JP2008537941A - Fc variants with optimized properties - Google Patents

Abstract

  The present invention relates to Fc variants with optimized properties, methods for their production, Fc polypeptides comprising Fc variants with optimized properties, and methods of using Fc variants with optimized properties.

Description

  USSN 60 / 667,197 filed March 31, 2005; USSN 60 / 705,378 filed August 3, 2005; USSN 60 / 723,294 filed October 3, 2005; and 2005 USSN 60 / 723,335 filed Oct. 3, and USSN 11 / 124,620, filed May 5, 2005, all of which are hereby incorporated by reference in their entirety. Claim the benefit of Section 119 (e) of the US Patent Act. The disclosure of US patent application Ser. No. 10 / 822,231 filed Mar. 26, 2004 is also expressly incorporated by reference in its entirety.

The present invention relates to Fc variant polypeptides with optimized properties, engineering methods for their production, and their application, particularly for therapeutic purposes.

BACKGROUND OF THE INVENTION Antibodies are immunological proteins that bind to a specific antigen. In most mammals, including humans and mice, antibodies are constructed from a pair of heavy and light chain polypeptide chains. Each chain consists of an individual immunoglobulin (Ig) domain, hence the generic name immunoglobulin is used for such proteins. Each chain consists of two different regions called the variable region (V region) and the constant region. The light chain variable region (VL) and heavy chain variable region (VH) exhibit significant sequence diversity between antibodies and are involved in binding to the target antigen. The constant region does not show much sequence diversity and is involved in the binding of many natural proteins, causing important biochemical events. In humans, there are five different antibody classes including IgA (including IgA1 and IgA2 subclasses), IgD, IgE, IgG (including IgG1, IgG2, IgG3 and IgG4 subclasses), and IgM. A striking feature between these antibody classes is their constant region, although slight differences may exist in the V region. FIG. 1, used herein as an example to describe the general structural characteristics of immunoglobulins, shows an IgG1 antibody. An IgG antibody is a tetrameric protein consisting of two heavy chains and two light chains. The IgG heavy chain represents V _H -CH1-CH2-CH3 from the N-terminus to the C-terminus (representing heavy chain variable domain, heavy chain constant domain 1, heavy chain constant domain 2, and heavy chain constant domain 3, respectively) ( In addition, VH-Cγ1-Cγ2-Cγ3 (also referred to as a heavy chain variable domain, a constant gamma 1 domain, a constant gamma 2 domain, and a constant gamma 3 domain, respectively) are connected in this order. It consists of an immunoglobulin domain. An IgG light chain is composed of two immunoglobulin domains linked in the order of V _L -C _L (representing a light chain variable domain and a light chain constant domain, respectively) from the N-terminus to the C-terminus.

The variable region of an antibody contains the antigen-binding determinants of the molecule and thus determines the specificity of the antibody for its target antigen. Variable regions are so named because they are most different in sequence from other antibodies in the same class. Most of the sequence diversity occurs in the complementarity determining region (CDR). There are a total of 6 CDRs, denoted V _H CDR1, V _H CDR2, V _H CDR3, V _L CDR1, V _L CDR2, and V _L CDR3, each 3 per heavy and light chain. The variable region other than the CDR is called a framework (FR) region. Although not as diverse as CDRs, sequence diversity occurs in the FR regions between different antibodies. In general, this characteristic structure of an antibody provides a stable scaffold (FR region) that allows substantial antigen binding diversity (CDR) to be investigated by the immune system to obtain specificity for a wide range of antigens. provide. Many high resolution structures are available for various variable region fragments from different organisms, some unbound with antigen and some in complex with antigen. The sequence and structural characteristics of antibody variable regions are well characterized (Morea et al., 1997, Biophys Chem 68: 9-16; Morea et al., 2000, Methods 20: 267-279, with reference to Conserved features of antibodies, which are encompassed herein in their entirety, allow the development of many antibody engineering techniques (see Maynard et al., 2000, Annu Rev Biomed Eng 2: 339-376). Are incorporated herein in their entirety). For example, a CDR from one antibody, such as a mouse antibody, can be transplanted into the framework region of another antibody, such as a human antibody. This method is referred to in the art as “humanization” and allows the production of less immunogenic antibody therapeutics derived from non-human antibodies. Fragment containing the variable region can exist lacking other regions of the antibody, including for example, _V H -Shiganma1 and _V H -C antigen-binding fragments comprising an _L (Fab), the _{V H} and _{V L} Variable fragments (Fv), single chain variable fragments (scFv) containing V _H and V _L linked together in the same chain, and a variety of other variable region fragments are included (Little et al., 2000, Immunol Today 21 : 364-370, which is incorporated herein by reference in its entirety).

  The Fc region of an antibody interacts with many Fc receptors and ligands and provides a series of important functions called effector functions. For IgG, as shown in FIG. 1, the Fc region includes Ig domains Cγ2 and Cγ3, and an N-terminal hinge leading to Cγ2. An important Fc receptor family for the IgG class is the Fc gamma receptor (FcγR). These receptors mediate communication between antibodies and cellular immune responses (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12: 181-220; Ravetch et al., 2001, Annu Rev Immunol 19: 275 -290, both of which are incorporated herein by reference in their entirety). In humans, this protein family includes FcγRIa (CD64) including isoforms FcγRIa, FcγRIb and FcγRIc; FcγRIIa (including allotypes H131 and R131), FcγRIIb (FcγRIIb-1 and FcγRIIb-2) And FcγRII (CD32) including FcγRIIIa (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) including FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (CD16), including FcγRIIc (Jefferis) et al., 2002, Immunol Lett 82: 57-65, which is incorporated herein in its entirety by reference). These receptors typically have an extracellular domain that mediates binding to Fc, a transmembrane region, and an intracellular domain that can mediate several signaling events within the cell. These receptors include monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and γγ T cells. It is expressed in a variety of immune cells, including. The formation of the Fc / FcγR complex captures these effector cells at the site of the bound antigen and typically causes intracellular signaling events as well as release of inflammatory mediators, B cell activation, endocytosis, phagocytosis Effects and subsequent important immune responses such as cytotoxic attack. The ability to mediate cytotoxic and phagocytic effector functions is a possible mechanism by which antibodies destroy target cells. The reaction mediated by non-specific cytotoxic cells that express FcγR recognizes the binding of the antibody to the target cell and then results in the lysis of the target cell is an antibody-dependent cell-mediated cytotoxicity (ADCC). (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12: 181-220; Ghetie et al., 2000, Annu Rev Immunol 18: 739-766; Ravetch et al., 2001, Annu Rev Immunol 19: 275-290, all of which are incorporated herein by reference in their entirety). The reaction mediated by non-specific cytotoxic cells expressing FcγR that recognize antibody binding to the target cell and then phagocytoses the target cell is antibody-dependent cell-mediated phagocytosis (ADCP). ). FcγRIIa (pdb accession code 1H9V, incorporated herein by reference in its entirety) (Sondermann et al., 2001, J Mol Biol 309: 737-749, incorporated herein by reference in its entirety. (Pdb accession code 1FCG, incorporated herein by reference in its entirety) (Maxwell et al., 1999, Nat Struct Biol 6: 437-442, incorporated herein by reference in its entirety. FcγRIIb (pdb accession code 2FCB, which is incorporated herein by reference in its entirety) (Sondermann et al., 1999, Embo J 18: 1095-1103, which is hereby incorporated by reference in its entirety. And FcγRIIIb (pdb accession code 1E4J, which is hereby incorporated in its entirety by reference) (Sondermann et al., 2000, Nature 406: 267-273, which is hereby incorporated by reference in its entirety. Included in the specification) Many structure of the extracellular domain of bets FcγR have been elucidated. All FcγRs bind to the same region on Fc at the N-terminus of the Cγ2 domain and the hinge, as shown in FIG. This interaction is structurally well characterized (Sondermann et al., 2001, J Mol Biol 309: 737-749, which is hereby incorporated by reference in its entirety) and is a cell of human FcγRIIIb Some structures of human Fc that bind to the outer domain (pdb accession code 1E4K, which is incorporated by reference in its entirety) (Sondermann et al., 2000, Nature 406: 267-273, (Incorporated herein in its entirety) (pdb accession codes 1IIS and 11IX, which is incorporated herein in its entirety by reference) (Radaev et al., 2001, J Biol Chem 276: 16469-16477, The entirety of which is hereby incorporated by reference), as well as the structure of the human IgE Fc / FcεRIα complex (pdb accession code 1F6A, which is hereby incorporated in its entirety by reference) (Garman et al. , 2000, Nature 406: 259-266 By reference in its entirety are incorporated herein) has been elucidated.

Different IgG subclasses have different affinities for FcγR, and IgG1 and IgG3 typically bind substantially better to the receptor than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett 82: 57-65, which is hereby incorporated by reference in its entirety). All FcγRs have different affinities but bind to the same region of IgG Fc: the high affinity binder FcγRI has a Kd10 ⁻⁸ M ⁻¹ for IgG1, while a low affinity receptor FcγRII and FcγRIII, which generally bind at 10 ⁻⁶ and 10 ⁻⁵ , respectively. The extracellular domains of FcγRIIIa and FcγRIIIb have 96% identity; however, FcγRIIIb does not have an intracellular signaling domain. Furthermore, FcγRI, FcγRIIa / c, and FcγRIIIa, characterized by having an intracellular domain with an immunoreceptor tyrosine-dependent activation motif (ITAM), are positive regulators of activation induced by immune complexes. However, FcγRIIb has an immunoreceptor tyrosine-dependent inhibition motif (ITIM) and is therefore inhibitory. Therefore, the former is called an activating receptor and FcγRIIb is called an inhibitory receptor. The receptors also differ in expression patterns and levels in various immune cells. Yet another complexity is the presence of many FcγR polymorphisms in the human proteome. A particularly relevant polymorphism with clinical significance is V158 / F158 FcγRIIIa. Human IgG1 binds with higher affinity to the V158 allotype than to the F158 allotype. This difference in affinity and its putative effect on ADCC and / or ADCP has been shown to be an important determinant of the effect of the anti-CD20 antibody rituximab (Rituxan®, BiogenIdec) . Patients with the V158 allotype respond favorably to rituximab treatment; however, patients with the low affinity F158 allotype rarely respond (Cartron et al., 2002, Blood 99: 754-758, in its entirety by reference). Is encompassed herein). About 10-20% of humans are V158 / V158 homozygous, 45% are V158 / F158 heterozygous, and 35-45% are F158 / F158 homozygous (Lehrnbecher et al ., 1999, Blood 94: 4220-4322; Cartron et al., 2002, Blood 99: 754-758, both of which are hereby incorporated by reference in their entirety). Thus, 80-90% of humans hardly respond, for example, they have at least one allele of F158 FcγRIIIa.

  The overlapping but distinct site on Fc shown in FIG. 1 functions as a junction site for complement protein C1q. Just as Fc / FcγR binding mediates ADCC, Fc / C1q binding mediates complement dependent cytotoxicity (CDC). C1q forms a complex with C1r and C1s, which are serine proteases, to form a C1 complex. C1q can bind 6 antibodies, but binding to 2 IgGs is sufficient to activate the complement cascade. Similar to the Fc and FcγR interaction, various IgG subclasses have different affinities for C1q, and typically IgG1 and IgG3 bind substantially more to FcγR than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett 82: 57-65, which is hereby incorporated by reference in its entirety). Currently there is no structure available for the Fc / C1q complex; however, mutagenesis experiments map the binding site on human IgG to C1q to a region containing residues D270, K322, K326, P329, P331 and E333. (Idusogie et al., 2000, J Immunol 164: 4178-4184; Idusogie et al., 2001, J Immunol 166: 2571-2575, both of which are hereby incorporated by reference in their entirety).

  The site on the Fc between the Cγ2 domain and the Cγ3 domain shown in FIG. 1 mediates interaction with the fetal receptor (neonatal receptor: FcRn), and the binding of the endocytic antibody from the endosome to the blood Recycled and reused (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12: 181-220; Ghetie et al., 2000, Annu Rev Immunol 18: 739-766, both of which are incorporated by reference in their entirety. Included in the specification). This process involves removal by renal filtration due to the large size of the full-length molecule, resulting in a convenient antibody serum half-life ranging from 1 to 3 weeks. The binding of Fc to FcRn also plays an important role in antibody transport. The binding site on Fc for FcRn is also the site where bacterial proteins A and G bind. Strong binding by these proteins is typically used as a means to purify antibodies by using protein A or protein G affinity chromatography during protein purification. Therefore, the suitability of this region on Fc is important for both clinical features of antibodies and their purification. Rat Fc / FcRn complex (Burmeister et al., 1994, Nature, 372: 379-383; Martin et al., 2001, Mol Cell 7: 867-877, both of which are hereby incorporated by reference in their entirety. And a complex of Fc and proteins A and G (Deisenhofer, 1981, Biochemistry 20: 2361-2370; Sauer-Eriksson et al., 1995, Structure 3: 265-278; Tashiro et al., 1995, The available structure of Curr Opin Struct Biol 5: 471-481, all incorporated herein by reference in its entirety, provides an understanding of the interaction of Fc with these proteins.

