US20070148170A1 - Fc Variants With Optimized Fc Receptor Binding Properties - Google Patents

Fc Variants With Optimized Fc Receptor Binding Properties Download PDF

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US20070148170A1
US20070148170A1 US11/538,406 US53840606A US2007148170A1 US 20070148170 A1 US20070148170 A1 US 20070148170A1 US 53840606 A US53840606 A US 53840606A US 2007148170 A1 US2007148170 A1 US 2007148170A1
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Prior art keywords
variant
variants
antibody
affinity
fcγriib
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John Desjarlais
Sher Karki
Gregory Lazar
John Richards
Gregory Moore
David Carmichael
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Xencor Inc
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Xencor Inc
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Priority claimed from US11/396,495 external-priority patent/US20060235208A1/en
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Priority to US11/538,406 priority Critical patent/US20070148170A1/en
Assigned to XENCOR, INC. reassignment XENCOR, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CARMICHAEL, DAVID F., DESJARLAIS, JOHN R., KARKI, SHER BAHADUR, LAZAR, GREGORY ALAN, MOORE, GREGORY L., RICHARDS, JOHN O.
Publication of US20070148170A1 publication Critical patent/US20070148170A1/en
Priority to US12/794,560 priority patent/US9040041B2/en
Priority to US14/210,236 priority patent/US20150071948A1/en
Priority to US15/406,588 priority patent/US20170166655A1/en
Priority to US15/883,006 priority patent/US20180360981A1/en
Abandoned legal-status Critical Current

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    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • C07K16/30—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
    • 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
    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention relates to Fc variants with optimized Fc receptor binding properties, engineering methods for their generation, and their application, particularly for therapeutic purposes.
  • Antibodies are immunological proteins that bind a specific antigen. Generally, antibodies are specific for targets, have the ability to mediate immune effector mechanisms, and have a long half-life in serum. Such properties make antibodies powerful therapeutics. Monoclonal antibodies are used therapeutically for the treatment of a variety of conditions including cancer, inflammation, and cardiovascular disease. There are currently over ten antibody products on the market and hundreds in development.
  • Antibodies have found widespread application in oncology, particularly for targeting cellular antigens selectively expressed on tumor cells with the goal of cell destruction.
  • There are a number of mechanisms by which antibodies destroy tumor cells including anti-proliferation via blockage of needed growth pathways, intracellular signaling leading to apoptosis, enhanced down regulation and/or turnover of receptors, CDC, ADCC, ADCP, and promotion of an adaptive immune response (Cragg et al., 1999, Curr Opin Immunol 11:541-547; Glennie et al., 2000, Immunol Today 21:403-410, both hereby entirely incorporated by reference).
  • Anti-tumor efficacy may be due to a combination of these mechanisms, and their relative importance in clinical therapy appears to be cancer dependent.
  • trastuzumab (Herceptin®, Genentech), an anti-HER2/neu antibody for treatment of metastatic breast cancer, has less efficacy.
  • any small improvement in mortality rate defines success.
  • Fc variants are typically engineered for optimal binding to human Fc ⁇ Rs.
  • experiments in animal models are important for ultimately developing a drug for clinical use in humans.
  • mouse models available for a variety of diseases are typically used to test properties such as efficacy, toxicity, and pharmacokinetics for a given drug candidate.
  • the present invention is directed to an Fc variant of a parent Fc polypeptide comprising at least a first and a second substitution.
  • the first and second substitutions are each at a position selected from group consisting of 234, 235, 236, 239, 267, 268, 293, 295, 324, 327, 328, 330, and 332 according to the EU index.
  • the Fc variant exhibits an increase in affinity for one or more receptors selected from the group consisting of Fc ⁇ RI, Fc ⁇ RIIa, and Fc ⁇ RIIIa as compared to the increase in a affinity of the Fc variant for the Fc ⁇ RIIb receptor.
  • the increases in affinities are relative to the parent polypeptide.
  • the present invention is further directed to methods of activating a receptor selected from the group consisting of Fc ⁇ RI, Fc ⁇ RIIa, and Fc ⁇ RIIIa relative to the Fc ⁇ RIIb receptor.
  • a cell that includes the Fc ⁇ RIIb receptor and one or more receptors selected from among Fc ⁇ RI, Fc ⁇ RIIa, and Fc ⁇ RIIIa is contacted with an Fc variant described above.
  • the method can be performed in vitro or in vivo.
  • the Fc variant exhibits an increase in affinity of the Fc variant for the Fc ⁇ RIIb receptor as compared to the increase in affinity for one or more activating receptors.
  • Activating receptors include Fc ⁇ RI, Fc ⁇ RIIa, and Fc ⁇ RIIIa. Increased affinities are relative to the parent polypeptide.
  • the first and second substitutions each at a position selected from group consisting of 234, 235, 236, 239, 267, 268, 293, 295, 324, 327, 328, 330 and 332 according to the EU index.
  • the present invention is further directed to methods of activating the Fc ⁇ RIIb receptor relative to a receptor selected from Fc ⁇ RI, Fc ⁇ RIIa, and Fc ⁇ RIIIa.
  • the method is accomplished by contacting cell that includes the Fc ⁇ RIIb receptor and one or more receptors selected from among Fc ⁇ RI, Fc ⁇ RIIa, and Fc ⁇ RIIIa with an Fc variant described above.
  • the method can be performed in vitro or in vivo.
  • the Fc variant has a reduced level of fucosylation relative to the parent Fc variant.
  • the Fc variant includes a glycosylated Fc region in which about 80-100% of the glycosylated Fc polypeptide in the composition having a mature core carbohydrate structure with no fucose.
  • the present invention also includes Fc variants of a parent mouse Fc polypeptide.
  • the Fc variant includes a substitution at a position selected from the group consisting of 236, 239, 268, 330, and 332.
  • the Fc variant includes a substitution selected from among 236A, 239D, 268E, 330Y, and 332E.
  • the present invention provides isolated nucleic acids encoding the Fc variants described herein.
  • the present invention provides vectors comprising the nucleic acids, optionally, operably linked to control sequences.
  • the present invention provides host cells containing the vectors, and methods for producing and optionally recovering the Fc variants.
  • the present invention provides novel Fc polypeptides, including antibodies, Fc fusions, isolated Fc, and Fc fragments, that comprise the Fc variants disclosed herein.
  • novel Fc polypeptides may find use in a therapeutic product.
  • the Fc polypeptides of the invention are antibodies.
  • compositions comprising Fc polypeptides that comprise the Fc variants described herein, and a physiologically or pharmaceutically acceptable carrier or diluent.
  • the present invention contemplates therapeutic and diagnostic uses for Fc polypeptides that comprise the Fc variants disclosed herein.
  • FIG. 1 Fc ⁇ R-dependent effector functions and potentially relevant Fc ⁇ Rs for select immune cell types that may be involved in antibody-targeted tumor therapy.
  • the third column presents interactions that may regulate activation or inhibition of the indicated cell type, with those that are thought to be particularly important highlighted in bold.
  • FIG. 2 Alignment of the amino acid sequences of the human IgG immunoglobulins IgG1, IgG2, IgG3, and IgG4.
  • FIG. 2 a provides the sequences of the CH1 (C ⁇ 1) and hinge domains (SEQ ID NOS: 21-24), and
  • FIG. 2 b provides the sequences of the CH2 (C ⁇ 2) (SEQ ID NOS: 25-28)and CH3 (C ⁇ 3) (SEQ ID NOS: 29-32) domains. Positions are numbered according to the EU index of the IgG1 sequence, and differences between IgG1 and the other immunoglobulins IgG2, IgG3, and IgG4 are shown in gray. Allotypic polymorphisms exist at a number of positions, and thus slight differences between the presented sequences and sequences in the prior art may exist. The possible beginnings of the Fc region are labeled, defined herein as either EU position 226 or 230.
  • FIG. 3 Common haplotypes of the human gamma1 ( FIG. 3 a ) and gamma2 ( FIG. 3 b ) chains.
  • FIG. 4 Sequence alignment of human Fc ⁇ Rs. Differences from Fc ⁇ RIIb are highlighted in gray, and positions at the Fc interface are indicated with an i. Numbering is shown according to both the 1IIS.pdb and 1E4K.pdb structures (SEQ ID NOS: 33-38).
  • FIG. 5 Structure of the Fc/Fc ⁇ R interface indicating differences between the Fc ⁇ RIIa and Fc ⁇ RIIb structures, and proximal Fc residues.
  • the structure is that of the 1E4K.pdb Fc/Fc ⁇ RIIIb complex.
  • Fc ⁇ R is represented by black ribbon and Fc is represented as gray ribbon.
  • Fc ⁇ R positions that differ between Fc ⁇ RIIa and Fc ⁇ RIIb are shown in gray, and proximal Fc residues to these Fc ⁇ R residues are shown in black.
  • FIG. 6 Binding of select anti-CD20 Fc variants to human R131 Fc ⁇ RIIa ( FIG. 6 a ) and Fc ⁇ RIIb ( FIG. 6 b ) as measured by competition AlphaScreenTM assay. In the presence of competitor antibody (Fc variant or WT) a characteristic inhibition curve is observed as a decrease in luminescence signal. The binding data were normalized to the maximum and minimum luminescence signal for each particular curve, provided by the baselines at low and high antibody concentrations respectively. The curves represent the fits of the data to a one site competition model using nonlinear regression.
  • FIG. 7 Summary of Fc ⁇ R binding properties of anti-CD20 Fc variants for binding to human Fc ⁇ RI, R131 Fc ⁇ RIIa, H131 Fc ⁇ RIIa, Fc ⁇ RIIb, and V158 Fc ⁇ RIIIa. Shown are the IC50s obtained from the AlphaScreen, and the Fold(IC50) relative to WT. Duplicate binding results, shown on separate lines, are provided for some variants.
  • FIG. 8 Binding of select anti-EGFR Fc variants to human Fc ⁇ RI, R131 and H131 Fc ⁇ RIIa, Fc ⁇ RIIb, and V158 Fc ⁇ RIIIa as measured by competition AlphaScreen assay.
  • FIG. 9 Summary of Fc ⁇ R binding properties of anti-EGFR Fc variants for binding to human Fc ⁇ RI, R131 Fc ⁇ RIIa, H131 Fc ⁇ RIIa, Fc ⁇ RIIb, and V158 Fc ⁇ RIIIa. Shown are the IC50s obtained from the AlphaScreen, and the Fold(IC50) relative to WT.
  • FIG. 10 Surface Plasmon Resonance (SPR) (BIAcore) sensorgrams of binding of select anti-EpCAM Fc variants to human R131 Fc ⁇ RIIa.
  • SPR Surface Plasmon Resonance
  • FIG. 11 Affinity data for binding of anti-EpCAM Fc variants to human Fc ⁇ RI, R131 and H131 Fc ⁇ RIIa, Fc ⁇ RIIb, V158 Fc ⁇ RIIIa, and F158 Fc ⁇ RIIIa as determined by SPR. Provided are the association (ka) and dissociation (kd) rate constants, the equilibrium dissociation constant (KD), the Fold KD relative to WT, and the negative log of the KD ( ⁇ log(KD)).
  • FIG. 12 Plot of the negative log of the KD for binding of select anti-EpCAM Fc variants to human Fc ⁇ RI, R131 Fc ⁇ RIIa, H131 Fc ⁇ RIIa, Fc ⁇ RIIb, and V158 Fc ⁇ RIIIa.
  • FIG. 11 Affinity data for binding of anti-EpCAM Fc variants to human Fc ⁇ RI, R131 and H131 Fc ⁇ RIIa, Fc ⁇ RIIb, V158 Fc ⁇ RIIIa, and F158 Fc ⁇ RIIIa as determined by SPR.
  • association (ka) and dissociation (kd) rate constants Provided are the association (ka) and dissociation (kd) rate constants, the equilibrium dissociation constant (KD), the Fold(KD) relative to the parent IgG (WT IgG1 or WT IgG(hybrid) and relative to WT IgG1, and the negative log of the KD ( ⁇ log(KD)).
  • FIG. 12 Plot of the negative log of the KD for binding of select anti-EpCAM Fc variants to human Fc ⁇ RI, R131 Fc ⁇ RIIa, H131 Fc ⁇ RIIa, Fc ⁇ RIIb, and V158 Fc ⁇ RIIIa.
  • FIG. 13 Affinity differences between activating and inhibitory Fc ⁇ Rs for select anti-EpCAM Fc variants.
  • FIG. 13 a shows the absolute affinity differences between the activating receptors and the inhibitory receptor Fc ⁇ RIIb.
  • the top graph shows the affinity differences between both isoforms of Fc ⁇ RIIa and Fc ⁇ RIIb, represented mathematically as [ ⁇ log(KD)Fc ⁇ RIIa] ⁇ [ ⁇ log(KD)Fc ⁇ RIIb]. Black represents logarithmic affinity difference between R131 Fc ⁇ RIIa and Fc ⁇ RIIb, and gray represents the logarithmic affinity difference between H131 Fc ⁇ RIIa and Fc ⁇ RIIb.
  • the bottom graph shows the affinity differences between both isoforms of Fc ⁇ RIIIa and Fc ⁇ RIIb, represented mathematically as [ ⁇ log(KD)Fc ⁇ RIIIa] ⁇ [ ⁇ log(KD)Fc ⁇ RIIb].
  • Black represents logarithmic affinity difference between V158 Fc ⁇ RIIIa and Fc ⁇ RIIb
  • gray represents the logarithmic affinity difference between F158 Fc ⁇ RIIIa and Fc ⁇ RIIb.
  • FIG. 13 b provides the fold affinity improvement of each variant for Fc ⁇ RIIa and Fc ⁇ RIIIa relative to the fold affinity improvement to Fc ⁇ RIIb.
  • RIIa represents R131Fc ⁇ RIIa
  • HIIa represents H131 Fc ⁇ RIIa
  • VIIIa represents V158 Fc ⁇ RIIIa
  • FIIIa represents F158 Fc ⁇ RIIIa
  • IIb represents Fc ⁇ RIIb.
  • this quantity is represented mathematically as Fold(KD) RIIa :Fold(KD) IIb or Fold(KD) RIIa /Fold(KD) IIb . See the Examples for a mathematical description of these quantities.
  • FIG. 13 c provides a plot of these data.
  • FIG. 16 Cell-based DC activation assay of anti-EpCAM Fc variants.
  • FIG. 16 a shows the quantitated receptor expression density on monocyte-derived dendritic cells measured with antibodies against Fc ⁇ RI (CD64), Fc ⁇ RIIa and Fc ⁇ RIIb (CD32), and Fc ⁇ RIIIa (CD16) using flow cytometry. “Control” indicates no antibody was used and is a negative control. The diagrams show the percentage of cells labeled with PE-conjugated antibody against the indicated Fc ⁇ R.
  • FIG. 16 b shows the dose-dependent TNF ⁇ release by dendritic cells in the presence of WT and Fc variant antibodies and EpCAM + LS180 target cells. The IgG1 negative control binds RSV and not EpCAM, and thus does not bind to the target cells.
