EP1871808A2 - VARIANTS Fc PRESENTANT DES PROPRIETES OPTIMISEES - Google Patents

VARIANTS Fc PRESENTANT DES PROPRIETES OPTIMISEES

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Publication number
EP1871808A2
EP1871808A2 EP06748967A EP06748967A EP1871808A2 EP 1871808 A2 EP1871808 A2 EP 1871808A2 EP 06748967 A EP06748967 A EP 06748967A EP 06748967 A EP06748967 A EP 06748967A EP 1871808 A2 EP1871808 A2 EP 1871808A2
Authority
EP
European Patent Office
Prior art keywords
antibody
variant
polypeptide
positions
variants
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06748967A
Other languages
German (de)
English (en)
Inventor
Gregory Alan Lazar
Wei Dang
John R. Desjarlais
Sher Bahadur Karki
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xencor Inc
Original Assignee
Xencor Inc
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Filing date
Publication date
Priority claimed from US11/124,620 external-priority patent/US8188231B2/en
Application filed by Xencor Inc filed Critical Xencor Inc
Publication of EP1871808A2 publication Critical patent/EP1871808A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]

Definitions

  • the present invention relates to Fc variant polypeptides with optimized properties, engineering methods for their generation, and their application, particularly for therapeutic purposes.
  • 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. Each chain is made up of individual immunoglobulin (Ig) domains, and thus the generic term immunoglobulin is used for such proteins. Each chain is made up of two distinct regions, referred to as the variable and constant regions. The light and heavy chain variable regions show significant sequence diversity between antibodies, and are responsible for binding the target antigen. The constant regions show less sequence diversity, and are responsible for binding a number of natural proteins to elicit important biochemical events.
  • IgA which includes subclasses IgAI and lgA2
  • IgD which includes subclasses IgAI and lgA2
  • IgE which includes subclasses IgG
  • IgM which includes subclasses IgGI , lgG2, lgG3, and lgG4
  • IgM IgM
  • Figure 1 shows an IgGI antibody, used here as an example to describe the general structural features of immunoglobulins.
  • IgG antibodies are tetrameric proteins composed of two heavy chains and two light chains.
  • 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 -CD1-CD2-CD3, 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 -CL, referring to the light chain variable domain and the light chain constant domain respectively.
  • 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 majority of sequence variability occurs in the complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the variable region outside of the CDRs is referred to as the framework (FR) region.
  • FR framework
  • 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.
  • the CDRs substantial antigen binding diversity
  • 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.
  • Fragments comprising the variable region can exist in the absence of other regions of the antibody, including for example the antigen binding fragment (Fab) comprising V ⁇ -C ⁇ 1 and VH-C L , the variable fragment (Fv) comprising V H and VL, the single chain variable fragment (scFv) comprising V H and VL linked together in the same chain, as well as a variety of other variable region fragments (Little et al., 2000, Immunol Today 21 :364-370, hereby entirely incorporated by reference).
  • Fab antigen binding fragment
  • Fv variable fragment
  • scFv single chain variable fragment
  • 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.
  • the Fc region As shown in Figure 1, 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 ⁇ Rlla (including allotypes H131 and R131), Fc ⁇ Rllb (including Fc ⁇ Rllb-1 and Fc ⁇ Rllb-2), and Fc ⁇ Rllc; and Fc ⁇ RIII (CD16), including isoforms Fc ⁇ Rllla (including allotypes V158 and F158) and Fc ⁇ Rlllb (including allotypes Fc ⁇ Rlllb-NA1 and Fc ⁇ Rlllb-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 1 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
  • ADCC antibody dependent cell-mediated cytotoxicity
  • ADCP antibody dependent cell-mediated phagocytosis
  • Fc ⁇ Rs bind the same region on Fc, at the N-terminal end of the C ⁇ 2 domain and the preceding hinge, shown in Figure 2. This interaction is well characterized structurally (Sondermann et al., 2001 , J MoI
  • the different IgG subclasses have different affinities for the Fc ⁇ Rs, with IgGI and lgG3 typically binding substantially better to the receptors than lgG2 and lgG4 (Jefferis et al., 2002, Immunol Lett 82:57-65, hereby entirely incorporated by reference). All Fc ⁇ Rs bind the same region on IgG Fc, yet with different affinities: the high affinity binder Fc ⁇ RI has a Kd for IgGI of 10 ⁇ 8 M "1 , whereas the low affinity receptors Fc ⁇ RII and Fc ⁇ RIII generally bind at 10 '8 and 10 '5 respectively.
  • Fc ⁇ Rllla and Fc ⁇ Rlllb are 96% identical, however Fc ⁇ Rlllb does not have a intracellular signaling domain.
  • Fc ⁇ RI, Fc ⁇ Rlla/c, and Fc ⁇ Rllla are positive regulators of immune complex-triggered activation, characterized by having an intracellular domain that has an immunoreceptor tyrosine-based activation motif (ITAM)
  • Fc ⁇ Rllb has an immunoreceptor tyrosine-based inhibition motif (ITIM) and is therefore inhibitory.
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • the receptors also differ in expression pattern and levels on different immune cells.
  • 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, hereby entirely incorporated by reference).
  • Approximately 10-20% of humans are V158/V158 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, Stood 99:754-758, both hereby entirely incorporated by reference).
  • 80-90% of humans are poor responders, e.g., they have at least one allele of the F158 Fc ⁇ Rllla.
  • FIG. 1 An overlapping but separate site on Fc, shown in Figure 1 , serves as the interface for the complement protein C1q.
  • Fc/Fc ⁇ R binding mediates ADCC
  • Fc/C1q binding mediates complement dependent cytotoxicity (CDC).
  • C1q forms a complex with the serine proteases C1r and C1s to form the C1 complex.
  • C1q is capable of binding six antibodies, although binding to two IgGs is sufficient to activate the complement cascade.
  • IgG subclasses Similar to Fc interaction with Fc ⁇ Rs, different IgG subclasses have different affinity for C1q, with IgGI and lgG3 typically binding substantially better to the Fc ⁇ Rs than lgG2 and lgG4 (Jefferis et al., 2002, Immunol Lett 82:57-65, hereby entirely incorporated by reference).
  • the binding site for FcRn on Fc is also the site at which the bacterial proteins A and G bind.
  • the tight binding by these proteins is typically exploited as a means to purify antibodies by employing protein A or protein G affinity chromatography during protein purification.