  One important feature of the Fc region is the conserved N-linked glycosylation that occurs at N297 shown in FIG. As well described, this carbohydrate or oligosaccharide plays an important structural and functional role for antibodies, and is one of the fundamental reasons why antibodies must be produced using mammalian expression systems. Without wishing to be limited to one theory, the structural purpose of this carbohydrate is to stabilize or solubilize Fc, to determine a specific angle or flexibility level between the Cγ3 and Cγ2 domains It is believed that the two Cγ2 domains may not aggregate together across the central axis, or a combination thereof. Efficient Fc binding to FcγR and C1q requires this modification, and changes in the composition of N297 carbohydrates or their removal affects binding to these proteins (Umana et al., 1999, Nat Biotechnol 17: 176-180; Davies et al., 2001, Biotechnol Bioeng 74: 288-294; Mimura et al., 2001, J Biol Chem 276: 45539-45547 .; Radaev et al., 2001, J Biol Chem 276: 16478- 16483; Shields et al., 2001, J Biol Chem 276: 6591-6604; Shields et al., 2002, J Biol Chem 277: 26733-26740; Simmons et al., 2002, J Immunol Methods 263: 133-147, All of which are incorporated herein by reference in their entirety). The carbohydrate does not make any specific binding with FcγR (Radaev et al., 2001, J Biol Chem 276: 16469-16477, which is hereby incorporated by reference in its entirety), but with Fc / FcγR binding It is shown that the functional role of the N297 carbohydrate that mediates can play a structural role in determining Fc conformation. This is supported by the collection of crystal structures of four different Fc glycoforms, which shows that the composition of the oligosaccharide affects the Cγ2 conformation resulting in Fc / FcγR binding (Krapp et al., 2003, J Mol Biol 325: 979-989, which is incorporated herein by reference in its entirety).

  The characteristics of the antibodies described above—specificity for the target, ability to mediate immune effector mechanisms, and long half-life in serum—makes the antibody a powerful therapeutic agent. Monoclonal antibodies are used therapeutically for the treatment of various conditions including cancer, inflammatory diseases and heart diseases. Currently, there are more than 10 commercially available antibody products and hundreds of antibody products under development. In addition to antibodies, antibody-like proteins that have found an expanding role in research and therapy are Fc fusions (Chamow et al., 1996, Trends Biotechnol 14: 52-60; Ashkenazi et al., 1997 Curr Opin Immunol 9: 195-200, both of which are incorporated herein by reference in their entirety). An Fc fusion is a protein in which one or more polypeptides are operably linked to an Fc. An Fc fusion binds the target binding region of a receptor, ligand or some other protein or protein domain to the Fc region of an antibody, thereby combining its preferred effector function and pharmacokinetics. The latter role is to mediate target recognition, and therefore it is functionally similar to the variable region of an antibody. Due to the structural and functional overlap of Fc fusions and antibodies, the description of the antibodies of the invention extends to Fc fusions.

  Antibodies have found wide application in oncology, particularly for targeting cellular antigens that are selectively expressed in tumor cells for the purpose of cell destruction. Antibodies comprising anti-proliferative effects by blocking the required proliferation pathways, apoptosis, enhanced down-regulation and / or intracellular signaling resulting in receptor turnover, CDC, ADCC, ADCP, and promotion of adaptive immune responses There are many possible mechanisms to destroy tumor cells (Cragg et al., 1999, Curr Opin Immunol 11: 541-547; Glennie et al., 2000, Immunol Today 21: 403-410, both see Are incorporated herein in their entirety). Antitumor effects are the result of a combination of these mechanisms, and their relative importance in clinical therapy appears to be cancer dependent. Despite having these anti-tumor means, the effectiveness of antibodies as anti-cancer agents is not satisfactory, especially they are expensive. Patient tumor response data indicates that monoclonal antibodies provide only a slight improvement in treatment success over conventional single agent cytotoxic chemotherapeutic agents. For example, only half of all recurrent mild non-Hodgkin lymphoma patients respond to the anti-CD20 antibody rituximab (McLaughlin et al., 1998, J Clin Oncol 16: 2825-2833, hereby incorporated by reference in its entirety. Included in the book). Of the 166 clinical patients, with a median response time of approximately 12 months, 6% showed a complete response and 42% showed a partial response. Trastuzumab (Herceptin®, Genentech), an anti-HER2 / neu antibody for the treatment of metastatic breast cancer, is less effective. The overall response rate with trastuzumab for 222 study patients was only 15%, including 8 complete responses and 26 partial responses, with a median response duration of 9 to 13 months and It was survival time (Cobleigh et al., 1999, J Clin Oncol 17: 2639-2648, which is hereby incorporated in its entirety by reference). In current anticancer therapies, any minor improvement in mortality defines success. Therefore, there is a considerable need to increase the ability of antibodies to destroy target cancer cells.

  A promising means to enhance the anti-tumor effects of antibodies is by enhancing their ability to mediate cytotoxic effector functions such as ADCC, ADCP and CDC. The importance of FcγR-mediated effector function for the anticancer activity of antibodies has been demonstrated in mice (Clynes et al., 1998, Proc Natl Acad Sci USA 95: 652-656; Clynes et al., 2000, Nat Med 6: 443-446, both of which are hereby incorporated by reference in their entirety), the affinity of the interaction between Fc and any FcγR is determined by the cytotoxicity targeted in the cell-based assay. Correlated (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, all incorporated herein by reference in its entirety). Furthermore, a correlation has been observed between clinical effects in humans and FcγRIIIa allotype high affinity polymorphism (V158) or low affinity polymorphism (F158) (Cartron et al., 2002, Blood 99: 754-758, which is incorporated herein in its entirety by reference). Taken together, these data suggest that an antibody that is optimized to bind to any FcγR mediates more effector function, thereby more effectively destroying the patient's cancer cells. The balance between receptor activation and inhibition is an important consideration, and optimal effector function has increased affinity for activating receptors such as FcγRI, FcγRIIa / c, and FcγRIIIa, but is inhibitory It can be generated by antibodies with reduced affinity for the receptor FcγRIIb. Furthermore, because FcγR can mediate antigen uptake and processing by antigen presenting cells, optimized FcγR affinity can also enhance the ability of antibody therapeutics to induce an adaptive immune response (Dhodapkar & Dhodapkar, 2005, Proc Natl Acad Sci USA, 102, 6243-6244, which is hereby incorporated by reference in its entirety). The importance of complement-mediated effector function for anti-cancer therapy of antibodies has not been well characterized. Antibodies optimized against CDC provide a way to investigate the role of complement in the clinical application of antibodies and may provide a potential mechanism for improving the tumoricidal ability of antibodies.

  In contrast to antibody therapies and applications where effector function contributes to clinical efficacy, for some antibodies and clinical applications, reducing or eliminating binding to one or more FcγRs, or without limitation, ADCC, ADCP It may be preferred to reduce or eliminate one or more FcγR or complement-mediated effector functions, including and / or CDC. This is often the case for therapeutic antibodies, whose mechanism of action involves blocking or suppressing rather than killing cells that carry the target antigen. In these cases, target cell reduction is undesirable and may be considered a side effect. For example, the ability of anti-CD4 antibodies to inhibit the CD4 receptor on T cells makes them effective anti-inflammatory drugs, and furthermore, their ability to capture FcγR receptors also prevents immune attack against target cells. Induces and results in T cell depletion (Reddy et al., 2000, J Immunol 164: 1925-1933, which is hereby incorporated by reference in its entirety). Effector function can also be a challenge for radiolabeled antibodies termed radioconjugates and antibodies complexed with toxins termed immunotoxins. Although these agents can be used to destroy cancer cells, the capture of immune cells by the interaction of Fc with FcγR brings healthy immune cells into close proximity to harmful payloads (radiation or toxins). Resulting in a reduction in normal lymphoid tissue with target cancer cells (Hutchins et al., 1995, Proc Natl Acad Sci USA 92: 11980-11984; White et al., 2001, Annu Rev Med 52: 125-145 , Both of which are incorporated herein by reference in their entirety). IgG isoforms that lack sufficient capture of complement or effector cells, such as IgG2 and IgG4, can be used in part to address this challenge. Fc variants that reduce or reduce 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; Armor 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; Armor 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 incorporated herein by reference in their entirety). However, the complete Fc ligand binding properties and effector function capabilities of these variants and their properties compared to the WT IgG isoform are not clear. There is a need for general and robust means for complete reduction of all FcγR binding and FcγR and complement-mediated effector functions. A further consideration is that other important antibody properties are not unstable. Fc variants not only reduce binding to FcγR and / or C1q, but also antibody stability, solubility and structural integrity, and other important Fc ligands such as FcRn and protein A and protein G Should be manufactured to maintain the ability to interact with.

  Recent successes have achieved obtaining Fc variants with altered binding to FcγR and C1q, and in some cases, these Fc variants have the ability to mediate FcγR and complement-mediated effector functions. USSN 10 / 672,280, USSN 10 / 822,231, USSN 11 / 124,620, filed May 5, 2005, and USSN 11 / 256,060, filed October 21, 2005, all by reference The entirety of which is encompassed herein). The Fc variants obtained in these experiments showed selectively improved binding to FcγR, increased ADCC, improved binding to complement protein C1q, decreased binding to FcγR, decreased to complement protein C1q. Various optimal enhancements of Fc ligand and effector functional properties including, but not limited to, binding, and other optimization properties are provided. The present invention aims to further characterize the properties of the Fc variants selected from these experiments and to use the data to produce new variants with optimized properties.

Summary of the Invention The present invention provides Fc variants with optimized properties. The optimized properties include altered binding to FcγR, altered antibody-dependent cell-mediated cytotoxicity, and altered complement-dependent cytotoxicity compared to the parent Fc polypeptide.

  In one embodiment, the Fc variants of the present invention improve binding to one or more FcγRs compared to the parent Fc polypeptide. In another embodiment, the Fc variants of the present invention improve antibody-dependent cell-mediated cytotoxicity compared to the parent Fc polypeptide. In a preferred embodiment, the Fc variant comprises positions 227, 234, 235, 236, 239, 246, 255, 258, 260, 264, 267, 268, 272, 281 Position, 282, 283, 284, 293, 295, 304, 324, 327, 328, 330, 332 and 335 (where the numbers are according to EU Index) It includes amino acid modification at one or more sites selected from the group consisting of

  In another embodiment, the Fc variants of the present invention improve complement dependent cytotoxicity compared to the parent Fc polypeptide. In a preferred embodiment, the Fc variant has positions 233, 234, 235, 239, 267, 268, 271, 272, 274, 276, 278, 281, 282, 284. , 285, 293, 300, 320, 322, 324, 326, 327, 328, 330, 331, 332, 333, 334, and 335 (where Numbers include amino acid modifications at one or more locations selected from the group consisting of:

  In another embodiment, the Fc variants of the present invention reduce binding to one or more FcγRs as compared to a parent Fc polypeptide. In another embodiment, the Fc variants of the present invention reduce antibody-dependent cell-mediated cytotoxicity compared to the parent Fc polypeptide. In another embodiment, the Fc variants of the present invention reduce complement dependent cytotoxicity as compared to a parent Fc polypeptide. In a preferred embodiment, the Fc variants of the present invention reduce binding to one or more FcγRs, reduce antibody-dependent cell-mediated cytotoxicity and complement-dependent, compared to the parent Fc polypeptide. Reduces cytotoxic effects. In a most preferred embodiment, the Fc variant has positions 232, 234, 235, 236, 237, 238, 239, 265, 267, 269, 270, 297, 299, It includes one or more amino acid modifications at positions selected from the group consisting of positions 325, 327, 328, 329, 330 and 331 (where the numbers are according to EU Index).

  The present invention provides a method for producing Fc variants having optimized properties. A further object of the present invention is to provide experimental production and screening methods to obtain optimized Fc variants.

  The present invention provides isolated nucleic acids that encode the Fc variants described herein. The present invention provides a vector comprising the nucleic acid operably linked to a regulatory sequence as desired. The present invention provides a host cell containing the vector, and a method for producing an Fc variant and optionally a method for recovering the Fc variant.

  The present invention provides novel Fc polypeptides comprising antibodies, Fc fusions, isolated Fc, and Fc fragments comprising the Fc variants described herein. The novel Fc polypeptides can find use in therapeutic products. In a most preferred embodiment, the Fc polypeptide of the present invention is an antibody.

  The invention provides a composition comprising an Fc polypeptide comprising an Fc variant described herein and a physiologically or pharmaceutically acceptable carrier or diluent.

  The present invention contemplates therapeutic and diagnostic uses for Fc polypeptides comprising the Fc variants described herein.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. Antibody structure and function. Humanized Fab structure from pdb accession code 1CE1 (James et al., 1999, J Mol Biol 289: 293-301, incorporated herein by reference in its entirety) and human IgG1Fc from pdb accession code 1DN2 1 shows a model of a full-length human IgG1 antibody modeled using the structure (DeLano et al., 2000, Science 287: 1279-1283, which is hereby incorporated by reference in its entirety). The flexible hinge connecting the Fab and Fc regions is not shown. IgG1 is a heterodimeric homodimer consisting of two light chains and two heavy chains. The Ig domains containing antibodies, including _VL and C _L of the light chain, and V _{H of} the heavy chain, C gamma 1 (Cγ1), C gamma 2 (Cγ2), and C gamma 3 (Cγ3) are shown. The Fc region is indicated. The binding sites of related proteins are shown, including antigen binding sites in the variable region and binding sites for FcγR, FcRn, C1q and proteins A and G in the Fc region.