  • FIG. 17 Binding of Fc variant antibodies comprising substitutions 298A, 326A, 333A, and 334A to human V158 Fc ⁇ RIIIa, F158 Fc ⁇ RIIIa, and Fc ⁇ RIIb as measured by competition AlphaScreen assay.
  • FIG. 17 a shows the legend for the data.
  • Antibodies in FIG. 17 b comprise the variable region of the anti-CD52 antibody alemtuzumab (Hale et al., 1990, Tissue Antigens 35:118-127; Hale, 1995, Immunotechnology 1:175-187), and antibodies in FIG. 17 c comprise the variable region of the anti-CD20 PRO70769 (PCT/US2003/040426).
  • FIG. 18 Preferred positions and substitutions of the invention that may be used to engineer Fc variants with selective Fc ⁇ R affinity.
  • FIG. 21 Binding of select anti-CD30 Fc variants to human V158 Fc ⁇ RIIIa as measured by competition AlphaScreen assay.
  • FIG. 22 Summary of V158 Fc ⁇ RIIIa binding properties of anti-CD30 Fc variants. Shown are the Fold-IC50s relative to WT as determined by competition AlphaScreen.
  • FIG. 23 Differences between human and mouse Fc ⁇ R biology.
  • FIG. 23 a shows the putative expression patterns of different Fc ⁇ Rs on various effector cell types. “yes” indicates that the receptor is expressed on that cell type. Inhibitory receptors in the human and mouse are shown in gray.
  • FIG. 23 b shows the % identity between the human (h) and mouse (m) Fc ⁇ R extracellular domains. Human receptors are shown in black and mouse receptors are shown in gray.
  • FIG. 24 Summary of human and mouse anti-EGFR antibodies constructed. For each variant are listed the variable region (Fv), constant light chain (CL), and constant heavy chain (CH).
  • Fv variable region
  • CL constant light chain
  • CH constant heavy chain
  • FIG. 25 Affinity data for binding of human and mouse anti-EGFR Fc variant antibodies to mouse Fc receptors Fc ⁇ RI, Fc ⁇ RII (Fc ⁇ RIIb), Fc ⁇ RIII, and Fc ⁇ RIV as determined by SPR. Provided are the equilibrium dissociation constant (KD), the Fold KD relative to WT, and the negative log of the KD ( ⁇ log(KD)) for each variant.
  • FIG. 26 Plot of the negative log of the KD for binding of human and mouse anti-EGFR Fc variant antibodies to mouse Fc receptors Fc ⁇ RI, Fc ⁇ RII (Fc ⁇ RIIb), Fc ⁇ RIII, and Fc ⁇ RIV.
  • FIG. 27 Amino acid sequences of variable light (VL) and heavy (VH) chains used in the present invention, including PRO70769 ( FIGS. 27 a and 27 b ), H4.40/L3.32 C225 ( FIGS. 27 c and 27 d ), H3.77/L3 17-1A ( FIGS. 27 e and 27 f ), and H3.69_V2/L3.71 AC10 ( FIGS. 27 g and 27 h ) (SEQ ID NOS: 1-8).
  • VL variable light
  • FIG. 28 Amino acid sequences of human constant light kappa ( FIG. 28 a ) and heavy ( FIGS. 28 b - 28 f ) chains used in the present invention (SEQ ID NOS: 9-20).
  • FIG. 29 Amino acid sequences of mouse constant light kappa ( FIG. 29 a ) and heavy ( FIGS. 29 b - 29 f chains of the present invention.
  • ADCC antibody dependent cell-mediated cytotoxicity
  • ADCP antibody dependent cell-mediated phagocytosis as used herein is meant the cell-mediated reaction wherein nonspecific cytotoxic cells that express Fc ⁇ Rs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.
  • amino acid modification herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence.
  • amino acid substitution or “substitution” herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid.
  • the substitution L328R refers to a variant polypeptide, in this case an Fc variant, in which the leucine at position 328 is replaced with arginine.
  • amino acid insertion or “insertion” as used herein is meant the addition of an amino acid at a particular position in a parent polypeptide sequence.
  • insert G >235-236 designates an insertion of glycine between positions 235 and 236.
  • amino acid deletion or “deletion” as used herein is meant the removal of an amino acid at a particular position in a parent polypeptide sequence.
  • Amino acids of the invention may be further classified as either isotypic or novel.
  • antibody herein is meant a protein consisting of one or more polypeptides substantially encoded by all or part of the recognized immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa ( ⁇ ), lambda ( ⁇ ), and heavy chain genetic loci, which together comprise the myriad variable region genes, and the constant region genes mu ( ⁇ ), delta ( ⁇ ), gamma ( ⁇ ), sigma ( ⁇ ), and alpha ( ⁇ ) which encode the IgM, IgD, IgG (IgG1, IgG2, IgG3, and IgG4), IgE, and IgA (IgA1 and IgA2) isotypes respectively.
  • Antibody herein is meant to include full length antibodies and antibody fragments, and may refer to a natural antibody from any organism, an engineered antibody, or an antibody generated recombinantly for experimental, therapeutic, or other purposes.
  • CDC complement dependent cytotoxicity
  • isotypic modification as used herein is meant an amino acid modification that converts one amino acid of one isotype to the corresponding amino amino acid in a different, aligned isotype.
  • isotypic modification an amino acid modification that converts one amino acid of one isotype to the corresponding amino amino acid in a different, aligned isotype.
  • IgG1 has a tyrosine and IgG2 a phenylalanine at EU position 296, a F296Y substitution in IgG2 is considered an isotypic modification.
  • novel modification as used herein is meant an amino acid modification that is not isotypic. For example, because none of the IgGs has a glutamic acid at position 332, the substitution I332E in IgG1, IgG2, IgG3, or IgG4 is considered a novel modification.
  • amino acid and “amino acid identity” as used herein is meant one of the 20 naturally occurring amino acids or any non-natural analogues that may be present at a specific, defined position.
  • effector 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 Fc ⁇ R-mediated effector functions such as ADCC and ADCP, and complement-mediated effector functions such as CDC.
  • effector 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' cells, natural killer (NK) cells, and ⁇ T cells, and may be from any organism including but not limited to humans, mice, rats, rabbits, and monkeys.
  • Fab or “Fab region” as used herein is meant the polypeptides that comprise the V H , CH1, V H , and C L immunoglobulin domains. Fab may refer to this region in isolation, or this region in the context of a full length antibody or antibody fragment.
  • Fc or “Fc region”, as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains.
  • IgA and IgM Fc may include the J chain.
  • Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (C ⁇ 2 and C ⁇ 3) and the hinge between Cgamma1 (C ⁇ 1) and Cgamma2 (C ⁇ 2).
  • Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat.
  • Fc may refer to this region in isolation, or this region in the context of an Fc polypeptide, as described below.
  • Fc polypeptide as used herein is meant a polypeptide that comprises all or part of an Fc region.
  • Fc polypeptides include antibodies, Fc fusions, isolated Fcs, and Fc fragments.
  • Fc fusion as used herein is meant a protein wherein one or more polypeptides is operably linked to Fc.
  • Fc fusion is herein meant to be synonymous with the terms “immunoadhesin”, “Ig fusion”, “Ig chimera”, and “receptor globulin” (sometimes with dashes) as used in the prior art (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200, both hereby entirely incorporated by reference).
  • An Fc fusion combines the Fc region of an immunoglobulin with a fusion partner, which in general may be any protein, polypeptide or small molecule.
  • the role of the non-Fc part of an Fc fusion i.e., the fusion partner, is to mediate target binding, and thus it is functionally analogous to the variable regions of an antibody.
  • Virtually any protein or small molecule may be linked to Fc to generate an Fc fusion.
  • Protein fusion partners may include, but are not limited to, the target-binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain.
  • Small molecule fusion partners may include any therapeutic agent that directs the Fc fusion to a therapeutic target.
  • Such targets may be any molecule, preferably an extracellular receptor that is implicated in disease.
  • Fc gamma receptor or “Fc ⁇ R” as used herein is meant any member of the family of proteins that bind the IgG antibody Fc region and are substantially encoded by the Fc ⁇ R genes. In humans this family includes but is not limited to Fc ⁇ RI (CD64), including isoforms Fc ⁇ RIa, Fc ⁇ RIb, and Fc ⁇ RIc; Fc ⁇ RII (CD32), including isoforms Fc ⁇ RIIa (including allotypes H131 and R131), Fc ⁇ RIIb (including Fc ⁇ RIIb-1 and Fc ⁇ RIIb-2), and Fc ⁇ RIIc; and Fc ⁇ RIII (CD16), including isoforms Fc ⁇ RIIIa (including allotypes V158 and F158) and Fc ⁇ RIIIb (including allotypes Fc ⁇ RIIIb-NA1 and Fc ⁇ RIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, hereby entirely incorporated by
  • An Fc ⁇ R may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys.
  • Mouse Fc ⁇ Rs include but are not limited to Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), Fc ⁇ RIII (CD16), and Fc ⁇ RIII-2 (CD16-2), as well as any undiscovered mouse Fc ⁇ Rs or Fc ⁇ R isoforms or allotypes.
  • Fc receptor or “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/Fc ligand complex.
  • Fc ligands include but are not limited to Fc ⁇ Rs, Fc ⁇ Rs, Fc ⁇ Rs, FcRn, C1q, C3, mannan binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral Fc ⁇ R.
  • Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors that are homologous to the Fc ⁇ Rs (Davis et al., 2002, Immunological Reviews 190:123-136, hereby entirely incorporated by reference). Fc ligands may include undiscovered molecules that bind Fc.
  • FcRH Fc receptor homologs
  • full length antibody as used herein is meant the structure that constitutes the natural biological form of an antibody, including variable and constant regions.
  • the full length antibody of the IgG isotype is a tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having one light and one heavy chain, each light chain comprising immunoglobulin domains V L and C L , and each heavy chain comprising immunoglobulin domains V H , C ⁇ 1, C ⁇ 2, and C ⁇ 3.
  • IgG antibodies may consist of only two heavy chains, each heavy chain comprising a variable domain attached to the Fc region.
  • IgG as used herein is meant a polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans this IgG comprises the subclasses or isotypes IgG1, IgG2, IgG3, and IgG4. In mice IgG comprises IgG1, IgG2a, IgG2b, IgG3.
  • immunoglobulin herein is meant a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include but are not limited to antibodies. Immunoglobulins may have a number of structural forms, including but not limited to full length antibodies, antibody fragments, and individual immunoglobulin domains.
  • immunoglobulin domain as used herein is meant a region of an immunoglobulin that exists as a distinct structural entity as ascertained by one skilled in the art of protein structure. Ig domains typically have a characteristic ⁇ -sandwich folding topology. The known Ig domains in the IgG isotype of antibodies are V H , C ⁇ 1, C ⁇ 2, C ⁇ 3, V L , and C L .
  • IgG immunoglobulin
  • IgG immunoglobulin a polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans this class comprises the subclasses or isotypes IgG1, IgG2, IgG3, and IgG4.
  • isotype as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.
  • the known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE.
  • parent polypeptide By “parent polypeptide”, “parent protein”, “precursor polypeptide”, or “precursor protein” as used herein is meant an unmodified polypeptide that is subsequently modified to generate a variant.
  • the parent polypeptide may be a naturally occurring polypeptide, or a variant or engineered version of a naturally occurring polypeptide.
  • Parent polypeptide may refer to the polypeptide itself, compositions that comprise the parent polypeptide, or the amino acid sequence that encodes it. Accordingly, by “parent Fc polypeptide” as used herein is meant an Fc polypeptide that is modified to generate a variant, and by “parent antibody” as used herein is meant an antibody that is modified to generate a variant antibody.
  • position as used herein is meant a location in the sequence of a protein. Positions may be numbered sequentially, or according to an established format, for example the EU index as in Kabat. For example, position 297 is a position in the human antibody IgG1.
  • polypeptide or “protein” as used herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
  • residue as used herein is meant a position in a protein and its associated amino acid identity.
  • Asparagine 297 also referred to as Asn297, also referred to as N297
  • Asn297 is a residue in the human antibody IgG1.
  • target antigen as used herein is meant the molecule that is bound specifically by the variable region of a given antibody.
  • a target antigen may be a protein, carbohydrate, lipid, or other chemical compound.
  • target cell as used herein is meant a cell that expresses a target antigen.
  • variable region as used herein is meant the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the V ⁇ , V ⁇ , and/or V H genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively.
  • variant polypeptide polypeptide variant
  • variant as used herein is meant a polypeptide sequence that differs from that of a parent polypeptide sequence by virtue of at least one amino acid modification.
  • the parent polypeptide may be a naturally occurring or wild-type (WT) polypeptide, or may be a modified version of a WT polypeptide.
  • variant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or the amino sequence that encodes it.
  • the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, e.g. from about one to about ten amino acid modifications, and preferably from about one to about five amino acid modifications compared to the parent.
  • the variant polypeptide sequence herein will preferably possess at least about 80% homology with a parent polypeptide sequence, and most preferably at least about 90% homology, more preferably at least about 95% homology. Accordingly, by “Fc variant” or “variant Fc” as used herein is meant an Fc sequence that differs from that of a parent Fc sequence by virtue of at least one amino acid modification.
  • An Fc variant may only encompass an Fc region, or may exist in the context of an antibody, Fc fusion, isolated Fc, Fc fragment, or other polypeptide that is substantially encoded by Fc.
  • Fc variant may refer to the Fc polypeptide itself, compositions comprising the Fc variant polypeptide, or the amino acid sequence that encodes it.
  • Fc polypeptide variant or “variant Fc polypeptide” as used herein is meant an Fc polypeptide that differs from a parent Fc polypeptide by virtue of at least one amino acid modification.
  • protein variant or “variant protein” as used herein is meant a protein that differs from a parent protein by virtue of at least one amino acid modification.
  • antibody variant or “variant antibody” as used herein is meant an antibody that differs from a parent antibody by virtue of at least one amino acid modification.
  • IgG variant or “variant IgG” as used herein is meant an antibody that differs from a parent IgG by virtue of at least one amino acid modification.
  • immunoglobulin variant or “variant immunoglobulin” as used herein is meant an immunoglobulin sequence that differs from that of a parent immunoglobulin sequence by virtue of at least one amino acid modification.
  • wild type or WT herein is meant an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations.
  • a WT protein, polypeptide, antibody, immunoglobulin, IgG, etc. has an amino acid sequence or a nucleotide sequence that has not been intentionally modified.
  • Antibodies are immunological proteins that bind a specific antigen. In most mammals, including humans and mice, antibodies are constructed from paired heavy and light polypeptide chains. The light and heavy chain variable regions show significant sequence diversity between antibodies, and are responsible for binding the target antigen. Each chain is made up of individual immunoglobulin (Ig) domains, and thus the generic term immunoglobulin is used for such proteins.
