immune complexes or specific antibody cross-linking serves to aggregate ITAMs along with receptor-associated kinases which facilitate ITAM phosphorylation.
  • ITAM phosphorylation serves as a docking site for Syk kinase, activation of which results in activation of downstream substrates (e.g., PI 3 K).
  • downstream substrates e.g., PI 3 K
  • the Fc ⁇ RIIB gene is expressed on B lymphocytes; its extracellular domain is
  • the present invention is based, in part, on the inventors' discovery of methods for engineering the Fc region of an antibody to confer one or more effector function activities to a parent antibody, which parent antibody does not exhibit the particular effector function activity at a detectable level when tested against a target cell.
  • Such methods of engineering include introducing one or more amino acid modifications (substitutions, deletions or insertions) in one or more portions of the Fc region, which modifications introduce a detectable level of the effector function activity in the modified antibody.
  • the modifications alter the parent antibody's affinity for certain Fc ⁇ R receptors (e.g., activating Fc ⁇ Rs, inhibitory Fc ⁇ Rs) and one or more effector functions, such as antibody-dependent cell mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell mediated cytotoxicity
  • the modifications confer homo-oligomerization activity to the parent Fc region such that oligomerization of the modified antibody cross-links cell-surface antigens, resulting in apoptosis, negative-growth regulation or cell killing.
  • modification of an Fc region of a chimeric 2B6 antibody surprisingly conferred an effector function activity (particularly, ADCC) on chimeric 2B6 antibodies, which normally exhibit no detectable ADCC in routine in vitro ADCC assays.
  • modification of an Fc region of a chimeric 4D5 antibody surprisingly improved the effector function activity (particularly, ADCC) of chimeric 4D5 antibodies in cells with low levels of antigen expression.
  • the inventors have further found that modification of an Fc region of rituximab (anti-CD20 monoclonal antibody) conferred effector function activity on the rituximab antibody in cells from a patient population whose cells were otherwise refractory to rituximab-induced effector function activity.
  • modification of an Fc region of rituximab conferred effector function activity on the rituximab antibody in cells from a patient population whose cells were otherwise refractory to rituximab-induced effector function activity.
  • the invention encompasses molecules, preferably polypeptides, and more preferably immunoglobulins (e.g., antibodies) comprising a variant Fc region having one or more amino acid modifications (e.g., substitutions, but also including deletions or insertions) in one or more Fc regions, relative to a parent molecule, which modifications confer a particular effector function activity on the modified molecule, as compared to the parent molecule which has little or no detectable activity of that effector function (as measured using standard in vitro methods known in the art and exemplified herein).
  • immunoglobulins e.g., antibodies
  • effector function activities that may be conferred using the methods of the invention include, but are not limited to, ADCC, antibody-dependent phagocytosis, phagocytosis, opsonization, opsonophagocytosis, cell binding, resetting, complement dependent cell mediated cytotoxicity (CDC).
  • Another aspect of the invention relates to molecules, preferably polypeptides, and, more preferably, immunoglobulins (e.g., antibodies) comprising a variant Fc region having one or more amino acid modifications (e.g., substitutions, deletions, insertions) in one or more portions, which modifications increase the affinity and avidity of the variant Fc region for an Fc ⁇ R (including activating and inhibitory Fc ⁇ Rs).
  • said one or more amino acid modifications increase the affinity of the variant Fc region for Fc ⁇ RIIIA and/or Fc ⁇ RIIA.
  • the variant Fc region further specifically binds Fc ⁇ RIIB with a lower affinity than does the Fc region of the comparable parent antibody (i.e., an antibody having the same amino acid sequence as the antibody of the invention except for the one or more amino acid modifications in the Fc region).
  • such modifications increase the affinity of the variant Fc region for Fc ⁇ RIIIA and/or Fc ⁇ RIIA and also enhance the affinity of the variant Fc region for Fc ⁇ RIIB relative to the parent antibody.
  • said one or more amino acid modifications increase the affinity of the variant Fc region for Fc ⁇ RIIIA and/or Fc ⁇ RIIA but do not alter the affinity of the variant Fc regions for Fc ⁇ RIIB relative to the Fc region of the parent antibody.
  • said one or more amino acid modifications enhance the affinity of the variant Fc region for Fc ⁇ RIIIA and Fc ⁇ RIIA but reduce the affinity for Fc ⁇ RIIB relative to the parent antibody.
  • the target antigen of the modified antibody is an Fc ⁇ R
  • the modified antibody exhibits Fc ⁇ R-binding or related activity in cells which express the target Fc ⁇ R at a density of 10,000 molecules/cell or less, at a density of 5000 molecules/cell or less, at a density of 1000 molecules /cell or less, at a density of 500 molecules or less, or at a density of 200 molecules or less (but at least 10, at least 50, at least 100 or at least 150 molecules/cell) .
  • the target antigen is an Fc ⁇ R
  • the increased binding to the on the cell surface may be mediated by the CDR region of the antibody to an epitope on the target Fc ⁇ R.
  • the invention encompasses molecules, e.g., antibodies, with altered affinities and avidities for one or more target Fc ⁇ Rs.
  • the antibodies of the invention with enhanced affinity and avidity for one or more target Fc ⁇ Rs are particularly useful in cellular systems (for example for research or diagnostic purposes) where the Fc ⁇ Rs are expressed at low levels, for example, tumor specific B cells with low levels of Fc ⁇ RIIB (e.g., non-Hodgkins lymphoma, CLL, and Burkitt's lymphoma).
  • the molecules of the invention with enhanced affinity and avidity for a particular target Fc ⁇ R are valuable as research and diagnostic tools by enhancing the sensitivity of detection of Fc ⁇ Rs which are normally undetectable due to a low level of expression.
  • the antibodies of the invention with enhanced affinity and avidity for Fc ⁇ Rs are particularly useful for the treatment, prevention or management of a cancer, or another disease or disorder, in a subject, wherein the Fc ⁇ Rs are expressed at low levels in the target cell populations.
  • Fc ⁇ R expression in cells is defined in terms of the density of such molecules per cell as measured using common methods known to those skilled in the art.
  • the molecules of the invention comprising variant Fc regions preferably also have an enhanced avidity and affinity and/or effector function in cells which express a target antigen to which the modified antibody immunospecifically binds, e.g., a, cancer antigen, at low density, for example, at a density of 30,000 to 20,000 molecules/cell, at a density of 20,000 to 10,000 molecules/cell, at a density of 10,000 to 5,000 molecules/cell, at a density of 5,000 to 1,000 molecules/cell, at a density of 1,000 to 200 molecules/cell or at a density of 200 molecules/cell or less.
  • a target antigen to which the modified antibody immunospecifically binds e.g., a, cancer antigen
  • the molecules of the invention have particular utility in treatment, prevention or management of a disease or disorder, such as cancer, in a sub-population of patients, wherein the target antigen is expressed at low levels in the target cell population, in particular, in patients refractory to treatment with an existing therapeutic antibody due to the low level of target antigen expression on the cancer or other cells associated with the disease or disorder to be treated, prevented or managed.
  • the invention encompasses engineering human, chimeric or humanized therapeutic antibodies in the Fc region by modifying one or more Fc region amino acids, which modifications alter the detectable affinity and avidity of the antibodies for one or more target antigens, e.g., Fc ⁇ R receptors or cancer antigens, and/or the detectable effector function activity or cell killing activity of the modified antibody.
  • said one or more modifications to the amino acids of the Fc region enhance the affinity and avidity of the antibody for one or more target antigens, e.g., Fc ⁇ R receptors or cancer antigens.
  • These therapeutic antibodies by virtue of the modifications of the invention, have increased efficacy in patients refractory to treatment with the parent antibody, due, in certain instances, to reduced levels of the expression of the target antigen, as well as in patients who respond to the parent antibody.
  • therapeutic antibodies engineered in accordance with the invention have enhanced therapeutic efficacy, in part, due to the ability of the Fc portion of the antibody to bind a target cell which expresses the particular Fc ⁇ Rs at reduced levels, for example, by virtue of the ability of the antibody to remain on the target cell longer due to an improved off rate for Fc-Fc ⁇ R interaction.
  • said one or more modifications to the amino acids of the Fc region modifies the affinity and avidity of the antibody for one or more Fc ⁇ R receptors.
  • the invention encompasses antibodies comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to the parent Fc region, which variant Fc region only binds one Fc ⁇ R, wherein said Fc ⁇ R is Fc ⁇ RIIIA.
  • the invention encompasses antibodies comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to the parent Fc region, which variant Fc region only binds one Fc ⁇ R, wherein said Fc ⁇ R is Fc ⁇ RIIA.
  • the invention encompasses antibodies comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to the parent Fc region, which variant Fc region only binds one Fc ⁇ R, wherein said Fc ⁇ R is Fc ⁇ RIIB.
  • the affinities and binding properties of the antibodies of the invention for the target antigen or an Fc ⁇ R are initially determined using in vitro assays (biochemical or immunological based assays) known in the art for determining antigen-antibody or Fc-Fc ⁇ R interactions (i.e., specific binding of an Fc region to an Fc ⁇ R), respectively, including but not limited to, ELISA assay, surface plasmon resonance assay or immunoprecipitation assay.
  • the binding properties of the molecules of the invention are also characterized by in vitro functional assays for determining one or more Fc ⁇ R mediated effector cell functions.
  • the antibodies of the invention have similar binding properties in in vivo models (such as those described and disclosed herein) as those in in vitro based assays.
  • the present invention does not exclude molecules of the invention that do not exhibit the desired phenotype in in vitro based assays but do exhibit the desired phenotype in vivo.
  • the invention also encompasses molecules, preferably polypeptides, and more preferably, immunoglobulins (e.g., antibodies) comprising a variant Fc region having one or more amino acid modifications (e.g., substitutions, deletions, insertions) in one or more portions, which modifications confer detectable effector function activity to the molecule not detectable in the parent molecule.
  • the parent molecule is an antibody.
  • the parent antibody is rituximab or humanized 2B6 (see U.S. Patent Application Publication 2004/0185045 and U.S.
  • the molecules of the invention with Fc modifications may exhibit enhanced therapeutic efficacy due to the introduction of homo-oligomerization activity in the Fc region, resulting in apoptosis, negative-growth regulation or cell killing associated with surface antigen cross-linking.
  • the invention encompasses methods and compositions for treatment, prevention or management of a cancer in a subject, comprising administering to the subject a therapeutically effective amount of one or more molecules comprising a variant Fc region engineered in accordance with the invention, which molecule further binds a cancer antigen.
  • the subject is human.
  • the molecules of the invention are modified rituximab, and are preferably used in the treatment of lymphoma, such as Non-Hodgkins lymphoma, or modified humanized 2B6 antibodiesengineered according to the methods of the invention, which modified antibodies possess the same indications as the parent antibodies.
  • Molecules of the invention comprising the variant Fc regions are particularly useful for the prevention, inhibition, reduction of growth or regression of primary tumors, or metastasis of cancer cells.
  • molecules of the invention enhance the efficacy of cancer therapeutics by i) enhancing antibody mediated effector function or ii) enhancing the apoptosis signaling, negative-growth regulation or cell killing associated with surface antigen cross-linking by introducing homo-oligomerization activity in the modified molecules, resulting in an enhanced rate of tumor clearance or an enhanced rated of tumor reduction or a combination thereof.
  • immunotherapeutics may be modifying in accordance with the invention to increase the potency of an antibody effector function activity, e.g., ADCC, CDC, phagocytosis, opsonization, etc.
  • antibody dependent cellular toxicity and/or phagocytosis e.g., of tumor cells
  • Molecules of the invention may render immunotherapy cancer treatment efficacious in a patient population by enhancing (or rendering detectable) at least one antibody-mediated effector function activity.
  • the efficacy of immunotherapy treatment is enhanced by rendering the complement dependent cascade activity detectable.
  • the efficacy of immunotherapy treatment is enhanced by rendering the phagocytosis and/or opsonization of the targeted cells, e.g. , tumor cells, detectable.
  • the efficacy of treatment is enhanced by enhancing antibody-dependent cell-mediated cytotoxicity ("ADCC") in destruction of the targeted cells, e.g., tumor cells, detectable. Determining whether such activity is detectable is done using routine assays known in the art and described herein.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to the parent Fc region such that the molecule has an enhanced effector activity, provided said one or more amino acid modifications includes substitutions at one or more positions.
  • the amino acid positions recited herein are numbered according to the EU index as set forth in Kabat et ah, Sequence of Proteins of Immunological Interest. 5 th Ed. Public Health Service, NHl, MD (1991), expressly incorporated herein by reference.
  • the variant Fc region has a leucine at position 247, a lysine at position 421, or a glutamic acid at position 270.
  • the variant Fc region has a leucine at position 247, a lysine at position 421 and a glutamic acid at position 270 (MgFc31/60); a threonine at position 392, a leucine at position 396, a glutamic acid at position 270, and a leucine at position 243 (MgFc38/60/F243L); a histidine at position 419, a leucine at position 396, and a glutamic acid at position 270 (MGFc51/60); an alanine at position 240, a leucine at position 396, and a glutamic acid at position 270 (MGFc52/60); a histidine at position 419, a leucine at position 396, a glutamic acid at position 270, and a leucine at position 243 (MGFc51/60/F243L); a lysine at position 255 and a leucine at position
  • the invention also encompasses methods for treating or preventing an infectious disease in a subject comprising administering a therapeutically or prophylactically effective amount of one or more molecules of the invention that bind an infectious agent or cellular receptor therefore.
  • infectious diseases that can be treated or prevented by the molecules of the invention are caused by infectious agents including but not limited to viruses, bacteria, fungi, protozae, and viruses.
  • the methods and/or molecules of the invention confer a therapeutic effect not detectable in the parent antibody or enhance the therapeutic effect of the parent antibody by i) enhancing or rendering detectable the antibody mediated effector function toward an infectious agent or ii) enhancing or rendering detectable the apoptosis signaling, negative-growth regulation or cell killing associated with surface antigen cross- linking by conferring homo-oligomerization activity in the modified molecules.
  • molecules of the invention comprising variant Fc regions have detectable antibody effector function towards an infectious agent, which was not detectable hi the parent molecule comprising a wild-type Fc region.
  • molecules of the invention enhance the efficacy of treatment of an infectious disease by enhancing or rendering detectable phagocytosis and/or opsonization of the infectious agent causing the infectious disease
  • molecules of the invention enhance the efficacy of treatment of an infectious disease by enhancing or rendering detectable ADCC of infected cells causing the infectious disease.
  • the invention encompasses characterization of the molecules of the invention
  • the invention encompasses characterizing the molecules of the invention for Fc ⁇ R-mediated effector cell function.
  • effector cell functions include but are not limited to, antibody-dependent cell mediated cytotoxicity (ADCC), phagocytosis, opsonization, opsonophagocytosis, CIq binding, and complement dependent cell mediated cytotoxicity (CDC).
  • ADCC antibody-dependent cell mediated cytotoxicity
  • phagocytosis phagocytosis
  • opsonization opsonization
  • opsonophagocytosis CIq binding
  • CDC complement dependent cell mediated cytotoxicity
  • Cell-based or cell free assays for determining effector cell function activity are routine and known to those skilled in the art and described herein.
  • the molecules of the invention can be assayed for Fc ⁇ R- mediated phagocytosis in human monocytes.
  • the Fc ⁇ R-mediated phagocytosis of the molecules of the invention may be assayed in other phagocytes, e.g., neutrophils (polymorphonuclear leuckocytes; PMN); human peripheral blood monocytes, monocyte- derived macrophages, which can be obtained using standard procedures known to those skilled in the art.
  • the molecules of the invention may be assayed using an antibody-dependent opsonophagocytosis assay (ADCP).
  • ADCP antibody-dependent opsonophagocytosis assay
  • the molecules of the invention can be assayed for Fc ⁇ R-mediated ADCC activity in effector cells, e.g., natural killer cells, using any of the standard methods known to those skilled in the art.
  • the molecules of the invention are characterized for antibody dependent cellular cytotoxicity (ADCC).
  • ADCC antibody dependent cellular cytotoxicity
  • the effector cells used in the ADCC assays of the invention are peripheral blood mononuclear cells (PBMC) that are purified from normal human blood, using standard methods known to one skilled in the art, e.g., using Ficoll-Paque density gradient centrifugation.
  • PBMC peripheral blood mononuclear cells
  • Preferred effector cells for use in the methods of the invention express different Fc ⁇ R activating receptors.
  • the invention encompasses, effector cells expressing Fc ⁇ RI, Fc ⁇ RIIA and Fc ⁇ RIIB, and monocyte derived primary macrophages derived from whole human blood expressing both FcyRIIIA and Fc ⁇ RIIB. Both the ratio of effector celhtarget cell and concentration of antibody to be used in the functional assays in accordance with the invention will be appreciated to be dependent of the particular assay and system to be tested.
  • the invention encompasses use of the effector cells in the functional assays of effector function activity at an effector cell:target cell ratio of 1:1, 10:1, 30:1, 60:1, 75:1 or 100:1.
  • the invention encompasses the use of antibody in the functional assays of effector function activity at an concentration of 0.2 ⁇ g/ml to 3 ⁇ g/ml, 0.5 ⁇ g/ml to 2 ⁇ g/ml or 0.5 ⁇ g/ml to 1 ⁇ g/ml.
  • the molecules of the invention may be assayed for
  • CIq binding which mediates complement dependent cytotoxicity (CDC).
  • a CIq binding ELISA may be performed.
  • a complement dependent cytotoxicity (CDC) assay may be performed using standard methods known in the art.
  • the Fc variants of the present invention may be combined with other Fc modifications known in the art.
  • the invention encompasses combining an Fc variant of the invention with other Fc modifications to provide additive, synergistic, or novel properties to the modified antibody.
  • the Fc variants of the invention enhance the phenotype of the modification with which they are combined. For example, if an Fc variant of the invention is combined with a mutant known to bind Fc ⁇ RIIIA with a higher affinity than a comparable wild type Fc region; the combination with a mutant of the invention results in a greater fold enhancement in Fc ⁇ RIIIA affinity.
  • the Fc variants of the present invention may be combined with other known Fc variants such as those disclosed in Duncan et al, 1988, Nature 332:563- 564; Lund et al., 1991, J. Immunol 147:2657-2662; Lund et al, 1992, MoI Immunol 29:53-59; Alegre et al, 1994, Transplantation 57:1537-1543; Hutchins et al, 1995, Proc Natl. Acad Sci U S A 92:11980-11984; Jefferis et al, 1995, Immunol Lett.
  • the present invention also encompasses antibodies that are homodimers or heterodimers of Fc regions.
  • Homodimeric or heterodimeric antibodies of the invention comprise variant Fc regions, wherein the two Fc chains have the same or different amino acid sequences, respectively.
  • each Fc chain of the heterodimeric antibody comprises one or more different amino acid modifications relative to the other chain.
  • one Fc chain of the heterodimeric antibody comprises the wild type Fc chain and the other Fc chain comprises one or more amino acid modifications relative to the wild type chain.
  • the present invention also includes polynucleotides that encode a molecule of the invention, including polypeptides and antibodies, identified by the methods of the invention.
  • the polynucleotides encoding the molecules of the invention may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
  • the invention relates to an isolated nucleic acid encoding a molecule of the invention.
  • the invention also provides a vector comprising said nucleic acid.
  • the invention further provides host cells containing the vectors or polynucleotides of the invention.
  • the invention further provides methods for the production of the molecules of the invention.
  • the molecules of the invention including polypeptides and antibodies, can be produced by any method known to those skilled in the art, in particular, by recombinant techniques.
  • antibodies of the invention are created by engineering mutations identified as conferring therapeutically effective, detectable, effector function activity into the Fc regions of antibodies which do not natively exhibit such activity.
  • the invention relates to a method for recombinantly producing a molecule of the invention, said method comprising: (i) culturing in a medium a host cell comprising a nucleic acid encoding said molecule, under conditions suitable for the expression of said molecule; and (ii) recovery of said molecule from said medium.
  • the invention also encompasses methods for improving the therapeutic efficacy of molecules, preferably polypeptides, and more preferably immunoglobulins (e.g., antibodies) comprising a variant Fc region having, which Fc regions have been engineered according to the methods of the invention, which engineering confers detectable effector function activity on the modified molecule, as compared to the parent molecule which exhibited little or no detectable activity of that effector function (as measured using standard in vitro methods known in the art and exemplified herein).
  • immunoglobulins e.g., antibodies
  • the invention provides pharmaceutical compositions comprising a molecule of the invention, e.g., a polypeptide comprising a variant Fc region, an immunoglobulin comprising a variant Fc region, a therapeutic antibody engineered in accordance with the invention, and a pharmaceutically acceptable carrier.
  • the invention additionally provides pharmaceutical compositions further comprising one or more additional therapeutic agents, including but not limited to anti-cancer agents, anti-inflammatory agents, immunomodulatory agents.
  • Fc region is used to define a C-terminal region of an IgG heavy chain.
  • the numbering of the residues in an IgG heavy chain is that of the EU index as in Kabat et ah, Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, NHl, MD (1991), expressly incorporated herein by references.
  • the "EU index as in Kabat” refers to the numbering of the human IgGl EU antibody.
  • An example of the amino acid sequence containing the human IgGl Fc region is SEQ ID NO: 11 and is shown in FIG. 1. SEQ ID NO: 11 and FIG.
  • the Fc domain extends from amino acid 231 to amino acid 447 (which corresponds to amino acid 16 to amino acid 232 as numbered in SEQ ID NO: 11 and FIG.l).
  • the Fc region of an IgG comprises two constant domains, CH2 and CH3.
  • CH2 domain of a human IgG Fc region usually extends from amino acids 231 to amino acid 341 according to the numbering system of Kabat (corresponding to amino acids 16 to 126 as numbered in SEQ ID NO: 11 and FIG. 1).
  • the CH3 domain of a human IgG Fc region usually extends from amino acids 342 to 447 according to the numbering system of Kabat (corresponding to amino acids 127 to 232 as numbered in SEQ ID NO: 11 and FIG. 1).
  • the CH2 domain of a human IgG Fc region (also referred to as "C ⁇ 2" domain) is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG.
  • the "hinge region” is generally defined as stretching from Glu216 to Pro230 of human IgGl (corresponding to amino acids 1-15 as numbered in SEQ ID NO: 11 and FIG. 1). Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain S-S binds in the same positions.
  • antibody refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelized antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked bispecific Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.
  • scFv single-chain Fvs
  • Fab fragments single chain antibodies
  • F(ab') fragments fragments
  • disulfide-linked bispecific Fvs sdFv
  • intrabodies and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site.
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 and IgA 2 ) or subclass.
  • the term "derivative" in the context of polypeptides or proteins refers to a polypeptide or protein that comprises an amino acid sequence which has been altered by the introduction of ammo acid residue substitutions, deletions or additions.