  • the fidelity of this region on Fc is important for both the clinical properties of antibodies and their purification.
  • a key feature of the Fc region is the conserved N-linked glycosylation that occurs at N297, shown in Figure 1.
  • 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. While not wanting to be limited to one theory, it is believed that the structural purpose of this carbohydrate may be to stabilize or solubilize Fc, determine a specific angle or level of flexibility between the C ⁇ 3 and C ⁇ 2 domains, keep the two C ⁇ 2 domains from aggregating with one another across the central axis, or a combination of these.
  • 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.
  • 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 possible 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. Despite this arsenal of anti-tumor weapons, the potency of antibodies as anti-cancer agents is unsatisfactory, particularly given their high cost.
  • Patient tumor response data show that monoclonal antibodies provide only a small improvement in therapeutic success over normal single-agent cytotoxic chemotherapeutics. For example, just half of all relapsed low-grade non-Hodgkin's lymphoma patients respond to the anti-CD20 antibody rituximab (McLaughlin et al., 1998, J CHn Oncol 16:2825-2833, hereby entirely incorporated by reference).
  • trastuzumab Herceptin®, Genentech
  • trastuzumab an anti-HER2/neu antibody for treatment of metastatic breast cancer
  • the overall response rate using trastuzumab for the 222 patients tested was only 15%, with 8 complete and 26 partial responses and a median response duration and survival of 9 to 13 months (Cobleigh et al., 1999, J Clin Oncol 17:2639-2648, hereby entirely incorporated by reference).
  • any small improvement in mortality rate defines success.
  • 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.
  • cytotoxic effector functions such as ADCC, ADCP, and CDC.
  • the importance of Fc ⁇ R-mediated effector functions for the anti-cancer activity of antibodies has been demonstrated in mice (Clynes et al., 1998, Proc Natl Acad Sci U S A 95:652-656; Clynes et al., 2000, Nat Med 6:443-446, both hereby entirely incorporated by reference), and the affinity of interaction between Fc and certain Fc ⁇ Rs correlates with targeted cytotoxicity in cell-based assays (Shields et al., 2001 , J Biol Chem 276:6591-6604; Presta et al., 2002, Biochem Soc Trans 30:487-490; Shields et al., 2002, J Biol Chem 277:26733-26740
  • Fc ⁇ Rs can mediate antigen uptake and processing by antigen presenting cells
  • optimized Fc ⁇ R affinity may also improve the capacity of antibody therapeutics to elicit an adaptive immune response (Dhodapkar & Dhodapkar, 2005, Proc Natl Acad Sci USA, 102, 6243-6244, hereby entirely incorporated by reference).
  • the importance of complement-mediated effector function for anti-cancer therapy of antibodies is not as well characterized.
  • Antibodies optimized for CDC would provide a way to investigate the role of complement in antibody clinical applications, and provide a potential mechanism for improving the tumor killing capacity of antibodies.
  • anti-CD4 antibodies For example, the ability of anti-CD4 antibodies to block CD4 receptors on T cells makes them effective anti-inflammatories, yet their ability to recruit Fc ⁇ R receptors also directs immune attack against the target cells, resulting in T cell depletion (Reddy et al., 2000, J Immunol 164:1925-1933, hereby entirely incorporated by reference). Effector function may also be a problem for radiolabeled antibodies, referred to as radioconjugates, and antibodies conjugated to toxins, referred to as immunotoxins.
  • Fc variants that reduce or ablate Fc ligand binding are also known in the art (Alegre et al., 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl Acad Sci USA 92:11980-11984; Armour et al., 1999, Eur J Immunol 29:2613-2624; Reddy et al., 2000, J Immunol 164:1925-1933; Xu et al., 2000, Cell Immunol 200:16-26; Shields et al., 2001 , J Biol Chem 276:6591- 6604; Armour et al., 1999, Eur J Immunol 29:2613-2624; US 6,194,551 ; US 5,885,573; PCT WO 99/58572; USSN 10/267,286, all hereby entirely incorporated by reference).
  • Fc variants should be engineered that not only ablate binding to Fc ⁇ Rs and/or C1q, but also maintain antibody stability, solubility, and structural integrity, as well as ability to interact with other important Fc ligands such as FcRn and proteins A and G.
  • Fc variants obtained in these studies provide a variety of optimal enhancements in Fc ligand and effector function properties, including but not limited to selectively improved binding to Fc ⁇ Rs, enhanced ADCC, improved binding to complement protein C1q, reduced binding to Fc ⁇ Rs, reduced binding to complement protein C1q, and other optimized properties.
  • the present invention aims to further characterize the properties of select Fc variants from these studies, and to utilize the data to generate novel variants with optimized properties.
  • the present invention provides Fc variants with optimized properties.
  • Said optimized properties include altered binding to Fc ⁇ R's, altered antibody dependent cell-mediated cytotoxicity, and altered complement dependent cytotoxicity relative to a parent Fc polypeptide.
  • the Fc variants of the present invention improve binding to one or more Fc ⁇ R's relative to a parent Fc polypeptide.
  • the Fc variants of the invention improve antibody dependent cell-mediated cytotoxicity relative to a parent Fc polypeptide.
  • said Fc variants comprise an amino acid modification at one or more positions selected from the group consisting of: 227, 234, 235, 236, 239, 246, 255, 258, 260, 264, 267, 268, 272, 281 , 282, 283, 284, 293, 295, 304, 324, 327, 328, 330, 332, 335, wherein numbering is according to the EU index.
  • the Fc variants of the present invention improve complement dependent cytotoxicity relative to a parent Fc polypeptide.
  • said Fc variants comprise an amino acid modification at one or more positions selected from the group consisting of: 233, 234, 235, 239, 267, 268, 271 , 272, 274, 276, 278, 281 , 282, 284, 285, 293, 300, 320, 322, 324,
  • the Fc variants of the present invention reduce binding to one or more Fc ⁇ Rs relative to a parent Fc polypeptide. In an alternate embodiment, the Fc variants of the invention reduce antibody dependent cell-mediated cytotoxicity relative to a parent Fc polypeptide. In an alternate embodiment, the Fc variants of the invention reduce complement dependent cytotoxicity relative to a parent Fc polypeptide. In a preferred embodiment, the Fc variants of the invention reduce binding to one or more Fc ⁇ Rs, reduce antibody dependent cell-mediated cytotoxicity, and reduce complement dependent cytotoxicity relative to a parent Fc polypeptide.