  • Ig immunoglobulin
  • Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one “light” (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa).
  • Human light chains are classified as kappa and lambda light chains.
  • Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • IgG has several subclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4.
  • IgM has subclasses, including, but not limited to, IgM1 and IgM2.
  • IgA has several subclasses, including but not limited to IgA1 and IgA2.
  • isotype as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.
  • the known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE.
  • the IgG heavy chain is composed of four immunoglobulin domains linked from N- to C-terminus in the order V H -CH1-CH2-CH3, referring to the heavy chain variable domain, heavy chain constant domain 1, heavy chain constant domain 2, and heavy chain constant domain 3 respectively (also referred to as V H -C ⁇ 1-C ⁇ 2-C ⁇ 3, referring to the heavy chain variable domain, constant gamma 1 domain, constant gamma 2 domain, and constant gamma 3 domain respectively).
  • the IgG light chain is composed of two immunoglobulin domains linked from N- to C-terminus in the order V L -C L , referring to the light chain variable domain and the light chain constant domain respectively.
  • the constant regions show less sequence diversity, and are responsible for binding a number of natural proteins to elicit important biochemical events.
  • the distinguishing features between these antibody classes are their constant regions, although subtler differences may exist in the V region.
  • variable region of an antibody contains the antigen binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen.
  • the variable region is so named because it is the most distinct in sequence from other antibodies within the same class.
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • three loops are gathered for each of the V domains of the heavy chain and light chain to form an antigen-binding site. Each of the loops is referred to as a complementarity-determining region (hereinafter referred to as a “CDR”), in which the variation in the amino acid sequence is most significant.
  • CDR complementarity-determining region
  • variable region outside of the CDRs is referred to as the framework (FR) region.
  • FR region The variable region outside of the CDRs.
  • sequence variability does occur in the FR region between different antibodies.
  • this characteristic architecture of antibodies provides a stable scaffold (the FR region) upon which substantial antigen binding diversity (the CDRs) can be explored by the immune system to obtain specificity for a broad array of antigens.
  • a number of high-resolution structures are available for a variety of variable region fragments from different organisms, some unbound and some in complex with antigen.
  • each chain defines a constant region primarily responsible for effector function.
  • Kabat et al. collected numerous primary sequences of the variable regions of heavy chains and light chains. Based on the degree of conservation of the sequences, they classified individual primary sequences into the CDR and the framework and made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, E. A. Kabat et al.).
  • immunoglobulin domains in the heavy chain.
  • immunoglobulin (Ig) domain herein is meant a region of an immunoglobulin having a distinct tertiary structure.
  • the heavy chain domains including, the constant heavy (CH) domains and the hinge domains.
  • the IgG isotypes each have three CH regions. Accordingly, “CH” domains in the context of IgG are as follows: “CH1” refers to positions 118-220 according to the EU index as in Kabat. “CH2” refers to positions 237-340 according to the EU index as in Kabat, and “CH3” refers to positions 341-447 according to the EU index as in Kabat.
  • Ig domain of the heavy chain is the hinge region.
  • hinge region or “hinge region” or “antibody hinge region” or “immunoglobulin hinge region” herein is meant the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 220, and the IgG CH2 domain begins at residue EU position 237.
  • the antibody hinge is herein defined to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1), wherein the numbering is according to the EU index as in Kabat.
  • the lower hinge is included, with the “lower hinge” generally referring to positions 226 or 230.
  • Fc regions are the Fc regions.
  • Fc or “Fc region”, as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain and in some cases, part of the hinge.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains.
  • Fc may include the J chain.
  • Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (C ⁇ 2 and C ⁇ 3) and the lower hinge region between Cgamma1 (C ⁇ 1) and Cgamma2 (C ⁇ 2).
  • the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat.
  • Fc may refer to this region in isolation, or this region in the context of an Fc polypeptide, as described below.
  • Fc polypeptide as used herein is meant a polypeptide that comprises all or part of an Fc region.
  • Fc polypeptides include antibodies, Fc fusions, isolated Fcs, and Fc fragments.
  • An Fc variant comprises one or more amino acid modifications relative to a parent Fc polypeptide, wherein the amino acid modification(s) provide one or more optimized properties.
  • An Fc variant of the present invention differs in amino acid sequence from its parent IgG by virtue of at least one amino acid modification.
  • Fc variants of the present invention have at least one amino acid modification compared to the parent.
  • the Fc variants of the present invention may have more than one amino acid modification as compared to the parent, for example from about one to fifty amino acid modifications, preferably from about one to ten amino acid modifications, and most preferably from about one to about five amino acid modifications compared to the parent.
  • sequences of the Fc variants and those of the parent Fc polypeptide are substantially homologous.
  • variant Fc variant sequences herein will possess about 80% homology with the parent Fc variant sequence, preferably at least about 90% homology, and most preferably at least about 95% homology. Modifications may be made genetically using molecular biology, or may be made enzymatically or chemically.
  • the Fc variants of the present invention may be substantially encoded by immunoglobulin genes belonging to any of the antibody classes.
  • the Fc variants of the present invention find use in antibodies or Fc fusions that comprise sequences belonging to the IgG class of antibodies, including IgG1, IgG2, IgG3, or IgG4.
  • FIG. 2 provides an alignment of these human IgG sequences.
  • the Fc variants of the present invention find use in antibodies or Fc fusions that comprise sequences belonging to the IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG, or IgM classes of antibodies.
  • the Fc variants of the present invention may comprise more than one protein chain. That is, the present invention may find use in an antibody or Fc fusion that is a monomer or an oligomer, including a homo- or hetero-oligomer.
  • the Fc variants of the invention are based on human IgG sequences, and thus human IgG sequences are used as the “base” sequences against which other sequences are compared, including but not limited to sequences from other organisms, for example rodent and primate sequences.
  • Fc variants may also comprise sequences from other immunoglobulin classes such as IgA, IgE, IgGD, IgGM, and the like. It is contemplated that, although the Fc variants of the present invention are engineered in the context of one parent IgG, the variants may be engineered in or “transferred” to the context of another, second parent IgG.
  • the amino acid sequence of a first IgG outlined herein is directly compared to the sequence of a second IgG. After aligning the sequences, using one or more of the homology alignment programs known in the art (for example using conserved residues as between species), allowing for necessary insertions and deletions in order to maintain alignment (i.e., avoiding the elimination of conserved residues through arbitrary deletion and insertion), the residues equivalent to particular amino acids in the primary sequence of the first Fc variant are defined.
  • Alignment of conserved residues preferably should conserve 100% of such residues. However, alignment of greater than 75% or as little as 50% of conserved residues is also adequate to define equivalent residues.
  • Equivalent residues may also be defined by determining structural homology between a first and second IgG that is at the level of tertiary structure for IgGs whose structures have been determined. In this case, equivalent residues are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the parent or precursor (N on N, CA on CA, C on C and O on O) are within about 0.13 nm and preferably about 0.1 nm after alignment.
  • the variant antibody may be engineered in another IgG1 parent antibody that binds a different antigen, a human IgG2 parent antibody, a human IgA parent antibody, a mouse IgG2a or IgG2b parent antibody, and the like.
  • the context of the parent Fc variant does not affect the ability to transfer the Fc variants of the present invention to other parent IgGs.
  • the Fc variants of the present invention are defined according to the amino acid modifications that compose them.
  • I332E is an Fc variant with the substitution I332E relative to the parent Fc polypeptide.
  • S239D/I332E/G236A defines an Fc variant with the substitutions S239D, I332E, and G236A relative to the parent Fc polypeptide.
  • the identity of the WT amino acid may be unspecified, in which case the aforementioned variant is referred to as 239D/332E/236A.
  • substitutions are provided is arbitrary, that is to say that, for example, S239D/I332E/G236A is the same Fc variant as G236A/S239D/I332E, and so on.
  • numbering is according to the EU index or EU numbering scheme (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, hereby entirely incorporated by reference).
  • the EU index or EU index as in Kabat or EU numbering scheme refers to the numbering of the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirely incorporated by reference).
  • the Fc region of an antibody interacts with a number of Fc receptors and ligands, imparting an array of important functional capabilities referred to as effector functions.
  • Fc comprises Ig domains C ⁇ 2 and C ⁇ 3 and the N-terminal hinge leading into C ⁇ 2.
  • An important family of Fc receptors for the IgG class are the Fc gamma receptors (Fc ⁇ Rs). These receptors mediate communication between antibodies and the cellular arm of the immune system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu Rev Immunol 19:275-290, both hereby entirely incorporated by reference).
  • this protein family includes Fc ⁇ RI (CD64), including isoforms Fc ⁇ RIa, Fc ⁇ RIb, and Fc ⁇ RIc; Fc ⁇ RII (CD32), including isoforms Fc ⁇ RIIa (including allotypes H131 and R131), Fc ⁇ RIIb (including Fc ⁇ RIIb-1 and Fc ⁇ RIIb-2), and Fc ⁇ RIIc; and Fc ⁇ RIII (CD16), including isoforms Fc ⁇ RIIIa (including allotypes V158 and F158) and Fc ⁇ RIIIb (including allotypes Fc ⁇ RIIIb-NA1 and Fc ⁇ RIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, hereby entirely incorporated by reference).
  • These receptors typically have an extracellular domain that mediates binding to Fc, a membrane spanning region, and an intracellular domain that may mediate some signaling event within the cell.
  • These receptors are expressed in a variety of immune cells including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, and ⁇ T cells.
  • NK natural killer
  • the ability to mediate cytotoxic and phagocytic effector functions is a potential mechanism by which antibodies destroy targeted cells.
  • the cell-mediated reaction wherein nonspecific cytotoxic cells that express Fc ⁇ Rs recognize bound antibody on a target cell and subsequently cause lysis of the target cell is referred to as antibody dependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu Rev Immunol 19:275-290, both hereby entirely incorporated by reference).
  • the cell-mediated reaction wherein nonspecific cytotoxic cells that express Fc ⁇ Rs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell is referred to as antibody dependent cell-mediated phagocytosis (ADCP).
  • ADCP antibody dependent cell-mediated phagocytosis
  • the different IgG subclasses have different affinities for the Fc ⁇ Rs, with IgG1 and IgG3 typically binding substantially better to the receptors than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett 82:57-65, hereby entirely incorporated by reference).
  • the Fc ⁇ Rs bind the IgG Fc region with different affinities: the high affinity binder Fc ⁇ RI has a Kd for IgG1 of 10 ⁇ 8 M ⁇ 1 , whereas the low affinity receptors Fc ⁇ RII and Fc ⁇ RIII generally bind at 10 ⁇ 6 and 10 ⁇ 5 respectively.
  • Fc ⁇ RIIIa and Fc ⁇ RIIIb are 96% identical, however Fc ⁇ RIIIb does not have a intracellular signaling domain.
  • Fc ⁇ RI, Fc ⁇ RIIa/c, and Fc ⁇ RIIIa are positive regulators of immune complex-triggered activation, characterized by having an intracellular domain that has an immunoreceptor tyrosine-based activation motif (ITAM)
  • Fc ⁇ RIIb has an immunoreceptor tyrosine-based inhibition motif (ITIM) and is therefore inhibitory.
  • IITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • Fc/Fc ⁇ R binding mediates ADCC
  • Fc/C1q binding mediates complement dependent cytotoxicity (CDC).
  • a site on Fc between the C ⁇ 2 and C ⁇ 3 domains mediates interaction with the neonatal receptor FcRn, the binding of which recycles endocytosed antibody from the endosome back to the bloodstream (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766, both hereby entirely incorporated by reference).
  • a key feature of the Fc region is the conserved N-linked glycosylation that occurs at N297.
  • This carbohydrate, or oligosaccharide as it is sometimes referred, plays a critical structural and functional role for the antibody, and is one of the principle reasons that antibodies must be produced using mammalian expression systems.
  • Fc variants of the present invention may be substantially encoded by genes from any organism, preferably mammals, including but not limited to humans, rodents including but not limited to mice and rats, lagomorpha including but not limited to rabbits and hares, camelidae including but not limited to camels, llamas, and dromedaries, and non-human primates, including but not limited to Prosimians, Platyrrhini (New World monkeys), Cercopithecoidea (Old World monkeys), and Hominoidea including the Gibbons and Lesser and Great Apes.
  • the Fc variants of the present invention are substantially human.
  • Gm polymorphism is determined by the IGHG1, IGHG2 and IGHG3 genes which have alleles encoding allotypic antigenic determinants referred to as G1m, G2m, and G3m allotypes for markers of the human IgG1, IgG2 and IgG3 molecules (no Gm allotypes have been found on the gamma 4 chain). Markers may be classified into ‘allotypes’ and ‘isoallotypes’. These are distinguished on different serological bases dependent upon the strong sequence homologies between isotypes. Allotypes are antigenic determinants specified by allelic forms of the Ig genes.
  • Allotypes represent slight differences in the amino acid sequences of heavy or light chains of different individuals. Even a single amino acid difference can give rise to an allotypic determinant, although in many cases there are several amino acid substitutions that have occurred. Allotypes are sequence differences between alleles of a subclass whereby the antisera recognize only the allelic differences.
  • An isoallotype is an allele in one isotype which produces an epitope which is shared with a non-polymorphic homologous region of one or more other isotypes and because of this the antisera will react with both the relevant allotypes and the relevant homologous isotypes (Clark, 1997, IgG effector mechanisms, Chem Immunol. 65:88-110; Gorman & Clark, 1990, Semin Immunol 2(6):457-66, both hereby entirely incorporated by reference).
  • G1m (1, 2, 3, 17) or G1m (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (b1, c3, b5, b0, b3, b4, s, t, g1, c5, u, v, g5)
  • G1m 1, 2, 3, 17 or G1m (a, x, f, z)
  • G2m (23) or G2m (n)
  • G3m 5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28
  • G3m b1, c3, b5, b0, b3, b4, s, t, g1, c5, u, v, g5)
  • Lefranc, et al. The human IgG subclasses: molecular analysis of structure, function and regulation. Pergamon, Oxford, pp. 43-78 (1990); Lefranc, G
  • FIG. 3 shows common haplotypes of the gamma chain of human IgG1 ( FIG. 3 a ) and IgG2 ( FIG. 3 b ) showing the positions and the relevant amino acid substitutions.
  • the Fc variants of the present invention may be substantially encoded by any allotype, isoallotype, or haplotype of any immunoglobulin gene.
  • the antibodies can be a variety of structures, including, but not limited to, antibody fragments, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as “antibody conjugates”), and fragments of each, respectively.
  • antibody mimetics sometimes referred to herein as “antibody mimetics”
  • chimeric antibodies humanized antibodies
  • antibody fusions sometimes referred to as “antibody conjugates”
  • the antibody is an antibody fragment.
  • antibodies that comprise Fc regions, Fc fusions, and the constant region of the heavy chain (CH1-hinge-CH2-CH3), again also including constant heavy region fusions.