  • derivative as used herein also refers to a polypeptide or protein which has been modified, Le, by the covalent attachment of any type of molecule to the polypeptide or protein.
  • an antibody may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.
  • a derivative polypeptide or protein may be produced by chemical modifications using techniques known to those of skill in the art, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.
  • a derivative polypeptide or protein derivative possesses a similar or identical function as the polypeptide or protein from which it was derived.
  • the term "derivative" in the context of a non-proteinaceous derivative refers to a second organic or inorganic molecule that is formed based upon the structure of a first organic or inorganic molecule.
  • a derivative of an organic molecule includes, but is not limited to, a molecule modified, e.g., by the addition or deletion of a hydroxyl, methyl, ethyl, carboxyl or amine group.
  • An organic molecule may also be esterified, alkylated and/or phosphorylated.
  • the terms “disorder” and “disease” are used interchangeably to refer to a condition in a subject.
  • autoimmune disease is used interchangeably with the term “autoimmune disorder” to refer to a condition in a subject characterized by cellular, tissue and/or organ injury caused by an immunologic reaction of the subject to its own cells, tissues and/or organs.
  • inflammatory disease is used interchangeably with the term “inflammatory disorder” to refer to a condition in a subject characterized by inflammation, preferably chronic inflammation.
  • Autoimmune disorders may or may not be associated with inflammation.
  • inflammation may or may not be caused by an autoimmune disorder.
  • certain disorders may be characterized as both autoimmune and inflammatory disorders.
  • cancer refers to a neoplasm or tumor resulting from abnormal uncontrolled growth of cells.
  • cancer explicitly includes, leukemias and lymphomas.
  • cancer refers to a benign tumor, which has remained localized.
  • cancer refers to a malignant tumor, which has invaded and destroyed neighboring body structures and spread to distant sites.
  • the cancer is associated with a specific cancer antigen that is expressed on cancer cells.
  • immunomodulatory agent and variations thereof refer to an agent that modulates a host's immune system.
  • an immunomodulatory agent is an immunosuppressant agent.
  • an immunomodulatory agent is an immunostimulatory agent.
  • Immunomodatory agents include, but are not limited to, small molecules, peptides, polypeptides, fusion proteins, antibodies, inorganic molecules, mimetic agents, and organic molecules.
  • epitope refers to a fragment of a polypeptide or protein or a non-protein molecule having antigenic or immunogenic activity in an animal, preferably in a mammal, and most preferably in a human.
  • An epitope having immunogenic activity is a fragment of a polypeptide or protein that elicits an antibody response in an animal.
  • An epitope having antigenic activity is a fragment of a polypeptide or protein to which an antibody immunospecifically binds as determined by any method well-known to one of skill in the art, for example by immunoassays. Antigenic epitopes need not necessarily be immunogenic.
  • fragment refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least contiguous 80 amino acid residues, at least contiguous 90 amino acid residues, at least contiguous 100 amino acid residues, at least contiguous 125 amino acid residues, at least 150 contiguous amino acid residues, at least contiguous 175 amino acid residues, at least contiguous 200 amino acid residues, or at least contiguous 250 amino acid residues of the amino acid sequence of another polypeptide.
  • a fragment of a polypeptide retains at least one function of
  • nucleic acids and “nucleotide sequences” include
  • DNA molecules e.g., cDNA or genomic DNA
  • RNA molecules e.g., mRNA
  • combinations of DNA and RNA molecules or hybrid DNA/RNA molecules e.g., DNA molecules, and analogs of DNA or RNA molecules.
  • analogs can be generated using, for example, nucleotide analogs, which include, but are not limited to, inosine or tritylated bases.
  • Such analogs can also comprise DNA or RNA molecules comprising modified backbones that lend beneficial attributes to the molecules such as, for example, nuclease resistance or an increased ability to cross cellular membranes.
  • a "therapeutically effective amount” refers to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder.
  • a therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., delay or minimize the spread of cancer.
  • a therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.
  • a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.
  • prophylactic agent and “prophylactic agents” refer to any agent(s) which can be used in the prevention of a disorder, or prevention of recurrence or spread of a disorder.
  • a prophylactically effective amount may refer to the amount of prophylactic agent sufficient to prevent the recurrence or spread of hyperproliferative disease, particularly cancer, or the occurrence of such in a patient, including but not limited to those predisposed to hyperproliferative disease, for example those genetically predisposed to cancer or previously exposed to carcinogens.
  • a prophylactically effective amount may also refer to the amount of the prophylactic agent that provides a prophylactic benefit in the prevention of disease.
  • a prophylactically effective amount with respect to a prophylactic agent of the invention means that amount of prophylactic agent alone, or in combination with other agents, that provides a prophylactic benefit in the prevention of disease.
  • the terms “prevent”, “preventing” and “prevention” refer to the prevention of the recurrence or onset of one or more symptoms of a disorder in a subject as result of the administration of a prophylactic or therapeutic agent.
  • the term “in combination” refers to the use of more than one prophylactic and/or therapeutic agents. The use of the term “in combination” does not restrict the order in which prophylactic and/or therapeutic agents are administered to a subject with a disorder.
  • a first prophylactic or therapeutic agent can be administered prior to ⁇ e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent to a subject with a disorder.
  • Effective function as used herein is meant a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include but are not limited to antibody dependent cell mediated cytotoxicity (ADCC), antibody dependent cell mediated phagocytosis (ADCP), and complement dependent cytotoxicity (CDC). Effector functions include both those that operate after the binding of an antigen and those that operate independent of antigen binding.
  • ADCC antibody dependent cell mediated cytotoxicity
  • ADCP antibody dependent cell mediated phagocytosis
  • CDC complement dependent cytotoxicity
  • Effector functions include both those that operate after the binding of an antigen and those that operate independent of antigen binding.
  • Effective cell as used herein is meant a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions.
  • Effector cells include but are not limited to monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans 1 cells, natural killer (NK) cells, and may be from any organism including but not limited to humans, mice, rats, rabbits, and monkeys.
  • Fc ligand as used herein is meant a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc-ligand complex.
  • Fc ligands include but are not limited to Fc ⁇ Rs, Fc ⁇ Rs, Fc ⁇ Rs, FcRn, CIq, C3, staphylococcal protein A, streptococcal protein G, and viral Fc ⁇ R.
  • Fc ligands may include undiscovered molecules that bind Fc.
  • FIG. 1 AMINO ACID SEQUENCE OF HUMAN IgGl HINGE-Fc REGION
  • Figure 1 shows the amino acid sequence of the human IgGl hinge-Fc region
  • amino acid residues shown in the figure, 1-232 correspond to amino acid residues 231 to 447 of the IgG heavy chain according to the numbering system of Kabat.
  • FIG. 2 SDS-PAGE ANALYSIS OF RECOMBINANT SOLUBLE Fc ⁇ R
  • FIG. 3 ELISA ASSAY OF RECOMBINANT SOLUBLE Fc ⁇ R
  • FIGs. 4 A and B CHARACTERIZATION OF Fc ⁇ RIIIA TETRAMERIC
  • Soluble tetrameric Fc ⁇ RIIIA complex binds soluble monomeric human IgG specifically. Binding of soluble tetrameric Fc ⁇ RIIIA to human IgG is blocked by 3G8 ( ⁇ ), a mouse anti-Fc ⁇ lllA monoclonal antibody; the 4-4-20 monoclonal antibody harboring the D265A mutation was not able to block the binding of soluble tetrameric Fc ⁇ RIIIA to aggregated human IgG ( ⁇ ).
  • Binding of soluble tetrameric Fc ⁇ RIIIA complex to soluble monomeric human IgG ( ⁇ ) is compared to the binding of monomeric soluble Fc ⁇ RIIIA to soluble monomeric human IgG ( ⁇ ).
  • FIGs. 5 A and B CHARACTERIZATION OF Fc ⁇ RIIIA TETRAMERIC
  • Fc ⁇ RIIIA Complex two Fc ⁇ RIIIA(filled shape) are joined by a monoclonal antibody DJ 130c (1 st Ab); the anti-mouse F(ab) 2 is conjugated to PE (circle).
  • FACS analysis of Fc ⁇ RIIIA bound to Fc coated beads (a) beads alone; (b) complex without Fc ⁇ RIIIA; (c) complex with Fc ⁇ RIIIA; (d) complex with Fc ⁇ RIIIA and LNK16.
  • FIG. 6 SCHEMATIC PRESENTATION OF Fc CONTAINING
  • the open box represents the hinge-CH2-CH3 domains; parallel vertical lines represent the CHl domain.
  • the N-terminal amino acids are shown.
  • the underlined residues correspond to the hinge region;
  • the * represents the Xpress epitope tag;
  • hatched boxes represent the Gly4-Ser linker, and the stippled boxes represent the Aga2p gene.
  • FIGS.7 A-H FACS ANALYSIS OF THE Fc FUSION PROTEINS ON THE YEAST CELL WALL
  • Fc antibody (FIGS. 6A-D) or with HP6017 (Sigma), a mouse anti-human IgGl Fc (CH3) specific monoclonal antibody (FIGS. 6E-H).
  • a and E represent vector alone; Panels B and F represent the CH1-CH3 construct; Panels C and G represent the GIF227; and Panels D and H represent the GIF 206 construct.
  • FIGs. 8A-C BINDING OF SOLUBLE TETRAMERIC Fc ⁇ RIIIA TO THE SURFACE DISPLAYED Fc FUSION PROTEINS
  • Cells containing pYDl-CHl (A); ⁇ YD-CHl-D265A (B); and pYD vector (C) were grown under conditions to express Aga2p fusion proteins on the cell surface.
  • Cells were incubated with Fc ⁇ RIIIA at 0.15 mM , 7.5 mM , and 7.5 mM, respectively, and analyzed by FACS.
  • FIG. 9 CHARACTERIZATION OF THE BINDING OF SOLUBLE
  • Fc ⁇ RIIIA tetrameric complex Binding of Fc ⁇ RIIIA tetrameric complex to Fc fusion proteins on the yeast cell surface was analyzed.
  • PE-conjugated Fc ⁇ RIIIA tetrameric complexes were pre-incubated with different concentrations of 3G8 ( ⁇ ), LNK (A) or an irrelevant isotype control ( ⁇ ), and subsequently incubated with the yeast cells. Cells were analyzed by FACS for PE fluorescence. The percent cells that bound the Fc ⁇ RIIIA tetrameric complex were plotted on the y-axis.
  • FIG. 10 EXAMPLE OF SORT GATE FOR SELECTING Fc MUTANTS
  • FIGs. 11 A-N FACS ANALYSIS OF SOME OF THE Fc MUTANTS IDENTIFIED HAVING AN INCREASED AFFINITY FOR Fc ⁇ RIIIA TETRAMERIC COMPLEXES
  • FIGS. 1OA and B represent cells harboring wild-type Fc
  • FIGS. 1OC and D represent mutant # 5
  • FIGS. 1OE and F represent mutant # 20
  • FIGS. 1OG and H represent mutant # 21;
  • FIGS. 1OA and B represent cells harboring wild-type Fc
  • FIGS. 1OC and D represent mutant # 5
  • FIGS. 1OE and F represent mutant # 20
  • FIGS. 1OG and H represent mutant # 21
  • FIGS. 10 1 and J represent mutant # 24; FIGS. 1OK and L represent mutant # 25; FIGS. 1OM and N represent mutant # 27.
  • Cells were stained with Fc ⁇ RIIIA tetrameric complex (FIGS. 10 A, C, E, G, I, K, and M) or Fc ⁇ RIIB tetrameric complex (FIGS. 1OB, D, F, H, J, L, and N).
  • FIGs. 12 A-B CHARACTERIZATION OF Fc MUTANTS IN THE 4-4-20 MONOCLONAL ANTIBODY BY ELISA
  • Fc domains from the pYD-CHl plasmids were cloned into the heavy chain of the chimeric 4-4-20 monoclonal antibody.
  • the 4-4-20 monoclonal antibody was expressed in 293 cells and supernatants were collected.
  • ELISA plates were coated with fluoresceine conjugated BSA to capture the chimeric 4-4-20 mutant antibodies.
  • Fc ⁇ RIIIA (A) and Fc ⁇ RIIB (B) receptors were then coated onto the ELISA plates to which the 4-4-20 monoclonal antibodies had been absorbed in order to determine the relative affinities of the variant receptors to the Fc domains. Mutants # 15 and # 29 were non-binding isolates included as controls.
  • FIG. 13 ADCC ACTIVITY OF MUTANTS IN THE 4-4-20
  • mutants containing mutant Fc regions were assessed for their ADCC activity, and compared to the ADCC activity of a wild type 4-4-20 antibody.
  • the mutants analyzed are as follows: MGFc-10 (K288N, A33OS, P396L), MGFc-26 (D265A), MGFc-27 (G316D, A378V, D399E), MGFc28 (N315I, A379M, D399E), MGFc29 (F243I, V379L, G420V), MGFc30 (F275V), MGFc-31 (P247L, N421K), MGFc-32 (D280E, S354F, A431D, L441I), MGFc-33 (K317N, F423 deleted), MGFc-34 (F241L, E258G), MGFc-35 (R255Q, K326E), MGFc-36 (K218R, G281D, G385R)
  • FIGs. 14 A and B ADCC ACTIVITY OF MUTANTS IN THE HER2/NEU HUMANIZED MONOCLONAL ANTIBODY
  • mutants containing mutant Fc regions were assessed for their ADCC activity and compared to the ADCC activity of a wild type Her2/neu antibody.
  • the mutants analyzed are as follows: MGFc-5 (V379M), MGFc-9 (F243I, V379L), MGFc-IO (K288N, A330S, P396L), MGFc-13 (K334E, T359N, T366S), MGFc-27 (G316D, A378V, D399E).
  • FIG. 15 CAPTURE OF CH 4-4-20 ANTIBODY ON
  • FIG. 16 SENSOGRAM OF REAL TIME BINDING OF Fc ⁇ RIIIA TO CH 4-4-
  • Binding of Fc ⁇ RIIIA to ch-4-4-20 antibodies carrying variant Fc regions was analyzed at 200 nM concentration. Responses were normalized at the level of ch-4-4-20 antibody obtained for wild-type.
  • Mutants used were as follows: Mut 6 (S219V), Mut 10 (P396L, A330S,
  • K288N Mut 18 (K326E); Mut 14 (K334E, K288N); Mut 11 (R255L, F243L); Mut 16 (F372Y); Mut 19 (K334N, K246I).
  • FIGs. 17 A-H ANALYSIS OF KINETIC PARAMETERS OF Fc ⁇ RIIIA
  • FIG. 18 SENSOGRAM OF REAL TIME BINDING OF Fc ⁇ RIIB-Fc FUSION
  • FIGs. 19 A-C ANALYSIS OF KINETIC PARAMETERS FcyRIIB-Fc FUSION
  • Mutants used were as follows: Mut 6 (S219V), Mut 10 (P396L, A33OS,
  • K288N Mut 18 (K326E); Mut 14 (K334E, K288N); Mut 11 (R255L, F243L); Mut 16 (F372Y); Mut 19 (K334N, K246I).
  • FIG. 20 RATIOS OF Koff (WT)/K off (MUT) FOR Fc ⁇ RIIIA-Fc PLOTTED
  • FIG. 21 COMPETITION WITH UNLABELED Fc ⁇ RIIIA
  • a kinetic screen was implemented to identify Fc region mutants with improved K off rates for binding Fc ⁇ RIIIA.
  • a library of Fc region variants containing P396L mutation was incubated with 0.1 ⁇ M biotinylated Fc ⁇ RIIIA-Linker-Avitag for one hour and then washed. Subsequently 0.8 uM unlabeled Fc ⁇ RIIIA was incubatd with the labeled yeast for different time points. Yeast was spun down and unlabeled Fc ⁇ RIIIA was removed, Receptor bound yeast was stained with SA (streptavidin):PE (phycoerythrin) for FACS analysis.
  • SA streptavidin
  • PE phytoerythrin
  • FIGs. 22 A-C FACS ANALYSIS BASED ON THE KINETIC SCREEN
  • Fc ⁇ RIIIA selection using magnetic beads The Fc ⁇ RIIB depletion by magnetic beads was repeated 5 times.
  • the resulting yeast population was analyzed and found to show greater than 50% cell staining with goat anti-human Fc and a very small percentage of cells stained with Fc ⁇ RIIIA. Subsequently cells were selected twice by FACS using 0.1 ⁇ M biotinylated Fc ⁇ RIIIA linker-avitag. Yeast cells were analyzed for both Fc ⁇ RIIIA and Fc ⁇ RIIB binding after each sort and compared to wild type binding.
  • Fc Mutants were selected from the Fc ⁇ RIIB depleted yeast population using biotinylated Fc ⁇ RIIIA 158F linker avitag monomer as a ligand.
  • the sort gate was set to select the top 0.25% Fc ⁇ RIIIA 158F binders.
  • the resulting enriched population was analyzed by FACS for binding to the different Fc ⁇ RIIIA (158F and 158V), Fc ⁇ RIIIB and Fc ⁇ RIIA (131R).
  • FIG. 24 RELATIVE RATES OF SKBR3 TARGET CELL LYSIS MEDIATED
  • 4D5 antibody with Fc mutants were divided by the rate of lysis mediated by wild type 4D5 antibody. Data from at least 2 independent assays were averaged and plotted on the histogram. For each Fc mutant data from two different antibody concentrations are shown. The antibody concentrations were chosen to flank the point along the curve at which lysis was -50%.
  • FIG. 25 RELATIVE RATES OF DAUDI CELL LYSIS MEDIATED BY
  • Relative rates of lysis was calculated for each Fc mutant tested. Lysis rates for 2H7 antibody with Fc mutants were divided by the rate of lysis mediated by wild type 2H7 antibody. Data from at least 1- 2 independent assays were averaged and plotted on the histogram. For each Fc mutant, data from two different antibody concentrations are shown The antibody concentrations were chosen based on the point along the curve at which lysis was -50%.
  • FIG. 26 SCHEME FOR LIBRARY PRODUCTION.
  • DNA strands are represented.
  • Forward arrows represent primers containing mutant codons.
  • Reverse arrow represent reverse gene specific oligo.
  • FIG.27 STRATEGY FOR PRODUCTION OF LIBRARIES BY BUILD A
  • the rectangular boxes represent the hinge, CH2, and CH3 domains, respectively.
  • the short black lines represent the double stranded oligos with 5' overhangs.
  • FIG. 28 NOVEL Fc MUTANTS IMPROVE PBMC MEDIATED ADCC IN
  • FIG. 29 NOVEL Fc MUTANTS IMPROVE PBMC MEDIATED ADCC IN
  • FIG. 30 Fc RECEPTOR PROFILES VIA FACS UPON CYTOKINE
  • Elutriated monocytes were cultured using specific cytokines in serum free media. Fc receptor profiles were assayed using FACS.
  • FIG. 31 IMPROVED TUMOR CELL KILLING USING FC MUTANTS IN
  • FIG. 32 COMPLEMENT DEPENDENT CYTOTOXICITY ASSAY FLOW
  • FIG.33 COMPLEMENT DEPENDENT CYTOTOXICITY ACTIVITY
  • Fc mutants that show enhanced binding to Fc ⁇ RIIIA also showed improved complement activity.
  • Anti-CD20 ChMAb over 3 orders of magnitude was titrated. Percent lysis was calculated as in as in FIG. 28.
  • FIG. 34 DECISION TREE FOR SELECTION OF Fc MUTANTS
  • FIG. 35 CIq BINDING TO 2B6 ANTIBODY
  • FIGs. 36 A-D CIq BINDING TO 2B6 MUTANT ANTIBODY.
  • FIGs. 37 A-D Fc VARIANTS WITH DECREASED BINDING TO FejRIIB
  • K D was analyzed at different concentrations of FcR; 40OnM CD16A 158V; 80OnM CD16A 158F; 20OnM CD32B; 20OnM CD32A 131H. Analysis was performed using separate K D using Biacore 3000 software.
  • FIGs. 38 A-D KINETIC CHARACTERISTICS OF 4D5 MUTANTS SELECTED FROM Fc)»iro DEPLETIONS/Fq «IIAH131 SELECTION
  • Binding of FcR to ch4D5 antibodies carrying different Fc mutations selected by CD32B depletion and CD32A Hl 31 screening strategy K D was analyzed at different concentrations of FcR; 40OnM CD16A 158V; 80OnM CD16A 158F; 20OnM CD32B; 20OnM CD32A 13 IH. Analysis was performed using separate K D using Biacore 3000 software.
  • FIG. 39 PLOT OF MDM ADCC DATA AGAINST THE K OFF DETERMINED
  • mutants are as follows: MgFc 25 (E333A, K334A, S298A); MgFc68
  • FIG. 40 A-D FC ⁇ R BINDING TO 4D5 MUTANT ANTIBODY, TRIPLE MUTATION
  • FIG. 42 A-E BINDING OF 4D5 VARIANT 31/60 TO HT29 CELLS
  • HER2/neu antibody ch4D5 variant 31/60 (P247L; N421K; D270E), to HT29 cells (low expression of HER2/neu).
  • Incubation with primary antibody was at 10 ⁇ g/ml (A), 1 ⁇ g/ml (B), 0.1 ⁇ g/ml (C), 0.001 ⁇ g/ml (D), or 0.001 ⁇ g/ml (E).
  • Wild-type ch4D5 and Synagis were used as controls.
  • PE-conjugated polyclonal F(ab) 2 goat anti-humanFC ⁇ R was used as the secondary antibody.
  • FIG. 43 A-B ADCC ACTIVITY OF MUTANTS IN THE ANTI-HER2/neu ANTIBODY, ch4D5
  • SKBR3 high expression of HER2/neu
  • HT29 low expression of HER2/neu cells lines were used as targets (panels A and B, respectively).
  • Effector to target ratio (E:T ratio) was 50:1 with 18 h incubation.
  • MGFc59/60 K370E; P396L; D270E
  • MGFc55/60 R255L; P396L; D270E
  • MGFc51/60 Q419H; P396L; D270E
  • MGFc55/60/F243L R255L; P396L; D270E; F243L
  • MGFc74/P396L F243L; R292P; V305I; P396L.
  • FIG. 44 A-B ADCC ACTIVITY OF MUTANTS IN THE ANTI-HER2/neu ANTIBODY, ch4D5
  • Ch4D5 antibodies containing mutant Fc regions were assessed for their ADCC activity and compared to the ADCC activity of wild type ch4D5.
  • SKBR3 high expression of HER2/neu
  • HT29 low expression of HER2/neu cells lines were used as targets (panels A and B, respectively).
  • Effector to target ratio was 75:1 with 18 h incubation.