  • said Fc variants comprise one or more amino acid modifications at a position selected from the group consisting of: 232, 234, 235, 236, 237, 238, 239, 265, 267, 269, 270, 297, 299, 325,
  • the present invention provides methods for engineering Fc variants with optimized properties. It is a further object of the present invention to provide experimental production and screening methods for obtaining optimized Fc variants.
  • the present invention provides isolated nucleic acids encoding the Fc variants described herein.
  • the present invention provides vectors comprising said 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. Said novel Fc polypeptides may find use in a therapeutic product. In a most preferred embodiment, 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 Antibody structure and function. Shown is a model of a full length human IgGI antibody, modeled using a humanized Fab structure from pdb accession code 1CE1 (James et al., 1999, J MoI Biol 289:293-301, hereby entirely incorporated by reference) and a human IgGI Fc structure from pdb accession code 1 DN2 (DeLano et al., 2000, Science 287:1279-1283, hereby entirely incorporated by reference). The flexible hinge that links the Fab and Fc regions is not shown. IgGI is a homodimer of heterodimers, made up of two light chains and two heavy chains.
  • the Ig domains that comprise the antibody are labeled, and include VL and C L for the light chain, and V H , Cgammai (C ⁇ 1), Cgamma2 (C ⁇ 2), and Cgamma3 (C ⁇ 3) for the heavy chain.
  • the Fc region is labeled. Binding sites for relevant proteins are labeled, including the antigen binding site in the variable region, and the binding sites for Fc ⁇ Rs, FcRn, C1q, and proteins A and G in the Fc region.
  • FIG. 1 The Fc/Fc ⁇ Rlllb complex structure 11IS. Fc is shown as a gray ribbon diagram, and Fc ⁇ Rlllb is shown as a black ribbon. The N297 carbohydrate is shown as black sticks.
  • Figures 3a - 3b Alignment of the amino acid sequences of the human IgG immunoglobulins IgGI , lgG2, lgG3, and lgG4.
  • Figure 3a provides the sequences of the CH1 (C ⁇ 1) and hinge domains
  • Figure 3b provides the sequences of the CH2 (C ⁇ 2) and CH3 (C ⁇ 3) domains.
  • Positions are numbered according to the EU index of the IgGI sequence, and differences between IgGI and the other immunoglobulins lgG2, lgG3, and lgG4 are shown in grey.
  • Polymorphisms exist at a number of positions (Kim et al., 2001 , J. MoI. Evol. 54:1-9, hereby entirely incorporated by reference), 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. 6 Binding to human V158 Fc ⁇ Rllla ( Figure 6a) and F158 Fc ⁇ Rllla ( Figure 6b) by select PRO70769 Fc variants as determined by the competition AlphaScreen 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.
  • Figure 7 Binding to human V158 Fc ⁇ Rllla and F158 Fc ⁇ Rllla by PRO70769 Fc variants as measured by competition AlphaScreen assay.
  • Figure 7a provides data for select variants
  • Figure 7b provides the IC50's and folds relative to WT PRO70769 IgGI .
  • Figure 8 Fc variants and Fc ⁇ R binding data. All Fc variants were constructed in the context of the variable region PRO70769 and either human IgGI or IgG(I /2) ELLGG.
  • Figure 8a provides the IC50's and fold IC50's relative to WT PRO70769 IgGI for binding to human activating receptors V158 and F158 Fc ⁇ Rllla, and the inhibitory receptor Fc ⁇ Rllb, as measured by competition AlphaScreen assay.
  • Figure 8b shows the AlphaScreen data for select variants.
  • Figure 9 Competition Surface Plasmon Resonance (SPR) experiment measuring binding affinities of I332E and S239D/I332E variants in the context of trastuzumab to human V158 Fc ⁇ Rllla.
  • Figure 9a provides the sensorgram raw data
  • Figure 9b provides a plot of the log of receptor concentration against the initial rate obtained at each concentration
  • Figure 9c provides the affinities obtained from the fits to these data as described in Example 1.
  • FIG. 10 Cell-based ADCC assays of select Fc variants in the context of the anti-CD20 antibody PRO70769. ADCC was measured by the release of lactose dehydrogenase using a LDH Cytotoxicity Detection Kit (Roche Diagnostic). CD20+ lymphoma WIL2-S cells were used as target cells and human PBMCs were used as effector cells. Shown is the dose-dependence of ADCC on antibody concentration for the indicated antibodies, normalized to the minimum and maximum fluorescence 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 sigmoidal dose-response model using nonlinear regression.
  • FIG. 11 Cell-based ADCC assay of select Fc variants in the context of PRO70769 IgGI in the absence and presence of serum levels of human IgG. ADCC was measured by the release of lactose dehydrogenase using a LDH Cytotoxicity Detection Kit (Roche Diagnostic). CD20+ lymphoma WIL2-S cells were used as target cells and human PBM ⁇ s were used as effector cells.
  • FIG. 12 Residues mutated in Fc variants designed to enhance CDC.
  • the structure of the human IgGI Fc region is shown (pdb accession code 1 E4K, Sondermann et al., 2000, Nature 406:267-273, hereby entirely incorporated by reference).
  • Black ball and sticks indicate residues D270, K322, P329, and P331 , which have been shown to be important in mediating binding to complement protein C1q, and grey sticks indicate residues that were mutated in the present invention to explore variants that affect CDC.
  • FIG. 13 Fc variants screened for complement-mediated cytotoxicity (CDC) and CDC data.
  • the variable region is that of the anti-CD20 antibody PRO70769, and the heavy chain constant region is IgGI unless noted IgG(I /2) ELLGG.
  • Fold CDC provides the relative CDC activity compared to WT PRO70769 lgG1.
  • FIG. 14 CDC assays of Fc variant anti-CD20 antibodies. The dose-dependence on antibody concentration of complement-mediated lysis is shown for the indicated PRO70769 antibodies against CD20+ WIL2-S lymphoma target cells. Lysis was measured using release of Alamar Blue, and data were normalized to the minimum and maximum fluorescence 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 sigmoidal dose-response model with variable slope using nonlinear regression.
  • Figure 15 Amino acid modifications that provide enhanced and reduced CDC, and positions that may be modified that may provide enhanced/modulated CDC. Positions are numbered according to the EU index.
  • FIG. 16 Fc variants screened for reduced Fc ⁇ R affinity, Fc ⁇ R-mediated effector function, and complement-mediated effector function.
  • the variable region is that of the anti-CD20 antibody PRO70769, and the heavy chain constant region is IgGI .