  FIG. Fc / FcγRIIIb complex structure 1IIS. Fc is shown as a gray ribbon and FcγRIIIb is shown as a black ribbon. N297 carbohydrate is shown as a black bar.

  3a-3b. Alignment of amino acid sequences of human IgG immunoglobulins IgG1, IgG2, IgG3 and IgG4. FIG. 3a provides the sequence and hinge domain of CH1 (Cγ1), and FIG. 3b provides the sequence of CH2 (Cγ2) and CH3 (Cγ3) domains. The positions are numbered according to the EU index of the IgG1 sequence and the differences between IgG1 and other immunoglobulins IgG2, IgG3 and IgG4 are shown in gray. Polymorphisms are present at multiple positions (Kim et al., 2001, J. Mol. Evol. 54: 1-9, which is hereby incorporated by reference in its entirety), and thus the sequence shown There may be slight differences from conventional sequences. Possible starting points for the Fc region are indicated and are described herein as either EU positions 226 or 230.

  FIG. The allotype and isoallotype positions of the gamma 1 chain of human IgG1 and the associated amino acid substitutions are shown (Gorman & Clark, 1990, Semin Immunol 2 (6): 457-66, which is hereby incorporated by reference in its entirety. ) For comparison, amino acids found at equivalent positions in human IgG2, IgG3 and IgG4 gamma chains are also shown.

  FIG. Fc variant and FcγR binding data. All Fc variants were constructed against the antibody PRO70769 IgG1. Fold indicates IC50 fold compared to WT PRO70769 IgG1 for binding to human V158 and F158 FcγRIIIa as measured by AlphaScreen competition assay.

  FIG. Binding of human V158 FcγRIIIa (FIG. 6a) and F158 FcγRIIIa (FIG. 6b) by selected PRO70769 Fc variants as determined by AlphaScreen competition assay. In the presence of a competing antibody (Fc variant or WT), a characteristic inhibition curve is observed as a decrease in luminescent signal. Binding data was normalized to the maximum and minimum luminescence signals for each particular curve provided by the baseline at low and high concentrations of antibody, respectively. The curve shows the fit of the data to a partial competition model using nonlinear regression.

  FIG. Binding of human V158 FcγRIIIa and F158 FcγRIIIa by PRO70769 Fc variants as measured by AlphaScreen competition assay. FIG. 7a shows data for selected mutants and FIG. 7b shows IC50 values and folds compared to WT PRO70769 IgG1.

  FIG. Fc variant and FcγR binding data. All Fc variants were constructed against the variable region PRO70769 and either human IgG1 or IgG (1/2) ELLGG. FIG. 8a shows IC50 values and IC50 fold compared to WT PRO70769 IgG1 for binding to human activated receptors V158 and F158 FcγRIIIa, and inhibitory receptor FcγRIIb as measured by AlphaScreen competition assay. FIG. 8b shows AlphaScreen data for selected mutants.

  FIG. Competition surface plasmon resonance (SPR) experiment measuring the binding affinity of I332E and S239D / I332E mutants in trastuzumab against human V158 FcγRIIIa. FIG. 9a shows sensorgram raw data, FIG. 9b shows a plot of the log value (logarithmic value) of the receptor concentration against the initial binding rate obtained at each concentration, and FIG. The affinities obtained from the fit to these data as described in Example 1 are shown.

  FIG. Cell-based ADCC assay of selected Fc variants in anti-CD20 antibody PRO70769. ADCC was measured by the release of lactose dehydrogenase using the LDH cytotoxicity detection kit (Roche Diagnostic). CD20 + lymphoma WIL2-S cells were used as target cells and human PBMC was used as effector cells. FIG. 5 shows the dose dependence of ADCC at the antibody concentration of a given antibody, normalized to the minimum and maximum fluorescence signals for each particular curve provided by the baseline at low and high concentrations of antibody, respectively. The curve shows the fit of the data to a sigmoidal dose response model using non-linear regression.

  FIG. Cell-based ADCC assay of selected Fc variants in PRO70769 IgG1 in the absence and presence of serum levels of human IgG. ADCC was measured by the release of lactose dehydrogenase using the LDH cytotoxicity detection kit (Roche Diagnostic). CD20 + lymphoma WIL2-S cells were used as target cells and human PBMC was used as effector cells.

  FIG. Residues mutated in Fc variants designed to enhance CDC. 1 shows the structure of a human IgG1 Fc region (pdb accession code 1E4K, Sondermann et al., 2000, Nature 406: 267-273, which is hereby incorporated by reference in its entirety). Black spheres and bars indicate residues D270, K322, P329 and P331 which have been shown to be important in mediating binding to complement protein C1q, gray bars affect CDC The residues mutated in the present invention in order to examine the mutants are shown.

  FIG. Fc variants and CDC data screened for complement-mediated cytotoxicity (CDC). The variable region is that of the anti-CD20 antibody PRO70769, the heavy chain constant region is IgG1, and unless otherwise stated, is IgG (1/2) ELLGG. CDC fold indicates relative CDC activity compared to WT PRO70769 IgG1.

  FIG. Fc variant CDC assay of anti-CD20 antibody. The dose dependence of antibody concentration of complement-mediated lysis is shown for the indicated PRO70769 antibody against CD20 + WIL2-S lymphoma target cells. Lysis was measured using the release of Alamar Blue, and the data is relative to the minimum and maximum fluorescence signals for each specific curve provided by the baseline at low and high concentrations of antibody, respectively. And standardized. The curve shows the fit of the data to a sigmoidal dose response model with a slope change using non-linear regression.

  FIG. Amino acid modifications that provide increased and decreased CDC, and positions that can be modified to provide increased / regulated CDC. The positions are numbered according to the EU index.

  FIG. Fc variants screened for reduced FcγR affinity, effector function mediated by FcγR, and effector function mediated by complement. The variable region is that of the anti-CD20 antibody PRO70769, and the heavy chain constant region is IgG1. The figure shows the IC50 fold for binding to human V158 FcγRIIIa and the EC50 fold for CDC activity compared to WT PRO70769 IgG1.

  FIG. Binding to human V158 FcγRIIIa by selection of PRO70769 Fc variants as determined by AlphaScreen competition assay.

  FIG. CDC assay of selected anti-CD20 antibody Fc variants against CD20 + WIL2-S lymphoma target cells. Dissolution was measured by Alamar Blue release.

  FIG. Cell-based ADCC activity of selected anti-CD20 antibody Fc variants against CD20 + lymphoma WIL2-S cells. Human PBMC were used as effector cells and lysis was measured by LDH release.

  FIG. Fc variants screened for reduced FcγR affinity, FcγR-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 IgG1. The figure shows the IC50 fold for binding to human V158 FcγRIIIa compared to WT, the IC50 fold for binding to human FcγRI compared to WT, and the EC50 fold for CDC activity compared to WT by two separate tests. Indicates.

  FIG. Binding of the low affinity human activated receptor V158 FcγRIIIa and the high affinity human activated receptor FcγRI by selected PRO70769 Fc variants as determined by AlphaScreen competition assay.

  FIG. CDC activity of selected PRO70769Fc variants against CD20 + WIL2-S lymphoma target cells. Dissolution was measured by the release of alamar blue.

  FIG. Cell-based ADCC activity of anti-Her2 Fc variants and WT IgG antibodies against Her2 / neu + SkBr-3 breast cancer target cells. Human PBMC were used as effector cells and lysis was measured by LDH release.

  FIG. Amino acid sequences of variable light chain (VL) and variable heavy chain (VH) used in the present invention, including PRO70769 (Figures 24a and 24b), trastuzumab (Figures 24c and 24d), and ipilimumab (Figures 24e and 24f).

  FIG. Amino acid sequences of human constant light chain kappa (FIG. 25a) and constant heavy chain (FIGS. 25b-25f) used in the present invention.

  FIG. A sequence showing a constant heavy chain sequence that may have reduced Fc ligand binding and effector function properties (FIG. 26a) and an improved anti-CTLA-4 antibody sequence (FIGS. 26b-26d). FIG. 26a shows a possible Fc variant constant heavy chain sequence with variable positions shown highlighted as X1, X2, X3, X4, X5, X6, X7 and X8. The table below the sequence shows WT amino acids and possible substituents at these positions. Improved antibody sequences can include one or more non-WT amino acids selected from this potential modifying group. FIG. 26b shows the anti-CTLA-4 antibody light chain sequence, and FIGS. 26c and 26d show the anti-CTLA-4 antibody heavy chain sequence with reduced Fc ligand binding and Fc-mediated effector function. These include the L235G / G236R IgG1 heavy chain (FIG. 26c) and the IgG2 heavy chain (FIG. 26d). The positions are numbered according to the EU index described in Kabat and therefore do not correspond to the sequential order in the sequence.

Detailed Description of the Invention In order that the present invention may be more fully understood, some definitions are set forth below. Such a definition is meant to encompass grammatical equivalents.

As described herein, “ ADCC ” or “ antibody-dependent cell-mediated cytotoxicity ” refers to non-specific cytotoxicity that recognizes an antibody bound on a target cell and then expresses FcγR resulting in lysis of the target cell. It means a reaction mediated by cells that are sex cells.

“ ADCP ” or “ antibody-dependent cell-mediated phagocytosis ” as described herein is a non-specific cell that expresses FcγR that recognizes the antibody bound on the target cell and then induces phagocytosis of the target cell. It means a reaction mediated by cells that are damaging cells.

As used herein, “ amino acid modification ” means an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. As used herein, “ amino acid substitution ” or “ substitution ” means a substitution of one amino acid for another at a particular position in the parent polypeptide sequence. For example, the L328R substitution means a mutant polypeptide in which leucine at position 328 is replaced with arginine in the Fc mutant in this case. As used herein, “ amino acid insertion ” or “ insertion ” means the addition of an amino acid at a particular position in the parent polypeptide sequence. For example, G> 235-236 insertion means insertion of glycine between positions 235 and 236. As used herein, “ amino acid deletion ” or “ deletion ” means the removal of an amino acid at a particular position in a parent polypeptide sequence. For example, G236- means a deletion of glycine at position 236. The amino acids of the invention can be further classified as either isotopes or novel.

“ CDC ” or “ complement dependent cytotoxicity ”, as described herein, is a reaction in which one or more complement protein components recognize an antibody bound on a target cell and then result in lysis of the target cell. Means.

As used herein, “ isotopic modification ” refers to an amino acid modification that converts one amino acid of one isoform to the corresponding different amino acid (aligned isoform). For example, IgG1 has a tyrosine at EU position 296 and IgG2 has phenylalanine, so F296Y substitution in IgG2 is considered an isotope modification.

As used herein, “ novel modifications ” refers to amino acid modifications that are not isoforms. For example, since IgG does not have a glutamic acid at position 332, an I332E substitution in IgG1, IgG2, IgG3, or IgG4 is considered a new modification.

As used herein, “ amino acid ” and “ amino acid identity ” means one of the 20 naturally occurring amino acids or any non-naturally occurring analog that may be present in a particular defined position. Do

As used herein, “ effector function ” refers to a biochemical event that results from the interaction of an Fc region of an antibody with an Fc receptor or ligand. Effector functions include effector functions mediated by FcγR such as ADCC and ADCP, and complement-mediated effector functions such as CDC.

As used herein, “ effector cells ” refers to cells of the immune system that express one or more Fc receptors and mediate one or more effector functions. Effector cells include monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and γδT cells But can be from any organism including but not limited to humans, mice, rats, rabbits and monkeys.

"Fab" or "Fab region" as used herein _refers to a polypeptide comprising a V H, CH1, _{V L} and _{C L} immunoglobulin domains. Fab can refer to this region alone or in a full-length antibody or antibody fragment.

As used herein, “ Fc ” or “ Fc region ” refers to a polypeptide comprising the constant region of an antibody, excluding the first constant region of an immunoglobulin domain. Thus, Fc comprises the remaining two constant regions of the IgA, IgD, and IgG immunoglobulin domains, and the remaining three constant regions of the IgE and IgM immunoglobulin domains, as well as the N-terminal flexible hinge of these domains. means. For IgA and IgM, Fc can include the J chain. For IgG, as shown in FIG. 1, Fc includes immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and a hinge between Cgamma1 (Cγ1) and Cgamma2 (Cγ2). Although the boundaries of the Fc region can vary, a human IgG heavy chain Fc region is usually defined to include residues C226 or P230 at its carboxyl terminus, where the numbers follow the EU index described in Kabat. Fc may refer to this isolated region or this region in an Fc polypeptide as described below. As used herein, “Fc polypeptide” means a polypeptide comprising all or part of an Fc region. Fc polypeptides include antibodies, Fc fusions, isolated Fc, and Fc fragments.