  • Specific antibody fragments include, but are not limited to, (i) the Fab fragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragment consisting of the VH and CH1 domains, (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward et al., 1989, Nature 341:544-546) which consists of a single variable, (v) isolated CDR regions, (vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al., 1988, Science 242:423-426, Huston et al., 1988, Proc.
  • scFv single chain Fv molecules
  • the antibodies of the invention multispecific antibody, and notably a bispecific antibody, also sometimes referred to as “diabodies”. These are antibodies that bind to two (or more) different antigens. Diabodies can be manufactured in a variety of ways known in the art (Holliger and Winter, 1993, Current Opinion Biotechnol. 4:446-449), e.g., prepared chemically or from hybrid hybridomas. In one embodiment, the antibody is a minibody. Minibodies are minimized antibody-like proteins comprising a scFv joined to a CH3 domain. Hu et al., 1996, Cancer Res. 56:3055-3061. In some cases, the scFv can be joined to the Fc region, and may include some or all of the hinge region.
  • the scaffold components can be a mixture from different species.
  • the antibody is an antibody
  • such antibody may be a chimeric antibody and/or a humanized antibody.
  • both “chimeric antibodies” and “humanized antibodies” refer to antibodies that combine regions from more than one species.
  • “chimeric antibodies” traditionally comprise variable region(s) from a mouse (or rat, in some cases) and the constant region(s) from a human.
  • “Humanized antibodies” generally refer to non-human antibodies that have had the variable-domain framework regions swapped for sequences found in human antibodies.
  • a humanized antibody the entire antibody, except the CDRs, is encoded by a polynucleotide of human origin or is identical to such an antibody except within its CDRs.
  • the CDRs some or all of which are encoded by nucleic acids originating in a non-human organism, are grafted into the beta-sheet framework of a human antibody variable region to create an antibody, the specificity of which is determined by the engrafted CDRs.
  • the creation of such antibodies is described in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus will typically comprise a human Fc region.
  • Humanized antibodies can also be generated using mice with a genetically engineered immune system. Roque et al., 2004, Biotechnol. Prog. 20:639-654. A variety of techniques and methods for humanizing and reshaping non-human antibodies are well known in the art (See Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and references cited therein).
  • Humanization methods include but are not limited to methods described in Jones et al., 1986, Nature 321:522-525; Riechmann et al.,1988; Nature 332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad.
  • Humanization or other methods of reducing the immunogenicity of nonhuman antibody variable regions may include resurfacing methods, as described for example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973.
  • the parent antibody has been affinity matured, as is known in the art. Structure-based methods may be employed for humanization and affinity maturation, for example as described in U.S. Ser. No. 11/004,590. Selection based methods may be employed to humanize and/or affinity mature antibody variable regions, including but not limited to methods described in Wu et al., 1999, J. Mol. Biol.
  • the antibody is a fully human antibody with at least one modification as outlined herein.
  • “Fully human antibody ” or “complete human antibody” refers to a human antibody having the gene sequence of an antibody derived from a human chromosome with the modifications outlined herein.
  • Fully human antibodies may be obtained, for example, using transgenic mice (Bruggemann et al., 1997, Curr Opin Biotechnol 8:455-458) or human antibody libraries coupled with selection methods (Griffiths et al., 1998, Curr Opin Biotechnol 9:102-108).
  • the antibodies of the invention are antibody fusion proteins (sometimes referred to herein as an “antibody conjugate”).
  • antibody fusions are Fc fusions, which join the Fc region with a conjugate partner.
  • Fc fusion as used herein is meant a protein wherein one or more polypeptides is operably linked to an Fc region.
  • Fc fusion is herein meant to be synonymous with the terms “immunoadhesin”, “Ig fusion”, “Ig chimera”, and “receptor globulin” (sometimes with dashes) as used in the prior art (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200).
  • An Fc fusion combines the Fc region of an immunoglobulin with a fusion partner, which in general can be any protein or small molecule. Virtually any protein or small molecule may be linked to Fc to generate an Fc fusion.
  • Protein fusion partners may include, but are not limited to, the variable region of any antibody, the target-binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain.
  • Small molecule fusion partners may include any therapeutic agent that directs the Fc fusion to a therapeutic target.
  • targets may be any molecule, preferably an extracellular receptor, that is implicated in disease.
  • an antibody-like protein that is finding an expanding role in research and therapy is the Fc fusion (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200, both hereby entirely incorporated by reference).
  • An Fc fusion is a protein wherein one or more polypeptides is operably linked to Fc.
  • An Fc fusion combines the Fc region of an antibody, and thus its favorable effector functions and pharmacokinetics, with the target-binding region of a receptor, ligand, or some other protein or protein domain. The role of the latter is to mediate target recognition, and thus it is functionally analogous to the antibody variable region. Because of the structural and functional overlap of Fc fusions with antibodies, the discussion on antibodies in the present invention extends also to Fc fusions.
  • antibody fusions include the fusion of the constant region of the heavy chain with one or more fusion partners (again including the variable region of any antibody), while other antibody fusions are substantially or completely full length antibodies with fusion partners.
  • a role of the fusion partner is to mediate target binding, and thus it is functionally analogous to the variable regions of an antibody (and in fact can be).
  • Virtually any protein or small molecule may be linked to Fc to generate an Fc fusion (or antibody fusion).
  • Protein fusion partners may include, but are not limited to, the target-binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain.
  • Small molecule fusion partners may include any therapeutic agent that directs the Fc fusion to a therapeutic target.
  • Such targets may be any molecule, preferably an extracellular receptor, that is implicated in disease.
  • the conjugate partner can be proteinaceous or non-proteinaceous; the latter generally being generated using functional groups on the antibody and on the conjugate partner.
  • linkers are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see, 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference).
  • Suitable conjugates include, but are not limited to, labels as described below, drugs and cytotoxic agents including, but not limited to, cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or active fragments of such toxins.
  • cytotoxic drugs e.g., chemotherapeutic agents
  • Suitable toxins and their corresponding fragments include diptheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin and the like.
  • Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antibodies, or binding of a radionuclide to a chelating agent that has been covalently attached to the antibody. Additional embodiments utilize calicheamicin, auristatins, geldanamycin, maytansine, and duocarmycins and analogs; for the latter, see U.S. 2003/0050331, hereby incorporated by reference in its entirety.
  • Covalent modifications of antibodies are included within the scope of this invention, and are generally, but not always, done post-translationally.
  • several types of covalent modifications of the antibody are introduced into the molecule by reacting specific amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues.
  • Cysteinyl residues most commonly are reacted with ⁇ -haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, ⁇ -bromo- ⁇ -(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.
  • Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0.
  • Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
  • tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane.
  • aromatic diazonium compounds or tetranitromethane Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Tyrosyl residues are iodinated using 125I or 131I to prepare labeled proteins for use in radioimmunoassay, the chloramine T method described above being suitable.
  • Carboxyl side groups are selectively modified by reaction with carbodiimides (R′—N ⁇ C ⁇ N—R′), where R and R′ are optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.
  • R′ is optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.
  • aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Derivatization with bifunctional agents is useful for crosslinking antibodies to a water-insoluble support matrix or surface for use in a variety of methods, in addition to methods described below.
  • Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis (succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane.
  • Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
  • reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
  • Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
  • Another type of covalent modification of the antibody comprises linking the antibody to various nonproteinaceous polymers, including, but not limited to, various polyols such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
  • amino acid substitutions may be made in various positions within the antibody to facilitate the addition of polymers such as PEG. See for example, U.S. Publication No. 2005/0114037, incorporated herein by reference in its entirety.
  • the covalent modification of the antibodies of the invention comprises the addition of one or more labels. In some cases, these are considered antibody fusions.
  • labelling group means any detectable label.
  • the labelling group is coupled to the antibody via spacer arms of various lengths to reduce potential steric hindrance.
  • spacer arms of various lengths to reduce potential steric hindrance.
  • Various methods for labelling proteins are known in the art and may be used in performing the present invention.
  • labels fall into a variety of classes, depending on the assay in which they are to be detected: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox active moieties; d) optical dyes; enzymatic groups (e.g. horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase); e) biotinylated groups; and f) predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.).
  • the labelling group is coupled to the antibody via spacer arms of various lengths to reduce potential steric hindrance.
  • Various methods for labelling proteins are known in the art and may be used in performing the present invention.
  • optical dyes including, but not limited to, chromophores, phosphors and fluorophores, with the latter being specific in many instances.
  • Fluorophores can be either “small molecule” fluores, or proteinaceous fluores.
  • fluorescent label any molecule that may be detected via its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueJ, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon green, the Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene, Oreg.), FITC, Rhod
  • Suitable proteinaceous fluorescent labels also include, but are not limited to, green fluorescent protein, including a Renilla, Ptilosarcus, or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol.
  • green fluorescent protein including a Renilla, Ptilosarcus, or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd
  • EYFP enhanced yellow fluorescent protein
  • luciferase Rhoplasminogen activatories, Inc.
  • ⁇ galactosidase Nolan et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:2603-2607
  • Renilla WO92/15673, WO95/07463, WO98/14605, WO98/26277, WO99/49019, U.S. Pat. Nos.
  • any antigen may be targeted by the Fc variants of the present invention, including but not limited to proteins, subunits, domains, motifs, and/or epitopes belonging to the following list of targets: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 Adenosine Receptor, A33, ACE, ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin
  • polypeptides including antibodies, are subjected to a variety of post-translational modifications involving carbohydrate moieties, such as glycosylation with oligosaccharides.
  • carbohydrate moieties such as glycosylation with oligosaccharides.
  • the species, tissue and cell type have all been shown to be important in the way that glycosylation occurs.
  • the extracellular environment through altered culture conditions such as serum concentration, may have a direct effect on glycosylation. (Lifely et al., 1995, Glycobiology 5(8): 813-822).
  • All antibodies contain carbohydrate at conserved positions in the constant regions of the heavy chain.
  • Each antibody isotype has a distinct variety of N-linked carbohydrate structures. Aside from the carbohydrate attached to the heavy chain, up to 30% of human IgGs have a glycosylated Fab region.
  • IgG has a single N-linked biantennary carbohydrate at Asn297 of the CH2 domain.
  • the IgG are heterogeneous with respect to the Asn297 linked carbohydrate.
  • the core oligosaccharide normally consists of GlcNAc 2 Man 3 GlcNAc, with differing numbers of outer residues.
  • carbohydrate moieties of the present invention will be described with reference to commonly used nomenclature for the description of oligosaccharides.
  • This nomenclature includes, for instance, Man, which represents mannose; GlcNAc, which represents 2-N-acetylglucosamine; Gal which represents galactose; Fuc for fucose; and Glc, which represents glucose.
  • Sialic acids are described by the shorthand notation NeuNAc, for 5-N-acetylneuraminic acid, and NeuNGc for 5-glycolylneuraminic.
  • glycosylation means the attachment of oligosaccharides (carbohydrates containing two or more simple sugars linked together e.g. from two to about twelve simple sugars linked together) to a glycoprotein.
  • oligosaccharide side chains are typically linked to the backbone of the glycoprotein through either N- or O-linkages.
  • the oligosaccharides of the present invention occur generally are attached to a CH2 domain of an Fc region as N-linked oligosaccharides.
  • N-linked glycosylation refers to the attachment of the carbohydrate moiety to an asparagine residue in a glycoprotein chain.
  • each of murine IgG1, IgG2a, IgG2b and IgG3 as well as human IgG1, IgG2, IgG3, IgG4, IgA and IgD CH2 domains have a single site for N-linked glycosylation at amino acid residue 297 (Kabat et al. Sequences of Proteins of Immunological Interest, 1991).
  • a “mature core carbohydrate structure” refers to a processed core carbohydrate structure attached to an Fc region which generally consists of the following carbohydrate structure GlcNAc(Fucose)-GlcNAc-Man-(Man-GlcNAc) 2 typical of biantennary oligosaccharides.
  • the mature core carbohydrate structure is attached to the Fc region of the glycoprotein, generally via N-linkage to Asn297 of a CH2 domain of the Fc region.
  • a “bisecting GlcNAc” is a GlcNAc residue attached to the ⁇ 1,4 mannose of the mature core carbohydrate structure.
  • the bisecting GlcNAc can be enzymatically attached to the mature core carbohydrate structure by a ⁇ (1,4)-N-acetylglucosaminyltransferase III enzyme (GnTIII).
  • GnTIII ⁇ (1,4)-N-acetylglucosaminyltransferase III enzyme
  • CHO cells do not normally express GnTIII (Stanley et al., 1984, J. Biol. Chem. 261:13370-13378), but may be engineered to do so (Umana et al., 1999, Nature Biotech. 17:176-180).
  • modified glycoforms or engineered glycoforms contemplates Fc variants that comprise modified glycoforms or engineered glycoforms.
  • modified glycoform or “engineered glycoform” as used herein is meant a carbohydrate composition that is covalently attached to an IgG, wherein the carbohydrate composition differs chemically from that of a parent IgG.
  • Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing Fc ⁇ R-mediated effector function.
  • the Fc variants of the present invention are modified to control the level of fucosylated and/or bisecting oligosaccharides that are covalently attached to the Fc region.
  • These techniques control the level of fucosylated and/or bisecting oligosaccharides that are covalently attached to the Fc region, for example by expressing an IgG in various organisms or cell lines, engineered or otherwise (for example Lec-13 CHO cells or rat hybridoma YB2/0 cells), by regulating enzymes involved in the glycosylation pathway (for example FUT8 [ ⁇ 1,6-fucosyltranserase] and/or ⁇ 1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by modifying carbohydrate(s) after the IgG has been expressed.
  • enzymes involved in the glycosylation pathway for example FUT8 [ ⁇ 1,6-fucosyltranserase] and/or ⁇ 1-4-N-acetylglucosaminyltransferase III [GnTIII]
  • Engineered glycoform typically refers to the different carbohydrate or oligosaccharide; thus an IgG variant, for example an antibody or Fc fusion, can include an engineered glycoform.
  • engineered glycoform may refer to the IgG variant that comprises the different carbohydrate or oligosaccharide.
  • a “parent Fc polypeptide” is a glycosylated Fc polypeptide having the same amino acid sequence and mature core carbohydrate structure as an engineered glycoform of the present invention, except that fucose is attached to the mature core carbohydrate structure. For instance, in a composition comprising the parent glycoprotein about 50-100% or about 70-100% of the parent glycoprotein comprises a mature core carbohydrate structure having fucose attached thereto.
  • the present invention provides a composition comprising a glycosylated Fc polypeptiden having an Fc region, wherein about 51-100% of the glycosylated Fc polypeptide in the composition comprises a mature core carbohydrate structure which lacks fucose, attached to the Fc region of the Fc polypeptide. More preferably, about 80-100% of the Fc polypeptide in the composition comprises a mature core carbohydrate structure which lacks fucose and most preferably about 90-99% of the Fc polypeptide in the composition lacks fucose attached to the mature core carbohydrate structure.
  • the Fc polypeptide in the composition both comprises a mature core carbohydrate structure that lacks fucose and additionally comprises at least one amino acid modification in the Fc region.