  • the figure provides the Fold IC50 for binding to human V158 Fc ⁇ Rllla and the Fold EC50 of CDC activity relative to WT PRO70769 IgGI .
  • FIG. Binding to human V158 Fc ⁇ Rllla by select PRO70769 Fc variants as determined by the competition AlphaScreen assay.
  • Figure 18 CDC assays of select Fc variant anti-CD20 antibodies against CD20+ W1L2-S lymphoma target cells. Lysis was measured by Alamar Blue release.
  • FIG. 19 Cell-based ADCC activity of select anti-CD20 Fc variants against CD20+ lymphoma WIL2-S cells. Human PBMCs were used as effector cells, and lysis was measured by LDH release.
  • FIG. 20 Fc variants screened for reduced Fc ⁇ R affinity, Fc ⁇ R-mediated effector function, and complement-mediated effector function.
  • the variable region is that of the anti-CD20 antibody PRO70769, and the heavy chain constant region is IgGL
  • the figure provides the Fold IC50 relative to WT for binding to human V158 Fc ⁇ Rllla by two separate experiments, the Fold IC50 relative to WT for binding to human Fc ⁇ RI, and the Fold EC50 relative to WT for CDC activity.
  • FIG. 21 Binding to the low affinity human activating receptor V158 Fc ⁇ Rllla and the high affinity human activating receptor Fc ⁇ RI by select PRO70769 Fc variants as determined by the competition AlphaScreen assay.
  • FIG. 23 Cell-based ADCC activity of anti-Her2 Fc variant and WT IgG antibodies against Her2/neu+ SkBr-3 breast carcinoma target cells. Human PBMCs were used as effector cells, and lysis was measured by LDH release.
  • FIG. 24 Amino acid sequences of variable light (VL) and heavy (VH) chains used in the present invention, including PRO70769 ( Figures 24a and 24b), trastuzumab ( Figures 24c and 24d), and ipilimumab ( Figures 24e and 24f).
  • Figure 25 Amino acid sequences of human constant light kappa (Figure 25a) and heavy ( Figures 25b - 25f) chains used in the present invention.
  • Figure 26 Sequences showing possible constant heavy chain sequences with reduced Fc ligand binding and effector function properties (Figure 26a), and sequences of improved anti-CTLA-4 antibodies ( Figures 26b - 26d).
  • Figure 26a shows potential Fc variant constant heavy chain sequences, with variable positions designated in bold as X1 , X2, X3, X4, X5, X6, X7, and X8.
  • the table below the sequence provides the WT amino acid and possible substitutions for these positions.
  • Improved antibody sequences may comprise one or more non-WT amino acid selected from this group of possible modifications.
  • Figure 26b provides the light chain sequence of an anti-CTLA-4 antibody
  • Figures 26c and 26d provide heavy chain sequences of anti-CTLA-4 antibodies with reduced Fc ligand binding and Fc-mediated effector function. These include an L235G/G236R IgGI heavy chain ( Figure 26c) and an lgG2 heavy chain ( Figure 26d). The positions are numbered according to the EU index as in Kabat, and thus do not correspond to the sequential order in the sequence.
  • 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 FcDRs 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.
  • 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.
  • G236- designates the deletion of glycine at position 236.
  • Amino acids of the invention may be further classified as either isotypic or novel.
  • 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 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.
  • IgGI has a tyrosine and lgG2 a phenylalanine at EU position 296, a F296Y substitution in lgG2 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 IgGI , lgG2, lgG3, or lgG4 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 DD 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 VH, CH1 , VH, and C
  • 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.
  • Fc may include the J chain.
  • Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (C ⁇ 2 and C ⁇ 3) and the hinge between Cgammai (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 Fes, 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 a/., 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.
  • a fusion partner which in general may be any protein, polypeptide 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 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, preferrably an extracellular receptor that is implicated in disease.
  • Fc gamma receptor or “FcDR” 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 FcDR 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 ⁇ Rlla (including allotypes H131 and R131), Fc ⁇ Rllb (including Fc ⁇ Rllb-1 and Fc ⁇ Rllb-2), and Fc ⁇ Rllc; and Fc ⁇ RIII (CD16), including isoforms Fc ⁇ Rllla (including allotypes V158 and F158) and Fc ⁇ Rlllb (including allotypes Fc ⁇ Rlllb-NA1 and Fc ⁇ Rlllb- NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, hereby
  • An Fc ⁇ R may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys.
  • Mouse FcDRs include but are not limited to FcDRI (CD64), FcDRII (CD32), FcDRIII (CD16), and FcDRIII-2 (CD16- 2), as well as any undiscovered mouse FcDRs or Fc ⁇ R isoforms or allotypes.
  • 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, C1 q, 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 FcDRs (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 antibodv 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 , CD1 , CD2, and CD3.
  • 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 IgGI , lgG2, lgG3, and lgG4. In mice IgG comprises IgGI , lgG2a, lgG2b, lgG3.
  • 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 G-sandwich folding topology. The known Ig domains in the IgG isotype of antibodies are V H , CD1 , CD2, CD3, V L , and C L .
  • IgG' or "IgG immunoglobulin” 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 class comprises the subclasses or isotypes IgGI , lgG2, lgG3, and lgG4.
  • 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 IgGI , lgG2, lgG3, lgG4, IgAI , lgA2, 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.
  • Said 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 IgGI .
  • 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 IgGI .
  • 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 VD, VD, and/or V H genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively.
  • variant polypeptide polypeptide variant
  • variant polypeptide 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 polyeptide 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 immunoglobluin” 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.
  • the present invention provides variant antibodies.
  • Traditional antibody structural units typically comprise a tetramer.
  • 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 IgGI , lgG2, lgG3, and lgG4.
  • IgM has subclasses, including, but not limited to, IgMI and lgM2.
  • 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 IgGI , lgG2, lgG3, lgG4, IgAI , lgA2, IgMI , lgM2, IgD, and IgE.
  • 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. In the variable region, 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
  • the carboxy-terminal portion of 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.
  • 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.
  • 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 IgGI) to 236 (G236 in IgGI), 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 (Cg2 and Cg3) and the lower hinge region between Cgammai (Cg1 ) and Cgamma2 (Cg2).
  • 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 Fes, and Fc fragments.
  • the antibodies are full length.
  • full length antibody herein is meant the structure that constitutes the natural biological form of an antibody, including variable and constant regions, including one or more modifications as outlined herein.