As used herein, an “ Fc fusion ” refers to a protein in which one or more polypeptides are operably linked to an Fc region. As used herein, Fc fusions are described in the prior art (Chamow et al., 1996, Trends Biotechnol 14: 52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9: 195-200, both in their entirety by reference. As used herein) and the terms “immunoadhesin”, “Ig fusion”, “Ig chimera”, and “receptor globulin” (sometimes with dashes). It is synonymous. An Fc fusion generally binds an Fc region of an immunoglobulin with a “ fusion partner ” that can be any protein, polypeptide or small molecule. The role of the non-Fc portion of the Fc fusion, the fusion partner, is to mediate target binding, and thus it is functionally similar to the variable region of an antibody. In fact, any protein or small molecule can bind Fc and create an Fc fusion. Protein fusion partners can include, but are not limited to, a target binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or other protein or protein domain. Small molecule fusion partners can include any therapeutic agent that directs the Fc fusion to a therapeutic target. Such a target can be any molecule, preferably an extracellular receptor involved in the disease.

As used herein, “ Fc gamma receptor ” or “ FcγR ” refers to a member of any protein family that binds to the IgG antibody Fc region and is substantially encoded by the FcγR gene. In humans, this family includes FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc And FcγRIII (CD16), including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82: 57-65, which is hereby incorporated by reference in its entirety), as well as any unknown human FcγR or FcγR isoform or allotype. Not. The FcγR can be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγR includes FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), and any undiscovered mouse FcγR or FcγR isotype or allotype, It is not limited to them.

As used herein, “ Fc ligand ” refers to 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, FcγR, FcγR, FcγR, FcRn, C1q, C3, mannan binding lectin, mannose receptor, staphylococcal protein A, staphylococcal protein G, and viral FcγR. Fc ligands also include the Fc receptor homologue (FcRH), a family of Fc receptors that are homologous to FcγR (Davis et al., 2002, Immunological Reviews 190: 123-136, which is hereby incorporated by reference in its entirety. Included in the specification). An Fc ligand may comprise an unknown molecule that binds to Fc.

As used herein, “ full-length antibody ” refers to the structure that constitutes the natural biological form of an antibody, including variable and constant regions. For example, in most mammals, including humans and mice, IgG isoform full-length antibodies are tetramers, two homologous pairs of two immunoglobulin chains, each pair having one light Chain and one heavy chain (each light chain comprises a light chain comprising immunoglobulin domains V _L and C _L , each heavy chain comprising immunoglobulin domains V _H , Cγ1, Cγ2 and Cγ3). Consists of homologous pairs. In some mammals, such as camels and llamas, IgG antibodies can be composed of only two heavy chains, each heavy chain comprising a variable domain attached to an Fc region.

As used herein, “ IgG ” refers to a polypeptide belonging to the class of antibodies substantially encoded by recognized immunoglobulin gamma genes. In humans, this IgG includes subclasses or isoforms IgG1, IgG2, IgG3 and IgG4. In mice, IgG includes IgG1, IgG2a, IgG2b, and IgG3.

As used herein, “ immunoglobulin (Ig) ” means a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include but are not limited to antibodies. Immunoglobulins can have many structural forms, including but not limited to full length antibodies, antibody fragments, and individual immunoglobulin domains.

As used herein, an “ immunoglobulin (Ig) domain ” refers to a region of an immunoglobulin that exists as a different structural element identified by one skilled in the art of protein structure. Ig domains typically have a characteristic β-sandwich folding topology. Known Ig domains in IgG isotype _{antibodies, V H, Cγ1, Cγ2,} Cγ3, a _{V L} and _{C L.}

As used herein, “ IgG ” or “ IgG immunoglobulin ” refers to a polypeptide belonging to the antibody class substantially encoded by a recognized immunoglobulin gamma gene. In humans, this class includes subclasses or isoforms IgG1, IgG2, IgG3 and IgG4. As used herein, “ isoform ” refers to any of the immunoglobulin subclasses defined by the chemical and antigenic properties of their constant regions. Known human immunoglobulin isoforms are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE.

As used herein, “ parent polypeptide ”, “ parent protein ”, “ precursor polypeptide ” or “ precursor protein ” refers to an unmodified polypeptide that is subsequently modified to create a variant. The parent polypeptide can be a naturally occurring polypeptide, or a variant or modified form of a naturally occurring polypeptide. A parent polypeptide can refer to the polypeptide itself, the parent polypeptide or a composition comprising the amino acid sequence that encodes it. Thus, a “ parent Fc polypeptide ” as used herein refers to an Fc polypeptide that is modified to create a variant, and a “ parent antibody ” as described herein is referred to as a modified antibody. It means the antibody to be produced.

As used herein, “ position ” means a position in a protein sequence. The positions can be numbered sequentially or according to an established format, for example the EU index described in Kabat. For example, position 297 is a position in human antibody IgG1.

As used herein, “ polypeptide ” or “ protein ” refers to at least two covalently linked amino acids and includes proteins, polypeptides, oligopeptides and peptides.

As used herein, “ residue ” refers to the identification of a position in a protein and its associated amino acid. For example, asparagine 297 (Asn297, also referred to as N297) is a residue in the human antibody IgG1.

As used herein, “ target antigen ” refers to a molecule that is specifically bound by the variable region of a given antibody. The target antigen can be a protein, carbohydrate, lipid, or other compound.

As used herein, “ target cell ” means a cell that expresses a target antigen.

As used herein, a “ variable region ” refers to one or more of the Vκ, Vλ, and / or V _H genes that are substantially encoded by kappa, lambda, and heavy chain immunoglobulin gene regions, respectively. It refers to the region of an immunoglobulin that contains an Ig domain.

As used herein, a “ variant polypeptide ”, “ polypeptide variant ” or “ variant ” means a polypeptide sequence that differs from a parent polypeptide sequence by at least one amino acid modification. The parent polypeptide can be a naturally occurring or wild type (WT) polypeptide, or can be a modified version of a WT polypeptide. Variant polypeptide can refer to the polypeptide itself, a composition comprising the polypeptide or the amino sequence that encodes it. Preferably, the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, eg, about 1 to about 10 amino acid modifications compared to the parent polypeptide, preferably about Has 1 to about 5 amino acid modifications. As used herein, a variant polypeptide sequence preferably has at least about 80% homology, most preferably at least about 90% homology, more preferably at least about 95% homology with the parent polypeptide sequence. Can have. Thus, as used herein, an “ Fc variant ” or “ variant Fc ” means an Fc sequence that differs from a parent Fc sequence by at least one amino acid modification. An Fc variant may comprise only the Fc region or may exist as an antibody, Fc fusion, isolated Fc, Fc fragment, or other polypeptide substantially encoded by Fc. Fc variant may refer to a composition comprising the Fc polypeptide itself, the Fc variant polypeptide or the amino acid sequence encoding it. As used herein, an “ Fc polypeptide variant ” or “ variant Fc polypeptide ” refers to an Fc polypeptide that differs from a parent Fc polypeptide by at least one amino acid modification. As used herein, a “ protein variant ” or “ variant protein ” refers to a protein that differs from a parent protein by at least one amino acid modification. As used herein, an “ antibody variant ” or “ variant antibody ” refers to an antibody that differs from a parent antibody by at least one amino acid modification. As used herein, an “ IgG variant ” or “ variant IgG ” refers to an antibody that differs from the parent IgG by at least one amino acid modification. As used herein, an “ immunoglobulin variant ” or “ variant immunoglobulin ” refers to an immunoglobulin sequence that differs from a parent immunoglobulin sequence by at least one amino acid modification.

As used herein, “ wild type or WT ” means an amino acid sequence or nucleotide sequence found in nature, including allelic variants. A WT protein, polypeptide, antibody, immunoglobulin, IgG, etc. has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

Antibodies Accordingly, the present invention provides variant antibodies.

  Classical antibody structural units typically include tetramers. Each tetramer typically has two homologous pairs of polypeptide chains (each pair has one “light” chain (typically having a molecular weight of about 25 kDa) and one “heavy”. "Having a chain (typically having a molecular weight of about 50-70 kDa)." Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. IgM has subclasses including but not limited to IgM1 and IgM2. Thus, as used herein, “isoform” refers to any of the immunoglobulin subclasses defined by the chemical and antigenic properties of their constant regions. Known human immunoglobulin isoforms are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD and IgE.

  The amino terminal site of each chain includes a variable region of about 100 to 110 or more amino acids that plays a major role in antigen recognition. In the variable region, three loops assemble into each heavy and light chain V domain to form an antigen binding site. Each loop is referred to as a complementarity determining region (hereinafter referred to as “CDR”), where mutations in the amino acid sequence are most important.

  The carboxy terminal site of each chain defines a constant region that plays a major role in effector function. Kabat et al. Collected a number of major sequences of the variable regions of the heavy and light chains. Based on the degree of conservation of the sequences, they classified individual major sequences into CDRs and frameworks and created a list (SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, EA Kabat see et al).

In the IgG subclass of immunoglobulins, there are several immunoglobulin domains in the heavy chain. As used herein, “ immunoglobulin (Ig) domain ” means a region of an immunoglobulin having a different three-dimensional structure. Of interest in the present invention are heavy chain domains comprising a heavy chain constant (CH) domain and a hinge domain. In IgG antibodies, each IgG isoform has 3 CH regions. Thus, the “CH” domain in IgG is: “CH1” indicates positions 118-220 according to the EU index described in Kabat. “CH2” indicates positions 237-340 according to the EU index described in Kabat, and “CH3” indicates positions 341-447 according to the EU index described in Kabat.

  Another type of heavy chain Ig domain is the hinge region. As used herein, “hinge” or “hinge region” or “antibody hinge region” or “immunoglobulin hinge region” refers to a flexible polypeptide comprising amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 220 and the IgG CH2 domain begins at EU position 237. Thus, with respect to IgG, antibody hinge is defined herein to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1), where the numbers are according to the EU index described in Kabat . In some embodiments, a “lower hinge” is included, typically in positions 226 or 230, eg, in the Fc region.

  Of particular interest in the present invention is the Fc region. As used herein, “Fc” or “Fc region” refers to a polypeptide that comprises the first constant region of an immunoglobulin domain, in some cases the constant region of the antibody, excluding part of the hinge. Thus, Fc means the remaining two constant regions of immunoglobulin domains IgA, IgD, and IgG, and the remaining three constant regions of immunoglobulin domains IgE and IgM, and the N-terminal flexible hinge of these domains . For IgA and IgM, Fc can include the J chain. With respect to IgG, as shown in FIG. 1, Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cg2 and Cg3) and a downstream hinge region between Cgamma1 (Cg1) and Cgamma2 (Cg2). Including. Although the boundaries of the Fc region can vary, a human IgG heavy chain Fc region is usually defined to include residues C226 or P230 at its carboxyl terminus, where the numbers follow the EU index described in Kabat. Fc can refer to this isolated region or this region in an Fc polypeptide, as described below. As used herein, “Fc polypeptide” means a polypeptide comprising all or part of an Fc region. Fc polypeptides include antibodies, Fc fusions, isolated Fc, and Fc fragments.

  In some embodiments, the antibody is full length. As used herein, “full-length antibody” means a structure that constitutes the natural biological form of an antibody, including variable and constant regions containing one or more modifications, as described herein.

  Alternatively, the antibodies are antibody fragments, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimics”), chimeric antibodies, respectively. , Humanized antibodies, antibody fusions (sometimes referred to herein as “antibody complexes”), and various structures, including but not limited to fragments.

Antibody Fragment In one embodiment, the antibody is an antibody fragment. Of particular interest are antibodies comprising an Fc region, an Fc fusion and a heavy chain constant region (CH1-hinge-CH2-CH3), and also comprising a heavy chain constant region fusion.

  Specific antibody fragments include (i) a Fab fragment consisting of VL, VH, CL and CH1 domains, (ii) an Fd fragment consisting of VH and CH1 domains, (iii) an Fv fragment consisting of VL and VH domains of a single antibody (Iv) a dAb fragment consisting of a single variable region (Ward et al., 1989, Nature 341: 544-546), (v) an isolated CDR region, (vi) an F (ab ′) 2 fragment, A bivalent fragment comprising two linked Fab fragments, (vii) a single chain Fv molecule (scFv), where the VH and VL domains are peptide linkers that bind the two domains and form an antigen binding site (Bird et al., 1988, Science 242: 423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883), (viii) bispecific mono This chain Fv dimer (PCT / US92 / 0996 ), And (ix) “diabodies” or “triabodies”, multivalent or multispecific fragments constructed by gene fusion (Tomlinson et. Al., 2000, Methods Enzymol. 326: WO94 / 13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-6448), etc. The antibody fragment may be modified, for example. The molecule can be stabilized by the incorporation of disulfide bridges that link the VH and VL domains (Reiter et al., 1996, Nature Biotech. 14: 1239-1245).