  • Mutants analyzed were MgFc31/60 (P247L; N421K; D270E) and MgFc71 (D270E; G316D; R416G).
  • FIG. 45 A-D BINDING OF MUTANTS IN THE MONOCLONAL ANTI-CD32B ANTIBODY ch2B6 TO DAUDI CELLS AND RAMOS CELLS [00100] FACS analysis was used to characterize the binding of monoclonal anti-
  • CD32B antibody ch2B6 variant 31/60 P247L; N421K; D270E), variant 71 (D270E; G316D; R416G) and variant 59/60 (K370E; P396L; D270E) to either Daudi cells (high expression of CD32B) or Ramos cells (low expression of CD32B).
  • Incubation with primary antibody was at 5 ⁇ g/ml (A), 0.5 ⁇ g/ml (B), 50 ng/ml (C), or 5 ng/ml (D). Wild-type ch2B6 and IgG (SYNAGIS) were used as controls.
  • PE-conjugated polyclonal F(ab) 2 goat anti-humanFC ⁇ R was used as the secondary antibody.
  • FIG.46 A-B ADCC ACTIVITY OF MUTANTS IN THE ANTI-CD32B ANTIBODY, ch2B6
  • Ch2B6 antibodies containing mutant Fc regions were assessed for their ADCC activity and compared to the ADCC activity of wild type 2B6.
  • the Ramos cell line (low expression of CD32B) was used as target.
  • Effector to target ratio (E:T ratio) was 75:1 with 18 h incubation.
  • FIG.47 A-C CDC ACTIVITY OF MUTANTS IN THE ANTI-CD32B ANTIBODY, ch2B6
  • Ch2B6 antibodies containing mutant Fc regions were assessed for their CDC activity and compared to the CDC activity of wild type ch2B6.
  • BL41 a Burkitt's lymphoma cell line
  • Ramos low expression of CD32B
  • FIG.48 A-B ADCC ACTIVITY OF MUTANTS IN THE ANTI-CD32B ANTIBODY, ch2B6
  • Ch2B6 antibodies containing mutant Fc regions were assessed for their ADCC activity and compared to the ADCC activity of wild type ch2B6.
  • the Daudi cell line high expression of CD32B
  • Effector to target ratio was 75:1 with 18 h incubation.
  • FIG.49 A-B FACS ANALYSIS OF THE BINDING OF THE ANTI-CD32B ANTIBODY, ch2B6, AND THE ANTI-CD20 ANTD3ODY, RITUXANTM, TO A TRANSGENIC CHO CELL LINE.
  • Cho cells were engineered to express both recombinant CD32B and recombinant CD20 on the cell surface. Following incubation and amplification in selective media, cells were analyzed by FACS. Cells were incubated in either FITC-conjugated wild- type 2B6 (A) or FITC-conjugated RituxanTM (B).
  • FIG. 50 A-B ADCC ACTIVITY OF MUTANTS IN THE ANTI-CD20 ANTD3ODY, RITUXANTM
  • FIG. 1 shows the ADCC activity of wild type RituxanTM and ch2B6.
  • a Cho cell line engineered to express both CD32B and CD20 was used as target. Effector to target ratio (E:T ratio) was 75:1 with 18 h incubation.
  • Figure A shows the ADCC activity of wild type ch2B6 and RituxanTM.
  • Figure B shows a comparison of the ADCC activity of wild type RituxanTM and RituxanTM comprising mutation variant MGFc55/60 (R255L; P396L; D270E).
  • FIG. 51 A-E COMPARISON OF BINDING AFFINITY AND KINETIC CHARACTERISTICS OF ch2B6 MUTANTS
  • FACS analysis was used to characterize the binding of mutant ch2B6 antibodies to Ramos cells (low expression of CD32B). Data were compared to a BIAcore analysis of the I -0If for the same variant antibodies. Mutants analyzed were MgFc55 (R255L; P396L), MgFc55/60 (R255L; P396L; D270E) and MgFc55/60/F243L (R255L; P396L; D270E; F243L). Wild-type ch2B6 was used as control.
  • Incubation with primary antibody was at 10 ⁇ g/ml (A), 1 ⁇ g/ml (B), 0.1 ng/ml (C), or 0.01 ng/ml (D).
  • PE-conjugated polyclonal F(ab) 2 goat anti-humanFC ⁇ R was used as the secondary antibody.
  • FIG. 52 A-C BINDING OF ACTIVATING RECEPTOR CD16A TO RAMOS CELLS OPSONIZED WITH MUTANT ch2B6 ANTIBODY [00107] FACS analysis was used to characterize the binding of activating receptor
  • CD16A to Ramos cells opsonized with mutant ch2B631/60 antibody P247L; N421K; D270E.
  • Opsonization with wild-type ch2B6, hu2B6YA (humanized 2B6 with YA substitution at positions 50,51 of antibody light-chain - eliminates glycosylation at position 50 of the light-chain protein), or antibody-free buffer was used as a control.
  • PE-conjugated polyclonal F(ab) 2 goat anti-humanFC ⁇ R was used as the secondary antibody.
  • FIG. 53 A-J BINDING OF MUTANTS IN THE MONOCLONAL ANTI-CD32B ANTIBODY ch2B6 TO DAUDI CELLS
  • CD32B antibody ch2B6 variant 31/60 P247L; N421K; D270E
  • hu2B6YA humanized 2B6 with YA substitution at positions 50,51 of antibody light-chain - eliminates glycosylation at position 50 of the light-chain protein
  • hu2B6YA31/60 to Daudi cells (high expression of CD32B).
  • Wild-type ch2B6, hu2B6, Synagis and ch2B6 AgIy (N297Q; aglycoslyated Fc, no FcR binding) were used as controls.
  • Incubation with primary antibody was at either 37 0 C (panels A-E) or 4 0 C (panels F-J) for 0.5 h and at a concentration of 10 ⁇ g/ml (A, F), 1 ⁇ g/ml (B, G), 0.1 ng/ml (C, H), 0.01 ⁇ g/ml (D, I), or 0.001 ⁇ g/ml (E, J).
  • PE-conjugated polyclonal F(ab) 2 goat anti-humanFC ⁇ R was used as the secondary antibody.
  • FIG. 54 A-E BINDING OF MUTANTS IN THE MONOCLONAL ANTI-CD32B ANTIBODY ch2B6 TO EL-4/CD32B CELLS
  • CD32B antibody ch2B6 variant 31/60 P247L; N421K; D270E
  • hu2B6YA humanized 2B6 with YA substitution at positions 50,51 of antibody light-chain - eliminates glycosylation at position 50 of the light-chain protein
  • hu2B6YA31/60 to EL-4/CD32B cells.
  • Wild-type ch2B6, hu2B6, Synagis and ch2B6 AgIy (N297Q; aglycoslyated Fc, no FcR binding) were used as controls.
  • Incubation with primary antibody was at 37 0 C for 0.5 h and at a concentration of 10 ⁇ g/ml (A), 1 ⁇ g/ml (B), 0.1 ⁇ g/ml (C), 0.01 ⁇ g/ml (D), or 0.001 ⁇ g/ml (E).
  • PE-conjugated polyclonal F(ab) 2 goat anti-humanFC ⁇ R was used as the secondary antibody.
  • FIG. 55 A-J BINDING OF MUTANTS IN THE MONOCLONAL ANTI-CD32B ANTD3ODY ch2B6 TO RAMOS CELLS
  • CD32B antibody ch2B6 variant 31/60 (P247L; N421K; D270E), hu2B6YA (humanized 2B6 with YA substitution at positions 50,51 of antibody light-chain - eliminates glycosylation at position 50 of the light-chain protein )or hu2B6YA31/60 to Ramos cells (low expression of CD32B).
  • Incubation with primary antibody was at either 37 0 C (panels A-E) or 4 0 C (panels F-J) for 0.5 h and at a concentration of 10 ⁇ g/ml (A, F), 1 ⁇ g/ml (B, G), 0.1 ng/ml (C, H), 0.01 ⁇ g/ml (D, I), or 0.001 ⁇ g/ml (E, J).
  • PE-conjugated polyclonal F(ab) 2 goat anti-humanFC ⁇ R was used as control.
  • FIG.56 ADCC ACTIVITY OF MUTANTS IN THE ANTI-CD32B
  • Ch2B6 antibodies containing mutant Fc regions were assessed for their ADCC activity and compared to the ADCC activity of wild type ch2B6.
  • the Ramos cell line (low expression of CD32B) was used as target.
  • Effector to target ratio (E:T ratio) was 75:1 with 18 h incubation.
  • FIG. 57 CDC ACTIVITY OF MUTANTS IN THE ANTI-CD32B ANTIBODY, ch2B6
  • Ch2B6 antibodies containing mutant Fc regions were assessed for their CDC activity and compared to the CDC activity of wild type ch2B6.
  • the Ramos cell line (low expression of CD32B) was used as target.
  • Effector to target ratio (E:T ratio) was 75: 1 with 18 h incubation.
  • FIG. 58 A-F ADCC ACTIVITY OF MODIFIED RITUXIMAB ANTIBODIES IN HUMAN PATIENTS TREATED WITH RITUXIMAB
  • Patient derived cells were used as target. Effector to target ratio (E:T ratio) was 30:1 and 10:1.
  • Mutants analyzed were MGFc55/60/300L (R255L; P396L; D270E; Y300L); MGFc51/60 (Q419H; P396L; D270E); MGFc52/60 (V240A; P396L; D270E); MGFc59/60 (K370E; P396L; D270E); MGFc38/60 (K392T;P396L; D270E); MGFc59 (K370E; P396L); MGFc51 (Q419H; P396L); MGFc31/60 (P247L; N421K; D270E); MGFc55/292G (R255L; P396L; D270E; R292G).
  • the present invention relates to engineering molecules, preferably polypeptides, and more preferably immunoglobulins (e.g., antibodies) to confer one or more effector function activities to the molecule, which effector functions the parent molecule does not have or has at low levels (e.g., not detectable in in vitro and/or in vivo assays known in the art), hi particular, the modified molecules, e.g., antibodies, of the invention comprise a variant Fc region, having one or more amino acid modifications (e.g., substitutions, but also including insertions or deletions) in one or more regions, which modifications confer at least one effector function, hi particular, the modifications alter the affinity and avidity of the variant Fc region for an Fc ⁇ R (e.g., activating Fc ⁇ Rs or inhibitory Fc ⁇ Rs) and thereby altering the activity of one or more effector functions.
  • an Fc ⁇ R e.g., activating Fc ⁇ Rs or inhibitory Fc ⁇ Rs
  • the modifications confer homo-oligomerization activity to the parent Fc region such that oligomerization of the modified antibody cross-links cell-surface antigens, resulting in apoptosis, negative-growth regulation or cell killing.
  • Effector function activities include, but are not limited to, antibody-dependent cell mediated cytotoxicity (ADCC), antibody-dependent phagocytosis, phagocytosis, opsonization, opsonophagocytosis, cell binding, rosetting, and complement dependent cell mediated cytotoxicity (CDC).
  • the modifications alter the affinity of the variant Fc region such that the variant Fc regions oligomerize and homo-oligomers of the modified antibody are formed, hi certain embodiments of the invention, the engineered molecule is not an anti- CD20 antibody, more particularly, does not compete for CD20 binding with rituximab or is not rituximab.
  • the present invention also relates to molecules (e.g., antibodies) comprising a variant Fc region having one or more amino acid modifications (e.g., substitutions, deletions, insertions) in one or more portions, which modifications increase the affinity and avidity of the variant Fc region for an Fc ⁇ R (including activating and inhibitory Fc ⁇ Rs).
  • said one or more amino acid modifications increase the affinity of the variant Fc region for Fc ⁇ RIIIA and/or Fc ⁇ RIIA.
  • the variant Fc region further specifically binds Fc ⁇ RIIB with a lower affinity than does the Fc region of the comparable parent antibody (Le., an antibody having the same amino acid sequence as the antibody of the invention except for the one or more amino acid modifications in the Fc region).
  • such modifications increase the affinity of the variant Fc region for Fc ⁇ RIIIA and/or Fc ⁇ RIIA and also enhance the affinity of the variant Fc region for Fc ⁇ RIEB relative to the parent antibody.
  • said one or more amino acid modifications increase the affinity of the variant Fc region for Fc ⁇ RIIIA and/or Fc ⁇ RIIA but do not alter the affinity of the variant Fc regions for Fc ⁇ RlIB relative to the Fc region of the parent antibody.
  • said one or more amino acid modifications enhance the affinity of the variant Fc region for Fc ⁇ RIIIA and Fc ⁇ RIIA but reduce the affinity for Fc ⁇ RIIB relative to the parent antibody. Increased affinity and/or avidity results in detectable binding to the Fc ⁇ R or Fc ⁇ R-related activity in cells that express low levels of the Fc ⁇ R when binding activity of the parent molecule (without the modified Fc region) can not be detected in the cells.
  • the modified molecule exhibits detectable binding in cells which express non- Fc ⁇ R receptor target antigens at a density of 30,000 to 20,000 molecules/cell, at a density of 20,000 to 10,000 molecules/cell, at a density of 10,000 to 5,000 molecules/cell, at a density of 5,000 to 1,000 molecules/cell, at a density of 1,000 to 200 molecules/cell or at a density of 200 molecules/cell or less ( but at least 10, 50, 100 or 150 molecules/cell).
  • said one or more modifications to the amino acids of the Fc region reduce the affinity and avidity of the antibody for one or more Fc ⁇ R receptors.
  • the invention encompasses antibodies comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild type Fc region, which variant Fc region only binds one Fc ⁇ R, wherein said Fc ⁇ R is Fc ⁇ RIIIA.
  • the invention encompasses antibodies comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild type Fc region, which variant Fc region only binds one Fc ⁇ R, wherein said Fc ⁇ R is Fc ⁇ RIIA.
  • the binding properties of the molecules of the invention are characterized by in vitro functional assays for determining one or more Fc ⁇ R mediator effector cell functions (See Section 5.2.7).
  • the affinities and binding properties of the molecules, e.g., antibodies, of the invention for an Fc ⁇ R can be determined using in vitro assays (biochemical or immunological based assays) known in the art for determining antibody-antigen or Fc-Fc ⁇ R interactions, i.e., specific binding of an antigen to an antibody or specific binding of an Fc region to an Fc ⁇ R, respectively, including but not limited to ELISA assay, surface plasmon resonance assay, immunoprecipitation assays (See Section 5.2.1).
  • the molecules of the invention have similar binding properties in in vivo models (such as those described and disclosed herein) as those in in vitro based assays.
  • the present invention does not exclude molecules of the invention that do not exhibit the desired phenotype in in vitro based assays but do exhibit the desired phenotype in vivo.
  • the molecules of the invention comprising a variant Fc region comprise at least one amino acid modification in the CH3 domain of the Fc region, which is defined as extending from amino acids 342-447. In other embodiments, the molecules of the invention comprising a variant Fc region comprise at least one amino acid modification in the CH2 domain of the Fc region, which is defined as extending from amino acids 231-341. In some embodiments, the molecules of the invention comprise at least two amino acid modifications, wherein one modification is in the CH3 region and one modification is in the CH2 region. The invention further encompasses amino acid modification in the hinge region. In a particular embodiment, the invention encompasses amino acid modification in the CHl domain of the Fc region, which is defined as extending from amino acids 216-230.
  • the invention encompasses molecules comprising a variant Fc region wherein said variant confers or has an increased ADCC activity and/or an increased binding to Fc ⁇ RIIA (CD32A), as measured using methods known to one skilled in the art and exemplified herein.
  • the ADCC assays used in accordance with the methods of the invention may be NK dependent or macrophage dependent.
  • the Fc variants of the present invention may be combined with other Fc modifications known in the art.
  • the invention encompasses combining an Fc variant of the invention with other Fc modifications to provide additive, synergistic, or novel properties to the modified antibody.
  • the Fc variants of the invention enhance the phenotype of the modification with which they are combined.
  • an Fc variant of the invention is combined with a mutant known to bind Fc ⁇ RIIIA with a higher affinity than a comparable wild type Fc region; the combination with a mutant of the invention results in a greater fold enhancement in Fc ⁇ RIIIA affinity.
  • the Fc variants of the present invention may be combined with any of the known Fc modifications in the art such as those disclosed in Tables 3 A and B below.
  • the invention encompasses molecules comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule has a conferred effector function (i.e., in a particular assay, the modified molecule has an effector function activity not detectable in the parent molecule) and/or an altered affinity for an Fc ⁇ R, provided that said variant Fc region does not have a substitution at positions that make a direct contact with Fc)R based on crystallographic and structural analysis of Fc-Fc)R interactions, such as those positions disclosed by Sondermann et al, 2000 ⁇ Nature, 406: 267-273 which is incorporated herein by reference in its entirety).
  • positions within the Fc region that make a direct contact with FeyR are amino acids 234-239 (hinge region), amino acids 265-269 (B/C loop), amino acids 297-299 (C 1 IE loop), and amino acids 327-332 (F/G) loop.
  • the molecules of the invention comprising variant Fc regions comprise modification of at least one residue that makes a direct contact with an Fc ⁇ R based on structural and crystallographic analysis.
  • the Fc ⁇ R interacting domain maps to the lower hinge region and select sites within the CH2 and CH3 domains of the IgG heavy chain.
  • Amino acid residues flanking the actual contact positions and amino acid residues in the CH3 domain play a role in IgG/Fc ⁇ R interactions as indicated by mutagenesis studies and studies using small peptide inhibitors, respectively (Sondermann et al, 2000 Nature, 406: 267-273; Melnhofer et al., 1981, Biochemistry, 20: 2361-2370; Shields et al, 2001, J. Biol. Chem. 276: 6591-6604; each of which is incorporated herein by reference in its entirety).
  • Direct contact refers to those amino acids that are within at least 1 A, at least 2 A, or at least 3 A of each other or within I A, 1.2 A, 1.5 A, 1.7 A or 2 A Van Der Waals radius.
  • An exemplary list of previously identified sites on the Fc that affect binding of Fc interacting proteins is listed in the Table 4 below.
  • the invention encompasses Fc variants that do not have any modifications at the sites listed below.
  • the invention encompasses Fc variants comprising amino acid modifications at one or more sites listed below in combination with other modifications disclosed herein such that such modification has a synergistic or additive effect on the property of the mutant.
  • Table 4 lists sites within the Fc region that have previously been identified to be important for the Fc-FcR interaction. Columns labeled FcR-Fc identifies the Fc chain contacted by the FcR. Letters identify the reference in which the data was cited.
  • C is Shields et al, 2001, J. Biol. Chem. 276: 6591-6604; D is Jefferis et al, 1995, Immunol. Lett. 44: 111-7; E is Hinton et al; 2004, J. Biol. Chem. 279(8): 6213-6; F is Idusogie et al. , 2000, J. Immunol. 164: 4178-4184; each of which is incorporated herein by reference in its entirety.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule has an enhanced effector function relative to a molecule comprising a wild-type Fc region, provided that said variant Fc region does not have or is not solely a substitution at any of positions 243, 255, 256, 258, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 300, 301, 303, 305, 307, 309, 312, 320, 322, 326, 329, 330, 332, 331, 333, 334, 335, 337, 338, 339, 340, 359, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438, 439.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule binds an Fc ⁇ R with an altered affinity relative to a molecule comprising a wild-type Fc region, provided that said variant Fc region does not have or is not solely a substitution at any of positions 243, 255, 258, 267, 269, 270, 276, 278, 280, 283, 285, 289, 292, 293, 294, 295, 296, 300, 303, 305, 307, 309, 320, 322, 329, 332, 331, 337, 338, 340, 373, 376, 416, 419, 434, 435, 437, 438, 439 and does not have an alanine at any of positions 256, 290, 298, 312, 326, 333, 334, 359, 360, or 430; an asparagine at
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region does not have or is not solely a substitution at any of positions 268, 269, 270, 272, 276, 278, 283, 285, 286, 289, 292, 293, 301, 303, 305, 307, 309, 320, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438 or 439 and does not have a histidine, glutamine, or tyrosine at position 280; a serine, glycine, threonine or tyrosine at position 290, an asparagine at position 294, a lysine at position 295; a proline at position 296; a proline, asparagine, aspartic acid, or valine at position 298; or a leucine or
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule binds an Fc ⁇ R with a reduced affinity relative to molecule comprising a wild-type Fc region provided that said variant Fc region does not have or is not solely a substitution at any of positions 243, 252, 254, 265, 268, 269, 270, 278, 289, 292, 293, 294, 295, 296, 298, 300, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438, or 439.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule binds an Fc ⁇ R with an enhanced affinity relative to a molecule comprising a wild-type Fc region provided that said variant Fc region does not have or is not solely a substitution at any of positions 280, 283, 285, 286, 290, 294, 295, 298, 300, 301, 305, 307, 309, 312, 315, 331, 333, 334, 337, 340, 360, 378, 398, or 430.
  • the invention encompasses molecule comprising a variant Fc region, wherein said variant Fc region does not include or are not solely a substitution at any of positions 330, 243, 247, 298, 241, 240, 244, 263, 262, 235, 269, or 328 and does not have a leucine at position 243, an asparagine at position 298, a leucine at position 241, and isoleucine or an alanine at position 240, a histidine at position 244, a valine at position 330, or an isoleucine at position 328.
  • Fc regions with enhanced effector function and/or altered affinities for activating and/or inhibitory receptors have one or more amino acid modifications, wherein said one or more amino acid modification is a substitution at position 288 with asaparagine, at position 330 with serine and at position 396 with leucine (MgFcl0)(See Table 5); or a substitution at position 334 with glutamic acid, at position 359 with asparagine, and at position 366 with serine (MgFcl3); or a substitution at position 316 with aspartic acid, at position 378 with valine, and at position 399 with glutamic acid (MgFc27); or a substitution at position 247 with leucine, and a substitution at position 421 with lysine (MgFc31); or a substitution at position 392 with threonine, and at position 396 with leucine (MgFc38); or a substitution at position 221 with glutamic acid, at position 270 with glutamic acid, at
  • the invention encompasses a molecule comprising a variant Fc region wherein said variant Fc region comprises a substitution at position 396 with leucine, at position 270 with glutamic acid and at position 243 with leucine.
  • the molecule further comprises one or more amino acid modification such as those disclosed herein.
  • the invention encompasses molecules comprising a variant Fc region having an amino acid modification at one or more of the following positions: 119, 125, 132, 133, 141, 142, 147, 149, 162, 166, 185, 192, 202, 205, 210, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 227, 229, 231, 232, 233, 235, 240, 241, 242, 243, 244, 246, 247, 248, 250, 251, 252, 253, 254, 255, 256, 258, 261, 262, 263, 268, 269, 270, 272, 274, 275, 276, 279, 280, 281, 282, 284, 287, 288, 289, 290, 291, 292, 293, 295, 298, 301, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 315,
  • mutations result in molecules that have been conferred an effector cell mediated function and, optionally, have an altered affinity for an Fc ⁇ R as determined using methods disclosed and exemplified herein and known to one skilled in the art.