  • the combination of engineered glycoform and amino acid modification provides optimal Fc receptor binding properties to the Fc polypeptide.
  • the Fc variants of the present invention may be optimized for a variety of Fc receptor binding properties.
  • An Fc variant that is engineered or predicted to display one or more optimized properties is herein referred to as an “optimized Fc variant”.
  • Properties that may be optimized include but are not limited to increased or reduced affinity for an Fc ⁇ R.
  • the Fc variants of the present invention are optimized to possess increased affinity for a human activating Fc ⁇ R, preferably Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIc, Fc ⁇ RIIIa, and Fc ⁇ RIIIb, most preferably Fc ⁇ RIIa and Fc ⁇ RIIIa.
  • the Fc variants are optimized to possess reduced affinity for the human inhibitory receptor Fc ⁇ RIIb.
  • Fc variants of the present invention provide increased affinity for one or more Fc ⁇ Rs, yet reduced affinity for one or more other Fc ⁇ Rs.
  • an Fc variant of the present invention may have increased binding to Fc ⁇ RI, Fc ⁇ RIIa, and/or Fc ⁇ RIIIa, yet reduced binding to Fc ⁇ RIIb.
  • the Fc variant with improved Fc receptor binding affinity may display from about 5 fold to about 1000 fold, e.g. from about 10 fold to about 500 fold improvement in Fc receptor binding affinity compared to the parent Fc polypeptide, where Fc receptor binding affinity is determined, for example, as disclosed in the Examples herein.
  • reduced affinity as compared to a parent Fc polypeptide as used herein is meant that an Fc variant binds an Fc receptor with significantly lower KA or higher KD than the parent Fc polypeptide.
  • a promising means for enhancing the anti-tumor potency of antibodies is via enhancement of their ability to mediate cytotoxic effector functions such as ADCC, ADCP, and CDC.
  • a critical set of data supporting the relevance of Fc ⁇ R-mediated effector functions in antibody therapeutic mechanism are the correlations observed between clinical efficacy in humans and their allotype of high and low affinity polymorphic forms of Fc ⁇ Rs.
  • human IgG1 binds with greater affinity to the V158 isoform of Fc ⁇ RIIIa than the F158 isoform.
  • This difference in affinity, and its effect Fc ⁇ R-mediated effector functions such as ADCC and/or ADCP, has been shown to be a significant determinant of the efficacy of the anti-CD20 antibody rituximab (Rituxan®, Biogenldec).
  • V158 allotype respond favorably to rituximab treatment; however, patients with the lower affinity F158 allotype respond poorly (Cartron et al., 2002, Blood 99:754-758; Weng & Levy, 2003, J Clin Oncol, 21(21):3940-3947, hereby entirely incorporated by reference).
  • Approximately 10-20% of humans are V158/N158 homozygous, 45% are V158/F158 heterozygous, and 35-45% of humans are F158/F158 homozygous (Lehrnbecher et al., 1999, Blood 94:4220-4232; Cartron et al., 2002, Blood 99:754-758, both hereby entirely incorporated by reference).
  • the Fc ⁇ RIIa polymorphism also correlated with clinical outcome following immunotherapy of neuroblastoma with a murine IgG3 anti-GD2 antibody and GMC-SF (Cheung et al., 2006 J Clin Oncol 24(18):1-6).
  • Murine IgG3 has higher affinity for the R131 isoform of human Fc ⁇ RIIa than the H131 form, and patients homozygous for R131 showed better response than H/H homozygous patients. Notably, this is the first documentation of a clinical correlation between Fc ⁇ R polymorphism and outcome in solid tumors, suggesting that the importance of Fc ⁇ R-mediated effector functions is not limited to antibodies targeting hematological cancers.
  • FIG. 1 shows the activating and inhibitory Fc ⁇ Rs that may be involved in regulating the activities of several immune cell types. Whereas NK cells only express the activating receptor Fc ⁇ RIIIa, all of the other cell types, including neutrophils, macrophages, and dendritic cells, express the inhibitory receptor Fc ⁇ RIIb, as well the other activating receptors Fc ⁇ RI and Fc ⁇ RIIa.
  • optimal effector function may result from an antibody that has increased affinity for activation receptors, for example Fc ⁇ RI, Fc ⁇ RIIa, and Fc ⁇ RIIIa, yet reduced affinity for the inhibitory receptor Fc ⁇ RIIb.
  • these other cells types can utilize Fc ⁇ Rs to mediate not only innate effector functions that directly lyse cells, for example ADCC, but can also phagocytose targeted cells and process antigen for presentation to other immune cells, events that can ultimately lead to the generation of adaptive immune response.
  • Fc variants that selectively ligate activating versus inhibitory receptors may affect DC processing, T cell priming and activation, antigen immunization, and/or efficacy against cancer (Dhodapkar & Dhodapkar, 2005, Proc Natl Acad Sci USA, 102, 6243-6244, entirely incorporated by reference).
  • Such variants may be employed as novel strategies for targeting antigens to the activating or inhibitory Fc ⁇ Rs on human DCs, macrophages, or other antigen presenting cells to generate target-specific immunity.
  • the present application is directed to Fc variants having differential specificity for various receptors.
  • the change in affinity for one or more receptors can be increased relative to a second receptor or group of receptors.
  • the present invention is directed to an Fc variant of a parent Fc polypeptide comprising at least a first and a second substitution.
  • the first and second substitutions are each at a position selected from group consisting of 234, 235, 236, 239, 267, 268, 293, 295, 324, 327, 328, 330, and 332 according to the EU index.
  • the Fc variant exhibits an increase in affinity for one or more receptors selected from the group consisting of Fc ⁇ RI, Fc ⁇ RIIa, and Fc ⁇ RIIIa as compared to the increase in a affinity of the Fc variant for the Fc ⁇ RIIb receptor.
  • the increases in affinities are relative to the parent polypeptide.
  • the Fc variant has increased affinity for the activating receptor as compared to the parent Fc polypeptide but has reduced affinity (i.e. a negative increase in affinity) for Fc ⁇ RIIb as compared to the parent Fc polypeptide.
  • the increase in affinity is greater for an activating receptor than it is for Fc ⁇ RIIb.
  • Other activating receptors are also contemplated.
  • the affinity for Fc ⁇ RI, Fc ⁇ RIIa, and Fc ⁇ RIIIa receptors is increased.
  • Table 1 illustrates several embodiments of human Fc receptor affinity profiles wherein the Fc variant provide selectively increased affinity for activating receptors relative to the inhibitory receptor Fc ⁇ RIIb.
  • Fc variants with such Fc receptor affinity profiles is to impart antibodies, Fc fusions, or other Fc polypeptides with enhanced Fc ⁇ R-mediated effector function and cellular activation, specifically for cells that express both activating and inhibitory receptors including but not limited to neutrophils, monocytes and macrophages, and dendritic cells.
  • the Fc variant exhibits an increase in affinity of the Fc variant for the Fc ⁇ RIIb receptor as compared to the increase in affinity for one or more activating receptors.
  • Activating receptors include Fc ⁇ RI, Fc ⁇ RIIa, and Fc ⁇ RIIIa. Increased affinities are relative to the parent polypeptide. The first and second substitutions each at a position selected from group consisting of 234, 235, 236, 239, 267, 268, 293, 295, 324, 327, 328, 330 and 332 according to the EU index.
  • the Fc variant has increased affinity for the activating receptor as compared to the parent Fc polypeptide but has reduced affinity (i.e.
  • Fc ⁇ RIIb a negative increase in affinity for Fc ⁇ RIIb as compared to the parent Fc polypeptide.
  • the increase in affinity is greater for Fc ⁇ RIIb than it is for the one or more activating receptors.
  • the affinity for Fc ⁇ RIIb is increased.
  • Table 2 illustrates several embodiments of human Fc receptor affinity profiles wherein the Fc variant provide selectively increased affinity for the inhibitory receptor Fc ⁇ RIIb relative to one or more activating receptors.
  • Fc variants with such Fc receptor affinity profiles is to impart antibodies, Fc fusions, or other Fc polypeptides with reduced Fc ⁇ R-mediated effector function and to inhibit cellular activation, specifically for cells that express the inhibitory receptor Fc ⁇ RIIb, including but not limited to neutrophils, monocytes and macrophages, dendritic cells, and B cells.
  • the Fc variants that provide selectively increased affinity for activating receptors or inhibitory receptor are murine antibodies, and said selective enhancements are to murine Fc receptors.
  • various embodiments provide for the generation of surrogate antibodies that are designed to be most compatible with mouse disease models, and may be informative for example in pre-clinical studies.
  • Fc ⁇ Rs The presence of different polymorphic forms of Fc ⁇ Rs provides yet another parameter that impacts the therapeutic utility of the Fc variants of the present invention.
  • specificity and selectivity of a given Fc variant for the different classes of Fc ⁇ Rs significantly affects the capacity of an Fc variant to target a given antigen for treatment of a given disease
  • the specificity or selectivity of an Fc variant for different polymorphic forms of these receptors may in part determine which research or pre-clinical experiments may be appropriate for testing, and ultimately which patient populations may or may not respond to treatment.
  • Fc variants of the present invention may be used to guide the selection of valid research and pre-clinical experiments, clinical trial design, patient selection, dosing dependence, and/or other aspects concerning clinical trials.
  • Fc variants of the invention may comprise modifications that modulate interaction with Fc receptors other than Fc ⁇ Rs, including but not limited to complement proteins, FcRn, and Fc receptor homologs (FcRHs).
  • FcRHs include but are not limited to FcRH1, FcRH2, FcRH3, FcRH4, FcRH5, and FcRH6 (Davis et al., 2002, Immunol. Reviews 190:123-136).
  • Fc receptor selectivity or specificity of a given Fc variant will provide different properties depending on whether it composes an antibody, Fc fusion, or Fc variants with a coupled fusion or conjugate partner.
  • Fc variants are used in therapeutic utilities based on their respective receptor specificities.
  • the utility of a given Fc variant for therapeutic purposes can depend on the epitope or form of the target antigen and the disease or indication being treated.
  • enhanced Fc ⁇ R-mediated effector functions may be preferable. This may be particularly favorable for anti-cancer Fc variants.
  • Fc variants can be used that comprise Fc variants that provide increased affinity for activating Fc ⁇ Rs and/or reduced affinity for inhibitory Fc ⁇ Rs.
  • Fc variants that provide differential selectivity for different activating Fc ⁇ Rs; for example, in some cases enhanced binding to Fc ⁇ RIIa and Fc ⁇ RIIIa may be desired, but not Fc ⁇ RI, whereas in other cases, enhanced binding only to Fc ⁇ RIIa may be preferred.
  • Fc variants that enhance both Fc ⁇ R-mediated and complement-mediated effector functions, whereas for other cases it may be advantageous to utilize Fc variants that enhance either Fc ⁇ R-mediated or complement-mediated effector functions.
  • Fc variants that provide enhanced binding to the inhibitory Fc ⁇ RIIb, yet WT level, reduced, or ablated binding to activating Fc ⁇ Rs. This may be particularly useful, for example, when the goal of an Fc variant is to inhibit inflammation or auto-immune disease, or modulate the immune system in some way.
  • the target of the Fc variants of the present invention is itself one or more Fc ligands.
  • Fc polypeptides of the invention can be utilized to modulate the activity of the immune system, and in some cases to mimic the effects of IVIg therapy in a more controlled, specific, and efficient manner.
  • IVIg is effectively a high dose of immunoglobulins delivered intravenously.
  • IVIg has been used to downregulate autoimmune conditions. It has been hypothesized that the therapeutic mechanism of action of IVIg involves ligation of Fc receptors at high frequency (J. Bayry et al., 2003, Transfusion Clinique et Bitechnik 10: 165-169; Binstadt et al., 2003, J Allergy Clin.
  • Ithrombocytopenia purpura Ithrombocytopenia purpura
  • immunoglobulins are harvested from thousands of donors, with all of the concomitant problems associated with non-recombinant biotherapeutics collected from humans.
  • An Fc variant of the present invention should serve all of the roles of IVIg while being manufactured as a recombinant protein rather than harvested from donors.
  • the immunomodulatory effects of IVIg may be dependent on productive interaction with one or more Fc ligands, including but not limited to Fc ⁇ Rs, complement proteins, and FcRn.
  • Fc variants of the invention with increased affinity for Fc ⁇ RIIb can be used to promote anti-inflammatory activity (Samuelsson et al., 2001, Science 291: 484-486) and or to reduce autoimmunity (Hogarth, 2002, Current Opinion in Immunology, 14:798-802).
  • Fc polypeptides of the invention with increased affinity for one or more Fc ⁇ Rs can be utilized by themselves or in combination with additional modifications to reduce autoimmunity (Hogarth, 2002, Current Opinion in Immunology, 14:798-802).
  • Fc variants of the invention with increased affinity for Fc ⁇ RIIa but reduced capacity for intracellular signaling can be used to reduce immune system activation by competitively interfering with Fc ⁇ RIIIa binding.
  • the context of the Fc variant impacts the desired specificity.
  • Fc variants that provide enhanced binding to one or more activating Fc ⁇ Rs may provide optimal immunomodulatory effects in the context of an antibody, Fc fusion, isolated Fc, or Fc fragment by acting as an Fc ⁇ R antagonist (van Mirre et al., 2004, J. Immunol. 173:332-339).
  • fusion or conjugation of two or more Fc variants may provide different effects, and for such an Fc polypeptide it may be optimal to utilize Fc variants that provide increased affinity for an inhibitory receptor.
  • the Fc variants of the present invention may be used as immunomodulatory therapeutics. Binding to or blocking Fc receptors on immune system cells may be used to influence immune response in immunological conditions including but not limited to idiopathic thrombocytopenia purpura (ITP) and rheumatoid arthritis (RA) among others.
  • ITP idiopathic thrombocytopenia purpura
  • RA rheumatoid arthritis
  • the Fc variants may provide enhanced binding to an Fc ⁇ R, including but not limited to Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa, Fc ⁇ RIIIb, and/or Fc ⁇ RI.
  • binding enhancements to Fc ⁇ RIIb would increase expression or inhibitory activity, as needed, of that receptor and improve efficacy.
  • blocking binding to activation receptors such as Fc ⁇ RIIIb or Fc ⁇ RI may improve efficacy.
  • modulated affinity of the Fc variants for FcRn and/or also complement may also provide benefits.
  • Fc variants that provide enhanced binding to the inhibitory receptor Fc ⁇ RIIb provide an enhancement to the IVIg therapeutic approach.
  • the Fc variants of the present invention that bind with greater affinity to the Fc ⁇ RIIb receptor than parent Fc polypeptide may be used.
  • Such Fc variants would thus function as Fc ⁇ RIIb agonists, and would be expected to enhance the beneficial effects of IVIg as an autoimmune disease therapeutic and also as a modulator of B-cell proliferation.
  • such Fc ⁇ RIIb-enhanced Fc variants may also be further modified to have the same or limited binding to other receptors.