  • 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.
  • 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 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.
  • 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 USSN 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. MoI. 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.
  • 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.
  • the antibody is a minibody.
  • Minibodies are minimized antibody-like proteins comprising a scFv joined to a CH3 domain.
  • the scFv can be joined to the Fc region, and may include some or all of the hinge region.
  • 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.
  • 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.
  • 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.
  • 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 0.1 M 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 1251 or 1311 to prepare labeled proteins for use in radioimmunoassay, the chloramine T method described above being suitable.
  • R and R' are optionally different alkyl groups, such as 1- cyclohexyl-3-(2-morpholinyl-4-ethyI) 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.
  • glycosylation Another type of covalent modification is glycosylation.
  • the IgG variants disclosed herein can be modified to include one or more engineered glycoforms.
  • engineered glycoform as used herein is meant a carbohydrate composition that is covalently attached to an IgG, wherein said 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 effector function.
  • Engineered glycoforms may be generated by a variety of methods known in the art (Umana 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); (US 6,602,684; USSN 10/277,370; USSN 10/113,929; PCT WO 00/61739A1; PCT WO 01/29246A1 ; PCT WO 02/31140A1; PCT WO 02/30954A1); (PotelligentTM technology [Biowa, Inc., Princeton, NJ]; GlycoMAbTM glycosylation engineering technology [GLYCART biotechnology AG, Zurich, Switzerland]).
  • 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.
  • glycosylation patterns can depend on both the sequence of the protein (e.g., the presence or absence of particular glycosylation amino acid residues, discussed below), or the host cell or organism in which the protein is produced. Particular expression systems are discussed below.
  • Glycosylation of polypeptides is typically either N-linked or O-linked.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tri-peptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • X is any amino acid except proline
  • O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxyIysine may also be used.
  • Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tri-peptide sequences (for N-linked glycosylation sites).
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites).
  • the antibody amino acid sequence is preferably altered through changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
  • Another means of increasing the number of carbohydrate moieties on the antibody is by chemical or enzymatic coupling of glycosides to the protein. These procedures are advantageous in that they do not require production of the protein in a host cell that has glycosylation capabilities for N- and O-linked glycosylation.
  • the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine.
  • Removal of carbohydrate moieties present on the starting antibody may be accomplished chemically or enzymatically.
  • Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N- acetylgalactosamine), while leaving the polypeptide intact.
  • Chemical degiycosylation is described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981 , Anal. Biochem. 118:131.
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol. 138:350. Glycosylation at potential glycosylation sites may be prevented by the use of the compound tunicamycin as described by Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation of protein-N-glycoside linkages.
  • 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.
  • Specific labels include 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, OR), FlTC, Rhodamine
  • 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
  • EYFP enhanced yellow fluorescent protein
  • luciferase lchiki et al., 1993, J. Immunol. 150:5408-5417
  • ⁇ 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. Patent Nos. 5292658, 5418155, 5683888, 5741668, 5777079, 5804387, 5874304, 5876995, 5925558). All of the above-cited references are expressly incorporated herein by reference.
  • antibody can 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 (D), lambda (D), and heavy chain genetic loci, which together comprise the myriad variable region genes, and the constant region genes mu (D), delta (D), gamma (D), sigma (D), and alpha (D) which encode the IgM, IgD, IgG (IgGI , lgG2, lgG3, and lgG4), IgE, and IgA (IgAI and lgA2) 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.
  • An Fc variant comprises one or more amino acid modifications relative to a parent Fc polypeptide, wherein said 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, preferrably 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 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/A330L/I332E defines an Fc variant with the substitutions S239D, A330L, and I332E relative to the parent Fc polypeptide. It is noted that the order in which substitutions are provided is arbitrary, that is to say that, for example, S239D/A330L/I332E is the same Fc variant as S239D/I332E/A330L, and so on.
  • EU index or EU 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).
  • 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.
  • the parent Fc polypeptide may be an antibody.
  • Parent antibodies may be fully human, obtained for example using transgenic mice (Bruggemann et al., 1997, Curr Opin Biotechnol 8:455- 458, hereby entirely incorporated by reference) or human antibody libraries coupled with selection methods (Griffiths et al., 1998, Curr Opin Biotechnol 9:102-108, hereby entirely incorporated by reference).
  • the parent antibody need not be naturally occurring.
  • the parent antibody may be an engineered antibody, including but not limited to chimeric antibodies and humanized antibodies (Clark, 2000, Immunol Today 21 :397-402, hereby entirely incorporated by reference).
  • the parent antibody may be an engineered variant of an antibody that is substantially encoded by one or more natural antibody genes.
  • the parent antibody has been affinity matured, as is known in the art.
  • the antibody has been modified in some other way, for example as described in USSN 10/339788, filed on March 3, 2003, hereby entirely incorporated by reference.
  • 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 IgGI, lgG2, lgG3, or lgG4.
  • Figure 3 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 IgAI and lgA2), 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.
  • 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 IgGI , lgG2 and lgG3 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).
  • Gm haplotypes 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, b ⁇ , 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. et al., 1979, Hum. Genet.: 50, 199-211 , both hereby entirely incorporated by reference). Allotypes that are inherited in fixed combinations are called Gm haplotypes.
  • Figure 4 shows the allotypes and isoaliotypes of the gammal chain of human IgGI showing the positions and the relevant amino acid substitutions (Gorman & Clark, 1990, Semin Immunol 2(6):457-66, hereby entirely incorporated by reference). For comparison the amino acids found in the equivalent positions in human lgG2, lgG3 and lgG4 gamma chains are also shown.
  • the Fc variants of the present invention may be substantially encoded by any allotype or isoallotype of any immunoglobulin gene.
  • the Fc variants of the present invention find use in antibodies or Fc fusions that comprise IgGI sequences that are classified as G1m(1), G1m(2), G1m(3), G1m(17), nG1m(1), nG1 m(2), and/or nG1 m(17).
  • the Fc variants of the present invention may comprise a Lys (G1m(17)) or Arg (G1m(3)) at position 214, an Asp356/Leu358 (G1m(1)) or Glu356/Met358 (nG1m(1)), and/or a GIy (G1m(2)) or Ala (nG1m(2)) at position 431.
  • 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 IgGI parent antibody that binds a different antigen, a human lgG2 parent antibody, a human IgA parent antibody, a mouse lgG2a or lgG2b 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.