  • the invention encompasses molecules comprising variant Fc regions consisting of or comprising any of the mutations listed in the table below in Table 5. TABLE 5. EXEMPLARY MUTATIONS
  • the invention encompasses molecules comprising variant Fc regions having more than two amino acid modifications.
  • a non-limiting example of such variants is listed in the table below (Table 6).
  • the invention encompasses mutations listed in Table 6 which further comprise one or more amino acid modifications such as those disclosed herein.
  • the variant Fc region has a leucine at position 247, a lysine at position 421 and a glutamic acid at position 270 (MgFc31/60); a threonine at position 392, a leucine at position 396, a glutamic acid at position 270, and a leucine at position 243 (MgFc38/60/F243L); a histidine at position 419, a leucine at position 396, and a glutamic acid at position 270 (MGFc51/60); a histidine at position 419, a leucine at position 396, a glutamic acid at position 270, and a leucine at position 243 (MGFc51/60/F243L); an alanine at position 240, a leucine at position 396, and a glutamic acid at position 270 (MGFc52/60); a lysine at position 255 and a leucine at position 240,
  • the molecules of the invention further comprise one or more glycosylation sites, so that one or more carbohydrate moieties are covalently attached to the molecule.
  • the molecules of the invention with one or more glycosylation sites and/or one or more modifications in the Fc region confer or have an enhanced antibody mediated effector function, e.g., enhanced ADCC activity, compared to a parent antibody.
  • the invention further comprises molecules comprising one or more modifications of amino acids that are directly or indirectly known to interact with a carbohydrate moiety of the antibody, including but not limited to amino acids at positions 241, 243, 244, 245, 245, 249, 256, 258, 260, 262, 264, 265, 296, 299, and 301.
  • Amino acids that directly or indirectly interact with a carbohydrate moiety of an antibody are known in the art, see, e.g., Jefferis et al, 1995 Immunology Letters, 44: 111-7, which is incorporated herein by reference in its entirety.
  • the invention encompasses molecules that have been modified by introducing one or more glycosylation sites into one or more sites of the molecules, preferably without altering the functionality of the molecules, e.g., binding activity to target antigen or Fc ⁇ R.
  • Glycosylation sites may be introduced into the variable and/or constant region of the molecules of the invention.
  • "glycosylation sites” include any specific amino acid sequence in an antibody to which an oligosaccharide ⁇ i.e., carbohydrates containing two or more simple sugars linked together) will specifically and covalently attach. Oligosaccharide side chains are typically linked to the backbone of an antibody via either N-or O-linkages.
  • N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue.
  • O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, e.g., serine, threonine.
  • the molecules of the invention may comprise one or more glycosylation sites, including N-linked and O-linked glycosylation sites. Any glycosylation site for N-linked or O-linked glycosylation known in the art may be used in accordance with the instant invention.
  • N-linked glycosylation site that is useful in accordance with the methods of the present invention is the amino acid sequence: Asn-X-Thr/Ser, wherein X may be any amino acid and Thr/Ser indicates a threonine or a serine.
  • a site or sites may be introduced into a molecule of the invention using methods well known in the art to which this invention pertains. See, for example, "In vitro Mutagenesis.” Recombinant DNA: A Short Course, J. D. Watson, et al. W.H. Freeman and Company, New York, 1983, chapter 8, pp. 106-116, which is incorporated herein by reference in its entirety.
  • An exemplary method for introducing a glycosylation site into a molecule of the invention may comprise: modifying or mutating an amino acid sequence of the molecule so that the desired Asn-X-Thr/Ser sequence is obtained.
  • the invention encompasses methods of modifying the carbohydrate content of a molecule of the invention by adding or deleting a glycosylation site. Methods for modifying the carbohydrate content of antibodies are well known in the art and encompassed within the invention, see, e.g., U.S. Patent No. 6,218,149; EP 0 359 096 Bl; U.S. Publication No. US 2002/0028486; WO 03/035835; U.S. Publication No.
  • the invention encompasses methods of modifying the carbohydrate content of a molecule of the invention by deleting one or more endogenous carbohydrate moieties of the molecule.
  • the invention encompasses shifting the glycosylation site of the Fc region of an antibody, by modifying positions adjacent to 297.
  • the invention encompasses modifying position 296 so that position 296 and not position 297 is glycosylated.
  • the present invention is based, in part, on the inventors' discovery of methods for engineering the Fc region of an antibody to confer an effector function activity to the antibody, which the parent antibody did not exhibit when tested against a target cell.
  • Such methods of engineering include introducing one or more amino acid modifications (substitutions, deletions or insertions) in one or more portions of the Fc region, which modifications introduce a detectable effector function activity on the parent antibody.
  • the modifications alter the parent antibody's affinity for certain Fc ⁇ R receptors (e.g., activating Fc ⁇ Rs, inhibitory Fc ⁇ Rs) and one or more effector functions, such as
  • ADCC alters the affinity of the variant Fc region such that the variant Fc regions oligomerize and homo-oligomers of the modified antibody are formed.
  • the inventors have found that modification of an Fc region of a chimeric 2B6 or 4D5 antibody (anti-FcyRIIB antibody) surprisingly conferred a particular effector function activity (ADCC) on chimeric 2B6 antibodies, which normally exhibit no detectable ADCC activity, and improved effector function activity (particularly ADCC) of chimeric 4D5 antibodies in cells with low levels of antigen expression.
  • modification of an Fc region of rituximab conferred effector function activity on the rituximab antibody in a patient population whose cells were otherwise refractory to rituximab- induced effector function activity.
  • rituximab anti-CD20 monoclonal antibody
  • the present invention contemplates other modifications of the Fc region amino acid sequence in order to generate an Fc region variant with one or more altered properties, e.g., enhanced effector function.
  • the invention contemplates deletion of one or more amino acid residues of the Fc region in order to, e.g. , reduce binding to an Fc ⁇ R.
  • the Fc region herein comprising one or more amino acid deletions will preferably retain at least about 80%, and preferably at least about 90%, and most preferably at least about 95%, of the wild type Fc region.
  • one or more properties of the molecules are maintained such as for example, non-immunogenicity, Fc ⁇ RIIIA binding, Fc ⁇ RIIA binding, or a combination of these properties.
  • the invention encompasses amino acid insertion to generate the Fc region variants, which variants have altered properties including enhanced effector function.
  • the invention encompasses introducing at least one amino acid residue, for example, one to two amino acid residues and preferably no more than 10 amino acid residues adjacent to one or more of the Fc region positions identified herein. In alternate embodiments, the invention further encompasses introducing at least one amino acid residue, for example, one to two amino acid residues and preferably no more than 10 amino acid residues adjacent to one or more of the Fc region positions known in the art as impacting Fc ⁇ R. interaction and/or binding.
  • the invention further encompasses incorporation of unnatural amino acids to generate the Fc variants of the invention.
  • unnatural amino acids such as those using the natural biosynthetic machinery to allow incorporation of unnatural amino acids into proteins, see, e.g., Wang et al, 2002 Chem. Comm. 1: 1-11; Wang et al, 2001, Science, 292: 498-500; van Hest et al, 2001. Chem. Comm. 19: 1897- 1904, each of which is incorporated herein by reference in its entirety.
  • Alternative strategies focus on the enzymes responsible for the biosynthesis of amino acyl-tRNA, see, e.g., Tang et al., 2001, J. Am. Chem.
  • effector function properties of the molecules of the invention are determined for one or more Fc ⁇ R mediator effector cell functions as described in Section 5.2.7.
  • the affinities and binding properties of the molecules of the invention for a target antigen or an Fc ⁇ R are initially determined using in vitro assays (biochemical or immunological based assays) known in the art for determining antibody-antigen or Fc-Fc ⁇ R interactions, Le., specific binding of an antibody to an antigen or an Fc region to an Fc ⁇ R, respectively, including but not limited to ELISA assay, surface plasmon resonance assay, immunoprecipitation assays ⁇ See Section 5.2.1).
  • the molecules of the invention have similar binding properties in in vivo models (such as those described and disclosed herein) as those in in vitro based assays.
  • the present invention does not exclude molecules of the invention that do not exhibit the desired phenotype in in vitro based assays but do exhibit the desired phenotype in vivo.
  • a representative flow chart of the screening and characterization of molecules of the invention is described in FIG. 34.
  • the invention encompasses molecules comprising a variant Fc region that binds with a greater affinity to one or more Fc ⁇ Rs. Such molecules preferably mediate effector function more effectively as discussed infra. In other embodiments, the invention encompasses molecules comprising a variant Fc region that bind with a weaker affinity to one or more Fc ⁇ Rs. m general, increased or added effector function would be directed to tumor and foreign cells.
  • the Fc variants of the present invention may be combined with other Fc modifications, including but not limited to other modifications that enhance effector function.
  • the invention encompasses combining an Fc variant of the invention with other Fc modifications to provide additive, synergistic, or novel properties in antibodies or Fc fusions.
  • the Fc variants of the invention enhance the phenotype of the modification with which they are combined. For example, if an Fc variant of the invention is combined with a mutant known to bind Fc ⁇ RIIIA with a higher affinity than a comparable molecule comprising a wild type Fc region; the combination with a mutant of the invention results in a greater fold enhancement in Fc ⁇ RIIIA affinity.
  • the Fc variants of the present invention may be combined with other known Fc variants such as those disclosed in Duncan et al, 1988, Nature 332:563-564; Lund etal, 1991, J. Immunol 147:2657-2662; Lund et al, 1992, MoI Immunol 29:53-59; Alegre et al, 1994, Transplantation 57:1537-1543; Hutchins et al. , 1995, Proc Natl. Acad Sci U S A 92:11980-11984; Jefferis et al, 1995, Immunol Lett.
  • the Fc variants of the present invention are incorporated into an antibody or Fc fusion that comprises one or more engineered glycoforms, Le., a carbohydrate composition that is covalently attached to an antibody comprising an Fc region, wherein said carbohydrate composition differs chemically from that of a parent antibody comprising an Fc region.
  • Engineered glycoforms may be useful for a variety of purposes, including, but not limited to, enhancing effector function.
  • Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example, DI N-acetylglucosaminyltransferase III (GnTIIl), by expressing an antibody comprising an Fc region in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the antibody comprising Fc region has been expressed.
  • Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et al, 1999, Nat.
  • the Fc variants of the present invention may be optimized for a variety of properties. Properties that may be optimized include, but are not limited to, conferred or enhanced effector function, enhanced or reduced affinity for an Fc ⁇ R, or conferred oligomerization activity.
  • the Fc variants of the present invention are optimized to possess enhanced affinity for a human activating Fc ⁇ R, preferably Fc ⁇ R, Fe)RIIA, Fc ⁇ RIIc, FcyRIIIA, and FcyRIIIB, most preferably Fe)RIIIA.
  • the Fc variants are optimized to possess reduced affinity for the human inhibitory receptor Fe)RIIB.
  • the Fc variants of the present invention are optimized to have reduced affinity for a human Fe)R, including but not limited to Fe)RI, Fc ⁇ RIIA, Fc ⁇ RIIB, Fe)RIIc, Fc ⁇ RIIIA, and FeyRIIIB. These embodiments are anticipated to provide antibodies and Fc fusions with enhanced therapeutic properties in humans, for example, reduced toxicity.
  • the Fc variants of the present invention possess a conferred effector function and/or enhanced or reduced affinity for Fe)Rs from non-human organisms, including, but not limited to, mice, rats, rabbits, and monkeys.
  • Fc variants that are optimized for effector function in a non-human or binding to a non-human Fe)R may find use in experimentation.
  • mouse models are available for a variety of diseases that enable testing of properties such as efficacy, toxicity, and pharmacokinetics for a given drug candidate.
  • cancer cells can be grafted or injected into mice to mimic a human cancer, a process referred to as xenografting.
  • Testing of antibodies or Fc fusions that comprise Fc variants that confer an effector function and/or are optimized for one or more mouse Fe)Rs may provide valuable information with regard to the efficacy of the antibody or Fc fusion, its mechanism of action, and the like.
  • the instant invention further contemplates Fc variants with altered binding affinity to the neonatal receptor (FcRn).
  • FcRn neonatal receptor
  • Fc region variants with improved affinity for FcRn are anticipated to have longer serum half-lives, and such antibodies will have useful applications in methods of treating mammals where long half-life of the administered polypeptide is desired, e.g., to treat a chronic disease or disorder.
  • Fc region variants with decreased FcRn binding affinity are expected to have shorter half-lives, and such antibodies may, for example, be administered to a mammal where a shortened circulation time may be advantageous, e.g., for in vivo diagnostic imaging or for polypeptides which have toxic side effects when left circulating in the blood stream for extended periods.
  • Fc region variants with decreased FcRn binding affinity are anticipated to be less likely to cross the placenta, and thus may be utilized in the treatment of diseases or disorders in pregnant women.
  • these variants may be combined with other known Fc modifications with altered FcRn affinity such as those disclosed in International Publication Nos. WO 98/23289; and WO 97/34631; and U.S. Patent No. 6,277,375, each of which is incorporated herein by reference in its entirety.
  • the invention encompasses any other method known in the art for generating molecules, e.g., antibodies, having an increased half -life in vivo, for example, by introducing one or more amino acid modifications (i.e., substitutions, insertions or deletions) into an IgG constant domain, or FcRn binding fragment thereof (preferably a Fc or hinge-Fc domain fragment).
  • molecules, e.g., antibodies, of the invention can be conjugated to albumin in order to make the antibody or antibody fragment more stable in vivo or have a longer half-life in vivo.
  • the techniques well-known in the art see, e.g., International Publication Nos. WO 93/15199, WO 93/15200, and WO 01/77137, and European Patent No. EP 413,622, all of which are incorporated herein by reference in their entirety.
  • the variant(s) described herein may be subjected to further modifications, often times depending on the intended use of the variant. Such modifications may involve further alteration of the amino acid sequence (substitution, insertion and/or deletion of amino acid residues), fusion to heterologous polypeptide(s) and/or covalent modifications. Such further modifications may be made prior to, simultaneously with, or following, the amino acid modification(s) disclosed herein which results in altered properties such as an enhanced binding to target antigen, conferred oligomerization activity, or enhanced effector function and/or alteration of Fc receptor binding.
  • the invention encompasses combining the amino acid modifications disclosed herein with one or more further amino acid modifications that confer or enhance additional effector functions, e.g., CIq binding and/or complement dependent cytoxicity function, of the Fc region as determined in vitro and/or in vivo.
  • additional effector functions e.g., CIq binding and/or complement dependent cytoxicity function
  • the further amino acid substitutions described herein will generally serve to confer the activity on a parent antibody that does not exhibit detectable levels of the activity or enhance the ability of the starting antibody to bind to CIq and/or complement dependent cytotoxicity (CDC) function.
  • the starting antibody may be unable to bind CIq and/or mediate CDC and may be modified according to the teachings herein such that it acquires these further effector functions.
  • antibodies with preexisting CIq binding activity may be modified such that one or both of these activities are enhanced.
  • the invention encompasses variant Fc regions with altered CDC activity without any alteration in CIq binding.
  • the invention encompasses variant Fc regions with altered CDC activity and altered CIq binding.
  • amino acid positions to be modified are generally selected from positions 270, 322, 326, 327, 329, 331, 333, and 334, where the numbering of the residues in an IgG heavy chain is that of the EU index as in Kabat et ah, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (199).
  • amino acid modifications may be combined with one or more Fc modifications disclosed herein to provide a synergistic or additive effect on CIq binding and/or CDC activity.
  • the invention encompasses Fc variants with altered CIq binding and/or complement dependent cytotoxicity (CDC) function comprising an amino acid substitution at position 396 with leucine and at position 255 with leucine; or an amino acid substitution at position 396 with leucine and at position 419 with histidine; an amino acid substitution at position 396 with leucine and at position 370 with glutamic acid; an ammo acid substitution at position 396 with leucine and at position 240 with alanine; an amino acid substitution at position 396 with leucine and at position 392 with threonine; an amino acid substitution at position 247 with leucine and at position 421 with lysine.
  • CDC complement dependent cytotoxicity
  • the invention encompasses any known modification of the Fc region which alters CIq binding and/or complement dependent cytotoxicity (CDC) function such as those disclosed in Idusogie et ah, 2001, J. Immunol. 166(4) 2571-5; Idusogie et al, J. Immunol. 2000 164(8): 4178-4184; each of which is incorporated herein by reference in its entirety.
  • the invention encompasses an Fc region with altered effector function, e.g., modified CIq binding and/or FcR binding and thereby altered CDC activity and/or ADCC activity.
  • the invention encompasses variant Fc regions with improved CIq binding and improved Fc ⁇ RIII binding; e.g.
  • the invention encompasses a variant Fc region with reduced CDC activity and/or reduced ADCC activity.
  • one may increase only one of these activities, and optionally also reduce the other activity, e.g. to generate an Fc region variant with improved ADCC activity, but reduced CDC activity and vice versa.
  • the invention encompasses molecules comprising a variant Fc region, having one or more amino acid modifications (e.g., substitutions) in one or more regions, wherein such modifications alter the affinity of the variant Fc region for an activating Fc ⁇ R.
  • molecules of the invention comprise a variant Fc region, having one or more amino acid modifications (e.g., substitutions) in one or more regions, which modifications increase the affinity of the variant Fc region for Fc ⁇ RIIIA and/or Fc ⁇ RIIA by at least 2-fold, relative to a comparable molecule comprising a wild-type Fc region.
  • molecules of the invention comprise a variant Fc region, having one or more amino acid modifications (e.g., substitutions) in one or more regions, which modifications increase the affinity of the variant Fc region for Fc ⁇ RIIIA and/or Fc ⁇ RIIA by greater than 2 fold, relative to a comparable molecule comprising a wild-type Fc region.
  • the one or more amino acid modifications increase the affinity of the variant Fc region for Fc ⁇ RIIIA and/or Fc ⁇ RIIA by at least 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, or 10-fold relative to a comparable molecule comprising a wild-type Fc region.
  • the one or more amino acid modifications decrease the affinity of the variant Fc region for Fc ⁇ RIIIA and/or Fc ⁇ RIIA by at least 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, or 10-fold relative to a comparable molecule comprising a wild-type Fc region.
  • fold increases are preferably determined by an ELISA or surface plasmon resonance assays.
  • the one or more amino acid modifications do not include or are not solely a substitution at any one of positions 329, 331, or 322 with any amino acid.
  • the one or more amino acid modifications do not include or are not solely a substitution with any one of alanine at positions 243, 256, 290, 298, 312, 333, 334, 359, 360, or 430; with lysine at position 330; with threonine at position 339; with methionine or arginine at position 320; with serine, asparagine, aspartic acid, or glutamic acid at position 326 with glutamine, glutamic acid, methionine, histidine, valine, or leucine at position 334.
  • the one or more amino acid modifications do not include or are not solely a substitution at any of positions 280, 290, 300, 294, or 295.
  • the one or more amino acid modifications do not include or are not solely a substitution at position 300 with leucine or isoleucine; at position 295 with lysine; at position 294 with asparagine; at position 298 with valine; aspartic acid proline, asparagine, or valine; at position 280 with histidine, glutamine or tyrosine; at position 290 with serine, glycine, threonine or tyrosine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said polypeptide specifically binds Fc ⁇ RIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region binds Fc ⁇ RIIA, provided that said variant Fc region does not have an alanine at any of positions 256, 290, 326, 255, 258, 267, 272, 276, 280, 283, 285, 286, 331, 337, 268, 272, or 430; an asparagine at position 268; a glutamine at position 272; a glutamine, serine, or aspartic acid at position 286; a serine at position 290; a methionine, glutamine, glutamic acid, or arginine at position 320; a glutamic acid at position 322; a serine, glutamic acid, or aspart
  • molecules of the invention comprise a variant Fc region, having one or more amino acid modifications (e.g., substitutions) in one or more regions, which modifications increase the affinity of the variant Fc region for Fc ⁇ RIIA by at least 2-fold, relative to a comparable molecule comprising a wild-type Fc region.
  • molecules of the invention comprise a variant Fc region, having one or more amino acid modifications (e.g., substitutions) in one or more regions, which modifications increase the affinity of the variant Fc region for Fc ⁇ RIIA by greater than 2 fold, relative to a comparable molecule comprising a wild-type Fc region.
  • the one or more amino acid modifications increase the affinity of the variant Fc region for Fc ⁇ RIIA by at least 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, or 10-fold relative to a comparable molecule comprising a wild-type Fc region.
  • the invention encompasses molecules comprising a variant Fc region, having one or more amino acid modifications (e.g., substitutions but also include insertions or deletions), which modifications increase the affinity of the variant Fc region for Fc ⁇ RIIIA and/or Fc ⁇ RIIA by at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 150%, and at least 200%, relative to a comparable molecule comprising a wild-type Fc region.