Chimeric and humanized antibodies In some embodiments, the backbone component can be a mixture from different species. As such, when the antibody in question is such an antibody, such antibody can be a chimeric antibody and / or a humanized antibody. In general, both “chimeric antibodies” and “humanized antibodies” refer to antibodies that combine regions from two or more species. For example, “chimeric antibodies” traditionally include variable region (s) from mouse (or in some cases, rat) and constant region (s) from human. “Humanized antibody” generally refers to non-human antibodies having variable domain framework regions substituted for sequences found in human antibodies. In general, in 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. CDRs, some or all of which are encoded by nucleic acids derived from non-human organisms, are grafted into the beta-sheet framework of the human antibody variable region to produce antibodies, the specificity of which is determined by the grafted CDRs. The The production of such antibodies is described, for example, in WO 92/11018, Jones, 1986, Nature 321: 522-525, Verhoeyen et al., 1988, Science 239: 1534-1536. “Backmutation” of selected acceptor framework residues to the corresponding donor residues is often necessary to regain the affinity missing in the initial grafted construct ( US5530101; US5585089; US5693761; US5693762; US6180370; US5859205; US5821337; US6054297; US6407213). A humanized antibody will also optimally comprise an immunoglobulin constant region, typically at least a portion 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). Various techniques and methods for humanization and remodeling of non-human antibodies are well known in the art (Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA) and references cited therein). Humanization methods include Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988; Nature 332: 323-329; Verhoeyen et al., 1988, Science, 239: 1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86: 10029-33; He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA 89: 4285 -9, Presta et al., 1997, Cancer Res. 57 (20): 4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA 88: 4181-4185; O'Connor et al. 1998, Protein Eng 11: 321-8, but is not limited thereto. Humanization or other methods for reducing the immunogenicity of non-human antibody variable regions include, for example, surface remodeling as described in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91: 969-973 ( resurfacing) method may be included. In one embodiment, the parent antibody has mature affinity, as is known in the art. Structure-based methods can be used for humanization and affinity maturation as described, for example, in USSN 11 / 004,590. Selection-based methods are described in Wu et al., 1999, J. Mol. Biol. 294: 151-162; Baca et al., 1997, J. Biol. Chem. 272 (16): 10678-10684; Rosok et al. , 1996, J. Biol. Chem. 271 (37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16 (10): 753-759, which can be used for humanization and / or affinity maturation of antibody variable regions, including but not limited to the methods described in US Pat. Other humanization methods are described in USSN 09 / 810,502; Tan et al., 2002, J. Immunol. 169: 1119-1125; De Pascalis et al., 2002, J. Immunol. 169: 3076-3084 It may involve transplantation of only a portion of a CDR, including but not limited to methods.

Bispecific Antibodies In one embodiment, multispecific antibodies, particularly bispecific antibodies, of the present invention may also be referred to as “biantibodies”. These are antibodies that bind to two (or more) different antigens. Double antibodies can be produced by various methods known in the art (Holliger and Winter, 1993, Current Opinion Biotechnol. 4: 446-449), eg chemically or hybrid hybridoma. ).

Minibodies In one embodiment, the antibody is a minibody. A minibody is a minimized antibody-like protein that contains an scFv bound to a CH3 domain (Hu et al., 1996, Cancer Res. 56: 3055-3061). In some cases, the scFv can bind to the Fc region and can include some or all of the hinge region.

Human antibodies In one embodiment, the antibody is a full-length human antibody having at least one modification as described herein. “Full-length human antibody” or “fully human antibody” means a human antibody having the gene sequence of an antibody derived from a human chromosome having the modifications described herein.

Antibody Fusion In one embodiment, an antibody of the invention is an antibody fusion protein (sometimes referred to herein as an “antibody complex”). One type of antibody fusion includes an Fc fusion that links Fc regions with binding partners. As used herein, an “ Fc fusion ” refers to a protein in which one or more polypeptides are operably linked to an Fc region. In this specification, Fc fusion is the term “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). Synonymous with “immunoadhesin”, “Ig fusion”, “Ig chimera”, and “receptor globulin” (sometimes with dashes). Fc fusions generally bind the Fc region of an immunoglobulin with a fusion partner that can be any protein or small molecule. In fact, any protein or small molecule can bind Fc and create an Fc fusion. Protein fusion partners can 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 other protein or protein domain. Small molecule fusion partners can include any therapeutic agent that directs the Fc fusion to a therapeutic target. Such a target may be any molecule involved in the disease, preferably an extracellular receptor.

  In addition to Fc fusions, antibody fusions comprise a fusion of the constant region of the heavy chain and one or more fusion partners (further including the variable region of any antibody), while other antibody fusions. Is a substantial or complete full-length antibody with a fusion partner. In one embodiment, the role of the fusion partner is to mediate target binding, and thus it is functionally similar to the variable region of an antibody (and may actually be an antibody variable region). In fact, any protein or small molecule can bind to Fc to create an Fc fusion (or antibody fusion). A protein fusion partner can include, but is not limited to, a 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 can include any therapeutic agent that directs the Fc fusion to a therapeutic target. Such a target may be any molecule involved in the disease, preferably an extracellular receptor.

  Binding partners can be proteinaceous or non-proteinaceous; the latter is generally made using functional groups on the antibody and binding partner. For example, linkers are known in the art; for example, homo- or hetero-bifunctional linkers are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200). Which is incorporated herein by reference).

  Suitable conjugates include, but are not limited to, the following labels, agents, and cytotoxic agents (eg, chemotherapeutic agents) or cytotoxic agents, or cytotoxic agents such as, but not limited to: Not. Suitable toxicants and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, cursin, crotin, phenomycin, enomycin and the like. Cytotoxic agents also include radiochemicals made by linking a radioisotope and an antibody, or by binding a radionuclide and a chelator that is covalently bound to the antibody. Further embodiments utilize calichemicin, auristatin, geldanamycin, maytansine, and duocarmycin and analogs; S. See 2003/0050331, which is hereby incorporated by reference in its entirety.

Covalent modification of antibodies
Covalent modifications of antibodies are encompassed within the scope of the invention and are generally, but not always, post-translational. For example, some types of covalent modification of an antibody can be performed by reacting specific amino acid residues of an antibody with an organic derivatizing agent that can react with a selected side chain or N- or C-terminal residue. Introduced in.

  Cysteinyl residues most commonly react with α-haloacetic acids (and corresponding amines) such as chloroacetic acid or chloroacetamide to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also include bromotrifluoroacetone, α-bromo-β- (5-imidozoyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide , P-chloromercury benzoate, 2-chloromercury-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

  Histidyl residues are derived by reaction at pH 5.5-7.0 with diethyl pyrocarbonate which reacts relatively specifically with histidyl side chains. Paraphenacyl bromide bromide is also useful; the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0.

  Lysinyl and the amino terminal residue are reacted with succinic anhydride or other carboxylic anhydride. Derivatization using these substances has the effect of reversing the charge of the ricinyl residue. Other reagents suitable for derivatizing alpha-amino containing residues include methylpicoline imidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzene sulfonic acid; O-methylisourea; Included are imidoesters such as diones, as well as transaminase catalyzed reactions with glyoxylic acid.

  Arginyl residues are modified by reaction with one or several conventional reactants (especially phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and ninhydrin). Derivatization of arginine residues must be performed under alkaline conditions because the guanidine functional group has a high pKa. In addition, these reactants can react with multiple groups of lysine as well as the arginine epsilon-amino group.

  Specific modifications of tyrosyl residues can be made with particular interest by introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. The tyrosyl acid group is iodinated with 125I or 131I to prepare a labeled protein for use in a suitable chloramine T method as described above, a radioimmunoassay.

  The carboxyl side group (aspartyl or glutamyl) is carbodiimide (R′N═C═N—R ′) where R and R ′ are optionally 1-cyclohexyl-3- (2-morpholinyl-4- Can be different alkyl groups such as ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4,4-dimethylpentyl) carbodiimide). Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

  Derivatization with bifunctional materials is useful for cross-linking antibodies to a water-insoluble support matrix or surface for use in various methods in addition to the methods described below. Commonly used crosslinking agents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters such as esters with 4-azidosalicylic acid, 3,3′-dithiobis Included are homobifunctional imide esters, including disuccinimidyl esters such as (succinimidyl propionate), and bifunctional maleimides such as bis-N-maleimide-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl) dithio] propioimidate yield photo-activated intermediates that can form crosslinks in the presence of light. Alternatively, a reactive water-insoluble matrix such as cyanogen bromide activated carbohydrate and US Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 247,642; 4,229,537; and 4,330,440 are used for protein immobilization.

  Glutaminyl and asparaginyl residues are often deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Both forms of these residues are within the scope of the present invention.

  Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of α-amino groups of lysine, arginine and histidine side chains (TE Creighton, Proteins: Structure and Molecular Properties , WH Freeman & Co., San Francisco, pp. 79-86 [1983]), acetylation of N-terminal amines, and amidation of any C-terminal carboxyl group.

Glycosylation Another type of covalent modification is glycosylation. In other embodiments, the IgG variants described herein can be modified to include one or more engineered glycoforms. As used herein, “modified sugar chain” refers to a carbohydrate composition that is covalently linked to IgG, where the carbohydrate composition is chemically different from that of the parent IgG. Modified sugar chains may be useful for a variety of purposes including, but not limited to, enhancing or decreasing effector function. Modified sugar chains can be made by various 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 WO00 / 61739A1; PCT WO01 / 29246A1; PCT WO02 / 31140A1; PCT WO02 / 30954A1; Potelligent ^™ technology [Biowa, Inc., Princeton, NJ]; GlycoMAb® glycosylation engineering technology [Glycart Biotechnology AG, Zuerich, Switzerland]). Many of these techniques are by, for example, expressing IgG in various organisms, or genetically modified or other method cell lines (eg, Lec-13 CHO cells or rat hybridoma YB2 / 0 cells), or By modulating an enzyme involved in the glycosylation pathway (eg FUT8 [α1,6-fucosyltransferase] and / or β1-4-N-acetylglucosaminyltransferase III [GnTIII]) or IgG is expressed After that, by modifying the carbohydrate (s) to control the fucosylation level of the oligosaccharide covalently bound to the Fc region and / or cleaving it into two. Modified sugar chains typically mean different carbohydrates or oligosaccharides; thus, IgG variants, such as antibodies or Fc fusions, can be included in modified sugar chains. Alternatively, modified sugar chains can refer to IgG variants that contain different carbohydrates or oligosaccharides. As is known in the art, the glycosylation pattern can depend on the sequence of the protein (eg, the presence or absence of certain glycosylated amino acid residues described below) or the host cell or organism that produces the protein. Specific expression systems are described below.

  Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the addition of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognized for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Is an array. Thus, the presence of any of these tripeptide sequences in the polypeptide provides a potential glycosylation site. O-linked glycosylation is a type of sugar (N-acetylgalactosamine, galactose, to hydroxyamino acids (most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine can also be used). Or addition of xylose).

  Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence to include one or more of the above tripeptide sequences (for N-linked glycosylation sites). The modification can also be made by addition or substitution of one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites). Briefly, the amino acid sequence of an antibody is encoded by a target polypeptide, preferably at a pre-selected base such that a change at the DNA level, in particular a codon can be translated into the desired amino acid. It is modified by mutating the DNA to be mutated.

  Another means of increasing the number of carbohydrate moieties on the antibody is by chemical or enzymatic coupling of glycosides to the protein. These methods 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. Depending on the coupling method used, the sugar (sugar) can be converted to (a) arginine and histidine, (b) a free carboxyl group, (c) a free sulfhydryl group such as part of cysteine, (d) serine, threonine or It can be attached to a free hydroxyl group such as part of hydroxyproline, (e) an aromatic residue such as phenylalanine, tyrosine, or part of triptolan, or (f) the amide group of glutamine. These methods are described in WO 87/05330 published September 11, 1987 and Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., Pp. 259-306.

  Removal of carbohydrate moieties present on the starting antibody can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to a trifluoromethanesulfonic acid compound, or equivalent compound. This treatment results in the cleavage of most or all sugar chains except the cross-linked sugar (N-acetylglucosamine or N-acetylgalactosamine), while the polypeptide remains intact. Chemical deglycosylation is described in Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259: 52, and Edge et al., 1981, Anal. Biochem. 118: 131. Enzymatic cleavage of the carbohydrate moiety on the polypeptide can be accomplished by the use of various endo- and exo-glycosidases as described in Thotakura et al., 1987, Meth. Enzymol. 138: 350. Glycosylation at potential glycosylation sites can be blocked by the use of tunicamycin compounds as described in Duskin et al., 1982, J. Biol. Chem. 257: 3105. Tunicamycin prevents the formation of protein-N-glycoside bonds.

  Other types of covalent modification of antibodies are described in US Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; In the method described in US Pat. No. 4,791,192 or US Pat. No. 4,179,337, the antibody can be prepared in various non-limiting ways including, but not limited to, various polyols such as polyethylene glycol, polypropylene glycol or polyoxyalkylene. Including binding to a proteinaceous polymer. In addition, as is known in the art, amino acid substitutions can be made at various sites within the antibody to facilitate the addition of polymers such as PEG. See, eg, US Patent Publication No. 2005/0114037, which is hereby incorporated by reference in its entirety.

Labeled antibodies In some embodiments, the covalent modification of the antibodies of the invention involves the addition of one or more labels. In some cases, these are considered antibody fusions.