  • the Fc variants with enhanced Fc ⁇ RIIb affinity may be combined with mutations that reduce or ablate to other receptors, thereby potentially further minimizing side effects during therapeutic use.
  • Such immunomodulatory applications of the Fc variants of the present invention may also be utilized in the treatment of oncological indications, especially those for which antibody therapy involves antibody-dependant cytotoxic mechanisms.
  • an Fc variant that enhances affinity to Fc ⁇ RIIb may be used to antagonize this inhibitory receptor, for example by binding to the Fc/Fc ⁇ RIIb binding site but failing to trigger, or reducing cell signaling, potentially enhancing the effect of antibody-based anti-cancer therapy.
  • Such Fc variants, functioning as Fc ⁇ RIIb antagonists may either block the inhibitory properties of Fc ⁇ RIIb, or induce its inhibitory function as in the case of IVIg.
  • Fc ⁇ RIIb antagonist may be used as co-therapy in combination with any other therapeutic, including but not limited to antibodies, acting on the basis of ADCC related cytotoxicity.
  • Fc ⁇ RIIb antagonistic Fc variants of this type are preferably isolated Fc or Fc fragments, although in alternate embodiments antibodies and Fc fusions may be used.
  • the Fc variants of the present invention may comprise modifications to reduce immunogenicity in humans.
  • the immunogenicity of an Fc variant of the present invention is reduced using a method described in U.S. Ser. No. 11/004,590, filed Dec. 3, 2004, hereby entirely incorporated by reference.
  • the Fc variants of the present invention are humanized (Clark, 2000, Immunol Today 21:397-402, hereby entirely incorporated by reference).
  • humanized antibody as used herein is meant an antibody comprising a human framework region (FR) and one or more complementarity determining regions (CDR's) from a non-human (usually mouse or rat) antibody.
  • the non-human antibody providing the CDR's is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor”.
  • Humanization relies principally on the grafting of donor CDRs onto acceptor (human) VL and VH frameworks (e.g., Winter et al, U.S. Pat. No. 5,225,539, hereby entirely incorporated by reference). This strategy is referred to as “CDR grafting”.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus will typically comprise a human Fc region.
  • an immunoglobulin constant region typically that of a human immunoglobulin
  • human Fc region typically comprise a human Fc region.
  • Humanization methods include but are not limited to methods described in Jones et al., 1986, Nature 321:522-525; Riechmann et al.,1988; Nature 332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad.
  • Humanization or other methods of reducing the immunogenicity of nonhuman antibody variable regions may include resurfacing methods, as described for example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973, hereby entirely incorporated by reference.
  • the parent antibody has been affinity matured, as is well known in the art. Structure-based methods may be employed for humanization and affinity maturation, for example as described in U.S. Ser. No. 11/004,590, hereby entirely incorporated by reference.
  • Selection based methods may be employed to humanize and/or affinity mature antibody variable regions, including but not limited to methods described in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759, all hereby entirely incorporated by reference.
  • Modifications to reduce immunogenicity may include modifications that reduce binding of processed peptides derived from the parent sequence to MHC proteins.
  • amino acid modifications may be engineered such that there are no or a minimal number of immune epitopes that are predicted to bind, with high affinity, to any prevalent MHC alleles.
  • Several methods of identifying MHC-binding epitopes in protein sequences are known in the art and may be used to score epitopes in an Fc variant of the present invention. See for example WO 98/52976; WO 02/079232; WO 00/3317; U.S. Ser. No. 09/903,378; U.S. Ser. No. 10/039,170; U.S. Ser. No.
  • Sequence-based information can be used to determine a binding score for a given peptide—MHC interaction (see for example Mallios, 1999, Bioinformatics 15: 432-439; Mallios, 2001, Bioinformatics 17: p942-948; Sturniolo et. al., 1999, Nature Biotech. 17: 555-561, all hereby entirely incorporated by reference).
  • the Fc variant of the present invention is conjugated or operably linked to another therapeutic compound.
  • the therapeutic compound may be a cytotoxic agent, a chemotherapeutic agent, a toxin, a radioisotope, a cytokine, or other therapeutically active agent.
  • the IgG may be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
  • the present invention provides methods for engineering, producing, and screening Fc variants.
  • the described methods are not meant to constrain the present invention to any particular application or theory of operation. Rather, the provided methods are meant to illustrate generally that one or more Fc variants may be engineered, produced, and screened experimentally to obtain Fc variants with optimized effector function.
  • a variety of methods are described for designing, producing, and testing antibody and protein variants in U.S. Ser. No. 10/672,280, U.S. Ser. No. 10/822,231, U.S. Ser. No. 11/124,620, and U.S. Ser. No. 11/256,060, all hereby entirely incorporated by reference.
  • a variety of protein engineering methods may be used to design Fc variants with optimized effector function.
  • a structure-based engineering method may be used, wherein available structural information is used to guide substitutions.
  • An alignment of sequences may be used to guide substitutions at the identified positions.
  • random or semi-random mutagenesis methods may be used to make amino acid modifications at the desired positions.
  • the Fc variant sequences are used to create nucleic acids that encode the member sequences, and that may then be cloned into host cells, expressed and assayed, if desired. These practices are carried out using well-known procedures, and a variety of methods that may find use in the present invention are described in Molecular Cloning—A Laboratory Manual, 3 rd Ed. (Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001), and Current Protocols in Molecular Biology (John Wiley & Sons), both entirely incorporated by reference.
  • the Fc variants of the present invention may be produced by culturing a host cell transformed with nucleic acid, preferably an expression vector, containing nucleic acid encoding the Fc variants, under the appropriate conditions to induce or cause expression of the protein.
  • a host cell transformed with nucleic acid preferably an expression vector, containing nucleic acid encoding the Fc variants
  • a wide variety of appropriate host cells may be used, including but not limited to mammalian cells, bacteria, insect cells, and yeast.
  • a variety of cell lines that may find use in the present invention are described in the ATCC cell line catalog, available from the American Type Culture Collection.
  • the methods of introducing exogenous nucleic acid into host cells are well known in the art, and will vary with the host cell used.
  • Fc variants are purified or isolated after expression.
  • Antibodies may be isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques, electrophoretic, immunological, precipitation, dialysis, filtration, concentration, and chromatofocusing techniques. As is well known in the art, a variety of natural proteins bind antibodies, for example bacterial proteins A, G, and L, and these proteins may find use in the present invention for purification. Purification can often be enabled by a particular fusion partner.
  • proteins may be purified using glutathione resin if a GST fusion is employed, Ni +2 affinity chromatography if a His-tag is employed, or immobilized anti-flag antibody if a flag-tag is used.
  • glutathione resin if a GST fusion is employed
  • Ni +2 affinity chromatography if a His-tag is employed
  • immobilized anti-flag antibody if a flag-tag is used.
  • Fc variants may be screened using a variety of methods, including but not limited to those that use in vitro assays, in vivo and cell-based assays, and selection technologies. Automation and high-throughput screening technologies may be utilized in the screening procedures. Screening may employ the use of a fusion partner or label, for example an immune label, isotopic label, or small molecule label such as a fluorescent or calorimetric dye.
  • a fusion partner or label for example an immune label, isotopic label, or small molecule label such as a fluorescent or calorimetric dye.
  • the functional and/or biophysical properties of Fc variants are screened in an in vitro assay.
  • the protein is screened for functionality, for example its ability to catalyze a reaction or its binding affinity to its target.
  • selection methods are those that select for favorable members of a library.
  • the methods are herein referred to as “selection methods”, and these methods find use in the present invention for screening Fc variants.
  • selection methods When protein libraries are screened using a selection method, only those members of a library that are favorable, that is which meet some selection criteria, are propagated, isolated, and/or observed.
  • selection methods are known in the art that may find use in the present invention for screening protein libraries.
  • Other selection methods that may find use in the present invention include methods that do not rely on display, such as in vivo methods.
  • a subset of selection methods referred to as “directed evolution” methods are those that include the mating or breading of favorable sequences during selection, sometimes with the incorporation of new mutations.
  • Fc variants are screened using one or more cell-based or in vivo assays.
  • purified or unpurified proteins are typically added exogenously such that cells are exposed to individual variants or pools of variants belonging to a library.
  • These assays are typically, but not always, based on the function of the Fc polypeptide; that is, the ability of the Fc polypeptide to bind to its target and mediate some biochemical event, for example effector function, ligand/receptor binding inhibition, apoptosis, and the like.
  • Such assays often involve monitoring the response of cells to the IgG, for example cell survival, cell death, change in cellular morphology, or transcriptional activation such as cellular expression of a natural gene or reporter gene.
  • such assays may measure the ability of Fc variants to elicit ADCC, ADCP, or CDC.
  • additional cells or components that is in addition to the target cells, may need to be added, for example serum complement, or effector cells such as peripheral blood monocytes (PBMCs), NK cells, macrophages, and the like.
  • PBMCs peripheral blood monocytes
  • NK cells macrophages, and the like.
  • additional cells may be from any organism, preferably humans, mice, rat, rabbit, and monkey.
  • Antibodies may cause apoptosis of certain cell lines expressing the target, or they may mediate attack on target cells by immune cells which have been added to the assay.
  • Methods for monitoring cell death or viability are known in the art, and include the use of dyes, immunochemical, cytochemical, and radioactive reagents. Transcriptional activation may also serve as a method for assaying function in cell-based assays.
  • cell-based screens are performed using cells that have been transformed or transfected with nucleic acids encoding the variants. That is, Fc variants are not added exogenously to the cells.
  • the immunogenicity of the Fc variants is determined experimentally using one or more cell-based assays. Several methods can be used for experimental confirmation of epitopes.
  • the biological properties of the Fc variants of the present invention may be characterized in cell, tissue, and whole organism experiments.
  • drugs are often tested in animals, including but not limited to mice, rats, rabbits, dogs, cats, pigs, and monkeys, in order to measure a drug's efficacy for treatment against a disease or disease model, or to measure a drug's pharmacokinetics, toxicity, and other properties.
  • the animals may be referred to as disease models.
  • Therapeutics are often tested in mice, including but not limited to nude mice, SCID mice, xenograft mice, and transgenic mice (including knockins and knockouts). Such experimentation may provide meaningful data for determination of the potential of the protein to be used as a therapeutic.
  • Any organism preferably mammals, may be used for testing.
  • monkeys can be suitable therapeutic models, and thus may be used to test the efficacy, toxicity, pharmacokinetics, or other property of the IgGs of the present invention. Tests of the in humans are ultimately required for approval as drugs, and thus of course these experiments are contemplated.
  • the IgGs of the present invention may be tested in humans to determine their therapeutic efficacy, toxicity, immunogenicity, pharmacokinetics, and/or other clinical properties.
  • the Fc variants of the present invention may find use in a wide range of products.
  • the Fc variant of the present invention is a therapeutic, a diagnostic, or a research reagent, preferably a therapeutic.
  • the Fc variant may find use in an antibody composition that is monoclonal or polyclonal.
  • the Fc variants of the present invention are used to kill target cells that bear the target antigen, for example cancer cells.
  • the Fc variants of the present invention are used to block, antagonize, or agonize the target antigen, for example for antagonizing a cytokine or cytokine receptor.
  • the Fc variants of the present invention are used to block, antagonize, or agonize the target antigen and kill the target cells that bear the target antigen.
  • an antibody comprising the Fc variant is administered to a patient to treat an antibody-related disorder.
  • a “patient” for the purposes of the present invention includes humans and other animals, preferably mammals and most preferably humans.
  • antibody related disorder or “antibody responsive disorder” or “condition” or “disease” herein are meant a disorder that may be ameliorated by the administration of a pharmaceutical composition comprising an Fc variant of the present invention.
  • Antibody related disorders include but are not limited to autoimmune diseases, immunological diseases, infectious diseases, inflammatory diseases, neurological diseases, pain, pulmonary diseases, hematological conditions, fibrotic conditions, and oncological and neoplastic diseases including cancer.
  • cancer and “cancerous” herein refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • examples of cancer include but are not limited to carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine tumors, mesothelioma, schwanoma, meningioma, adenocarcinoma, melanoma, and leukemia and lymphoid malignancies.
  • treatment as used herein is meant to include therapeutic treatment, as well as prophylactic, or suppressive measures for the disease, condition or disorder.
  • successful administration of a pharmaceutical composition comprising an Fc variant of the present invention prior to onset of the disease results in “treatment” of the disease.
  • successful administration of a pharmaceutical composition comprising an Fc variant of the present invention after clinical manifestation of the disease to combat the symptoms of the disease comprises “treatment” of the disease.
  • Treatment also encompasses administration of a pharmaceutical composition comprising an Fc variant of the present invention after the appearance of the disease in order to eradicate the disease.
  • Successful administration of a pharmaceutical composition comprising an Fc variant of the present invention after onset and after clinical symptoms have developed, with possible abatement of clinical symptoms and perhaps amelioration of the disease comprises “treatment” of the disease.
  • Those “in need of treatment” as used herein include mammals already having the disease or disorder, as well as those prone to having the disease or disorder, including those in which the disease or disorder is to be prevented.
  • a variety of diseases that may be treated using the Fc variants of the present invention are described in U.S. Ser. No. 11/124,620, filed May 5, 2005 and entitled “Optimized Fc Variants”, hereby expressly incorporated by reference.
  • an Fc variant of the present invention is the only therapeutically active agent administered to a patient.
  • the Fc variant of the present invention is administered in combination with one or more other therapeutic agents, including but not limited to cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitory agents, anti-hormonal agents, kinase inhibitors, anti-angiogenic agents, cardioprotectants, or other therapeutic agents, as well as pre- or post-surgery.
  • the IgG variants may be administered concomitantly with one or more other therapeutic regimens.
  • an Fc variant of the present invention may be administered to the patient along with surgery, chemotherapy, radiation therapy, or any or all of surgery, chemotherapy and radiation therapy.
  • the Fc variant of the present invention may be administered in conjunction with one or more antibodies, which may or may not comprise an Fc variant of the present invention.
  • the Fc variant of the present invention and one or more other anti-cancer therapies are employed to treat cancer cells ex vivo. It is contemplated that such ex vivo treatment may be useful in bone marrow transplantation and particularly, autologous bone marrow transplantation. It is of course contemplated that the Fc variants of the invention can be employed in combination with still other therapeutic techniques such as surgery. A variety of agents that may be co-administered with the Fc variants of the present invention are described in U.S. Ser. No. 11/124,620.
  • the IgG is administered with an anti-angiogenic agent.
  • anti-angiogenic agent as used herein is meant a compound that blocks, or interferes to some degree, the development of blood vessels.
  • the anti-angiogenic factor may, for instance, be a small molecule or a protein, for example an antibody, Fc fusion, or cytokine, that binds to a growth factor or growth factor receptor involved in promoting angiogenesis.
  • the anti-angiogenic factor herein is an antibody that binds to Vascular Endothelial Growth Factor (VEGF).