  • amino acid modifications e.g., substitutions but also include insertions or deletions
  • the one or more amino acid modifications which increase the affinity of the variant Fc region comprise a substitution at position 347 with histidine, and at position 339 with valine; or a substitution at position 425 with isoleucine and at position 215 with phenylalanine; or a substitution at position 408 with isoleucine, at position 215 with isoleucine, and at position 125 with leucine; or a substitution at position 385 with glutamic acid and at position 247 with histidine; or a substitution at position 348 with methionine, at position 334 with asparagine, at position 275 with isoleucine, at position 202 with methionine, and at position 147 with threonine; or a substitution at position 275 with isoleucine, at position 334 with asparagine, and at position 348 with methionine; or a substitution at position 279 with leucine and at position 395 with serine; or a substitution at position
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises a substitution at position 243 with isoleucine and at position 379 with leucine, such that said molecule binds Fc)RIIIA with about a 1.5 fold higher affinity than a comparable molecule comprising the wild type Fc region binds Fc ⁇ RIIIA, as determined by an ELISA assay.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises a substitution at position 288 with asparagine, at position 330 with serine, and at position 396 with leucine, such that said molecule binds Fc ⁇ RIIIA with about a 5 fold higher affinity than a comparable molecule comprising the wild type Fc region binds Fc ⁇ RIIIA, as determined by an ELISA assay.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises a substitution at position 243 with leucine and at position 255 with leucine such that said molecule binds Fc ⁇ RIIIA with about a 1 fold higher affinity than a comparable molecule comprising the wild type Fc region binds Fc ⁇ RIIIA, as determined by an ELISA assay.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises a substitution at position 334 with glutamic acid, at position 359 with asparagine, and at position 366 with serine, such that said molecule binds Fc ⁇ RIIIA with about a 1.5 fold higher affinity than a comparable molecule comprising the wild type Fc region binds Fc ⁇ RIIIA, as determined by an ELISA assay, hi a specific embodiment, the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises a substitution at position 288 with methionine and at position 334 with glutamic acid, such that said molecule binds Fc ⁇ RIIIA with about a 3 fold higher affinity than a comparable molecule comprising the wild type Fc region binds Fc ⁇ RIIIA, as determined by an ELISA assay.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises a substitution at position 316 with aspartic acid, at position 378 with valine, and at position 399 with glutamic acid, such that said molecule binds Fc ⁇ RIIIA with about a 1.5 fold higher affinity than a comparable molecule comprising the wild type Fc region binds Fc ⁇ RIIIA, as determined by an ELISA assay, hi a specific embodiment, the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises a substitution at position 315 with isoleucine, at position 379 with methionine, and at position 399 with glutamic acid, such that said molecule binds Fc ⁇ RIIIA with about a 1 fold higher affinity than a comparable molecule comprising the wild type Fc region binds Fc ⁇ RIIIA, as determined by an ELISA assay.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises a substitution at position 243 with isoleucine, at position 379 with leucine, and at position 420 with valine, such that said molecule binds Fc ⁇ RIIIA with about a 2.5 fold higher affinity than a comparable molecule comprising the wild type Fc region binds Fc ⁇ RIIIA, as determined by an ELISA assay.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises a substitution at position 247 with leucine, and at position 421 with lysine, such that said molecule binds Fc ⁇ RIIIA with about a 3 fold higher affinity than a comparable molecule comprising the wild type Fc region binds Fc ⁇ RIIIA, as determined by an ELISA assay.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises a substitution at position 392 with threonine and at position 396 with leucine such that said molecule binds Fc ⁇ RIIIA with about a 4.5 fold higher affinity than a comparable molecule comprising the wild type Fc region binds Fc ⁇ RIIIA, as determined by an ELISA assay.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises a substitution at position 293 with valine, at position 295 with glutamic acid, and at position 327 with threonine, such that said molecule binds Fc ⁇ RIIIA with about a 1.5 fold higher affinity than a comparable molecule comprising the wild type Fc region binds Fc ⁇ RIIIA, as determined by an ELISA assay.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises a substitution at position 268 with asparagine and at position 396 with leucine, such that said molecule binds Fc ⁇ RIIIA with about a 2 fold higher affinity than a comparable molecule comprising the wild type Fc region binds Fc ⁇ RIIIA, as determined by an ELISA assay.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises a substitution at position 319 with phenylalanine, at position 352 with leucine, and at position 396 with leucine, such that said molecule binds Fc ⁇ RIIIA with about a 2 fold higher affinity than a comparable molecule comprising the wild type Fc region binds Fc ⁇ RIIIA, as determined by an ELISA assay.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 396 with histidine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 248 with methionine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said polypeptide specifically binds Fc ⁇ RIIIA with a similar affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 392 with arginine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a similar affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 315 with isoleucine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a similar affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 132 with isoleucine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a similar affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 162 with valine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild- type Fc region, wherein said at least one amino acid modification comprises substitution at position 396 with leucine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable polypeptide comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 379 with methionine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 219 with tyrosine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 282 with methionine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 401 with valine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 222 with asparagine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 334 with glutamic acid.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 377 with phenylalanine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 334 with isoleucine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 247 with leucine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 326 with glutamic acid.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 372 with tyrosine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 224 with leucine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 275 with tyrosine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 398 with valine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 334 with asparagine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 400 with proline.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 407 with isoleucine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a greater affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 372 with tyrosine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a similar affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 366 with asparagine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a reduced affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 414 with asparagine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a reduced affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 225 with serine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with a reduced affinity than a comparable molecule comprising the wild-type Fc region, wherein said at least one amino acid modification comprises substitution at position 377 with asparagine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one ammo acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with about a 2 fold greater affinity than a comparable molecule comprising the wild-type Fc region as determined by an ELISA assay, wherein said at least one amino acid modification comprises substitution at position 379 with methionine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one ammo acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIIA with about a 1.5 fold greater affinity than a comparable molecule comprising the wild-type Fc region as determined by an ELISA assay, wherein said at least one amino acid modification comprises substitution at position 248 with methionine.
  • the molecules of the invention have an altered affinity for Fc ⁇ RIIIA and/or Fc ⁇ RIIA as determined using in vitro assays (biochemical or immunological based assays) known in the art for determining Fc-Fc ⁇ R interactions, Le., specific binding of an Fc region to an Fc ⁇ R including but not limited to ELISA assay, surface plasmon resonance assay, immunoprecipitation assays (See Section 5.2.1).
  • the binding properties of these molecules with altered affinities for activating Fc ⁇ R receptors are also correlated to their activity as determined by in vitro functional assays for determining one or more Fc ⁇ R mediator effector cell functions, e.g., molecules with variant Fc regions with enhanced affinity for Fc ⁇ RIIIA have a conferred or an enhanced ADCC activity.
  • the molecules of the invention that have an altered binding property for an activating Fc receptor, e.g., Fc ⁇ RHIA in an in vitro assay also have an altered binding property in in vivo models (such as those described and disclosed herein).
  • the present invention does not exclude molecules of the invention that do not exhibit an altered Fc ⁇ R binding in in vitro based assays but do exhibit the desired phenotype in vivo.
  • the molecules of the invention comprise a variant
  • Fc region having one or more amino acid modifications (Le., substitutions) in one or more regions, which one or more modifications increase the affinity of the variant Fc region for Fc ⁇ RIIIA and decrease the affinity of the variant Fc region for Fc ⁇ RIIB, relative to a comparable molecule comprising a wild-type Fc region which binds Fc ⁇ RIIIA and Fc ⁇ RIIB witn wild-type attinity.
  • the one or more amino acid modifications do not include or are not solely a substitution with alanine at any of positions 256, 298, 333, 334, 280, 290, 294, 298, or 296; or a substitution at position 298 with asparagine, valine, aspartic acid, or proline; or a substitution 290 with serine.
  • the one or more amino acid modifications increases the affinity of the variant Fc region for Fc ⁇ RIIIA by at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 200%, at least 300%, at least 400% and decreases the affinity of the variant Fc region for Fc ⁇ RIIB by at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 200%, at least 300%, at least 400%.
  • the molecule of the invention comprising a variant
  • Fc region with an enhanced affinity for Fc ⁇ RIIIA and a lowered affinity or no affinity for Fc ⁇ RIIB, as determined based on an ELISA assay and/or an ADCC based assay using ch-4- 4-20 antibody carrying the variant Fc region comprises a substitution at any of the following: at position 275 with isoleucine, at position 334 with asparagine, and at position 348 with methionine; or a substitution at position 279 with leucine and at position 395 with serine; or a substitution at position 246 with threonine and at position 319 with phenylalanine; or a substitution at position 243 with leucine, at position 255 with leucine, and at position 318 with lysine; or a substitution at position 334 with glutamic acid, at position 359 with asparagine and at position 366 with serine; or a substitution at position 334 with glutamic acid and at position 380 with aspartic acid; or a substitution at position 256 with
  • the molecule of the invention comprising a variant
  • Fc region with an enhanced affinity for Fc ⁇ RIIIA and a lowered affinity or no affinity for Fc ⁇ RIIB as determined based on an ELISA assay and/or an ADCC based assay using ch-4- 4-20 antibody carrying the variant Fc region comprises a substitution at position 379 with methionine; at position 219 with tyrosine; at position 282 with methionine; at position 401 with valine; at position 222 with asparagine; at position 334 with isoleucine; at position 334 with glutamic acid; at position 275 with tyrosine; at position 398 with valine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIB with about a 3 fold lower affinity than a comparable molecule comprising the wild-type Fc region as determined by an ELISA assay, wherein said at least one amino acid modification comprises substitution at position 288 with asparagine, at position 330 with serine, and at position 396 with leucine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIB with about a 10-15 fold lower affinity than a comparable molecule comprising the wild-type Fc region as determined by an ELISA assay, wherein said at least one amino acid modification comprises substitution at position 316 with aspartic acid, at position 378 with valine, and at position 399 with glutamic acid.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIB with about a 10 fold lower affinity than a comparable molecule comprising the wild-type Fc region as determined by an ELISA assay, wherein said at least one amino acid modification comprises substitution at position 315 with isoleucine, at position 379 with methionine, and at position 399 with glutamic acid.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIB with about a 7 fold lower affinity than a comparable molecule comprising the wild-type Fc region as determined by an ELISA assay, wherein said at least one amino acid modification comprises substitution at position 243 with isoleucine, at position 379 with leucine, and at position 420 with valine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIB with about a 3 fold lower affinity than a comparable molecule comprising the wild-type Fc region as determined by an ELISA assay, wherein said at least one amino acid modification comprises substitution at position 392 with threonine and at position 396 with leucine.
  • the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIB with about a 5 fold lower affinity than a comparable molecule comprising the wild-type Fc region as determined by an ELISA assay, wherein said at least one amino acid modification comprises substitution at position 268 with asparagine and at position 396 with leucine.
  • the invention also encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule specifically binds Fc ⁇ RIIB with about a 2 fold lower affinity than a comparable molecule comprising the wild-type Fc region as determined by an ELISA assay, wherein said at least one amino acid modification comprises substitution at position 319 with phenylalanine, at position 352 with leucine, and at position 396 with leucine.
  • the invention encompasses molecules comprising variant Fc regions, having one or more amino acid modifications, which modifications increase the affinity of the variant Fc region for Fc ⁇ RIIIA and Fc ⁇ RIIB by at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 200%, at least 300%, at least 400% and decreases the affinity of the variant Fc region for Fc ⁇ RIIB by at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 200%, at least 300%, at least 400%.
  • the molecule of the invention comprising a variant Fc region with an enhanced affinity for Fc ⁇ RIIIA and an enhanced affinity for Fc ⁇ RIIB (as determined based on an ELISA assay and/or an ADCC based assay using ch-4-4-20 antibody carrying the variant Fc region as described herein) comprises a substitution at position 415 with isoleucine and at position 251 with phenylalanine; or a substitution at position 399 with glutamic acid, at position 292 with leucine, and at position 185 with methionine; or a substitution at position 408 with isoleucine, at position 215 with isoleucine, and at position 125 with leucine; or a substitution at position 385 with glutamic acid and at position 247 with histidine; or a substitution at position 348 with methionine, at position 334 with asparagine, at position 275 with isoleucine, at position 202 with methionine and at position 147 with th
  • the invention encompasses molecules, e.g. , immunoglobulins comprising Fc variants with altered effector functions, preferably, added effector functions, i.e., where the variants exhibit detectable levels of one or more effector functions that are not detectable in the parent antibody.
  • immunoglobulins comprising Fc variants mediate effector function more effectively in the presence of effector cells as determined using assays known in the art and exemplified herein.
  • the Fc variants of the invention may be combined with other known Fc modifications that enhance effector function, such that the combination has an additive, synergistic effect.
  • the Fc variants of the invention have conferred or enhanced effector function in vitro and/or in vivo.
  • the immunoglobulins of the invention have an enhanced Fc ⁇ R-mediated effector function as determined using ADCC activity assays disclosed herein.
  • effector functions that could be mediated by the molecules of the invention include, but are not limited to,. CIq binding, complement-dependent cytotoxicity, antibody-dependent cell mediate cytotoxicity (ADCC), phagocytosis, etc.
  • the effector functions of the molecules of the invention can be assayed using standard methods known in the art, examples of which are disclosed in Section 5.2.7.
  • the immunoglobulins of the invention comprising a variant Fc region mediate ADCC where the parent molecule does not exhibit detectable levels of ADCC activity or induces ADCC 2- fold more effectively, than an immunoglobulin comprising a wild-type Fc region.
  • the immunoglobulins of the invention comprising a variant Fc region mediate ADCC where the parent molecule does not exhibit detectable levels of ADCC activity or induces ADCC at least 4- fold, at least 8-fold, at least 10-fold, at least 100-fold, at least 1000-fold, at least 10 4 -fold, at least 10 s -fold more effectively, than an immunoglobulin comprising a wild-type Fc region.
  • the immunoglobulins of the invention have altered CIq binding activity.
  • the immunoglobulins of the invention mediate CIq binding activity where the parent molecule does not exhibit detectable levels of CIq binding activity or has at least 2-fold, at least 4- fold, at least 8-fold, at least 10-fold, at least 100-fold, at least 1000-fold, at least 10 4 -fold, at least 10 5 -fold higher CIq binding activity than an immunoglobulin comprising a wild-type Fc region.
  • the immunoglobulins of the invention have altered complement dependent cytotoxicity.
  • the immunoglobulins of the invention have complement dependent cytotoxicity where the parent molecule does not exhibit detectable levels of complement dependent cytotoxicity or enhances complement dependent cytotoxicity to levels greater than an immunoglobulin comprising a wild-type Fc region.
  • the immunoglobulins of the invention have at least 2-fold, at least 4- fold, at least 8-fold, at least 10-fold, at least 100-fold, at least 1000-fold, at least 10 4 -fold, at least 10 5 -fold higher complement dependent cytotoxicity than an immunoglobulin comprising a wild-type Fc region.
  • immunoglobulins of the invention have phagocytosis activity where the parent molecule does not exhibit detectable levels of phagocytosis activity or have enhanced phagocytosis activity relative to an immunoglobulin comprising a wild-type Fc region, as determined by standard assays known to one skilled in the art or disclosed herein.
  • the immunoglobulins of the invention have at least 2-fold, at least 4- fold, at least 8-fold, at least 10-fold higher phagocytosis activity relative to an immunoglobulin comprising a wild-type Fc region.
  • the invention encompasses an immunoglobulin comprising a variant Fc region with one or more amino acid modifications, such that the immunoglobulin has an effector function, e.g., antibody dependent cell mediated cytotoxicity or phagocytosis, where the parent molecule does not exhibit detectable levels of the effector function or has an enhanced effector function,.
  • an effector function e.g., antibody dependent cell mediated cytotoxicity or phagocytosis
  • the one or more amino acid modifications which increase the ADCC activity of the immunoglobulin comprise a substitution at position 379 with methionine; or a substitution at position 243 with isoleucine and at position 379 with leucine; or a substitution at position 288 with asparagine, at position 330 with serine, and at position 396 with leucine; or a substitution at position 243 leucine and at position 255 with leucine; or a substitution at position 334 with glutamic acid, at position 359 with asparagine, and at position 366 with serine; or a substitution at position 288 with methionine and at position 334 with glutamic acid; or a substitution at position 334 with glutamic acid and at position 292 with leucine; or a substitution at position 316 with aspartic acid, at position 378 with valine, and at position 399 with glutamic acid; or a substitution at position 315 with isoleucine, at position 379 with methionine, and
  • the variant Fc region has a leucine at position 247, a lysine at position 421 and a glutamic acid at position 270 (MgFc31/60); a threonine at position 392, a leucine at position 396, a glutamic acid at position 270, and a leucine at position 243 (MgFc38/60/F243L); a histidine at position 419, a leucine at position 396, and a glutamic acid at position 270 (MGFc51/60); a histidine at position 419, a leucine at position 396, a glutamic acid at position 270, and a leucine at position 243 (MGFc51/60/F243L); an alanine at position 240, a leucine at position 396, and a glutamic acid at position 270 (MGFc52/60); a lysine at position 255 and a leucine at
  • the one or more amino acid modifications which confers or increases the ADCC activity of the immunoglobulin is any of the mutations listed below, in table 7. TABLE 7. AMESfO ACID MODIFICATIONS WHICH CONFER OR INCREASE
  • the starting molecule may be unable to bind CIq and/or mediate CDC and may be modified according to the teachings herein such that it acquires these further effector functions.
  • molecules with preexisting CIq binding activity, optionally further having the ability to mediate CDC may be modified such that one or both of these activities are enhanced.
  • one can design an Fc region with altered effector function e.g., by modifying or conferring CIq binding and/or FcR binding and thereby changing CDC activity and/or ADCC activity.
  • the invention encompasses molecules with specific variants of the Fc region that have been identified using the methods of the invention from a yeast library of mutants after 2nd-4th-round of sorting are listed in Table 8.
  • Table 8 summarizes the various mutants that were identified using the methods of the invention.
  • the mutants were assayed using an ELISA assay for determining binding to Fc ⁇ RIIIA and Fc ⁇ RIIB.
  • the mutants were also tested in an ADCC assay, by cloning the Fc variants into a ch 4-4-20 antibody using methods disclosed and exemplified herein.
  • Bolded items refer to experiments, in which the ch4-4-20 were purified prior the ADCC assay.
  • the antibody concentration used was standard for ADCC assays, in the range 0.5 ⁇ g/mL - 1.0 ⁇ g/mL.
  • the invention provides modified molecules with variant Fc regions, having one or more amino acid modifications, which one or more amino acid modifications confer an effector function and/or increase the affinity of the molecule for Fc ⁇ RIIIA and/or Fc ⁇ RIIA.
  • modified molecules include IgG molecules that naturally contain Fc ⁇ R binding regions (e.g., Fc ⁇ RIIIA and/or Fc ⁇ RIIB binding region), or immunoglobulin derivatives that have been engineered to contain an Fc ⁇ R binding region (e.g., Fc ⁇ RIIIA and/or Fc ⁇ RIIB binding region).
  • the modified molecules of the invention include any immunoglobulin molecule that binds, preferably, immunospecifically, i.e., competes off non-specific binding as determined by immunoassays well known in the art for assaying specific antigen-antibody binding, an antigen and contains an Fc ⁇ R binding region (e.g., a Fc ⁇ RIIIA and/or Fc ⁇ RIIB binding region).
  • immunoglobulin molecule that binds, preferably, immunospecifically, i.e., competes off non-specific binding as determined by immunoassays well known in the art for assaying specific antigen-antibody binding, an antigen and contains an Fc ⁇ R binding region (e.g., a Fc ⁇ RIIIA and/or Fc ⁇ RIIB binding region).
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, bi-specific, multi-specific, human, humanized, chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, disulfide-linked Fvs, Fc fusions, and fragments containing either a VL or VH domain or even a complementary determining region (CDR) that specifically binds an antigen, in certain cases, engineered to contain or fused to an Fc ⁇ R binding region.
  • CDR complementary determining region
  • the molecules of the invention comprise portions of an Fc region.
  • portion of an Fc region refers to fragments of the Fc region, preferably a portion with effector activity and/or Fc ⁇ R binding activity (or a comparable region of a mutant lacking such activity).
  • the fragment of an Fc region may range in size from 5 amino acids to the entire Fc region minus one amino acids.
  • the portion of an Fc region may be missing up to 10, up to 20, up to 30 amino acids from the N- terminus or C-terminus.
  • the IgG molecules of the invention are preferably IgGl subclass of IgGs, but may also be any other IgG subclasses of given animals.
  • the IgG class includes IgGl, IgG2, IgG3, and IgG4; and mouse IgG includes IgGl, IgG2a, IgG2b, IgG2c and IgG3.
  • the immunoglobulins may be from any animal origin including birds and mammals.
  • the antibodies are human, rodent (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken.
  • "human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example, in U.S. Patent No.
  • the antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide or may be specific for heterologous epitopes, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al, J. Immunol., 147:60- 69, 1991; U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny etal., J. Immunol, 148:1547-1553, 1992.
  • Multispecific antibodies have binding specificities for at least two different antigens. While such molecules normally will only bind two antigens (i.e. bispecific antibodies, BsAbs), antibodies with additional specificities such as trispecific antibodies are encompassed by the instant invention. Examples of BsAbs include without limitation those with one arm directed against a tumor cell antigen and the other arm directed against a cytotoxic molecule. [00184] Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al, Nature, 305:537-539 (1983); which is incorporated herein by reference in its entirety).
  • the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHl) containing the site necessary for light chain binding, present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co- transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields.
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation.
  • a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains ⁇ e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones ⁇ e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies include cross-linked or "heteroconjugate” antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089).
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared. See, e.g., Tutt et al, 1991, J. Immunol. 147: 60, which is incorporated herein by reference.
  • the antibodies of the invention include derivatives that are otherwise modified, Le., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding antigen and/or generating an anti-idiotypic response.
  • the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a constant region derived from a human immunoglobulin.
  • Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science, 229:1202, 1985; Oi et al, BioTechniques, 4:214 1986; Gillies et al, J. Immunol. Methods, 125:191-202, 1989; U.S. Patent Nos.
  • Humanized antibodies are antibody molecules from non-human species that bind the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions and constant domains from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. See, e.g., Queen et al, U.S. Patent No. 5,585,089;
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology, 28(4/5):489-498, 1991; Studnicka et al, Protein Engineering, 7(6):805-814, 1994; Roguska et al., ProcNatl Acad.
  • Human antibodies are particularly desirable for therapeutic treatment of human patients.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741, each of which is incorporated herein by reference in its entirety.
  • Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • the invention encompasses engineering human or humanized therapeutic antibodies (e.g., tumor specific monoclonal antibodies) in the Fc region, by modification (e.g., substitution, insertion, deletion) of at least one amino acid residue, which modification confers an effector function activity, e.g., enhanced ADCC activity, phagocytosis activity, etc., as determined by standard assays known to those skilled in the art.
  • the engineered therapeutic antibodies may further have increased affinity of the Fc region for Fc ⁇ RHIA and/or Fc ⁇ RIIA. In other embodiments, the engineered therapeutic antibodies may exhibit oligomerization activity mediated by the variant Fc region.
  • the invention relates to engineering human or humanized therapeutic antibodies (e.g., tumor specific monoclonal antibodies) in the Fc region, by modification of at least one amino acid residue, which modification increases the affinity of the Fc region for Fc ⁇ RIIIA and/or Fc ⁇ RIIA and further decreases the affinity of the Fc region for Fc ⁇ RIIB.
  • the invention encompasses engineering an monoclonal antibody by modification (e.g., substitution, insertion, deletion) of at least one amino acid residue in the Fc region which modification confers an effector function as determined by standard assays known in the art and disclosed and exemplified herein.
  • modification of the monoclonal antibody increases the affinity of the Fc rgion for Fc ⁇ RIIIA and/or Fc ⁇ RIIA. In another specific embodiment, modification of the monoclonal antibody may also further decrease the affinity of the Fc region for Fc ⁇ RIIB.