  The term “labeling group” means any detectable label. In some embodiments, a labeling group is attached to the antibody via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used in the practice of the present invention.

  In general, labels are classified into various classes depending on the analysis in which they are detected: a) isotope labels that can be radioisotopes or heavy isotopes; b) magnetic labels (eg, magnetic particles) C) a redox active moiety; d) an optical dye; an enzyme group (eg, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) a biotinylated group; and f) a predetermined reporter recognized by a secondary reporter. Polypeptide epitopes (eg, leucine zipper base pair sequences, secondary antibody binding sites, metal binding domains, epitope tags, etc.). In some embodiments, a labeling group is attached to the antibody via spacer arms of various lengths to reduce potential steric hindrance. A variety of methods for labeling proteins are known in the art and can be used in the practice of the invention.

  Particular labels include optical dyes, including but not limited to chromophores, fluorophores and fluorophores, and in many instances are the latter in particular. Fluorophores can be either “small molecule” phosphors or proteinaceous phosphors.

  “Fluorescent label” means any molecule that can be detected by its inherent fluorescent properties. Suitable fluorescent labels include fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malacite green, stilbene, yellow fluorescent dye (Lucifer Yellow), Cascade BlueJ, Texas Red, IAEDANS , EDANS, BODIPY FL, LC Red 640, Cy5, Cy5.5, LC Red 705, Oregon green, Alexa-Fluor dye (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 546, 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 68 ), Cascade Blue, Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene, OR), FITC, rhodamine, and Texas Red (Pierce, Rockford, IL), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, PA), but not limited to. Optical dyes containing suitable fluorophores are described in Richard P. Haugland's Molecular Probes Handbook, which is expressly incorporated herein by reference.

  Suitable proteinaceous fluorescent labels also include green fluorescent proteins (Chalfie et al., 1994, Science 263: 802-805), including Renilla, Ptilosarcus, or Aequorea GFP. 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. 6: 178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol. 150). : 5408-5417), β-galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. USA 85: 2603-2607) and Renilla (WO92 / 15673, WO95 / 07463, WO98 / 14605, WO 98/26277, WO 99/49019, U.S. Pat.Nos. 5,292,658, 5,418,155, 5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995, No. 5925558), but is not limited thereto. All of the above cited references are expressly incorporated herein by reference.

  In any variant, an antibody can refer to a protein consisting of one or more polypeptides substantially encoded by all or part of a recognized immunoglobulin gene. For example, immunoglobulin genes recognized in humans include the myriad variable region genes and constant region genes, IgM, IgD, IgG (IgG1, IgG2, IgG3 and IgG4), IgE, and IgA (IgA1 and IgA2) isoforms. Includes kappa (κ), lambda (λ) and heavy chain gene regions, including mu (μ), delta (δ), gamma (γ), sigma (σ) and alpha (α) encoded by each type It is. As used herein, antibody is meant to include full-length antibodies and antibody fragments, natural antibodies from any organism, modified antibodies, or recombinantly produced for experimental, therapeutic or other purposes. Antibody may mean.

  Fc variants contain one or more amino acid modifications compared to the parent Fc polypeptide, wherein the amino acid modification (s) provide one or more optimization properties. The Fc variants of the present invention are amino acid sequences that differ from their parent IgG by at least one amino acid modification. Thus, the Fc variants of the present invention have at least one amino acid modification compared to the parent Fc polypeptide. Alternatively, the Fc variants of the present invention have 2 or more amino acid modifications, such as about 1 to 5 amino acid modifications, preferably about 1 to 10 amino acid modifications, most preferably compared to the parent Fc polypeptide. Can have from about 1 to about 5 amino acid modifications. Thus, the sequence of the Fc variant and the sequence of the parent Fc polypeptide are substantially homologous. For example, herein, a variant Fc variant sequence has about 80% homology with the parent Fc variant sequence, preferably at least about 90% homology, most preferably at least about 95% homology. Can have. Modifications can be made genetically using molecular biology, or enzymatically or chemically.

  The Fc variants of the present invention are defined according to the amino acid modifications that constitute them. Thus, for example, I332E is an Fc variant containing an I332E substitution compared to the parent Fc polypeptide. Similarly, S239D / A330L / I332E defines Fc variants that contain S239D, A330L and I332E substitutions compared to the parent Fc polypeptide. It is noted that the order in which substitutions are provided is arbitrary, ie, for example, S239D / A330L / I332E is the same Fc variant as S239D / I332E / A330L and the like. For all positions considered in the present invention, the numbering follows 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, which is hereby incorporated in its entirety by reference). EU index or EU numbering scheme as described in EU index or Kabat means EU antibody numbering (Edelman et al., 1969, Proc Natl Acad Sci USA 63: 78-85, which is incorporated herein by reference in its entirety. Included in the specification).

  Fc variants of the present invention include Rabbits including but not limited to rodents, rabbits and hares including but not limited to any organism, preferably including mammals, mice and rats, including but not limited to humans ( lagomorpha), including but not limited to camels, llamas and dromedaries, as well as Prosimian, Platyrrhini (New World monkey), Cercopithecoidea (Old World monkey) And can be substantially encoded by genes derived from non-human primates including but not limited to Gibbons and human superfamily including small and large apes. In the most preferred embodiments, the Fc variants of the present invention are substantially human.

  The parent Fc polypeptide can be an antibody. The parent antibody can be, for example, a transgenic mouse (Bruggemann et al., 1997, Curr Opin Biotechnol 8: 455-458, which is hereby incorporated by reference in its entirety) or a human antibody live integrated with a selection method. It can be a full-length human antibody obtained using a rally (Griffiths et al., 1998, Curr Opin Biotechnol 9: 102-108, which is hereby incorporated by reference in its entirety). The parent antibody need not be naturally occurring. For example, parent antibodies are produced by genetic recombination, including but not limited to chimeric antibodies and humanized antibodies (Clark, 2000, Immunol Today 21: 397-402, which is hereby incorporated by reference in its entirety). Antibody. The parent antibody can be a genetically engineered antibody variant substantially encoded by one or more natural antibody genes. In one embodiment, the parent antibody has mature affinity, as is known in the art. Alternatively, the antibody has been modified in several other ways, for example as described in USSN 10/339788, filed Mar. 3, 2003, which is hereby incorporated by reference in its entirety.

  The Fc variants of the present invention can be substantially encoded by immunoglobulin genes belonging to any antibody class. In a preferred embodiment, the Fc variants of the present invention are used for antibodies or Fc fusions comprising sequences belonging to the IgG class of antibodies, including IgG1, IgG2, IgG3 or IgG4. FIG. 3 provides an alignment of these human IgG sequences. In another embodiment, the Fc variants of the invention are used in antibodies or Fc fusions comprising sequences belonging to the IgA (including IgA1 and IgA2 subclass), IgD, IgE, IgG or IgM antibody classes. The Fc variants of the present invention may contain more than one protein chain. That is, the present invention may be used with antibodies or Fc fusions that are monomers or oligomers including homo- or hetero-oligomers.

  As is well known in the art, immunoglobulin polymorphisms exist in the human population. The Gm polymorphism encodes an allotype antigenic determinant called G1m, G2m and G3m allotypes (Gm allotypes are not found on the gamma 4 chain) for markers of human IgG1, IgG2 and IgG3 molecules Determined by IGHG1, IGHG2 and IGHG3 genes with alleles. Markers can be classified as 'allotype' and 'isoallotype'. These are distinguished by different serological bases depending on the strong sequence homology between isoforms. Allotypes are antigenic determinants specified by the allelic form of the Ig gene. Allotypes show slight differences in the amino acid sequence of different individual heavy or light chains. Even single amino acid differences can form allotypic determinants, but in many cases, several amino acid substitutions have occurred. Allotypes are sequence differences between subclass alleles in which the serum recognizes only allelic differences. An isoallotype is an allele in one isoform that produces an epitope that is shared with a non-polymorphic homology region of one or more other isoforms, so that serum contains related allotypes and related homologous isoforms. (Clark, 1997, IgG effector mechanisms, Chem Immunol. 65: 88-110; Gorman & Clark, 1990, Semin Immunol 2 (6): 457-66, both of which are hereby incorporated by reference in their entirety. Included in the book).

  The allelic form of human immunoglobulin is well characterized (WHO Review of the notation for the allotypic and related markers of human immunoglobulins. J Immunogen 1976, 3: 357-362; WHO Review of the notation for the allotypic and related 1976, Eur. J. Immunol. 6, 599-601; Loghem E van, 1986, Allotypic markers, Monogr Allergy 19: 40-51, all incorporated herein by reference in their entirety. ). In addition, other polymorphisms have been characterized (Kim et al., 2001, J. Mol. Evol. 54: 1-9, which is hereby incorporated by reference in its entirety). Currently, 18 Gm allotypes are known: G1m (1, 2, 3, 17) or G1m (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (b1, c3, b5, b0, b3, b4, s, t, g1, c5, u, v, g5) (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 of which are incorporated herein by reference in their entirety). Allotypes inherited in a fixed combination are called Gm haplotypes.

  FIG. 4 shows allotypes and isoallotypes of the gamma 1 chain of human IgG1 showing position and related amino acid substitutions (Gorman & Clark, 1990, Semin Immunol 2 (6): 457-66, which is hereby incorporated by reference in its entirety. Included). For comparison, amino acids found at equivalent positions in the human IgG2, IgG3 and IgG4 gamma chains are also shown.

  The Fc variants of the present invention can be substantially encoded by any allotype or isoallotype of any immunoglobulin gene. In a preferred embodiment, the Fc variants of the present invention are G1m (1), G1m (2), G1m (3), G1m (17), nG1m (1), nG1m (2), and / or nG1m (17). Used for antibodies or Fc fusions containing IgG1 sequences to be classified. Therefore, in the IgG1 isoform, the Fc variants of the present invention include Lys (G1m (17)) or Arg (G1 m (3)) at position 214 and Asp356 / Leu358 (G1m (1)) or Glu356 / at position 431. Met358 (nG1m (1)) and / or Gly (G1m (2)) or Ala (nG1m (2)) are included.

  In a most preferred embodiment, the Fc variants of the present invention are based on human IgG sequences, so that human IgG sequences include sequences from other organisms, such as rodents and primates, to which other sequences are compared. , But not limited to, used as a “base” sequence. Fc variants may also include sequences from other immunoglobulin classes such as IgA, IgE, IgD, IgM and the like. It is contemplated that the Fc variants of the present invention are produced in the context of one parent IgG, but the variant may be produced or “converted” in the context of another, second parent IgG. This generally determines the “equivalent” or “corresponding” residues and substituents between the first and second IgG based on the sequence or structural identity between the first and second IgG sequences. Is done. In order to establish homology, the amino acid sequence of the first IgG outlined herein is directly compared to the sequence of the second IgG. One or more homology alignment programs known in the art, taking into account the insertions and deletions necessary to maintain alignment (ie, avoiding removal of conserved residues by any deletions and insertions) Using (eg, using residues conserved between species), after aligning the sequence, residues equivalent to a particular amino acid are defined in the first sequence of the first Fc variant. Conserved residue alignments should preferably conserve 100% of such residues. However, alignments of more than 75% or as little as 50% of conserved residues are also suitable for defining equivalent residues. Equivalent residues can also be defined by determining the structural homology between the first and second IgG at the tertiary structure level of the IgG whose structure has been determined. In this case, the equivalent residues are those in which the atomic coordinates of two or more main chain atoms (N and N, CA and CA, C and C, and O and O) of a specific amino acid residue of the parent or precursor are aligned. Later, it is defined to be within about 0.13 nm, preferably about 0.1 nm. Alignment is achieved after the best model is oriented and positioned to give maximum overlap of atomic coordinates of non-hydrogen protein atoms of the protein. It has been found by the present invention that it should be conveyed regardless of how equivalent or corresponding residues are determined and regardless of the identity of the parent IgG from which the IgG is made. The Fc variant can be modified to any second parent IgG that has structural homology with the critical sequence or Fc variant. Thus, for example, when a variant antibody is made by using the methods described above or other methods for determining equivalent residues (where the parent antibody is human IgG1), the variant antibody is: It can be modified to another IgG1 parent antibody that binds a different antigen, a human IgG2 parent antibody, a human IgA parent antibody, a mouse IgG2a or IgG2b parent antibody, and the like. Furthermore, as described above, the parent Fc variant does not affect the ability to convert the Fc variant of the present invention into another parent IgG.