  • VEGF Vascular Endothelial Growth Factor
  • the IgG is administered with a therapeutic agent that induces or enhances adaptive immune response, for example an antibody that targets CTLA-4.
  • the IgG is administered with a tyrosine kinase inhibitor.
  • tyrosine kinase inhibitor as used herein is meant a molecule that inhibits to some extent tyrosine kinase activity of a tyrosine kinase.
  • the Fc variants of the present invention are administered with a cytokine.
  • cytokine as used herein is meant a generic term for proteins released by one cell population that act on another cell as intercellular mediators.
  • compositions are contemplated wherein an Fc variant of the present invention and one or more therapeutically active agents are formulated.
  • Formulations of the Fc variants of the present invention are prepared for storage by mixing the IgG having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980, hereby entirely incorporated by reference), in the form of lyophilized formulations or aqueous solutions.
  • the formulations to be used for in vivo administration are preferably sterile. This is readily accomplished by filtration through sterile filtration membranes or other methods.
  • the Fc variants and other therapeutically active agents disclosed herein may also be formulated as immunoliposomes, and/or entrapped in microcapsules.
  • the concentration of the therapeutically active Fc variant in the formulation may vary from about 0.001 to 100 weight %. In certain embodiments, the concentration of the IgG is in the range of 0.003 to 1.0 molar.
  • a therapeutically effective dose of the Fc variant of the present invention may be administered.
  • therapeutically effective dose herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. Dosages may range from 0.001 to 100 mg/kg of body weight or greater, for example 0.1, 1, 10, or 50 mg/kg of body weight, with 1 to 10 mg/kg being preferred.
  • Administration of the pharmaceutical composition comprising an Fc variant of the present invention may be done in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, intranasally, intraotically, transdermally, topically (e.g., gels, salves, lotions, creams, etc.), intraperitoneally, intramuscularly, intrapulmonary (e.g., AERx® inhalable technology commercially available from Aradigm, or Inhance® pulmonary delivery system commercially available from Inhale Therapeutics), vaginally, parenterally, rectally, or intraocularly.
  • intravenously intravenously, intranasally, intraotically, transdermally, topically
  • topically e.g., gels, salves, lotions, creams, etc.
  • intraperitoneally e.g., intramuscularly, intrapulmonary (e.g., AERx® inhalable technology commercially available from Aradigm, or Inhance® pulmonary
  • FIG. 4 shows an alignment of the sequences of the human Fc ⁇ Rs, highlighting the differences from Fc ⁇ RIIb and positions at the Fc interface. The analysis indicates that although there is extensive homology among the human Fc ⁇ Rs, there are significant differences. Particularly relevant are differences at the Fc binding interface that may be capitalized on to engineer selective Fc variants.
  • FIG. 4 shows that there are 3 or 4 differences between Fc ⁇ RIIb and Fc ⁇ RIIa (depending on allotype) that distinguish binding of these receptors to the Fc region ( FIG. 4 ).
  • Fc ⁇ RIIa is Gln, Fc ⁇ RIIb is Lys
  • 131 Fc ⁇ RIIa is either His or Arg depending on the allotype, Fc ⁇ RIIb is an Arg
  • 132 Fc ⁇ RIIa is Leu, Fc ⁇ RIIb is Ser
  • 160 Fc ⁇ RIIa is Phe, Fc ⁇ RIIb is Tyr.
  • Fc ⁇ R numbering here is according to that provided in the 1E4K pdb structure for Fc ⁇ RIIIb. Mapping of these differences onto the Fc/Fc ⁇ RIIIb complex ( FIG.
  • Fc positions 235-239, 265-270, 295-296, 298-299, 325-330, and 332 are positions that may be modified to obtain Fc variants with selectively increased affinity Fc ⁇ RIIa relative to Fc ⁇ RIIb.
  • a similar analysis can be carried out for selectively altering affinity to one or more of the other activating receptors relative to the inhibitory receptor, for example for selectively improving affinity for Fc ⁇ RIIIa relative to Fc ⁇ RIIb, or conversely for selectively improving affinity for Fc ⁇ RIIb relative to Fc ⁇ RIIIa.
  • G236S provides a selective enhancement to Fc ⁇ RII's (Fc ⁇ RIIa, Fc ⁇ RIIb, and Fc ⁇ RIIc) relative to Fc ⁇ RI and Fc ⁇ RIIIa, with a somewhat greater enhancement to Fc ⁇ RIIa relative to Fc ⁇ RIIb and Fc ⁇ RIIc.
  • G236A is highly selectively enhanced for Fc ⁇ RIIa, not only with respect to Fc ⁇ RI and Fc ⁇ RIIIa, but also over Fc ⁇ RIIb and Fc ⁇ RIIc.
  • Selective enhancements and reductions are observed for a number of Fc variants, including a number of substitutions occurring at the analyzed above, namely 235-239, 265-270, 295-296, 298-299, 325-330, and 332. Although substitutions at some of these positions have been characterized previously (U.S. Pat. No. 5,624,821; Lund et al., 1991, J Immunol 147(8):2657-2662; U.S. Pat. No.
  • Fc variants were engineered in the context of the anti-CD20 antibody PRO70769 (PCT/US2003/040426, hereby entirely incorporated by reference).
  • the genes for the variable regions of PRO70769 (SEQ IDs NO:1 and NO:2, FIGS. 27 a and 27 b ) were constructed using recursive PCR, and subcloned into the mammalian expression vector pcDNA3. 1Zeo (Invitrogen) comprising the full length light kappa (C ⁇ ) and heavy chain IgG1 constant regions. Amino acid substitutions were constructed in the variable region of the antibody in the pcDNA3.
  • Plasmids containing heavy chain gene VH-CH1-CH2-CH3 (wild-type or variants) were co-transfected with plasmid containing light chain gene (VL-C ⁇ ) into 293T cells. Media were harvested 5 days after transfection, and antibodies were purified from the supernatant using protein A affinity chromatography (Pierce).
  • Binding affinity to human Fc ⁇ Rs by Fc variant anti-CD20 antibodies was measured using a competitive AlphaScreenTM assay.
  • the AlphaScreen is a bead-based luminescent proximity assay. Laser excitation of a donor bead excites oxygen, which if sufficiently close to the acceptor bead will generate a cascade of chemiluminescent events, ultimately leading to fluorescence emission at 520-620 nm.
  • the AlphaScreen was applied as a competition assay for screening the antibodies. Wild-type IgG1 antibody was biotinylated by standard methods for attachment to streptavidin donor beads, and tagged Fc ⁇ R was bound to glutathione chelate acceptor beads.
  • wild-type antibody and Fc ⁇ R interact and produce a signal at 520-620 nm.
  • Addition of untagged antibody competes with wild-type Fc/Fc ⁇ R interaction, reducing fluorescence quantitatively to enable determination of relative binding affinities.
  • Fc/Fc ⁇ R binding In order to screen for Fc/Fc ⁇ R binding, the extracellular regions of human Fc ⁇ Rs were expressed and purified. The extracellular regions of these receptors were obtained by PCR from clones obtained from the Mammalian Gene Collection (MGC), or generated de novo using recursive PCR. To enable purification and screening, receptors were fused C-terminally with either a His tag, or with His-glutathione S-Transferase (GST). Tagged Fc ⁇ Rs were transfected into 293T cells, and media containing secreted receptor were harvested 3 days later and purified using Nickel chromatography. Additionally, some His-tagged Fc ⁇ Rs were purchased commercially from R&D Systems.
  • FIG. 6 show the data for binding of select antibody variants to the human receptors R131 Fc ⁇ RIIa ( FIG. 6 a ) and Fc ⁇ RIIb ( FIG. 6 b ).
  • the data were fit to a one site competition model using nonlinear regression, and these fits are represented by the curves in the figure. These fits provide the inhibitory concentration 50% (IC50) (i.e. the concentration required for 50% inhibition) for each antibody, thus enabling the relative binding affinities relative to WT to be determined.
  • IC50 inhibitory concentration 50%
  • single variants do not necessarily completely provide favorable Fc ⁇ R affinities (see for example Table 1).
  • the single variant G236A provides selectively improved affinity to Fc ⁇ RIIa relative to Fc ⁇ RIIb, it is reduced in affinity for both the other activating receptors Fc ⁇ RI and Fc ⁇ RIIIa.
  • combination of this substitution with other modifications that provide increased affinity to these other activating receptors, for example I332E results in an Fc variant with a promising Fc ⁇ R affinity profile, namely increased affinity for Fc ⁇ RIIa and Fc ⁇ RIIIa relative to the inhibitory receptor Fc ⁇ RIIb.
  • FIG. 8 shows binding data for the Fc variants to human Fc ⁇ RI, R131 Fc ⁇ RIIa, H131 Fc ⁇ RIIa, Fc ⁇ RIIb, and V158 Fc ⁇ RIIIa.
  • FIG. 9 provides the IC50's and Fold IC50's relative to WT for fits to these binding curves for all of the anti-EGFR antibody Fc variants tested. The data indicate that it is possible to combine modifications at the aforementioned positions to generate variants with selectively improved affinity for one or more human activating receptors relative to the human inhibitory receptor Fc ⁇ RIIb.
  • Binding affinity to human Fc ⁇ Rs by Fc variant anti-EpCAM antibodies was measured using surface plasmon resonance (SPR), also referred to as BIAcore. SPR measurements were performed using a BIAcore 3000 instrument (BIAcore, Uppsala Sweden). Running buffer was 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20 (HBS-EP, BIAcore), and chip regeneration buffer was 10 mM glycine-HCl pH 1.5. 100 nM WT or variant anti-EpCAM antibody was bound to the protein A/G CM5 chip in HBS-EP at 1 ⁇ l/min for 5 min.
  • SPR surface plasmon resonance
  • Fc ⁇ R-His analyte 50 ⁇ l Fc ⁇ R-His analyte, in serial dilutions between 30 and 1000 nM, was injected in HBS-EP at 25 ⁇ l/min for 2 minutes association, followed by a dissociation phase with buffer alone. Data were normalized for baseline response, obtained from a cycle with antibody and buffer alone. Response sensorgrams were fit to a 1:1 Langmuir binding model within BIAevaluation software, providing the association (ka) and dissociation (kd) rate constants, and the equilibrium dissociation constant (KD).
  • FIG. 10 shows SPR sensorgrams for binding of select anti-EpCAM Fc variants to human R131 Fc ⁇ RIIa.
  • FIG. 11 shows kinetic and equilibrium constants obtained from the fits of the SPR data for all of the receptors, well as the calculated Fold(KD) relative to WT and the negative log of the KD ( ⁇ log(KD).
  • FIG. 12 provides a plot of the negative log of the KD for binding of select anti-EpCAM Fc variants to the set of human Fc ⁇ Rs.
  • greater ⁇ log(KD) on the y-axis corresponds to tighter affinity for the receptor.
  • the activating versus inhibitory Fc ⁇ R affinity differences are plotted for Fc ⁇ RIIa vs. Fc ⁇ RIIb and Fc ⁇ RIIIa vs. Fc ⁇ RIIb.
  • each variant the ⁇ log(KD) for its binding to Fc ⁇ RIIb is subtracted from the ⁇ log(KD) for it binding to the activating receptor, providing a direct measure of Fc ⁇ R selectivity of the variants.
  • all variants comprising the G236A substitution including I332E/G236A, S239D/I332E/G236A, and I332E/H268E/G236A have favorable Fc ⁇ RIIa:Fc ⁇ RIIb selectivity relative to, respectively, the I332E, S239D/I332E, and I332E/H268E variants alone.
  • the results show that suboptimal G236A substitution can be combined with other substitutions that have favorable Fc ⁇ R affinities to generate Fc variants with the most optimal Fc ⁇ R affinity profiles.
  • the selective enhancement in affinity for Fc ⁇ RIIa relative to Fc ⁇ RIIb provided by an Fc variant is defined as Fold(KD) Fc ⁇ RIIa :Fold(KD) Fc ⁇ RIIb , also written as Fold(KD) Fc ⁇ RIIa /Fold(KD) Fc ⁇ RIIb .
  • FIG. 13 b provides these values for both R131 and H131 isoforms of Fc ⁇ RIIa (RIIa and HIIa for brevity), and for both V158 and F158 isoforms of Fc ⁇ RIIIa (VIIIa and FIIIa for brevity).
  • FIG. 13 c provides a plot of these data. The results show that the Fc variants of the invention provide up to 9-fold selective enhancements in affinity for binding to the activating receptor Fc ⁇ RIIa relative to the inhibitory receptor Fc ⁇ RIIb, and up to 4-fold selective enhancements in affinity for binding to the activating receptor Fc ⁇ RIIIa relative to the inhibitory receptor Fc ⁇ RIIb.
  • a central goal of improving the activating Fc ⁇ R vs. inhibitory Fc ⁇ R profile of an antibody or Fc fusion was to enhance its Fc ⁇ R-mediated effector function in vitro and ultimately in vivo.
  • in vitro cell-based ADCC assays were run using human PBMCs as effector cells. ADCC was measured by the release of lactose dehydrogenase using a LDH Cytotoxicity Detection Kit (Roche Diagnostic). Human PBMCs were purified from leukopacks using a ficoll gradient, and the EpCAM + target gastric adenocarcinoma line LS180.
  • Target cells were seeded into 96-well plates at 10,000 cells/well, and opsonized using Fc variant or WT antibodies at the indicated final concentration. Triton X100 and PBMCs alone were run as controls. Effector cells were added at 40:1 PBMCs:target cells, and the plate was incubated at 37° C. for 4 hrs. Cells were incubated with the LDH reaction mixture, and fluorescence was measured using a FusionTM Alpha-FP (Perkin Elmer). Data were normalized to maximal (triton) and minimal (PBMCs alone) lysis, and fit to a sigmoidal dose-response model. FIG. 14 provides these data for select Fc variant antibodies.
  • the G236A variant mediates reduced ADCC relative to WT, due likely to its reduced affinity for Fc ⁇ RIIIa and/or Fc ⁇ RI.
  • ADCC in PBMCs is potentially dominated by NK cells, which express only Fc ⁇ RIIIa, although in some cases they can express Fc ⁇ RIIc.
  • the reduced ADCC of the G236A single variant is consistent with its reduced affinity for this receptor.
  • combination of the G236A substitution with modifications that improve affinity for these activating receptors, for example including but not limited to substitutions at 332 and 239, provide substantially improved ADCC relative to the parent WT antibody.
  • Monocyte-derived effector cells express not only Fc ⁇ RIIIa, but also Fc ⁇ RI, Fc ⁇ RIIa, and the inhibitory receptor Fc ⁇ RIIb.
  • Macrophages are phagocytes that act as scavengers to engulf dead cells, foreign substances, and other debris.
  • macrophages are professional antigen presenting cells (APCs), taking up pathogens and foreign structures in peripheral tissues, then migrating to secondary lymphoid organs to initiate adaptive immune responses by activating naive T-cells.