  • the invention encompasses a modified molecule comprising an Fc chain with a substitution at position 255 with leucine, at position 396 with leucine, at position 270 with glutamic acid, and at position 300 with leucine; or a substitution at position 419 with histidine, at position 396 with leucine, and at position 270 with glutamic acid; or a substitution at position 240 with alanine, at position 396 with leucine, and at position 270 with glutamic acid; or a substitution at position 370 with glutamic acid, at position 396 with leucine, and at position 270 with glutamic acid; or a substitution at position 392 with threonine, at position 396 with leucine, and at position 270 with glutamic acid; or a substitution at position 370 with glutamic acid and at position 396 with leucine; or a substitution at position 419 with histidine and at position 396 with leucine; or a substitution at position 2
  • the variant Fc region has a leucine at position 247, a lysine at position 421 and a glutamic acid at position 270 (MgFc31/60); a threonine at position 392, a leucine at position 396, a glutamic acid at position 270, and a leucine at position 243 (MgFc38/60/F243L); a histidine at position 419, a leucine at position 396, and a glutamic acid at position 270 (MGFc51/60); a histidine at position 419, a leucine at position 396, a glutamic acid at position 270, and a leucine at position 243 (MGFc51/60/F243L); an alanine at position 240, a leucine at position 396, and a glutamic acid at position 270 (MGFc52/60); a lysine at position 255 and a leucine at
  • Molecules of the invention comprising variant Fc regions may be recombinantly fused or chemically conjugated (including both covalently and non- covalently conjugations) to heterologous polypeptides (Le., an unrelated polypeptide; or portion thereof, preferably at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids of the polypeptide) to generate fusion proteins.
  • heterologous polypeptides Le., an unrelated polypeptide; or portion thereof, preferably at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids of the polypeptide
  • the fusion does not necessarily need to be direct, but may occur through linker sequences.
  • molecules of the invention comprising variant Fc regions may be conjugated to a therapeutic agent or a drug moiety that modifies a given biological response.
  • Therapeutic agents or drug moieties are not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin (Le., PE-40), or diphtheria toxin, ricin, gelonin, and pokeweed antiviral protein, a protein such as tumor necrosis factor, interferons including, but not limited to, ⁇ -interferon (IFN- ⁇ ), ⁇ -interferon (IFN- ⁇ ), nerve growth factor (NGF), platelet derived growth factor (PDGF), tissue plasminogen activator (TPA), an apoptotic agent (e.g., TNF- ⁇ , TNF- ⁇ , AIM I as disclosed in PCT Publication No.
  • a toxin such as abrin, ricin A, pseudomonas exotoxin (Le., PE-40), or diphtheria toxin, ricin, gelonin, and pokeweed antiviral protein
  • a protein such as tumor necrosis factor
  • interferons including, but not
  • WO 97/33899 AIM II (see, PCT Publication No. WO 97/34911), Fas Ligand (Takahashi et al, J. Immunol., 6:1567-1574, 1994), and VEGI (PCT Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent (e.g.
  • angiostatin or endostatin or a biological response modifier such as, for example, a lymphokine (e.g., interleukin-1 ("IL- 1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), and granulocyte colony stimulating factor (“G-CSF”), macrophage colony stimulating factor, (“M-CSF'), or a growth factor (e.g., growth hormone (“GH”); proteases, or ribonucleases.
  • a lymphokine e.g., interleukin-1 ("IL- 1"), interleukin-2 ("IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), and granulocyte colony stimulating factor (“G-CSF”), macrophage colony stimulating factor, (“M-CSF'), or a growth factor (e.g., growth hormone (“GH”); protea
  • Molecules of the invention can be fused to marker sequences, such as a peptide to facilitate purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
  • a pQE vector QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311
  • hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell, 37:767 1984) and the "flag” tag (Knappik et al, Biotechniques, 17(4):754-761, 1994).
  • HA hemagglutinin
  • DNA shuffling may be employed to alter the activities of molecules of the invention (e.g., antibodies with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al, 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol.
  • Molecules of the invention comprising variant Fc regions, or the nucleic acids encoding the molecules of the invention, may be further altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • One or more portions of a polynucleotide encoding a molecule of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc.
  • the present invention also encompasses molecules of the invention comprising variant Fc regions conjugated to a diagnostic or therapeutic agent or any other molecule for which serum half-life is desired to be increased and/or targeted to a particular subset of cells.
  • the molecules of the invention can be used diagnostically to, for example, monitor the development or progression of a disease, disorder or infection as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the molecules of the invention to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions.
  • the detectable substance may be coupled or conjugated either directly to the molecules of the invention or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
  • Such diagnosis and detection can be accomplished by coupling the molecules of the invention to detectable substances including, but not limited to, various enzymes, enzymes including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic group complexes such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent materials such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent material such as, but not limited to, luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin, and aequorin; radioactive material such as, but not limited to, bismuth ( 213 Bi), carbon ( 14 C), chromium (
  • Molecules of the invention comprising a variant Fc region may be conjugated to a therapeutic moiety such as a cytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agent or a radioactive element (e.g., alpha-emitters, gamma-emitters, etc.).
  • cytotoxin e.g., a cytostatic or cytocidal agent
  • a therapeutic agent e.g., a therapeutic agent
  • a radioactive element e.g., alpha-emitters, gamma-emitters, etc.
  • Cytotoxins or cytotoxic agents include any agent that is detrimental to cells.
  • Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC), and anti-mitotic agents (e.g., vin
  • a molecule of the invention can be conjugated to therapeutic moieties such as a radioactive materials or macrocyclic chelators useful for conjugating radiometal ions (see above for examples of radioactive materials).
  • the macrocyclic chelator is l,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA) which can be attached to the antibody via a linker molecule.
  • DOTA l,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid
  • linker molecules are commonly known in the art and described in Denardo et al , 1998, Clin Cancer Res. 4:2483-90; Peterson et al, 1999, Bioconjug. Chem.
  • the molecule of the invention is an antibody comprising a variant Fc region
  • it can be administered with or without a therapeutic moiety conjugated to it, administered alone, or in combination with cytotoxic factor(s) and/or cytokine(s) for use as a therapeutic treatment.
  • an antibody of the invention can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
  • Antibodies of the invention may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
  • Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the affinities and binding properties of the molecules of the invention for an Fc ⁇ R are initially determined using in vitro assays (biochemical or immunological based assays) known in the art for determining Fc-Fc ⁇ R interactions, Le. , specific binding of an Fc region to an Fc ⁇ R including but not limited to ELISA assay, surface plasmon resonance assay, immunoprecipitation assays.
  • the binding properties of the molecules of the invention are also characterized by in vitro functional assays for determining one or more Fc ⁇ R mediator effector cell functions.
  • the antibodies of the invention have similar binding properties in in vivo models (such as those described and disclosed herein) as those in in vitro based assays.
  • screening and identifying molecules comprising variant Fc regions with altered Fc ⁇ R affinities are done using the yeast display technology as described herein in combination with one or more biochemical based assays, preferably in a high throughput manner.
  • screening and identifying molecules comprising variant Fc regions with altered Fc ⁇ R affinities are done using the yeast display technology as described herein in combination with one or more functional based assays, preferably in a high throughput manner.
  • the functional based assays can be any assay known in the art for characterizing one or more Fc ⁇ R mediated effector cell functions such as those described herein in Section 5.2.7.
  • Non-limiting examples of effector cell functions include but are not limited to, antibody- dependent cell mediated cytotoxicity (ADCC), antibody-dependent phagocytosis, phagocytosis, opsonization, opsonophagocytosis, cell binding, rosetting, CIq binding, and complement dependent cell mediated cytotoxicity.
  • ADCC antibody- dependent cell mediated cytotoxicity
  • phagocytosis phagocytosis
  • opsonization opsonophagocytosis
  • cell binding rosetting
  • CIq binding complement dependent cell mediated cytotoxicity
  • the term "specific binding" of an Fc region to an Fc ⁇ R refers to an interaction of the Fc region and a particular Fc ⁇ R which has an affinity constant of at least about 150 nM, in the case of monomeric Fc ⁇ RIIIA and at least about 60 nM in the case of dimeric FeyRIIB as determined using, for example, an ELISA or surface plasmon resonance assay (e.g., a BIAcoreTM).
  • the affinity constant of an Fc region for monomeric Fc ⁇ RIIIA may be 150 nM, 200 nM or 30OnM.
  • the affinity constant of an Fc region for dimeric Fc ⁇ RIIB may be 60 nM, 80 nM, 90 nM, or 100 nM.
  • Dimeric Fc ⁇ RIIB for use in the methods of the invention may be generated using methods known to one skilled in the art.
  • the extracellular region of Fc ⁇ RIIB is covalently linked to a heterologous polypeptide which is capable of dimerization, so that the resulting fusion protein is a dimer, e.g., see, U.S. Application No. 60/439,709 filed on January 13, 2003 (Attorney Docket No. 11183-005-888), which is incorporated herein by reference in its entirety.
  • a specific interaction generally is stable under physiological conditions, including, for example, conditions that occur in a living individual such as a human or other vertebrate or invertebrate, as well as conditions that occur in a cell culture such conditions as used for maintaining and culturing mammalian cells or cells from another vertebrate organism or an invertebrate organism.
  • screening for and identifying molecules comprising variant Fc regions and altered Fc ⁇ R affinities comprise: displaying the molecule comprising a variant Fc region on the yeast surface; and characterizing the binding of the molecule comprising the variant Fc region to a Fc ⁇ R (one or more), using a biochemical assay for determining Fc-Fc ⁇ R interaction, preferably, an ELISA based assay.
  • the molecule comprising a variant Fc region has been characterized for its interaction with one or more Fc ⁇ Rs and determined to have an altered affinity for one or more Fc ⁇ Rs, by at least one biochemical based assay, e.g., an ELISA assay, the molecule maybe engineered into a complete immunoglobulin, such as a molecule, using standard recombinant DNA technology methods known in the art, and the immunoglobulin comprising the variant Fc region expressed in mammalian cells for further biochemical characterization.
  • a biochemical based assay e.g., an ELISA assay
  • the immunoglobulin into which a variant Fc region of the invention is introduced can be any immunoglobulin including, but not limited to, polyclonal antibodies, monoclonal antibodies, bispecific antibodies, multi- specific antibodies, humanized antibodies, and chimeric antibodies.
  • a variant Fc region is introduced into an immunoglobulin specific for a cell surface receptor, a tumor antigen, or a cancer antigen.
  • the immunoglobulin into which a variant Fc region of the invention is introduced may specifically bind a cancer or tumor antigen for example, including, but not limited to, KS 1/4 pan-carcinoma antigen (Perez and Walker, 1990, J. Immunol.
  • melanoma antigen gp75 (Vijayasardahl et al, 1990, /. Exp. Med. 171(4): 1375-1380), high molecular weight melanoma antigen (HMW-MAA) (Natali et al, 1987, Cancer 59: 55-63; Mittelman et al, 1990, J. Clin. Invest. 86: 2136- 2144), prostate specific membrane antigen, carcinoembryonic antigen (CEA) (Foon et al, 1994, Proc. Am. Soc. CHn. Oncol.
  • HMW-MAA high molecular weight melanoma antigen
  • CEA carcinoembryonic antigen
  • polymorphic epithelial mucin antigen such as: CEA, TAG-72 (Yokata et al, 1992, Cancer Res. 52: 3402-3408), CO17-1A (Ragnhammar et al, 1993, Int. J. Cancer 53: 751-758); GICA 19-9 (Herlyn et al, 1982, /. Clin. Immunol.
  • ganglioside GM2 Livingston et al, 1994, 7. Clin. Oncol. 12: 1036-1044
  • ganglioside GM3 Hoon et al, 1993, Cancer Res. 53: 5244-5250
  • tumor-specific transplantation type of cell-surface antigen TSTA
  • virally-induced tumor antigens including T-antigen DNA tumor viruses and Envelope antigens of RNA tumor viruses
  • oncofetal antigen-alpha-fetoprotein such as CEA of colon
  • bladder tumor oncofetal antigen Hellstrom et al, 1985, Cancer. Res.
  • differentiation antigen such as human lung carcinoma antigen L6, L20 (Hellstrom et al , 1986, Cancer Res. 46: 3917-3923), antigens of fibrosarcoma, human leukemia T cell antigen-Gp37 (Bhattacharya-Chatterjee et al, 1988, J. oflmmun. 141:1398- 1403), neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR (Epidermal growth factor receptor), HER2 antigen (pl85 HER2 ), polymorphic epithelial mucin (PEM) (Hilkens et al, 1992, Trends in Bio. Chem. Sci.
  • PEM polymorphic epithelial mucin
  • malignant human lymphocyte antigen-APO-1 (Bernhard et al, 1989, Science 245: 301-304), differentiation antigen (Feizi, 1985, Nature 314: 53-57) such as I antigen found in fetal erythrocytes, primary endoderm I antigen found in adult erythrocytes, preimplantation embryos, I(Ma) found in gastric adenocarcinomas, Ml 8, M39 found in breast epithelium, SSEA-I found in myeloid cells, VEP8, VEP9, MyI, VIM-D5, Di56-22 found in colorectal cancer, TRA-1-85 (blood group H), C 14 found in colonic adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten, Le y found in embryonal carcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells , E 1 series (blood group B) found in pancre
  • a variant Fc region of the invention is introduced into an anti-fluoresceine monoclonal antibody, 4-4-20 (Kranz et al, 1982, J. Biol. Chem. 257(12): 6987-6995; which is incorporated herein by reference in its entirety).
  • a variant Fc region of the invention is introduced into a mouse-human chimeric anti-CD20 monoclonal antibody, 2H7, which recognizes the CD20 cell surface phosphoprotein on B cells (Liu et al, 1987, Journal of Immunology, 139: 3521-6; which is incorporated herein by reference in its entirety).
  • the monoclonal antibody is not an anti-CD20 antibody.
  • a variant Fc region of the invention is introduced into a humanized antibody (Ab4D5) against the human epidermal growth factor receptor 2 (pl85 HER2) as described by Carter et al (1992, Proc. Natl. Acad. Sci. USA 89: 4285-9; which is incorporated herein by reference in its entirety).
  • a variant Fc region of the invention is introduced into a humanized anti- TAG72 antibody (CC49) (Sha et al, 1994 Cancer Biother. 9(4): 341-9; which is incorporated herein by reference in its entirety).
  • a variant Fc region of the invention is introduced into Rituxan (humanized anti-CD20 antibody; rituximab) (International Patnet Publication No. WO 02/096948; which is incorporated herein by reference in its entirety ) which is used for treating lymphomas.
  • Rituxan humanized anti-CD20 antibody; rituximab
  • International Patnet Publication No. WO 02/096948 International Patnet Publication No. WO 02/096948; which is incorporated herein by reference in its entirety
  • the invention encompasses engineering an anti-Fc ⁇ RIIB antibody including but not limited to any of the antibodies disclosed in U.S. Provisional Application No. 60/403,266 filed on August 12, 2002; U.S. Application No. 10/643,857 filed on August 14, 2003 (having Attorney Docket No. 011183-010-999); the U.S. Provisional Application No. 60/562,804 (having Attorney Docket No. 011183-014- 888) that was filed on April 16, 2004; U.S. Provisional Application No. 60/569,882 (having Attorney Docket No. 011183-013-888) that was filed on May 10, 2004 and U.S. Provisional Application Nos.
  • modification e.g., substitution, insertion, deletion
  • anti-Fc ⁇ RIIB antibodies that may be engineered in accordance with the methods of the invention are 2B6 monoclonal antibody having ATCC accession number PTA-4591 and 3H7 having ATCC accession number PTA-4592, 1D5 monoclonal antibody having ATCC accession number PTA-5958, 1F2 monoclonal antibody having ATCC accession number PT A-5959, 2D11 monoclonal antibody having ATCC accession number PTA-5960, 2El monoclonal antibody having ATCC accession number PTA-5961 and 2H9 monoclonal antibody having ATCC accession number PTA-5962 (all deposited at 10801 University Boulevard, Manassas, VA 02209-2011), which are incorporated herein by reference.
  • modification of the anti- FcyRIIB antibody may also further decrease the affinity of the Fc region for Fc ⁇ RIEB.
  • the engineered anti-Fc ⁇ RIIB antibody may further have an enhanced effector function as determined by standard assays known in the art and disclosed and exemplified herein.
  • a variant Fc region of the invention is introduced into a therapeutic monoclonal antibody specific for a cancer antigen or cell surface receptor including but not limited to, ErbituxTM (also known as IMC-C225) (ImClone Systems Inc.), a chimerized monoclonal antibody against EGFR; HERCEPTIN® (Trastuzumab) (Genentech, CA) which is a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer; REOPRO® (abciximab) (Centocor) which is an anti-glycoprotein Ilb/IIIa receptor on the platelets for the prevention of clot formation; ZENAP AX® (daclizumab) (Roche Pharmaceuticals, Switzerland) which is an immunosuppressive, humanized anti-CD25 monoclonal antibody for the prevention of acute renal allograft rejection.
  • ErbituxTM also known as IMC-C225
  • HERCEPTIN® Trastuzumab) (
  • a humanized anti-CD 18 F(ab') 2 (Genentech); CDP860 which is a humanized anti-CD 18 F(ab') 2 (Celltech, UK); PRO542 which is an anti- HIV gpl20 antibody fused with CD4 (Progenics/Genzyme Transgenics); C14 which is an anti-CD14 antibody (ICOS Pharm); a humanized anti-VEGF IgGl antibody (Genentech); OVAREXTM which is a murine anti-CA 125 antibody (Altarex); PANOREXTM which is a murine anti-17-IA cell surface antigen IgG2a antibody (Glaxo Wellcome/Centocor); IMC- C225 which is a chimeric anti-EGFR IgG antibody (ImClone System); VITAXINTM which is a humanized anti- ⁇ V ⁇ 3 integrin antibody (Applied Molecular Evolution/Medlmmune); Campath 1H/LDP-03 which is a humanized anti-
  • LYMPHOCIDETM which is a humanized anti-CD22 IgG antibody (Immunomedics); Smart IDlO which is a humanized anti-HLA antibody (Protein Design Lab); ONCOLYMTM (Lym-1) is a radiolabeled murine anti-HLA DR antibody (Techniclone); anti-CDlla is a humanized IgGl antibody (Genetech/Xoma); ICM3 is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC- 114 is a primatized anti- CD80 antibody (IDEC Pharm/Mitsubishi); ZEVALINTM is a radiolabelled murine anti- CD20 antibody (IDEC/Schering AG); IDEC-131 is a humanized anti-CD40L antibody (EDEC/Eisai); EDEC-151 is a primatized anti-CD4 antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody (
  • Corsevin M is a chimeric anti-Factor VII antibody (Centocor). In certain embodiments, the antibody is not RITUXANTM.
  • the variant Fc regions of the invention can be further characterized using one or more biochemical assays and/or one or more functional assays, preferably in a high throughput manner.
  • the variant Fc regions of the inventions are not introduced into an immunoglobulin and are further characterized using one or more biochemical based assays and/or one or more functional assays, preferably in a high throughput manner.
  • the one or more biochemical assays can be any assay known in the art for identifying immunoglobulin- antigen or Fc-Fc)R interactions, including, but not limited to, an ELISA assay, and surface plasmon resonance-based assay, e.g., BIAcore assay, for determining the kinetic parameters of immunoglobulin-antigen or Fc-Fc ⁇ R interaction. Characterization of target antigen binding affinity or assessment of target antigen density on a cell surface may be assessed by methods well known in the art such as Scatchard analysis or by the use of kits as per manufacturer's instructions, such as QuantumTM Simply Cellular ® (Bangs Laboratories, Inc., Fishers, IN).
  • the one or more functional assays can be any assay known in the art for characterizing one or more Fc ⁇ R mediated effector cell function as known to one skilled in the art or described herein.
  • the immunoglobulins comprising the variant Fc regions are assayed in an ELISA assay for binding to one or more Fc ⁇ Rs, e.g., Fc ⁇ RIIIA, Fc ⁇ RIIA, Fc ⁇ RIIA; followed by one or more ADCC assays.
  • the immunoglobulins comprising the variant Fc regions are assayed further using a surface plasmon resonance-based assay, e.g., BIAcore. Surface plasmon resonance-based assays are well known in the art, and are further discussed in Section 5.2.7, and exemplified herein in Example 6.8.
  • An exemplary high throughput assay for characterizing immunoglobulins comprising variant Fc regions may comprise: introducing a variant Fc region of the invention, e.g., by standard recombinant DNA technology methods, in a 4-4-20 antibody; characterizing the specific binding of the 4-4-20 antibody comprising the variant Fc region to an Fc ⁇ R (e.g., FcyRIIIA, Fc ⁇ RIIB) in an ELISA assay; characterizing the 4-4-20 antibody comprising the variant Fc region in an ADCC assay (using methods disclosed herein) wherein the target cells are opsonized with the 4-4-20 antibody comprising the variant Fc region; the variant Fc region may then be cloned into a second immunoglobulin, e.g., 4D5, 2H7, and that second immunoglobulin characterized in an ADCC assay, wherein the target cells are opsonized with the second antibody comprising the variant Fc region.
  • the second antibody comprising the variant Fc region is then be
  • a variant Fc region of the invention binds Fc ⁇ RIIIA and/or
  • Fc ⁇ RIIA with a higher affinity than a wild type Fc region as determined in an ELISA assay.
  • a variant Fc region of the invention binds Fc ⁇ RIIIA and/or Fc)RIIA with a higher affinity and binds Fc ⁇ RIIB with a lower affinity than a wild type Fc region as determined in an ELISA assay.
  • the variant Fc region binds Fc)RIIIA and/or Fc ⁇ RIIA with at least 2-fold higher, at least 4-fold higher, more preferably at least 6-fold higher, most preferably at least 8 to 10-fold higher affinity than a wild type Fc region binds Fc ⁇ RIIIA and/or Fc)RIIA and binds Fc)RIIB with at least 2-fold lower, at least 4-fold lower, more preferably at least 6-fold lower, most preferably at least 8 to 10- fold lower affinity than a wild type Fc region binds Fc ⁇ RIIB as determined in an ELISA assay.
  • the immunoglobulin comprising the variant Fc regions may be analyzed at any point using a surface plasmon based resonance based assay, e.g., BIAcore, for defining the kinetic parameters of the Fc-Fc ⁇ R interaction, using methods disclosed herein and known to those of skill in the art.
  • a surface plasmon based resonance based assay e.g., BIAcore
  • the Kd of a variant Fc region of the invention for binding to a monomelic Fc)RIIIA and/or Fc ⁇ RIIA as determined by BIAcore analysis is about 100 nM, preferably about 70 nM, most preferably about 40 nM.; and the Kd of the variant Fc region of the invention for binding a dimeric Fc ⁇ RIIB is about 80 nM, about 100 nM, more preferably about 200 nM.
  • the immunoglobulin comprising the variant Fc regions is further characterized in an animal model for interaction with an Fc ⁇ R.
  • Preferred animal models for use in the methods of the invention are, for example, transgenic mice expressing human FeyRs, e.g., any mouse model described in U.S. Patent No. 5,877,397, and 6,676,927 which are incorporated herein by reference in their entirety.