  Virtually all antigens, including but not limited to proteins, subunits, domains, motifs and / or epitopes belonging to the following target list can be targeted by the Fc variants of the present invention: 17 -IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B , Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5 Adressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 osteogenin ( Osteogenin), BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK) -3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE- DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, Cathepsin C / DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X / Z / P, C L, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3 CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, D22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40,

CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148 CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT- 1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXC L10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-related antigen, DAN, DCC, DcR3, DC-SIGN, complement control factor accelerating factor), des (1-3) -IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2 , EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-se Lectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10 G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13) CDMP-2), GDF-7 (BMP-12, CDMP) 3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap),

HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3) ), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human heart myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM -3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2 IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL -15, IL-18, IL-18R, IL-23, interferon (INF) -alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin alpha 4 / beta 7, integrin Rufa 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 14, kallikrein 14, kallikrein L, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF) -1), potential TGF-1, potential TGF-1 bp1, LBP, LDGF, LECT2, lefti, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, IGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG , MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13,

MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Mueller tube suppression Substance, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF -Beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF , PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory multinuclear virus (RSV) F, RSV Fgp, Ret, Riumide factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF , SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (eg, T cell receptor alpha / Beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan specific, TGF-beta RI (ALK-5) ), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, thrombin, thymus Ck-1, thyroid Stimulating hormone, Tie, TIMP, TIQ, group Factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF- alpha, TNF- alpha beta, TNF- beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4),

TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (TNKSF11RS (TNKSF11A) OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR T75RSR18F) AITR), TNFRSF19 (TROY TAJ, TRADE), NFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60), TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF4, TNF RIII, TNF RIII, TNFC4) , TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1RSDR6, IL1F6137R ), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRRST23 (DcTRAIL R1 TNFRH1) ), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-) 3 ligands, DR3 ligands), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGIT) , TNFSF1A (TNF-a connectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1CD7, APT1CD7, APT1CD7) ), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor associated antigen CA125, tumor associated antigen expressing Lewis Y related carbohydrate, TWEAK, TXB2, Ung, uP R, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, and hormones and growth factors Receptor for.

The present invention provides Fc variants that are optimized for a variety of therapeutically relevant properties. An Fc variant that is incorporated or predicted to exhibit one or more optimization properties is referred to herein as an “ optimized Fc variant ”. Properties that can be optimized include, but are not limited to, increased or decreased affinity for FcγR. In a preferred embodiment, the Fc variants of the invention are optimized to have increased affinity for human activated FcγR, preferably FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and FcγRIIIb, most preferably FcγRIIIa. In another preferred embodiment, the Fc variant is optimized to have reduced affinity for the human inhibitory receptor FcγRIIb. These preferred embodiments are expected to provide IgG polypeptides with increased therapeutic properties, for example in humans. In another aspect, the Fc variants of the present invention are optimized to have reduced or reduced affinity for human FcγR, including but not limited to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. . These aspects are intended to provide IgG polypeptides with enhanced therapeutic properties in humans, such as reduced effector function and reduced toxicity. In other embodiments, the Fc variants of the present invention provide increased affinity for one or more FcγRs but decreased affinity for one or more other FcγRs. For example, the Fc variants of the present invention may have increased binding to FcγRIIIa but decreased binding to FcγRIIb. Alternatively, the Fc variants of the invention may have increased binding to FcγRIIa and FcγRI, but decreased binding to FcγRIIb. In yet another aspect, the Fc variants of the present invention may have increased affinity for FcγRIIb, but may have decreased affinity for one or more activated FcγRs.

  Preferred embodiments include optimization of binding to human FcγR, but in another embodiment, the Fc variants of the present invention include non-human organisms, including but not limited to rodents and non-human primates. Has increased or decreased affinity for the derived FcγR. Fc variants that are optimized for binding to non-human FcγR can be used in experiments. For example, mouse models are available for various diseases that allow examination of properties such as effects, toxicity, and pharmacokinetics for a given drug candidate. As is known in the art, cancer cells, a method called xenotransplantation, can be transplanted or injected into mice to mimic human cancer. Testing Fc variants, including Fc variants that are optimized for one or more mouse FcγRs, may provide useful information regarding the effectiveness of the protein, its mechanism of action, and the like. The Fc variants of the present invention can also be optimized to increase the functionality and / or solubility properties of glycosylated forms. In preferred embodiments, the glycosylated Fc variants of the present invention bind Fc ligands with a stronger affinity than the glycosylated form of the parent Fc variant. The Fc ligands include FcγR, C1q, FcRn, and proteins A and G, but are not limited to any origin, including but not limited to human, mouse, rat, rabbit or monkey Preferably, it may be derived from a human. In another preferred embodiment, the Fc variant is optimized to be more stable and / or soluble than the glycosylated form of the parent Fc variant.

  The Fc variants of the present invention may include modifications that are regulated to interact with Fc ligands other than FcγR, including but not limited to complement proteins, FcRn, and Fc receptor homologs (FcRH). . FcRH includes, but is not limited to, FcRH1, FcRH2, FcRH3, FcRH4, FcRH5, and FcRH6 (Davis et al., 2002, Immunol. Reviews 190: 123-136, hereby incorporated by reference in its entirety. Included).

  Preferably, the Fc ligand specificity of the Fc variants of the present invention can determine its therapeutic utility. The usefulness of a given Fc variant for therapeutic purposes can vary depending on the epitope or form of the target antigen and the disease or indication being treated. For some targets and indications, enhanced FcγR-mediated effector function is preferred. This is particularly preferred for anticancer Fc variants. Thus, Fc variants can be used, including Fc variants that provide increased affinity for activated FcγR and / or reduced affinity for inhibitory FcγR. Utilizing Fc variants that provide different selectivity for different activated FcγRs for some targets and indications may be further beneficial; for example, in some cases increased with FcγRIIa and FcγRIIIa Although binding is desirable, it is not desirable for FcγRI, while in other cases, increased binding only with FcγRIIa is preferred. For any target and indication, it is preferred to utilize Fc variants that enhance both FcγR and complement-mediated effector functions, whereas in other cases, FcγR or complement-mediated effectors It is convenient to utilize Fc variants that increase either function. For some targets or cancer indications, one or more effector functions are reduced or reduced, eg, by eliminating binding to C1q, one or more FcγR, FcRn, or one or more other Fc ligands. Is convenient. For other targets and indications, it is preferred to utilize Fc variants that provide increased or decreased binding of inhibitory FcγRIIb, but decreased or decreased binding of WT levels to activated FcγR. This can be particularly useful, for example, when the purpose of the Fc variant is to prevent inflammatory or autoimmune diseases, or to modulate the immune system in several ways.

  A clearly important parameter that determines the most beneficial selectivity of a given Fc variant for treating a given disease is the Fc variant, for example which type Fc variant is used. Thus, the Fc ligand selectivity or specificity of a given Fc variant varies depending on whether it comprises an antibody, Fc fusion, or Fc variant with a bound fusion or complex partner. Can provide. For example, toxicants, radionuclides, or other complexes are less toxic to normal cells if the Fc variant that contains them reduces or reduces binding to one or more Fc ligands. As another example, use Fc variants with increased affinity for activated FcγRs that bind to and inhibit activated FcγRs to block inflammatory or autoimmune diseases It is preferable. Conversely, Fc variants that contain two or more Fc regions with increased FcγRIIb affinity can co-associate with this receptor on the surface of immune cells, thereby preventing the growth of these cells. In some cases, an Fc variant can bind its target antigen on one cell type and further bind to an FcγR on a cell other than the target antigen, while in other cases it As convenient for binding to FcγR on the same cell surface. For example, if an antibody targets an antigen on a cell that expresses one or more FcγRs, it may be beneficial to utilize an Fc variant that increases or decreases binding to FcγR on the surface of the cell. obtain. This is possible, for example, when Fc variants are used as anticancer agents, where the co-binding of target antigen and FcγR on the same cell surface results in intracellular inhibition leading to growth inhibition, apoptosis, or other antiproliferative effects. Promotes signal transduction events. Alternatively, co-binding of antigen and FcγR on the same cell is convenient when Fc variants are used to modulate the immune system in several ways, where the co-binding of target antigen and FcγR is Provides several proliferative or antiproliferative effects. Similarly, an Fc variant comprising two or more Fc regions may be beneficial to an Fc variant that modulates FcγR selectivity or specificity and co-binds FcγRs on the same cell surface.

  The presence of different polymorphic forms of FcγR provides yet another parameter that affects the therapeutic availability of the Fc variants of the present invention. Although the specificity and selectivity of a given Fc variant for a different class of FcγR significantly influences the ability of an Fc variant to target a given antigen for treatment of a given disease, these receptors The specificity or selectivity of Fc variants for different polymorphisms of whether or not a study or preclinical experiment may be appropriate for the study, and ultimately the patient population may or may not respond to treatment You can decide in part. Thus, the specificity or selectivity of the Fc variants of the present invention for Fc ligand polymorphisms, including but not limited to FcγR, C1q, FcRn and FcRH polymorphisms, can be effectively investigated and preclinical experiments, clinical trial plans. Can be used to guide selection of aspects related to patient selection, dose dependence, and / or other clinical trials.

Modifications can be made to improve IgG stability, solubility, functionality or clinical use. In preferred embodiments, the Fc variants of the present invention may include modifications to reduce immunogenicity in humans. In a most preferred embodiment, the immunogenicity of the Fc variants of the present invention is determined by the method described in USSN 11 / 004,590, filed December 3, 2004, which is hereby incorporated by reference in its entirety. Use to decrease. In another embodiment, the Fc variants of the present invention are humanized (Clark, 2000, Immunol Today 21: 397-402, which is hereby incorporated by reference in its entirety). As used herein, a “ humanized ” antibody refers to an antibody comprising a human framework region (FR) and one or more complementarity determining regions (CDRs) derived from a non-human (usually mouse or rat) antibody. . Non-human antibodies displaying CDRs are termed “donors” and human immunoglobulins presenting frameworks are termed “acceptors”. Humanization is primarily by transplantation of donor CDRs onto acceptor (human) VL and VH frameworks (eg, Winter et al, US 5225539, which is hereby incorporated by reference in its entirety). This strategy is referred to as “CDR grafting”. “Backmutation” of selected acceptor framework residues to the corresponding donor residues is often necessary to regain the affinity that the originally transplanted construct lacks (US5530101; US5585089; US5639371). US Pat. No. 5,693,762; US Pat. No. 6,180,370; US Pat. No. 5,859,205; US Pat. No. 5,821,337; US Pat. No. 6,054,297, and US Pat. A humanized antibody also optimally comprises at least a portion of an immunoglobulin constant region, typically at least a portion of a human immunoglobulin, and thus typically may comprise a human Fc region. Various techniques and methods for humanization and remodeling of non-human antibodies are well known in the art (Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, See Elsevier Science (USA), and references cited therein, all of which are incorporated herein by reference in their entirety). Humanization methods include Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988; Nature 332: 323-329; Verhoeyen et al., 1988, Science, 239: 1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86: 10029-33; He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA 89: 4285 -9, Presta et al., 1997, Cancer Res. 57 (20): 4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA 88: 4181-4185; O'Connor et al. , 1998, Protein Eng 11: 321-8, all of which are incorporated herein by reference in their entirety, but are not limited thereto. Humanization methods or other methods that reduce the immunogenicity of non-human antibody variable regions include, for example, Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91: 969-973 (in its entirety by reference). Surface reshaping methods, such as those described herein) may be included. In one embodiment, the parent antibody has mature affinity, as is well known in the art. Structure-based methods can be used for humanization and affinity maturation as described, for example, in USSN 11 / 004,590, which is hereby incorporated by reference in its entirety. Selection-based methods are described in Wu et al., 1999, J. Mol. Biol. 294: 151-162; Baca et al., 1997, J. Biol. Chem. 272 (16): 10678-10684; Rosok et al. , 1996, J. Biol. Chem. 271 (37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16 (10): 753-759, all of which are incorporated herein by reference in their entirety, including but not limited to humanization and / or affinity maturation of antibody variable regions. Can be used. Other humanization methods include USSN 09 / 810,510; Tan et al., 2002, J. Immunol. 169: 1119-1125; De Pascalis et al., 2002, J. Immunol. 169: 3076-3084 (all Which may be involved in transplantation of only a portion of a CDR, including, but not limited to, the methods described in.

  Modifications that reduce immunogenicity can include modifications that reduce binding of the processing peptide from the parent sequence to the MHC protein. For example, amino acid modifications can be made such that there are no or a minimal number of immune epitopes that are predicted to bind with high affinity to any common MHC allele. Several methods for identifying MHC binding epitopes in protein sequences are known in the art and can be used to count epitopes in the Fc variants of the present invention. For example, WO 98/52976; WO 02/079232; WO 00/3317; USSN 09 / 903,378; USSN 10 / 039,170; USSN 60 / 222,697; USSN 10 / 754,296; PCT WO 01/18223; and PCT WO 02 / Mallios, 1999, Bioinformatics 15: 432-439; Mallios, 2001, Bioinformatics 17: 942-948; Sturniolo et al., 1999, Nature Biotech. 17: 555-561; WO98 / 59244; WO02 / 069232; WO02 / 77187; Marshall et al., 1995, J. Immunol. 154: 5927-5933; and Hammer et al., 1994, J. Exp. Med. 180: 2353-2358 (all herein incorporated by reference in their entirety. See). Sequence-based information can be used to determine the binding score for a given peptide-MHC interaction (eg, Mallios, 1999, Bioinformatics 15: 432-439; Mallios, 2001, Bioinformatics 17: p942-948; Sturniolo et al. al., 1999, Nature Biotech. 17: 555-561, all of which are incorporated herein by reference in their entirety).