  • 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, 4-1 BB, 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
  • the present invention provides Fc variants that are optimized for a variety of therapeutically relevant 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 enhanced or reduced affinity for an Fc ⁇ R.
  • the Fc variants of the present invention are optimized to possess enhanced affinity for a human activating Fc ⁇ R, preferably Fc ⁇ RI, Fc ⁇ Rlla, Fc ⁇ Rllc, Fc ⁇ Rllla, and Fc ⁇ Rlllb, most preferably Fc ⁇ Rllla.
  • the Fc variants are optimized to possess reduced affinity for the human inhibitory receptor Fc ⁇ Rllb.
  • Fc variants of the present invention are optimized to have reduced or ablated affinity for a human Fc ⁇ R, including but not limited to Fc ⁇ RI, Fc ⁇ Rlla, Fc ⁇ Rllb, Fc ⁇ Rllc, Fc ⁇ Rllla, and Fc ⁇ Rlllb. These embodiments are anticipated to provide IgG polypeptides with enhanced therapeutic properties in humans, for example reduced effector function and reduced toxicity. In other embodiments, Fc variants of the present invention provide enhanced 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 enhanced binding to Fc ⁇ Rllla, yet reduced binding to Fc ⁇ Rllb.
  • an Fc variant of the present invention may have enhanced binding to Fc ⁇ Rlla and Fc ⁇ RI, yet reduced binding to Fc ⁇ Rllb.
  • an Fc variant of the present invention may have enhanced affinity for Fc ⁇ Rllb, yet reduced affinity to one or more activating Fc ⁇ Rs.
  • Preferred embodiments comprise optimization of binding to a human Fc ⁇ R, however in alternate embodiments the Fc variants of the present invention possess enhanced or reduced affinity for Fc ⁇ Rs from nonhuman organisms, including but not limited to rodents and non-human primates.
  • Fc variants that are optimized for binding to a nonhuman Fc ⁇ R may find use in experimentation.
  • mouse models are available for a variety of diseases that enable testing of properties such as efficacy, toxicity, and pharmacokinetics for a given drug candidate.
  • cancer cells can be grafted or injected into mice to mimic a human cancer, a process referred to as xenografting.
  • Fc variants that comprise Fc variants that are optimized for one or more mouse Fc ⁇ Rs, may provide valuable information with regard to the efficacy of the protein, its mechanism of action, and the like.
  • the Fc variants of the present invention may also be optimized for enhanced functionality and/or solution properties in aglycosylated form.
  • the aglycosylated Fc variants of the present invention bind an Fc ligand with greater affinity than the aglycosylated form of the parent Fc variant.
  • Said Fc ⁇ gands include but are not limited to Fc ⁇ Rs, C1q, FcRn, and proteins A and G, and may be from any source including but not limited to human, mouse, rat, rabbit, or monkey, preferably human.
  • the Fc variants are optimized to be more stable and/or more soluble than the aglycosylated form of the parent Fc variant.
  • Fc variants of the invention may comprise modifications that modulate interaction with Fc ligands other than Fc ⁇ Rs, including but not limited to complement proteins, FcRn, and Fc receptor homologs (FcRHs).
  • FcRHs include but are not limited to FcRHI , FcRH2, FcRH3, FcRH4, FcRH5, and FcRH6 (Davis et al., 2002, Immunol. Reviews 190:123-136, hereby entirely incorporated by reference).
  • the Fc ligand specificity of the Fc variant of the present invention will determine its therapeutic utility.
  • the utility of a given Fc variant for therapeutic purposes will 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 may be used that comprise Fc variants that provide enhanced 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 ⁇ Rlla and Fc ⁇ Rllla may be desired, but not Fc ⁇ Rl, whereas in other cases, enhanced binding only to Fc ⁇ Rlla 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 ⁇ Rllb, 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.
  • Fc ligand selectivity or specifity 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.
  • toxin, radionucleotide, or other conjugates may be less toxic to normal cells if the Fc variant that comprises them has reduced or ablated binding to one or more Fc ligands.
  • an Fc variant with enhanced affinity for activating Fc ⁇ Rs such as to bind these Fc ⁇ Rs and prevent their activation.
  • an Fc variant that comprises two or more Fc regions with enhanced Fc ⁇ Rllb affinity may co-engage this receptor on the surface of immune cells, thereby inhibiting proliferation of these cells.
  • an Fc variants may engage its target antigen on one cell type yet engage Fc ⁇ Rs on separate cells from the target antigen, in other cases it may be advantageous to engage Fc ⁇ Rs on the surface of the same cells as the target antigen.
  • an antibody targets an antigen on a cell that also expresses one or more Fc ⁇ Rs
  • antigen and Fc ⁇ R co-engagement on the same cell may be advantageous when the Fc variant is being used to modulate the immune system in some way, wherein co-engagement of target antigen and Fc ⁇ R provides some proliferative or anti-proliferative effect.
  • Fc variants that comprise two or more Fc regions may benefit from Fc variants that modulate Fc ⁇ R selectivity or specifity to co-engage Fc ⁇ Rs on the surface of the same cell.
  • 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 signficantly 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.
  • 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 USSN 11/004,590, filed December 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, US 5225539, 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.
  • Humanization methods include but are not limited to methods described in Jones et al., 1986, Nature 321 :522-525; Riechmann et a/., 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.
  • 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 USSN 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. MoI. 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.
  • 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 variants of the present invention comprise one or more engineered glycoforms.
  • engineered qlvcoform as used herein is meant a carbohydrate composition that is covalently attached to an IgG, wherein said 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 effector function.
  • Engineered glycoforms may be generated by a variety of methods known in the art (Umana 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); (US 6,602,684; USSN 10/277,370; USSN 10/113,929; PCT WO 00/61739A1 ; PCT WO 01 /29246A1 ; PCT WO 02/31140A1 ; PCT WO 02/30954A1 ); (PotelligentTM technology [Biowa, Inc., Princeton, NJ]; GlycoMAb® glycosylation engineering technology [GLYCART biotechnology AG, Zurich, Switzerland], all hereby entirely incorporated by reference).
  • Engineered glycoform typically refers to the different carbohydrate or oligosaccharide; thus an Fc variant, for example an antibody or Fc fusion, may comprise an engineered glycoform.
  • engineered glycoform may refer to the Fc variant that comprises the different carbohydrate or oligosaccharide.
  • 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 USSN 10/672,280, USSN 10/822,231 , USSN 11/124,620, and USSN 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 colorimetric 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 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.
  • the Fc variants of the present invention may be used for various therapeutic purposes.