  • APCs professional antigen presenting cells
  • macrophages express the range of Fc ⁇ Rs, and thus their activation and function may be dependent on engagement of antibody immune complexes with receptors other than only Fc ⁇ RIIIa.
  • a cell-based ADCP assay was carried out to evaluate the capacity of the Fc variants to mediate phagocytosis.
  • Monocytes were purified from PBMCs and differentiated into macrophages in 50 ng/ml M-CSF for 5 days.
  • Quantitated receptor expression density of Fc ⁇ RI (CD64), Fc ⁇ RIIa and Fc ⁇ RIIb (CD32), and Fc ⁇ RIIIa (CD16) on these cells was determined with standard flow cytometry methods using PE (orange)—labeled anti-Fc ⁇ Rs and biotinylated PE-Cy5—labeled antibodies against macrophage markers CD11b and CD14.
  • PE-conjugated anti-CD64 (Clone 10.1) was purchased from eBioscience, PE-conjugated anti-CD32 (Clone 3D3) and PE-conjugated anti-CD16 (Clone 3G8) were purchased from BD Bioscience.
  • Biotinylated anti-CD14 (TUK4) was purchased from Invitrogen, and biotinylated anti-CD11b (Clone ICRF44) was purchased from BD Bioscience. Secondary detection was performed with streptavidin PE-Cy5 obtained from Biolegend. Cytometry was carried out on a Guava Personal Cell Analysis-96 (PCA-96) System (Guava Technologies).
  • PCA-96 Guava Personal Cell Analysis-96
  • 15 a shows that the monocyte-derived macrophages (MDM) express high levels of Fc ⁇ RII (99%) and Fc ⁇ RIII (81%), and moderate (45%) levels of Fc ⁇ RI.
  • MDM monocyte-derived macrophages
  • target EpCAM + LS180 cells were labeled with PKH26 and plated in a 96-well round bottom plate at 25 000 cells/well.
  • Antibodies WT and Fc variants
  • WT and Fc variants were added to wells at indicated concentrations, and antibody opsinized cells were incubated for approximately 30 minutes prior to the addition of effector cells.
  • Monocyte derived macrophages MDM were added to each well at approximately 4:1 effector to target ratio, and the cells were incubated overnight. Cells were washed and treated with HyQtase.
  • MDM were stained with biotinylated CD11b and CD14, followed by a secondary stain with Streptavidin PE-Cy5. Cells were fixed in 1% paraformaldehyde and read on the Guava flow cytometer.
  • FIG. 15 b shows the results of an ADCP assay of select anti-EpCAM Fc variants in the presence of macrophages.
  • FIG. 15 c show a repeat experiment with some of these variants. The data show that the improved Fc ⁇ RII:Fc ⁇ RIIb profile of the I332E/G236A variant relative to the I332E single variant provides enhanced phagocytosis. Interestingly, G236A does not improve phagocytosis of the S239D/I332E variant.
  • the inhibitory receptor Fc ⁇ RIIb the affinity for which is greater in the S239D/I332E and S239D/I332E/G236A variants relative to the I332E and I332E/G236A variants, establishes an absolute threshold of activation/repression. That is, regardless of how much affinity to Fc ⁇ RIIa is improved, at a certain level of Fc ⁇ RIIb engagement cellular activation and effector function is inhibited.
  • DCs Dendritic cells
  • APCs professional antigen presenting cells
  • Immature DCs endocytose either free or complexed antigens in the periphery, and this stimulus induces their maturation and migration to secondary lymphoid organs.
  • DC-derived cytokines play a crucial role in shaping the adaptive response via determining polarization of T-cells towards either the Th1 or the Th2 phenotype (Bajtay et al., 2006, Immunol Letters 104: 46-52).
  • Human DCs can express the various Fc ⁇ Rs depending on their source and activation state (Bajtay et al., 2006, Immunol Letters 104: 46-52).
  • immature monocyte-derived DCs express primarily Fc ⁇ RIIa and Fc ⁇ RIIb.
  • DCs Dendritic cells
  • GM-CSF 1000 Units/ml or 100 ng/ml
  • IL4 500 Units/ml or 100 ng/ml
  • Fc ⁇ RIIa and Fc ⁇ RIIb CD32
  • Fc ⁇ RIIIa CD16
  • PE-conjugated anti-CD64 (Clone 10.1) was purchased from eBioscience, PE-conjugated anti-CD32 (Clone 3D3) and PE-conjugated anti-CD16 (Clone 3G8) were purchased from BD Bioscience. Cytometry was carried out on the Guava.
  • FIG. 16 a shows that the DCs used express high levels of Fc ⁇ RII (94.7%), low to moderate levels of Fc ⁇ RIII (37.2%), and low to no Fc ⁇ RI (7.3%).
  • FIG. 16 b shows the dose response curves for TNF ⁇ release by DCs in the presence of WT and Fc variant antibodies.
  • the data show that DC activation is correlated roughly with the Fc ⁇ RIIa:Fc ⁇ RIIb affinity ratio ( FIG. 13 ), consistent with the literature and the dominant expression of Fc ⁇ RII receptors on the DCs used in the present assay.
  • I332E and S239D/I332E mediate DC activation comparable with or lower than WT, in agreement with their Fc ⁇ RIIa:Fc ⁇ RIIb affinity profile.
  • the cell-based results indicate that the most optimal engineered Fc ⁇ R profile is selectively improved affinity for both Fc ⁇ RIIa and Fc ⁇ RIII relative to the inhibitory receptor Fc ⁇ RIIb, for example as provided by the combination of S239D, I332E, and G236A substitutions.
  • Fc variants that selectively improve binding to one or more human activating receptors relative to Fc ⁇ RIIb, or selectively improve binding to Fc ⁇ RIIb relative to one or more activating receptors may comprise a substitution, as described herein, selected from the group consisting of 234G, 234I, 235D, 235E, 235I, 235Y, 236A, 236S, 239D, 267D, 267E, 267Q, 268D, 268E, 293R, 295E, 324G, 324I, 327H, 328A, 328F, 328I, 330I, 330L, 330Y, 332D, and 332E.
  • substitutions that may also be combined include other substitutions that modulate Fc ⁇ R affinity and complement activity, including but not limited to 298A, 298T, 326A, 326D, 326E, 326W, 326Y, 333A, 333S, 334L, and 334A (U.S. Pat. No. 6,737,056; Shields et al, Journal of Biological Chemistry, 2001, 276(9):6591-6604; U.S. Pat. No. 6,528,624; Idusogie et al., 2001, J. Immunology 166:2571-2572).
  • Preferred variants that may be particularly useful to combine with variants of the present invention include those that comprise the substitutions 298A, 326A, 333A, and 334A.
  • AlphaScreen data measuring the binding of Fc variants comprising these substitutions to the human activating receptors V158 and F158 Fc ⁇ RIIIa and the inhibitory receptor Fc ⁇ RIIb are shown in FIG. 17 .
  • Additional substitutions that may be combined with the Fc ⁇ R selective variants of the present invention 247L, 255L, 270E, 392T, 396L, and 421K U.S. Ser. No. 10/754,922; U.S. Ser. No. 10/902,588), and 280H, 280Q, and 280Y (U.S. Ser. No. 10/370,749), all of which are herein expressly incorporated by reference
  • Fc variants of the present invention may be combined with Fc variants that alter FcRn binding.
  • variants that increase Fc binding to FcRn include but are not limited to: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Jounal of Immunology 176:346-356, U.S. Ser. No. 11/102621, PCT/US2003/033037, PCT/US2004/011213, U.S. Ser. No. 10/822300, U.S. Ser. No.
  • Fc Variants Comprising Amino Acid Modifications and Engineered Glycoforms that Provide Selective Fc ⁇ R Affinity
  • An alternative method to amino acid modification for modulating Fc ⁇ R affinity of an Fc polypeptide is glycoform engineering.
  • antibodies are post-translationally modified at position 297 of the Fc region with a complex carbohydrate moiety. It is well known in the art that this glycosylation plays a role in the functional fidelity of the Fc region with respect to binding Fc ligands, particularly Fc ⁇ Rs and complement.
  • Fc polypeptide compositions that comprise a mature core carbohydrate structure which lacks fucose have improved Fc ⁇ R affinity relative to compositions that comprise carbohydrate that is fucosylated (Uma ⁇ a et al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473); (U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No.
  • Example 1 The data provided in Example 1 suggest that combination of glycoform engineering with Fc ⁇ R selective amino acid modifications may provide Fc variants with selectively improved affinity for one or more activating receptors relative to the inhibitory receptor Fc ⁇ RIIb.
  • Lec13 refers to the lectin-resistant Chinese Hamster Ovary (CHO) mutant cell line which displays a defective fucose metabolism and therefore has a diminished ability to add fucose to complex carbohydrates. That cell line is described in Ripka & Stanley, 1986, Somatic Cell & Molec. Gen. 12(1):51-62; and Ripka et al., 1986, Arch. Biochem. Biophys.
  • Lec13 cells are believed lack the transcript for GDP-D-mannose-4,6-dehydratase, a key enzyme for fucose metabolism.
  • GDP-D-mannose-4,6-dehydratase generates GDP-mannose-4-keto-6-D-deoxymannose from GDP-mannose, which is then converted by the FX protein to GDP-L-fucose.
  • fucosylated oligosaccharides is dependent on the GDP-L-fucose donor substrates and fucosyltransferase(s).
  • the Lec13 CHO cell line is deficient in its ability to add fucose, but provides IgG with oligosaccharide which is otherwise similar to that found in normal CHO cell lines and from human serum (Jefferis, R. et al., 1990, Biochem. J. 268, 529-537; Raju, S. et al., 2000, Glycobiology 10, 477-486; Routier, F. H., et al., 1997, Glycoconj. J. 14, 201-207).
  • Normal CHO and HEK293 cells add fucose to IgG oligosaccharide to a high degree, typically from 80-98%, and IgGs from sera are also highly fucosylated (Jefferis, R.
  • FIG. 19 provides the equilibrium constants obtained from the fits of the SPR data for all of the receptors, as well as the calculated fold KD relative to WT and the negative log of the KD ( ⁇ log(KD).
  • Lec13 cell line is not meant to limit the present invention to that particular mode of reducing fucose content.
  • a variety of other methods are known in the art for controlling the level of fucosylated and/or bisecting oligosaccharides that are covalently attached to the Fc region, including but not limited to expression in various organisms or cell lines, engineered or otherwise (for example Lec13 CHO cells or rat hybridoma YB2/0 cells), regulation of enzymes involved in the glycosylation pathway (for example FUT8 [ ⁇ 1,6-fucosyltranserase] and/or ⁇ 1-4-N-acetylglucosaminyltransferase III [GnTIII]), and modification of modifying carbohydrate(s) after the IgG has been expressed (Uma ⁇ a et al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et
  • FIG. 21 shows binding data for select Fc variants to human V158 Fc ⁇ RIIIa.
  • FIG. 22 provides the Fold IC50's relative to WT for fits to these binding curves for all of the anti-CD30 antibody Fc variants tested.
  • FIG. 23 a shows the putative expression patterns of different Fc ⁇ Rs on various effector cell types
  • FIG. 23 b shows the % identity between the human and mouse Fc ⁇ R extracellular domains.
  • Fc ⁇ RIV is thought to be the true ortholog of human Fc ⁇ RIIIa, and the two receptors are 64% identical ( FIG. 23 b ).
  • human Fc ⁇ RIIIa is expressed on NK cells
  • mouse Fc ⁇ RIV is not.
  • mouse Fc ⁇ RIII The receptor that is expressed on mouse NK cells is Fc ⁇ RIII, which shows substantially lower homology to human Fc ⁇ RIIIa (49%).
  • mouse Fc ⁇ RIII is 93% homologous to the mouse inhibitory receptor Fc ⁇ RIIb, a pair that is potentially analogous to human Fc ⁇ RIIa and Fc ⁇ RIIb (93% identical).
  • the expression pattern of mouse Fc ⁇ RIII differs from that of human Fc ⁇ RIIa.
  • the most optimal human antibody in humans with respect to Fc ⁇ R-mediated effector function likely does not have the optimal Fc ⁇ R affinity profile for the murine receptors. Accordingly, Fc variant antibodies having optimized affinity for human Fc receptors may not provide optimal enhancements in mice, and thus may provide misleading results.
  • the most optimal mouse Fc ⁇ R affinity profile is likely provided by the most naturally optimal mouse IgG or IgGs, for example mouse IgG2a and/or IgG2b. Accordingly, engineering of mouse IgGs for optimized affinity for mouse Fc ⁇ Rs may provide the most informative results in in vivo experiments. In this way Fc-optimized mouse IgGs may find use as surrogate Fc-optimized antibodies in preclinical mouse models.
  • the present invention provides mouse IgG antibodies optimized for binding to mouse Fc ⁇ Rs.
  • Fc substitutions were constructed in the context of mouse IgG1, mouse IgG2a, mouse IgG2b, and human IgG1 ( FIG. 29 ).
  • DNA encoding murine IgGs were obtained as IMAGE clones from the American Type Culture Collection (ATCC).
  • Antibodies were constructed with the variable region of the anti-EGFR antibody H4.40/L3.32 C225 (SEQ IDs NO:3 and NO:4, FIGS. 27 c and 27 d ) as disclosed in U.S. Ser. No. 60/778,226, filed Mar. 2, 2006, entitled “Optimized anti-EGFR antibodies”, herein expressly incorporated by reference).
  • Antibody variants were constructed in the pcDNA3. 1Zeo vector, expressed in 293T cells, and purified as described above.
  • FIG. 24 lists the mouse and human IgG variants that were engineered.
  • FIG. 25 shows equilibrium constants obtained from the fits of the SPR data for the set of murine Fc ⁇ Rs. Also presented is the calculated fold KD relative to WT murine IgG2a, potentially the most potent natural murine IgG antibody with respect to Fc ⁇ R-mediated effector function (Hamaguchi et al., 2005, J Immunol 174: 4389-4399).
  • FIG. 26 shows a plot of the negative log of the KD for binding of human and mouse anti-EGFR Fc variant antibodies to mouse Fc receptors Fc ⁇ RI, Fc ⁇ RIIb, Fc ⁇ RIII, and Fc ⁇ RIV.
  • the variants provide remarkable enhancements in binding to the murine activating receptors, particularly Fc ⁇ RIV, currently thought to be one of the most relevant receptors for mediating antibody-dependent effector functions in murine xencograft models (Nimmerjahn & Ravetch, 2005, Science 310:1510-1512).

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EP1931709A2 (de) 2008-06-18
AU2006299429A1 (en) 2007-04-12
DK1931709T3 (en) 2017-03-13
US20080206867A1 (en) 2008-08-28
EP1931709B1 (de) 2016-12-07
AU2006299429B2 (en) 2012-02-23
US9040041B2 (en) 2015-05-26
WO2007041635A3 (en) 2008-11-13
US20100249382A1 (en) 2010-09-30
WO2007041635A2 (en) 2007-04-12
CA2624189A1 (en) 2007-04-12

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