  • Transgenic mice for use in the methods of the invention include, but are not limited to, nude knockout Fc ⁇ RIIIA mice carrying human Fc ⁇ RIIIA; nude knockout Fc ⁇ RIIIA mice carrying human Fc ⁇ RIIA; nude knockout Fc ⁇ RIIIAmice carrying human Fc ⁇ RIIB and human Fc ⁇ RIIIA; nude knockout Fc ⁇ RIIIA mice carrying human Fc ⁇ RIIB and human Fc ⁇ RIIA; nude knockout Fc ⁇ RIIIA and Fc ⁇ RIIA mice carrying human Fc ⁇ RIIIA and Fc ⁇ RIIA and nude knockout Fc ⁇ RIIIA, Fc ⁇ RIIA and Fc ⁇ RIIB mice carrying human Fc ⁇ RIIIA, Fc ⁇ RIIA and Fc ⁇ RIIB mice carrying human Fc ⁇ RIIIA, Fc ⁇ RIIA and Fc ⁇ RIIB.
  • the present invention encompasses engineering methods to generate Fc variants including but not limited to computational design strategies, library generation methods, and experimental production and screening methods. These strategies may be applied individually or in various combinations to engineer the Fc variants of the instant invention.
  • the engineering methods of the invention comprise methods in which amino acids at the interface between an Fc region and the Fc ligand are not modified.
  • Fc ligands include but are not limited to Fc ⁇ Rs, CIq, FcRn, C3, mannose receptor, protein A, protein G, mannose receptor, and undiscovered molecules that bind Fc.
  • Amino acids at the interface between an Fc region and an Fc ligand is defined as those amino acids that make a direct and/ or indirect contact between the Fc region and the ligand, play a structural role in determining the conformation of the interface, or are within at least 3 angstroms, preferably at least 2 angstroms of each other as determined by structural analysis, such as x-ray crystallography and molecular modeling
  • the amino acids at the interface between an Fc region and an Fc ligand include those amino acids that make a direct contact with an Fc ⁇ R based on crystallographic and structural analysis of Fc-Fc ⁇ R interactions such as those disclosed by Sondermann et al, (2000, Nature, 406: 267-273; which is incorporated herein by reference in its entirety).
  • positions within the Fc region that make a direct contact with Fc ⁇ R are amino acids 234-239 (hinge region), amino acids 265-269 (B/C loop), amino acids 297-299 (C '/E loop), and amino acids 327- 332 (F/G) loop.
  • the molecules of the invention comprising variant Fc regions comprise modification of at least one residue that does not make a direct contact with an Fc ⁇ R based on structural and crystallographic analysis, e.g., is not within the Fc- Fc ⁇ R binding site.
  • the engineering methods of the invention do not modify any of the amino acids as identified by Shields et al,. which are located in the CH2 domain of an Fc region proximal to the hinge region, e.g., Leu234-Pro238; Ala327, Pro329, and affect binding of an Fc region to all human Fc ⁇ Rs.
  • the invention encompasses Fc variants with altered
  • Fc ⁇ R affinities and/or altered effector functions such that the Fc variant does not have an amino acid modification at a position at the interface between an Fc region and the Fc ligand.
  • Fc variants in combination with one or more other amino acid modifications which are at the interface between an Fc region and the Fc ligand have a further impact on the particular altered property, e.g. altered Fc ⁇ R affinity.
  • Modifying amino acids at the interface between Fc and an Fc ligand may be done using methods known in the art, for example based on structural analysis of Fc-ligand complexes.
  • variants can be engineered that sample new interface conformations, some of which may improve binding to the Fc ligand, some of which may reduce Fc ligand binding, and some of which may have other favorable properties.
  • new interface conformations could be the result of, for example, direct interaction with Fc ligand residues that form the interface, or indirect effects caused by the amino acid modifications such as perturbation of side chain or backbone conformations
  • the invention encompasses engineering Fc variants comprising any of the amino acid modifications disclosed herein in combination with other modifications in which the conformation of the Fc carbohydrate at position 297 is altered.
  • the invention encompasses conformational and compositional changes in the N297 carbohydrate that result in a desired property, for example increased or reduced affinity for an Fc ⁇ R. Such modifications may further enhance the phenotype of the original amino acid modification of the Fc variants of the invention.
  • Fc region is reengineered to eliminate the structural and functional dependence on glycosylation.
  • This design strategy involves the optimization of Fc structure, stability, solubility, and/or Fc function (for example affinity of Fc for one or more Fc ligands) in the absence of the N297 carbohydrate.
  • positions that are exposed to solvent in the absence of glycosylation are engineered such that they are stable, structurally consistent with Fc structure, and have no tendency to aggregate.
  • Approaches for optimizing aglycosylated Fc may involve but are not limited to designing amino acid modifications that enhance aglycosylated Fc stability and/or solubility by incorporating polar and/or charged residues that face inward towards the Cg2-Cg2 dimer axis, and by designing amino acid modifications that directly enhance the aglycosylated Fc- • Fc ⁇ R. interface or the interface of aglycosylated Fc with some other Fc ligand.
  • the Fc variants of the present invention may be combined with other Fc modifications, including but not limited to modifications that enhance effector function.
  • the invention encompasses combining an Fc variant of the invention with other Fc modifications to provide additive, synergistic, or novel properties in antibodies or Fc fusions. Such modifications may be in the CHl, CH2, or CH3 domains or a combination thereof.
  • the Fc variants of the invention enhance the property of the modification with which they are combined. For example, if an Fc variant of the invention is combined with a mutant known to bind Fc ⁇ RIIIA with a higher affinity than a comparable molecule comprising a wild type Fc region; the combination with a mutant of the invention results in a greater fold enhancement in Fc ⁇ RIIIA affinity.
  • the Fc variants of the present invention may be combined with other known Fc variants such as those disclosed in Duncan et al, 1988, Nature 332:563-564; Lund et al, 1991, J. Immunol 147:2657-2662; Lund et al, 1992, MoI Immunol 29:53-59; Alegre et al, 1994, Transplantation 57:1537-1543; Hutchins et al. , 1995, Proc Natl. Acad Sci U S A 92:11980-11984; Jefferis et al, 1995, Immunol Lett.
  • An Fc ⁇ R-Fc binding assay was developed for determining the binding of the molecules of the invention comprising variant Fc regions to Fc ⁇ R, which allowed detection and quantization of the interaction, despite the inherently weak affinity of the receptor for its ligand, e.g., in the micro molar range for Fc ⁇ RIIB and Fc ⁇ RIIIA.
  • the method involves the formation of an Fc ⁇ R complex that has an improved avidity for an Fc region, relative to an uncompleted Fc ⁇ R.
  • the preferred molecular complex is a tetrameric immune complex, comprising: (a) the soluble region of Fc ⁇ R ⁇ e.g., the soluble region of Fc ⁇ RIIIA, Fc ⁇ RIIA or Fc ⁇ RIIB); (b) a biotinylated 15 amino acid AVITAG sequence (AVITAG) operably linked to the C-terminus of the soluble region of Fc ⁇ R (e.g.
  • SA-PE streptavidin-phycoerythrin
  • the fusion protein is biotinylated enzymatically, using for example, the E.coli Bir A enzyme, a biotin ligase which specifically biotinylates a lysine residue in the 15 amino acid AVITAG sequence, hi a specific embodiment of the invention, 85% of the fusion protein is biotinylated, as determined by standard methods known to those skilled in the art, including but not limited to streptavidin shift assay.
  • the biotinylated soluble Fc ⁇ R proteins are mixed with SA-PE in a IX SA-PE:5X biotinylated soluble Fc ⁇ R molar ratio to form a tetrameric Fc ⁇ R complex.
  • molecules comprising Fc regions bind the tetrameric Fc ⁇ R complexes, formed according to the methods of the invention, with at least an 8-fold higher affinity than the monomeric uncomplexed Fc ⁇ R.
  • the binding of polypeptides comprising Fc regions to the tetrameric Fc ⁇ R complexes may be determined using standard techniques known to those skilled in the art, such as for example, fluorescence activated cell sorting (FACS), radioimmunoassays, ELISA assays, etc.
  • FACS fluorescence activated cell sorting
  • the invention encompasses the use of the immune complexes formed according to the methods described above, for determining the functionality of molecules comprising an Fc region in cell-based or cell-free assays.
  • the reagents may be provided in an assay kit,
  • a packaged combination of reagents for assaying the ability of molecules comprising variant Fc regions to bind Fc ⁇ R tetrameric complexes are also contemplated for use in the methods of the invention, e.g., fusion proteins formed as described in U.S. Provisional Application 60/439,709, filed on January 13, 2003 (Attorney Docket No. 11183-005-888); which is incorporated herein by reference in its entirety.
  • the instant invention encompasses construction of multiple libraries based on both genetic and structural data known in the art or being developed.
  • the method described and exemplified herein incorporates building individual libraries that contain mutants testing all 20 amino acid changes at between 3-6 residues in the Fc region.
  • the complete set of mutations will be assembled in all possible combinations of mutations.
  • the number of independent mutations generated is based on the number of sites being saturated during library assembly (Table 9 below). Library size will determine the choice of primary screen and therefore the choice of vector for initial cloning steps.
  • the instant invention encompasses construction of combinatorial libraries, focusing on a limited number of critical residues (e.g., 3-6). Using a library of randomly mutagenized IgGl Fc and the screening assays described and exemplified herein Fc variants will be identified. In the initial rounds, the best 5 mutations, based on both FcR binding profile and functional activity will be selected. It will take 20 5 individual mutants to cover all possible amino acid changes and their combinations at five locations. A library with at least 10-fold coverage for each mutant will be generated. In addition regions will be chosen based on available information, e.g., crystal structure data, Mouse/Human isotype Fc ⁇ R binding differences, genetic data, and additional sites identified by mutagenesis.
  • the biggest disadvantage of current site directed mutagenic protocols is production of bias populations, over-representing variations in some regions and under- representing or completely lacking mutations in others.
  • the present invention overcomes this problem by generating unbiased arrays of desirable Fc mutants using a well-developed gene building technology to eliminate the bias introduced in library construction by PCR based approaches such as overlapping PCR and inverted PCR.
  • the key distinctions of the approach of the present invention are: 1) Employment of equimolar mix of 20 individual oligos for every targeted codon instead of degenerated primers. This way each amino acid is represented by a single, most used codon, whereas degenerated primers over represent those amino acids encoded by more codons over those encoded by fewer codons. 2)
  • An exemplary protocol comprises of the following steps: 1) phosphorylated oligos, representing desirable changes at one or several locations, all complementary to the same strand, added to the template along with a thermostable, 5'>3' exonuclease deficient, DNA polymerase and ligase (FIG. 26 a). 2) assembled mix undergoes a number of polymerization/ligation cycles, sufficient to generate desirable amount of product.
  • Use of a 5'>3' exonuclease deficient DNA polymerase insures integrity of the primer sequence and its phosphate residue, when a thermostable ligase assembles individual primer-extended fragments into a contiguous single-stranded chain.
  • a fraction of the mutant library will be sequenced to determine the rate of mutant codon incorporation.
  • the number of fragments sequenced will be based on the number of target sites mutated and library validation will be determined by the observed rate of mutation at targeted sites (Table 10).
  • the rate of vector without inserts should be less than 2 %.
  • the rate of mutation at non- targeted sites should be less than 8%.
  • Libraries containing clones with >90% correct inserts will allow us to maintain screening timelines.
  • the invention encompasses overlapping or inverted PCR for construction of libraries.
  • individual primers for each codon will be used rather than degenerative primers.
  • a similar validation scheme as disclosed supra will be employed.
  • a Qpix-2 clone picker robot will be used for picking colonies into 96 deep well plates. Culture growth will be done using a magnetic levitation stirrer, capable of incubating 12 plates and resulting in dense growth in 12 -16 hr at 37° C.
  • a Qiagen miniprep robot will be used to perform DNA preps at the rate of 4 96 well plates in 2.5 hrs. By overlapping tasks 5 such libraries could be constructed in 9 months with 1 FTE.
  • Affinity maturation requires the assembly of a new set of combinations of mutations, from a preselected mutant pool or members of a gene family, which can be enriched by a selection protocol. The process is repeated several times until the isolation of a mutant with the desired phenotype is achieved.
  • the disadvantage of the current enzymatic approach, DNA shuffling, to accomplish this process is bias which can be introduced due to specific sites within gene that are hot spots for nucleases, dominance of specific mutants in the final reassembled pool and loss of some of the original mutants in the final pool.
  • a build-a-gene (B AG) technology will be used to generate a highly complex library of Fc mutants containing random amino acid changes at all potential locations that may be important for receptor(s) binding.
  • Sets of degenerated oligos covering specific regions of the IgG Fc will be used (See FIG. 27).
  • Oligos will be -30 nt and degenerate oligos synthesized to change one (4 oligos) or two AAs (8 oligos) will be constructed. The oligos are designed to be overlapping with no gaps. It will take -200 oligos to accommodate all single AA changes and -2000 to change two AAs per oligonucleotide.
  • All 2000+ oligos will be used individually and in combinations to generate arrays of Fc mutants using the protocol outlined above (A.20).
  • Large libraries will be cloned into vectors that will allow for screening using yeast surface display. This approach utilizes a magnetic bead selection followed by flow cytometry and has been successfully applied to libraries with a complexity >10 9 (Feldhaus et al., 2003, Nat. Biotech. 21(2): 163-170; which is incorporated herein by reference in its entirety). This limits the number of sites to test at any one pool to 7, resulting in -1.3 x 10 9 possible mutations/pool.
  • PCR amplified fragments will be analyzed by electrophoresis to determine the length of the final PCR products. The reaction will be characterized as successful if >99% of the PCR products are of the expected length. A fraction of the mutant library will be sequenced to determine the rate of mutant codon incorporation. The number of fragments sequenced will be based on the number of target sites mutated and library validation will be determined by the observed rate of mutation at targeted sites (Table 10). The rate of vectors without inserts should be less than 2 %. The rate of mutation at non-targeted sites should be less than 8%.
  • the ability to generate the desired level of efficiency of mutagenesis by this approach will be determined by sequencing of a subset of clones.
  • the alternative to BAG will be using a "DNA shuffle" protocol. This requires pooling all of the mutants, single, double, triple, etc.
  • Fc regions will be amplified by PCR using flanking primers that selectively amplify the mutated region of the Fc, -700 bp.
  • Novel mutants are constructed by reshuffling of mutations in the Fc via DNAseI treatment of the amplified DNA and isolation of 150-200 bp fragments (see, e.g., Stemmer et ah, 1994, Proc. Natl. Acad. Sci.
  • Fragments will be religated, PCR amplified with nested primers and cloned into the yeast surface display vector, pYDl. The recombined library will be reselected in the yeast Fc display screen as described and exemplified herein.
  • BAG libraries will utilize most of the same equipment as the combinatorial library. However cloning will be in a vector suitable for yeast surface display and will not require arraying of individual clones as the yeast surface display will initially be employed for enrichment of large libraries. Subsequent to the appropriate level of enrichment individual clones will be arrayed.
  • An initial library of molecules comprising variant Fc regions is produced using any random based mutagenesis techniques known in the art. It will be appreciated by one of skill in the art that amino acid sequence variants of Fc regions may be obtained by any mutagenesis technique known to those skilled in the art. Some of these techniques are briefly described herein, however, it will be recognized that alternative procedures may produce an equivalent result.
  • molecules of the invention comprising variant Fc regions are prepared by error-prone PCR as exemplified in Example 6, infra ⁇ See Leung et al, 1989, Technique, 1:11). It is especially preferred to have error rates of 2-3 bp/Kb for use in the methods of the invention.
  • Mutagenesis may be performed in accordance with any of the techniques known in the art including, but not limited to, synthesizing an oligonucleotide having one or more modifications within the sequence of the Fc region of an antibody or a polypeptide comprising an Fc region ⁇ e.g., the CH2 or CH3 domain) to be modified.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 30 to about 45 nucleotides or more in length is preferred, with about 10 to about 25 or more residues on both sides of the junction of the sequence being altered.
  • a number of such primers introducing a variety of different mutations at one or more positions may be used to generated a library of mutants.
  • site-directed mutagenesis is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide.
  • An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically.
  • This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as T7 DNA polymerase, in order to complete the synthesis of the mutation-bearing strand.
  • DNA polymerizing enzymes such as T7 DNA polymerase
  • This heteroduplex vector is then used to transform or transfect appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
  • the technique typically employs a phage vector which exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the M 13 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.
  • PCRTM with commercially available thermostable enzymes such as Taq DNA polymerase may be used to incorporate a mutagenic oligonucleotide primer into an amplified DNA fragment that can then be cloned into an appropriate cloning or expression vector.
  • PCRTM employing a thermostable ligase in addition to a thermostable polymerase may also be used to incorporate a phosphorylated mutagenic oligonucleotide into an amplified DNA fragment that may then be cloned into an appropriate cloning or expression vector ⁇ see e.g., Michael, Biotechniques, 16(3):410-2, 1994, which is hereby incorporated by reference in its entirety).
  • Another method for preparing variants for use in the invention is cassette mutagenesis based on the technique described by Wells et al. (1985, Gene, 34: 315).
  • the starting material is the plasmid comprising the desired DNA encoding the protein to be mutated (e.g., the DNA encoding a polypeptide comprising an Fc region).
  • the codon(s) in the DNA sequence to be mutated are identified; there must be a unique restriction endonuclease site on each side of the identified mutations site(s). If no such restriction site exits, it may be generated by oligonucleotide directed mutagenesis.
  • the plasmid is cut at these sites and linearized.
  • a double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the mutation is synthesized using standard procedures known to those skilled in the art.
  • the double stranded oligonucleotide is referred to as the cassette.
  • This cassette is designed to have 3' and 5' ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. [00247]
  • Other methods known to those of skill in the art for producing sequence variants of the Fc region of an antibody or polypeptides comprising an Fc region can be used.
  • recombinant vectors encoding the amino acid sequence of the constant domain of an antibody or a fragment thereof may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • mutagenic agents such as hydroxylamine
  • yeast surface display technology for review see Boder and Wittrup, 2000, Methods in Enzymology, 328: 430-444, which is incorporated herein by reference in its entirety
  • yeast surface display is a genetic method whereby polypeptides comprising Fc mutants are expressed on the yeast cell wall in a form accessible for interacting with Fc ⁇ R.
  • Yeast surface display of the mutant Fc containing polypeptides of the invention may be performed in accordance with any of the techniques known to those skilled in the art. See U.S. Patent No.'s 6,423,538; 6,114,147; and 6,300,065, all of which are incorporated herein by reference in their entirety. See Boder et al , 1997 Nat. Biotechnol , 15:553-7; Boder et al. , 1998 Biotechnol. Prog. , 14:55-62; Boder et al, 2000 Methods EnzymoL, 328:430-44; Boder et al, 2000 Proc. Natl Acad.
  • Yeast Surface Display will be used to enrich libraries containing >10 7 independent clones. This approach will provide the ability to enrich large libraries >20-fold in single sort. Fc mutant libraries with >10,000 independent mutants (4 or more sites) will be cloned into the appropriate vectors for yeast surface display and enriched by FACS sorting until ⁇ 8000 mutants are able to be tested by other biochemical and functional assays as described below.
  • the invention provides methods for constructing an Fc mutant library in yeast for displaying molecules comprising Fc regions, which have been mutated as described in Section 5.2.2.
  • the Fc mutant libraries for use in the methods of the invention contain at least 10 7 cells, up to 10 9 cells.
  • One exemplary method for constructing a Fc library for use in the methods of the invention comprises the following: nucleic acids encoding molecules comprising Fc regions are cloned into the multiple cloning site of a vector derived from a yeast replicating vector, e.g., pCT302; such that the Fc encoding nucleic acids are expressed under the control of the GALl galactose- inducible promoter and in-frame with a nucleotide sequence encoding Aga2p, the mating agglutinin cell wall protein.
  • a yeast replicating vector e.g., pCT302
  • nucleic acids encoding molecules comprising Fc regions are cloned C-terminal to the Aga2p coding region, such that a Fc-region Aga2p fusion protein is encoded.
  • a fusion protein comprising the Aga2p protein and polypeptides comprising Fc regions will be secreted extracellularly and displayed on the cell wall via disulfide linkage to the Agalp protein, an integral cell wall protein, using the preferred construct of the invention.
  • the constructs may further comprise nucleotide sequences encoding epitope tags.
  • Any epitope tag nucleotide coding sequence known to those skilled in the art can be used in accordance with the invention, including, but not limited to nucleotide sequences encoding hemagglutinin (HA), c-myc Xpress TAG, His - TAG, or V5TAG.
  • HA hemagglutinin
  • c-myc Xpress TAG His - TAG
  • V5TAG V5TAG.
  • the presence of the fusion protein on the yeast cell surface may be detected using FACS analysis, confocal fluorescence microscopy or standard immunostaining methods, all of which are known to those skilled in the art.
  • the presence of the Fc fusion proteins of the invention on the yeast cell surface are detected using Fc-specific monoclonal antibodies (CH3 specific), including but not limited to IgGl Fc-specific monoclonal antibody, HP6017 (Sigma), JL512 (Immunotech), and any antibody disclosed in Partridge et ah, 1986, Molecular Immunology, 23 (12): 1365- 72, which is incorporated herein by reference in its entirety.
  • the presence of the Fc fusion proteins of the invention are detected by immunofluorescent labeling of epitope tags using techniques known to those skilled in the art.
  • nucleotide sequences encoding epitope tags to flank the nucleic acids encoding the Fc fusion proteins, as an internal control, to detect if the fusion proteins are displayed on the cell wall in a partially proteolyzed form.
  • the invention encompasses screening the yeast display libraries using immunological based assays including but not limited to cell based assays, solution based assays, and solid phase based assays.
  • the invention encompasses identification of Fc mutants with altered Fc ⁇ R affinities using affinity maturation methods which are known to those skilled in the art and encompassed herein. Briefly, affinity maturation creates novel alleles by randomly recombining individual mutations present in a mutant library, see, e.g., Hawkins et al, 1992, /. MoI. Biol.
  • the invention encompasses using mutations that show increased Fc ⁇ R. binding as a baseline to construct new mutant libraries with enhanced phenotypes.
  • a population of IgGl Fc mutants enriched by yeast surface display for increased binding to an Fc ⁇ R, e.g., Fc ⁇ RIIIA, may be selected.
  • Fc regions can be amplified by PCR using flanking primers that selectively amplify the mutated region of the Fc, which is about -700 bp using methods known to one skilled in the art and exemplified or disclosed herein. Novel mutants can thus be constructed by reshuffling of mutations in the Fc region for example via DNAseI treatment of the amplified DNA and isolation of fragments using methods such as those disclosed by Stemmer et al, 1994 P roc. Natl. Acad. ScL USA 91: 10747-51, which is incorporated herein by reference in its entirety.