  • 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.
  • 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.
  • 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 preferred 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 said 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 a preferred embodiment, 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 10mg/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, intranasaliy, 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, intranasaliy, 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 Inhal
  • Figure 5 provides a list of these Fc variants.
  • this variant set comprises a number of insertions.
  • “Insert L > 235-236 / I332E” refers to a double mutant comprising the substitution I332E and an insertion of leucine between residues 235 and 236.
  • Plasmids containing heavy chain gene (VH-CH1-CH2-CH3) (wild-type or variants) were co-transfected with plasmid containing light chain gene (VL-CK) into 293T cells. Media were harvested 5 days after transfection, and antibodies were purified from the supernatant using protein A affinity chromatography (Pierce). Select Fc variants were also expressed in the context of alemtuzumab.
  • Binding affinity to human Fc ⁇ Rs by IgG 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 IgGI 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.
  • Figures 6 provides competitive AlphaScreen data for binding of select PRO70769 Fc variants to the human activating receptors V158 Fc ⁇ Rllla ( Figure 6a) and F158 Fc ⁇ Rllla ( Figure 6b).
  • 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.
  • Figure 5 provides the IC50's and Fold IC50's relative to WT for fits to these binding curves.
  • WT and variant trastuzumab antibodies were expressed and purified as described above.
  • data were acquired on a BIAcore 3000 instrument (BIAcore).
  • V158 Fc ⁇ Rllla-His-GST was captured using immobilized anti-GST antibody, blocked with recombinant GST, and binding to antibody/receptor competition analyte was measured.
  • Anti-GST antibody was covalently coupled to a CM5 sensor using the BIAcore GST Capture Kit. Flow cell 1 of every sensor chip was coupled with ethanolamine as a control of unspecific binding and to subtract bulk refractive index changes online.
  • Running buffer was HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCI, 3 mM EDTA, 0.005% v/v Surfactant P20, BIAcore), and chip regeneration buffer was Glycine 1.5 (10 mM glycine-HCI, pH 1.5, BIAcore).
  • Glycine 1.5 10 mM glycine-HCI, pH 1.5, BIAcore.
  • 1 ⁇ M V158 Fc ⁇ Rllla-His-GST was bound to the anti-GST CM5 chip in HBS-EP at 1 ⁇ l/min for 5 minutes. The surface was blocked with 5 ⁇ M recombinant GST (Sigma) injected at 1 ⁇ l/minute for 2 minutes.
  • V158 Fc ⁇ Rllla-His-GST 100 nM wild-type or variant trastuzumab antibody was combined with V158 Fc ⁇ Rllla-His-GST in serial dilutions between 4 and 1000 nM and incubated for at least two hours at room temperature.
  • the competition mixture was injected over the V158 Fc ⁇ Rllla-His-GST/recombinant GST surface for 30 seconds association in HBS-EP at 50 ⁇ l/minute. A cycle with antibody but no competing receptor provided a baseline response.
  • R - ⁇ -([A 0 ]- l0 x - K D ) + 4(K D 2 + 2 ⁇ W)(K D ) + (10 x ) 2 + 2[A 0 ]K 0 - 2[A 0 )IO* +[A 0 ] 2 ) 2IA 0 ] with:
  • R 0 reflects the rate of binding between antibody and immobilized receptor (in the absence of competing receptor), and because of their different receptor affinities was calculated separately for WT, I332E, and S239D/I332E antibodies.
  • the formula for the initial rate R is derived from the definition of K 0 for a single binding site:
  • 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 CD20+ target lymphoma cell line WIL2-S was obtained from ATCC.
  • 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.
  • the ADCC assays were repeated in the presence of a biologically relevant (1 mg/ml) concentration of IgG purified from human serum (purchased commercially from Jackson lmmunoresearch Lab, Inc.). These data are provided in Figure 11.
  • the efficacy of the WT anti-CD20 antibody is not only reduced, but completely ablated in the presence of serum level IgG.
  • the Fc variant antibodies although significantly reduced, still show substantial capacity to mediate killing against the target cell line.
  • Figure 12 shows a structure of the human IgGI Fc region with this epicenter mapped.
  • Select amino acid modifications disclosed in USSN 10/672,280, USSN 10/822,231 , USSN 11/124,620, and USSN 11/256,060, all hereby entirely incorporated by reference, that are structurally proximal to these four residues were investigated to explore variants that may mediate increased affinity for C1q and and/or provide enhanced CDC.
  • Variants that previously showed enhanced Fc ⁇ R affinity and Fc ⁇ R-mediated effector function were included in this set of variants to characterize their complement properties.
  • This variant library is provided in Figure 13.
  • variants were constructed as described above in the context of the anti-CD20 antibody PRO70769 (variable region) and either IgGI or lgG(1/2) ELLGG as the heavy chain constant region. Variants were expressed and purified as described above.
  • a cell-based assay was used to measure the capacity of the Fc variants to mediate CDC. Lysis was measured using release of Alamar Blue to monitor lysis of Fc variant and WT PRO70769 -opsonized WIL2-S lymphoma cells by human serum complement. Target cells were washed 3x in 10% FBS medium by centrifugation and resuspension, and WT or variant rituximab antibody was added at the indicated final concentrations.
  • Human serum complement (Quidel) was diluted 50% with medium and added to antibody-opsonized target cells. Final complement concentration was 1/6 th original stock. Plates were incubated for 2 hrs at 37 0 C, Alamar Blue was added, cells were cultured for two days, and fluorescence was measured. Representative data from this assay are shown in Figure 14. 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 data were fit to a sigmoidal dose-response with variable slope model using nonlinear regression, and these fits are represented by the curves in the figure. These fits provide the effective concentration 50% (EC50) (i.e.
  • modifications that show 0.5 fold and lower relative CDC include 235D, 239D, 284D, 322H, 322T, 322Y, 327R, 330E, 3301, 330L, 330N, 330V, 331 D, and 331 L, 332E ( Figure 15). These modifications provide further valuable structure activity relationship (SAR) information that may be used to guide further design of variants for enhanced CDC.
  • SAR structure activity relationship
  • Fc ⁇ R- and complement- binding A previously unconsidered advantage of ablated Fc ⁇ R- and complement- binding is that in cases where effector function is not needed, binding to Fc ⁇ R and complement may effectively reduce the active concentration of drug. Binding to Fc ligands may localize an antibody or Fc fusion to cell surfaces or in complex with serum proteins wherein it is less active or inactive relative to when it is free (uncomplexed).
  • Fc ⁇ R- receptors may be one mechanism of antibody turnover, and can mediate uptake and processing by antigen presenting cells such as dendritic cells and macrophages. This may affect affect the pharmacokinetics (or in vivo half-life) of the antibody or Fc fusion and its immunogenicity, both of which are critical parameters of clinical performance.
  • Figure 17 shows AlphaScreen data for binding of select Fc variants to human V158 Fc ⁇ Rllla, and Figure 16 provides their Fold IC ⁇ O's relative to WT PRO70769 IgGI .
  • the variants were also investigated for their capacity to mediate complement- mediated lysis against CD20+ WIL2-S lymphoma target cells using the CDC assay described above.
  • Figure 18 provides CDC data for select Fc variants, and Figure 16 provides their Fold EC50's relative to WT PRO70769 IgGI . Based on the results of these experiments, select Fc variants were characterized for their capacity to mediate Fc ⁇ R-mediated effector function.
  • An ADCC assay using human PBMCs as effector cells and WIL2-S lymphoma cells as target cells was carried out as described above.
  • Figure 19 shows these ADCC data for select variants.
  • modification at a number of positions provide reduced or ablated Fc ⁇ R affinity, reduced Fc ⁇ R-mediated effector function, and reduced complement-mediated effector function.
  • modifications at some positions may provide reduced CDC but WT Fc ⁇ R affinity.
  • 235D, 330L, 330N, and 330R display such behavior.
  • modification at some positions including but not limited to 236 and 299, may provide reduced Fc ⁇ R affinity but WT level CDC. For example 236I and 299A show these properties.
  • WT IgGI antibody As well as lgG2 and lgG4 antibody versions, an aglycosylated variant N297S, and two variants previously characterized as having reduced effector function: L234A/L235A (Xu et al., 2000, Cellular Immunology 200:16-26; USSN 10/267,286, hereby entirely incorporated by reference) and E233P/L234V/L235A/G236- (Armour et al., 1999, Eur J Immunol 29:2613-2624, hereby entirely incorporated by reference).
  • L234A/L235A Xu et al., 2000, Cellular Immunology 200:16-26; USSN 10/267,286, hereby entirely incorporated by reference
  • E233P/L234V/L235A/G236- Armour et al., 1999, Eur J Immunol 29:2613-2624, hereby entirely incorporated by reference).
  • trastuzumab variant region sequences provided in Figures 24c and 24d.
  • trastuzumab robustly provides a substantial signal in ADCC assays against Her2+ expressing cell lines, and therefore provides a stringent test of the Fc variants for reducing/ablating effector function.
  • L235G, G236R, G237K, N325L, N325A, L328R, L235G/G236R, G236R/G237K, G236R/N325L, G236R/L328R, G237K/N325L, L235G/G236R/G237K, and G236R/G237K/L328R were constructed in the context of trastuzumab IgGI .
  • WT IgGI ,WT lgG2, and WT lgG4 antibody versions were constructed as well.
  • An ADCC assay was carried out as described above, except the Her2+ breast carcinoma cell line SkBr-3 was used as target cells.
  • Figure 23 provides the results of the ADCC experiments. The data indicate that some of the variants completely ablate ADCC. Additionally, although lgG2 also appears to mediate no ADCC, lgG4 does show a significant level of ADCC.
  • amino acid modifications 232G, 234G, 234H, 235D, 235G, 235H, 236I, 236N, 236P, 236R, 237K, 237L, 237N, 237P, 238K, 239R, 265G, 267R, 269R, 270H, 297S, 299A, 299I, 299V, 325A, 325L, 327R, 328R, 329K, 330I, 330L, 330N, 330P, 330R, 330S, and 331 L provide significantly reduced Fc ligand binding properties and/or effector function.
  • amino acid modifications that compose these variants are capable of reducing binding to both Fc ⁇ Rllla and Fc ⁇ RI, and reducing CDC by greater than 10 fold.
  • the data show that human lgG2 has significantly reduced Fc ⁇ R-affinity, Fc ⁇ R-mediated effector function, and complement-mediated effector function relative to human lgG4.
  • reduced Fc ⁇ R affinity and/or effector function may be optimal for Fc polypeptides for which Fc ligand binding or effector function leads to toxicity and/or reduced efficacy.
  • Fc ligand binding or effector function leads to toxicity and/or reduced efficacy.
  • antibodies that target CTLA-4 block inhibition of T-celi activation and are effective at promoting anti-tumor immune response, but destruction of T cells via antibody mediated effector functions may be counterproductive to mechanism of action and/or potentially toxic. Indeed toxicity has been observed with clinical use of the anti-CTLA-4 antibody ipilimumab (Maker et al., 2005, Ann Surg Oncol 12:1005-16, hereby entirely incorporated by reference).
  • the optimized antibody sequences sequences comprise at least one non-WT amino acid selected from the group consisting of X 1 , X 2 , X 3 , X 4 , X 5 , Xe, X 7 , and X 8 .
  • an improved anti- CTLA-4 antibody sequence comprising the L235G and G236R modifications in the IgGI constant region are provided in Figures 26b and 26c.
  • lgG2 and lgG4 can also be used to reduce Fc ligand binding and Fc-mediated effector function.
  • Figures 26b and 26d provide the sequences of improved anti-CTLA-4 lgG2 antibody sequences.
  • an anti-CTLA-4 here is solely an example, and is not meant to constrain application of the Fc variants to this antibody or any other particular Fc polypeptide.
  • Other exemplary applications for reduced Fc ligand binding and/or effector function include but are not limited to anti-TNF ⁇ antibodies, including for example infliximab and adalimumab, anti-VEGF antibodies, including for example bevacizumab, anti- ⁇ 4-integrin antibodies, including for example natalizumab, and anti-CD32b antibodies, including for example those described in USSN 10/643,857, hereby entirely incorporated by reference.

Abstract

La présente invention concerne des variants Fc présentant des propriétés optimisées, des procédés de production associés, des polypeptides Fc contenant lesdits variants, ainsi que des méthodes d'utilisation de ces variants Fc présentant des propriétés optimisées.
EP06748967A 2005-03-31 2006-03-31 VARIANTS Fc PRESENTANT DES PROPRIETES OPTIMISEES Withdrawn EP1871808A2 (fr)

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US72333505P 2005-10-03 2005-10-03
US72329405P 2005-10-03 2005-10-03
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