  • Fragments can then be religated, PCR amplified with nested primers and cloned into the yeast display vector, e.g., pYDl using methods known to one skilled in the art.
  • the recombined library can then be reselected in the yeast Fc display screen.
  • K D decreases, below 10 nM, conditions can be established to allow for further increases in affinity based on the reduction of the off rate of the Fc)RIIIA ligand from the Fc receptor using methods known in the art such as those disclosed in Boder et al., 1998, Biotechnol. Prog. 14: 55-62, which is incorporated herein by reference in its entirety.
  • the invention encompasses a kinetic screen of the yeast library.
  • a kinetic screen may be established by labeling of the Fc displaying cells to saturation with a labeled ligand, e.g., a fluorescent ligand followed by incubation with an excess of non-labeled ligand for a predetermined period. After termination of the reaction by the addition of excess buffer (e.g., IX PBS, 0.5 mg/ml BSA) cells will be analyzed by FACS and sort gates set for selection. After each round of enrichment individual mutants can be tested for fold increases in affinity and sequenced for diversity.
  • the in vitro recombination process can be repeated. In some embodiments, the in vitro recombination process is repeated at least 3 times.
  • Selection of the Fc variants of the invention may be done using any Fc ⁇ R including but not limited to polymorphic variants of Fc ⁇ R.
  • selection of the Fc variants is done using a polymorphic variant of Fc ⁇ RIIIA which contains a phenylalanine at position 158.
  • selection of the Fc variants is done using a polymorphic variant of Fc ⁇ RIIIA which contains a valine at position 158.
  • Fc ⁇ RIIIA 158V displays a higher affinity for IgGl than 158F and an increased ADCC activity (see, e.g., Koene et al, 1997, Blood, 90:1109-14; Wu et al, 1997, /. Clin.
  • the invention encompasses screening yeast libraries based on Fc ⁇ RIIB depletion and Fc ⁇ RIIIA selection so that Fc mutants are selected that not only have an enhanced affinity for Fc ⁇ RIIIIA but also have a reduced affinity for Fc ⁇ RIIB.
  • Yeast libraries may be enriched for clones that have a reduced affinity for Fc)RIIB by sequential depletion methods, for example, by incubating the yeast library with magnetic beads coated with Fc ⁇ RIIB.
  • Fc ⁇ RIIB depletion is preferably carried out sequentially so that the library is enriched in clones that have a reduced affinity for Fc)RIIB.
  • the Fc ⁇ RIIB depletion step results in a population of cells so that only 30% , preferably only 10%, more preferably only 5%, most preferably less than 1% bind Fc ⁇ RIIB. In some embodiments, Fc ⁇ RIIB depletion is carried out in at least 3 cycles, at least 4 cycles, at least 6 cycles.
  • the Fc ⁇ RIIB depletion step is preferably combined with an Fc ⁇ RIIIIA selection step, for example using FACS sorting so that Fc variants with an enhanced affinity for Fc ⁇ RIIIIA are selected.
  • the invention encompasses characterization of the mutant Fc fusion proteins that are displayed on the yeast surface cell wall, according to the methods described in Section 5.2.3.
  • One aspect of the invention provides a method for selecting mutant Fc fusion proteins with a desirable binding property, specifically, the ability of the mutant Fc fusion protein to bind Fc ⁇ RIIIA and/or Fc ⁇ RIIA with a greater affinity than a comparable polypeptide comprising a wild-type Fc region binds Fc ⁇ RIIIA and/or Fc ⁇ RIIA.
  • the invention provides a method for selecting mutant Fc fusion proteins with a desirable binding property, specifically, the ability of the mutant Fc fusion protein to bind Fc ⁇ RIIIA and/or Fc ⁇ RIIA with a greater affinity than a comparable polypeptide comprising a wild-type Fc region binds Fc ⁇ RIIIA and/or Fc ⁇ RIIA, and further the ability of the mutant Fc fusion protein to bind Fc ⁇ RIIB with a lower affinity than a comparable polypeptide comprising a wild-type Fc region binds Fc ⁇ RIIB.
  • the methods of the invention can be used for identifying and screening any mutations in the Fc regions of molecules, with any desired binding characteristic.
  • Yeast cells displaying the mutant Fc fusion proteins can be screened and characterized by any biochemical or immunological based assays known to those skilled in the art for assessing binding interactions.
  • FACS fluorescence activated cell sorting
  • Flow sorters are capable of rapidly examining a large number of individual cells that contain library inserts (e.g., 10-100 million cells per hour) (Shapiro et at, Practical Flow Cytometry, 1995).
  • ligand concentration Le., Fc ⁇ RIIIA tetrameric complex
  • kinetic competition time or FACS stringency
  • Fc fusion proteins with specific binding properties e.g., higher affinity for Fc ⁇ RIIIA compared to a comparable polypeptide comprising a wild-type Fc region.
  • Flow cytometers for sorting and examining biological cells are well known in the art. Known flow cytometers are described, for example, in U.S. Patent Nos.
  • yeast cells are analyzed by fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • the FACS analysis of the yeast cells is done in an iterative manner, at least twice, at least three times, or at least 5 times. Between each round of selection cells are regrown and induced so the Fc regions are displayed on the maximum number of yeast cell surfaces.
  • this iterative process helps enrich the population of the cells with a particular phenotype, e.g., high binding to Fc ⁇ RIIIA.
  • screening for Fc variants of the invention comprises a selection process that has multiple rounds of screening, e.g., at least two rounds of screening.
  • screening for Fc variants that have an enhanced affinity for Fc ⁇ RIIIA may comprise the following steps: in the first round of screening, a library of yeast cells, e.g., a naive library of 10 7 cells is enriched by FACS, preferably in an iterative manner, using for example labeled tetrameric Fc ⁇ RIIIA to select for Fc variants that have an enhanced affinity for Fc ⁇ RIIIA; the variant Fc region that is selected with the desired phenotype, e.g., enhanced binding to Fc ⁇ RIIIA, is then introduced into an antibody, e.g., a 4-4-20 antibody, and the engineered antibody is assayed using a secondary screen, e.g., ELISA for binding to an Fc ⁇ R.
  • a secondary screen e.g., ELISA for binding to an Fc ⁇ R.
  • a single mutation library may be generated based on the first screen so that the Fc region harbors the variant displaying the enhanced affinity for Fc ⁇ RIIIA; and enriched by FACS using for example labeled monomeric Fc ⁇ RIIIA in both the presence and absence of unlabeled receptor; and the variant Fc region is then introduced into an antibody, e.g., a.4-4-20 antibody, and the engineered antibody is assayed using a secondary screen, e.g., ELISA for binding to an Fc ⁇ R.
  • an antibody e.g., a.4-4-20 antibody
  • a secondary screen e.g., ELISA for binding to an Fc ⁇ R.
  • the secondary screen may further comprise characterizing the antibodies comprising Fc variants in an ADCC or BIAcore based assay using methods disclosed herein [00262]
  • the invention encompasses FACS screening of the mutant yeast library under equilibrium or kinetic conditions. When the screening is performed under equilibrium conditions, an excess of the yeast library carrying Fc mutants is incubated with Fc ⁇ RIIIA, preferably labeled Fc ⁇ RIIIA at a concentration 5-10 fold below the Kd, for at least one hour to allow binding of Fc mutants to Fc ⁇ RIIIA under equilibrium conditions.
  • the mutant yeast library is incubated with labeled Fc ⁇ RIIIA; the cells are then incubated with equimolar unlabeled Fc ⁇ RIIIA for a pre-selected time, bound Fc ⁇ RIIIA is then monitored.
  • FACS One exemplary method of analyzing the yeast cells expressing mutant Fc fusion proteins with FACS is costaining the cells with Fc ⁇ RIIIA-tetrameric complex which has been labeled with a fluorescent label such as, PE and an anti-Fc antibody, such as F(ab) 2 anti-Fc which has been fluorescently labeled.
  • Fluorescence measurements of a yeast library produced according to the methods of the invention preferably involves comparisons with controls; for example, yeast cells that lack the insert encoding molecules comprising an Fc region (negative control).
  • the flow sorter has the ability not only to measure fluorescence signals in cells at a rapid rate, but also to collect cells that have specified fluorescent properties. This feature may be employed in a preferred embodiment of the invention to enrich the initial library population for cells expressing Fc fusion proteins with specific binding characteristics, e.g., higher affinity for Fc ⁇ RIIIA compared to a comparable polypeptide comprising a wild-type Fc region.
  • yeast cells are analyzed by FACS and sort gates established to select for cells that show the highest affinity for Fc ⁇ RIIIA relative to the amount of Fc expression on the yeast cell surface.
  • sort gates established to select for cells that show the highest affinity for Fc ⁇ RIIIA relative to the amount of Fc expression on the yeast cell surface.
  • four consecutive sorts are established, wherein the gates for each successive sort is 5.5%, 1%, 0.2%, and 0.1%.
  • the yeast display library formed according to the methods of the invention be over-sampled by at least 10-fold to improve the probability of isolating rare clones (e.g., analyze ⁇ 10 cells from a library of 10 7 clones).
  • 2-5 sorts are established to select for cells of the desired phenotype. Sort gates can be established empirically by one skilled in the art.
  • mutant Fc fusion proteins displayed on the yeast cell surface are screened using solid phase based assays, for example assays using magnetic beads, e.g. , supplied by Dynal, preferably in a high through put manner for binding to an Fc ⁇ R, e.g., Fc ⁇ RIIIA.
  • magnetic bead assays may be used to identify mutants with enhanced affinity for Fc ⁇ RIIIA and/or reduced affinity for Fc ⁇ RIIB.
  • An exemplary assay to identify mutants with enhanced affinity for Fc ⁇ RIIIA and reduced affinity for Fc ⁇ RIIB may comprise selecting mutants by a sequential solid phase depletion using magnetic beads coated with Fc ⁇ RIIB followed by selection with magnetic beads coated with Fc ⁇ RIIIA.
  • one assay may comprise the following steps: incubating the library of yeast cells generated in accordance with the methods of the invention with magnetic beads coated with Fc ⁇ RIIB; separating yeast cells bound to beads from the non bound fraction by placing the mixture in a magnetic field, removing the non- bound yeast cells and placing them in a fresh media; binding the yeast cells to beads coated with Fc ⁇ RIIIA, separating yeast cells bound to beads from the non bound fraction by placing the mixture in a magnetic field, removing the non-bound yeast cells; removing the bound cells by rigorous vortexing; growing the recovered cells in glucose containing media; re-inducing in selective media containing galactose. The selection process is repeated at least once. Inserts containing the Fc domain are then amplified using common methodologies known in the art, e.g., PCR, and introduced into an antibody by methods already described for further characterization.
  • a non- yeast based system is used to characterize the binding properties of the molecules of the invention.
  • One exemplary system for characterizing the molecules of the invention comprises a mammalian expression vector containing the heavy chain of the anti-fluorescein monoclonal antibody 4-4-20, into which the nucleic acids encoding the molecules of the invention with variant Fc regions are cloned.
  • the resulting recombinant clone is expressed in a mammalian host cell line (i.e., human kidney cell line 293H), and the resulting recombinant immunoglobulin is analyzed for binding to Fc ⁇ R using any standard assay known to those in the art, including but not limited to ELISA and FACS.
  • Molecules of the present invention may be characterized in a variety of ways.
  • molecules of the invention comprising modified Fc regions may be assayed for the ability to immunospecifically bind to a ligand, e.g., Fc ⁇ RIIIA tetrameric complex.
  • a ligand e.g., Fc ⁇ RIIIA tetrameric complex.
  • Such an assay may be performed in solution (e.g., Houghten, Bio/Techniques, 13:412-421, 1992), on beads (Lam, Nature, 354:82-84, 1991, on chips (Fodor, Nature, 364:555-556, 1993), on bacteria (U.S. Patent No. 5,223,409), on spores (U.S. Patent Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (Cull et al, Proc. Natl.
  • Fc regions e.g., therapeutic antibodies
  • have been identified in the yeast display system to have the desired phenotype see Section 5.1
  • an antigen e.g., cancer antigen and cross-reactivity with other antigens (e.g., Fc ⁇ R) by any method known in the art.
  • Immunoassays which can be used to analyze immunospecific binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiornetric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
  • competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays
  • the binding affinity of the molecules of the present invention comprising modified Fc regions to a ligand, e.g., Fc ⁇ R tetrameric complex and the off-rate of the interaction can be determined by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled ligand, such as tetrameric Fc ⁇ R (e.g., 3 H or 125 I) with a molecule of interest (e.g., molecules of the present invention comprising modified Fc regions) in the presence of increasing amounts of unlabeled ligand, such as tetrameric Fc ⁇ R, and the detection of the molecule bound to the labeled ligand.
  • labeled ligand such as tetrameric Fc ⁇ R (e.g., 3 H or 125 I)
  • a molecule of interest e.g., molecules of the present invention comprising modified Fc regions
  • the affinity of the molecule of the present invention for the ligand and the binding off -rates can be determined from the saturation data by scatchard analysis.
  • BIAcore kinetic analysis is used to determine the binding on and off rates of molecules of the present invention to a ligand such as Fc ⁇ R.
  • BIAcore kinetic analysis comprises analyzing the binding and dissociation of a ligand from chips with immobilized molecules (e.g., molecules comprising modified Fc regions) on their surface.
  • sequencing reactions Any of a variety of sequencing reactions known in the art can be used to directly sequence the molecules of the invention comprising variant Fc regions. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert (Proc. Natl. Acad. ScL USA, 74:560, 1977) or Sanger (Proc. Natl. Acad. ScL USA, 74:5463, 1977). It is also contemplated that any of a variety of automated sequencing procedures can be utilized (Bio/Techniques, 19:448, 1995), including sequencing by mass spectrometry
  • the invention encompasses characterization of the molecules of the invention (e.g., an antibody comprising a variant Fc region identified by the yeast display technology described supra; or therapeutic monoclonal antibodies engineered according to the methods of the invention) using assays known to those skilled in the art for identifying the effector cell function of the molecules.
  • the invention encompasses characterizing the molecules of the invention for Fc ⁇ R-mediated effector cell function.
  • effector cell functions include but are not limited to, antibody-dependent cell mediated cytotoxicity, phagocytosis, opsonization, opsonophagocytosis, CIq binding, and complement dependent cell mediated cytotoxicity. Any cell-based or cell free assay known to those skilled in the art for determining effector cell function activity can be used (For effector cell assays, see Perussia et al, 2000, Methods MoI. Biol. 121: 179-92; Baggiolini et al, 1998 Experientia, 44(10): 841-8; Lehmann et ah, 2000 /. Immunol.
  • the molecules of the invention can be assayed for Fc ⁇ R- mediated phagocytosis in human monocytes.
  • the Fc ⁇ R-mediated phagocytosis of the molecules of the invention may be assayed in other phagocytes, e.g., neutrophils (polymorphonuclear leuckocytes; PMN); human peripheral blood monocytes, monocyte-derived macrophages, which can be obtained using standard procedures known to those skilled in the art (e.g., see Brown EJ. 1994, Methods Cell Biol., 45: 147-164).
  • the function of the molecules of the invention is characterized by measuring the ability of THP-I cells to phagocytose fluoresceinated IgG-opsonized sheep red blood cells (SRBC) by methods previously described (Tridandapani et al, 2000, /. Biol. Chem. 275: 20480-7).
  • an exemplary assay for measuring phagocytosis of the molecules of the invention comprising variant Fc regions with enhanced affinities for Fc ⁇ RIIIA comprises of: treating THP-I cells with a molecule of the invention or with a control antibody that does not bind to Fc ⁇ RIIIA, comparing the activity levels of said cells, wherein a difference in the activities of the cells (e.g.
  • rosetting activity the number of THP-I cells binding IgG-coated SRBC
  • adherence activity the total number of SRBC bound to THP-I cells
  • phagocytic rate would indicate the functionality of the molecule of the invention. It can be appreciated by one skilled in the art that this exemplary assay can be used to assay any of the molecules identified by the methods of the invention.
  • ADCP antibody-dependent opsonophagocytosis assay
  • a target bioparticle such as Escherichia c ⁇ / ⁇ -labeled FITC (Molecular Probes) or Staphylococcus aureus-F ⁇ TC with (i) wild-type 4-4-20 antibody, an antibody to fluorescein (See Bedzyk et al, 1989, J.
  • THP-I cells are analyzed by FACS for expression of Fc ⁇ R on the cell surface.
  • THP-I cells express both CD32A and CD64.
  • CD64 is a high affinity Fc ⁇ R that is blocked in conducting the ADCP assay in accordance with the methods of the invention.
  • the THP-I cells are preferably blocked with 100 ⁇ g/mL soluble IgGl or 10% human serum.
  • the gate is preferably set on THP-I cells and median fluorescence intensity is measured.
  • the ADCP activity for individual mutants is calculated and reported as a normalized value to the wild type chMab 4-4-20 obtained.
  • the opsonized particles are added to THP-I cells such that the ratio of the opsonized particles to THP-I cells is 30:1 or 60:1.
  • the ADCP assay is conducted with controls, such as E. coli-FlTC in medium, E.
  • E. coli-FI ⁇ C and THP-I cells (to serve as Fc ⁇ R-independent ADCP activity), E. coli-FTTC, THP-I cells and wild-type 4-4-20 antibody (to serve as Fc ⁇ R-dependent ADCP activity), E coli-V ⁇ C, THP-I cells, 4-4-20 D265A (to serve as the background control for Fc ⁇ R- dependent ADCP activity).
  • the molecules of the invention can be assayed for
  • Fc ⁇ R-mediated ADCC activity in effector cells e.g., natural killer cells
  • effector cells e.g., natural killer cells
  • any of the standard methods known to those skilled in the art See e.g., Perussia et at, 2000, Methods MoI. Biol. 121: 179-92; Weng et al, 2003, J. Clin. Oncol. 21:3940-3947).
  • An exemplary assay for determining ADCC activity of the molecules of the invention is based on a 51 Cr release assay comprising of: labeling target cells with [ 51 Cr]Na 2 CrO 4 (this cell-membrane permeable molecule is commonly used for labeling since it binds cytoplasmic proteins and although spontaneously released from the cells with slow kinetics, it is released massively following target cell necrosis); opsonizing the target cells with the molecules of the invention comprising variant Fc regions; combining the opsonized radiolabeled target cells with effector cells in a microtitre plate at an appropriate ratio of target cells to effector cells; incubating the mixture of cells for 16-18 hours at 37°C; collecting supernatants; and analyzing radioactivity.
  • % lysis ( experimental cpm - target leak cpm)/(detergent lysis cpm - target leak cpm) x 100%.
  • % lysis (ADCC- AICC)/(maximum release-spontaneous release).
  • a graph can be generated by varying either the target: effector cell ratio or antibody concentration.
  • the molecules of the invention are characterized for antibody dependent cellular cytotoxicity (ADCC) see, e.g., Ding et al, Immunity, 1998, 8:403-11; which is incorporated herein by reference in its entirety.
  • ADCC antibody dependent cellular cytotoxicity
  • the effector cells used in the ADCC assays of the invention are peripheral blood mononuclear cells (PBMC) that are preferably purified from normal human blood, using standard methods known to one skilled in the art, e.g. , using Ficoll- Paque density gradient centrifugation.
  • PBMC peripheral blood mononuclear cells
  • Preferred effector cells for use in the methods of the invention express different Fc ⁇ R activating receptors.
  • the invention encompasses, effector cells, THP-I, expressing Fc ⁇ RI, Fc ⁇ RIIA and Fc ⁇ RIIB, and monocyte derived primary macrophages derived from whole human blood expressing both Fc ⁇ RIIIA and Fc ⁇ RIIB, to determine if Fc antibody mutants show increased ADCC activity and phagocytosis relative to wild type IgGl antibodies.
  • THP-I The human monocyte cell line, activates phagocytosis through expression of the high affinity receptor Fc ⁇ RI and the low affinity receptor Fc ⁇ RIIA (Fleit et al., 1991, J. Leuk. Biol. 49: 556). THP-I cells do not constitutively express Fc ⁇ RIIA or Fc ⁇ RIIB. Stimulation of these cells with cytokines effects the FcR expression pattern
  • Cytokine induced expression of Fc ⁇ R on the cell surface provides a system to test both activation and inhibition in the presence of Fc ⁇ RIIB. If THP-I cells are unable to express the Fc ⁇ RIIB the invention also encompasses another human monocyte cell line, U937. These cells have been shown to terminally differentiate into macrophages in the presence of IFN ⁇ and TNF (Koren et al., 1979, Nature 279: 328-331).
  • Fc ⁇ R dependent tumor cell killing is mediated by macrophage and NK cells in mouse tumor models (Clynes et al, 1998, PNAS USA 95: 652-656).
  • the invention encompasses the use of elutriated monocytes from donors as effector cells to analyze the efficiency Fc mutants to trigger cell cytotoxicity of target cells in both phagocytosis and ADCC assays.
  • Expression patterns of Fc ⁇ RI, Fc ⁇ RIIIA, and Fc ⁇ RIIB are affected by different growth conditions.
  • Fc ⁇ R expression from frozen elutriated monocytes, fresh elutriated monocytes, monocytes maintained in 10% FBS, and monocytes cultured in FBS + GM-CSF and or in human serum may be determined using common methods known to those skilled in the art. For example, cells can be stained with Fc ⁇ R specific antibodies and analyzed by FACS to determine FcR profiles. Conditions that best mimic macrophage in vivo Fc ⁇ R expression is then used for the methods of the invention. [00279] In some embodiments, the invention encompasses the use of mouse cells especially when human cells with the right Fc ⁇ R profiles are unable to be obtained.
  • the invention encompasses the mouse macrophage cell line RAW264.7(ATCC) which can be transfected with human Fc ⁇ RIIIA and stable transfectants isolated using methods known in the art, see, e.g., Ralph et al, J. Immunol. 119: 950-4). Transfectants can be quantitated for Fc ⁇ RIIIA expression by FACS analysis using routine experimentation and high expressors can be used in the ADCC assays of the invention.
  • the invention encompasses isolation of spleen peritoneal macrophage expressing human Fc ⁇ R. from knockout transgenic mice such as those disclosed herein.
  • Lymphocytes may be harvested from peripheral blood of donors (PBM) using a Ficoll-Paque gradient (Pharmacia). Within the isolated mononuclear population of cells the majority of the ADCC activity occurs via the natural killer cells (NK) containing Fc ⁇ RIIIA but not Fc ⁇ RIIB on their surface. Results with these cells indicate the efficacy of the mutants on triggering NK cell ADCC and establish the reagents to test with elutriated monocytes.
  • PBM peripheral blood of donors
  • NK natural killer cells
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