In one embodiment, the Fc variants of the present invention comprise one or more engineered glycoforms. As used herein, “ modified sugar chain ” refers to a carbohydrate composition that is covalently linked to IgG, where the carbohydrate composition is chemically different from that of the parent IgG. Modified sugar chains may be useful for a variety of purposes including, but not limited to, enhancing or decreasing effector function. Modified sugar chains can be made by various 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); PCT WO00 / 61739A1; PCT WO01 / 29246A1; PCT WO02 / 31140A1; PCT WO02 / 30954A1); (Potelligent ^™ technology [Biowa, Inc., Princeton, NJ]; GlycoMAb® glycosylation engineering technology [GLYCART Biotechnology AG, Zuerich, Switzerland]) (all incorporated herein by reference in their entirety). Many of these techniques are involved, for example, by expressing IgG in genetically modified or other various organisms or cell lines (eg, Lec-13 CHO cells or rat hybridoma YB2 / 0 cells) or involved in glycosylation pathways Carbohydrates (eg, after FUT8 [α1,6-fucosyltransferase] and / or β1-4-N-acetylglucosaminyltransferase III [GnTIII]) or after IgG is expressed By modifying the fucosylation level of the oligosaccharide covalently linked to the Fc region and / or cleaving it into two. Modified sugar chains are typically referred to as different carbohydrates or oligosaccharides; therefore, Fc variants, such as antibodies or Fc fusions, can include modified sugar chains. Alternatively, modified sugar chains can be referred to as Fc variants that contain different carbohydrates or oligosaccharides.

  In another embodiment, the Fc variants of the present invention are bound to or operably linked to another therapeutic compound. The therapeutic compound can be a cytotoxic agent, chemotherapeutic agent, toxicant, radioisotope, cytokine, or other therapeutically active agent. IgG can bind to one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene, or a copolymer of polyethylene glycol and polypropylene glycol.

  The present invention provides methods for producing, producing and screening Fc variants. The described method does not imply that the present invention is bound by any particular application or theory of operation. Rather, the provided methods generally indicate that one or more Fc variants can be manufactured, produced, and screened to experimentally obtain Fc variants with optimized effector functions. Intended to explain. Various methods are available for antibody design, production, and testing, and protein variants, USSN 10 / 672,280, USSN 10 / 822,231, USSN 11 / 124,620, and USSN 11 / 256,060 (all by reference). The entirety of which is incorporated herein.

  Various protein production methods can be used to design Fc variants with optimized effector functions. In one embodiment, a structure-based manufacturing method can be used, where the structural information available is used to induce substitution. Sequence alignment can be used to induce substitution at the indicated position. Alternatively, random or semi-random mutagenesis methods can be used to produce amino acid modifications at the desired positions.

  Methods for producing and screening Fc variants are well known in the art. General methods for antibody molecular biology, expression, purification, and screening are described by Antibody Engineering, Duebel & Kontermann, Springer-Verlag, Heidelberg, 2001; and Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5: 683. -689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2: 339-76, all of which are incorporated herein by reference in their entirety. See also the methods described in USSN 10 / 672,280, USSN 10 / 822,231, USSN 11 / 124,620, and USSN 11 / 256,060, all of which are incorporated herein by reference in their entirety. thing.

In one embodiment of the invention, the Fc variant sequence is used to produce a nucleic acid that encodes a member sequence and thus can be cloned, optionally expressed, and analyzed in a host cell. These embodiments, known methods, and 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 Both are performed using a variety of methods that may be used in the present invention, as described in their entirety herein by reference. By culturing a host cell transformed with a nucleic acid, preferably an expression vector, comprising a nucleic acid encoding the Fc variant under conditions suitable to induce or cause expression of the protein of the Fc variant of the invention. Can be produced. A variety of suitable host cells can be used, including but not limited to mammalian cells, bacteria, insect cells and yeast. For example, various cell lines that can be used in the present invention are described in the ATCC cell line catalog (available through the American Type Culture Collection). Methods for introducing foreign nucleic acids into host cells are known in the art and may vary depending on the host cell used.

In a preferred embodiment, the Fc variant is purified or isolated after expression. Antibodies can be isolated or purified by a variety of methods known to those skilled in the art. Standard purification methods include chromatographic, electrophoretic, immunological, precipitation, dialysis, filtration, concentration, and chromatographic fractionation techniques. As is known in the art, a variety of natural proteins such as bacterial proteins A, G, and L bind to antibodies and these proteins can be used in the present invention for purification. Often, purification may be possible with a particular fusion partner. For example, purifying proteins using glutathione resin when GST fusion is used, Ni ⁺² affinity chromatography when His tag is used, or using immobilized anti-flag antibody when flag tag is used. Can do. For general guidance in suitable purification techniques, Antibody Purification:. Principles and Practice , 3 rd Ed, Scopes, Springer-Verlag, NY, 1994 ( by reference in its entirety is being incorporated herein) see .

  Fc variants can be screened using a variety of methods including, but not limited to, methods used in in vitro analysis, in vivo and cell-based assays, and selection techniques. Automated high performance screening techniques can be used for screening methods. Screening can employ the use of fusion partners or labels, such as immunolabels, isotope labels, or small molecule labels such as fluorescent or colorimetric dyes.

  In preferred embodiments, the functional and / or biophysical characteristics of the Fc variants are screened in in vitro analyses. In a preferred embodiment, the protein is screened for functionality, such as its ability to catalyze the reaction or its binding affinity to its target.

As is known in the art, some screening methods include methods for selecting preferred members of a library. The methods are referred to herein as “ selection methods ” and these methods are used in the present invention to screen for Fc variants. When screening protein libraries using selection methods, only those library members that are grown, isolated and / or observed are preferred as meeting some selection criteria. Various selection methods that can be used for screening protein libraries in the present invention are known in the art. Other selection methods that can be used in the present invention include non-presentation methods such as in vivo methods. Some of the selection methods, referred to as “directed evolution” methods, include preferred sequence mating or breading during selection, sometimes including the insertion of new mutations. is there.

  In preferred embodiments, Fc variants are screened using one or more cell based assays or in vivo assays. For such analysis, purified or unpurified protein is typically added extracellularly so that the cells are exposed to individual mutants or a collection of mutants belonging to a library. These analyzes are generally, but not always, based on the function of the Fc polypeptide; that is, it binds to its target and several biochemical events such as effector function, ligand / receptor binding inhibition, apoptosis, etc. Based on the ability of Fc polypeptides to mediate. Such analysis often involves observing cellular responses to IgG, eg, cell survival, cell death, changes in cell morphology, or transcriptional activation such as cellular expression of a native or reporter gene. For example, such an assay can measure the ability of an Fc variant to induce ADCC, ADCP, or CDC. For some analyses, it may be necessary to add additional cells or components, such as serum complement, or effector cells such as peripheral blood monocytes (PBMC), NK cells, macrophages, in addition to the target cells. Such added cells can be from any organism, preferably humans, mice, rats, rabbits and monkeys. The antibody can cause apoptosis of any cell line that expresses the target or can mediate attack on the target cell by immune cells added to the analysis. Methods for observing cell death or cell survival are known in the art and include the use of dyes, immunochemical reactive agents, cytochemical reactive agents, and radioactive reactive agents. Transcriptional activation can also be a method for analyzing function in cell-based assays. Alternatively, cell-based screening is performed using cells transformed or transfected with a nucleic acid encoding the variant. That is, the Fc variant is not added extracellularly to the cells.

  In a preferred embodiment, the immunogenicity of the Fc variant is determined experimentally using one or more cell based assays. Several methods can be used for experimental confirmation of epitopes.

  The biological characteristics of the Fc variants of the present invention can be characterized in cell, tissue and whole organism experiments. As is known in the art, drugs are often used to measure the effects of drugs for treatment on a disease or disease model, or to measure the pharmacokinetics, toxicity and other properties of drugs, Test in animals including but not limited to rabbits, dogs, cats, pigs and monkeys. The animal can be referred to as a disease model. Therapeutic agents are often tested in mice including, but not limited to, nude mice, SCID mice, xenograft mice, and transgenic mice (including knock-in and knock-out). Such experiments can provide meaningful data for determining the potential of proteins used as therapeutic agents. Any organism, preferably a mammal, can be used for the test. For example, because it is genetically similar to humans, monkeys can be a suitable therapeutic model and can therefore be used to test the effects, toxicity, pharmacokinetics, or other properties of the IgGs of the invention. Tests in humans are ultimately needed for drug approval and, of course, these experiments are contemplated. Thus, IgGs of the invention can be tested in humans to determine their therapeutic efficacy, toxicity, immunogenicity, pharmacokinetics, and / or other clinical properties.

  The Fc variants of the present invention can be used in a variety of products. In one embodiment, the Fc variant of the present invention is a therapeutic agent, diagnostic agent, or experimental reaction agent, preferably a therapeutic agent. Fc variants can be used in antibody compositions that are monoclonal or polyclonal. In a preferred embodiment, the Fc variants of the present invention are used to kill target cells having a target antigen, such as cancer cells. In another embodiment, the Fc variants of the present invention are used to block, antagonize or agonize a target antigen, for example to antagonize a cytokine or cytokine receptor. In another preferred embodiment, the Fc variants of the present invention are used to block, antagonize or agonize the target antigen, and kill target cells with the target antigen.

The Fc variants of the present invention can be used for various therapeutic purposes. In a preferred embodiment, an antibody comprising an Fc variant is administered to a patient to treat an antibody related disease. “ Patient ” for this purpose includes humans and other animals, preferably mammals, most preferably humans. As used herein, “ antibody-related disease ” or “ antibody- responsive disease ” or “ condition ” or “ disease ” means a disease that can be alleviated by administration of a pharmaceutical composition comprising an Fc variant of the present invention. Antibody-related diseases include autoimmune diseases, immunological diseases, infectious diseases, inflammatory diseases, neurological diseases, pain, lung diseases, hematological conditions, fibrotic conditions, and neoplastic and neoplastic diseases including cancer Is included, but is not limited thereto. As used herein, “ cancer ” and “ cancerous ” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine tumors, mesothelioma, schwanoma, meningioma, adenocarcinoma, melanoma, and leukemia and This includes but is not limited to lymphoid malignancies. Other conditions that can be treated include rheumatoid arthritis, juvenile rheumatoid arthritis, Crohn's disease, ulcerative colitis, Sjorgren's disease, multiple sclerosis, ankylosing spondylitis, asthma, allergies and allergic conditions Including, but not limited to, graft versus host disease. The term “ treatment ” as used herein is meant to include therapeutic treatment as well as prophylactic or inhibitory means of the disease, condition or disorder. Thus, for example, successful administration of a pharmaceutical composition comprising an Fc variant of the present invention prior to the onset of the disease results in a “treatment” of the disease. As another example, successful administration of a pharmaceutical composition comprising an Fc variant of the present invention after a clinical symptom of the disease to combat the symptom of the disease is included in the “treatment” of the disease. “Treatment” also includes administration of a pharmaceutical composition comprising an Fc variant of the present invention after the appearance of the disease to eradicate the disease. Successful administration of a pharmaceutical composition comprising an Fc variant of the present invention after onset and after the onset of clinical symptoms, with the possibility of elimination of clinical symptoms and the amelioration of the disease, is included in the “treatment” of the disease . As used herein, the term “ in need of treatment ” includes mammals already having the disease or disorder including the disease or disorder to be prevented, as well as mammals prone to have the disease or disorder It is.

  In one embodiment, the Fc variant of the present invention is the only therapeutically active agent administered to a patient. Alternatively, Fc variants of the present invention include cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitors, antihormonal agents, kinase inhibitors, angiogenesis inhibitors, cardioprotectants, or other therapeutic agents, It is administered in combination with one or more other therapeutic agents that are not so limited, as well as before or after surgery. IgG variants can be administered in combination with one or more other treatment regimens. For example, the Fc variants of the present invention may be administered to a patient in combination with surgery, chemotherapy, radiation therapy, or any or all of surgery, chemotherapy and radiation therapy. In one embodiment, the Fc variants of the present invention can be co-administered with one or more antibodies that may or may not include the Fc variants of the present invention. In accordance with another aspect of the present invention, the Fc variants of the present invention and one or more other anti-cancer therapeutic agents are used for the treatment of cancer cells ex vivo. It is believed that such ex vivo treatment may be useful for bone marrow transplantation and particularly autologous bone marrow transplantation. Of course, it is contemplated that the Fc variants of the present invention can be used in combination with other therapeutic techniques, such as surgery.

A variety of other therapeutic agents can be used for co-administration with the Fc variants of the present invention. In one embodiment, IgG is administered in combination with an angiogenesis inhibitor. As used herein, “ angiogenesis inhibitor ” means a compound that inhibits or prevents some formation of blood vessels. Angiogenesis inhibitors can be, for example, small molecules or proteins that bind to growth factors or growth factor receptors involved in promoting angiogenesis, such as antibodies, Fc fusions or cytokines. A preferred angiogenesis inhibitor herein is an antibody that binds to vascular endothelial growth factor (VEGF). In another embodiment, IgG is administered with a therapeutic agent that induces or augments an adaptive immune response, such as an antibody that targets CTLA-4. In another embodiment, IgG is administered with a tyrosine kinase inhibitor. As used herein, “ tyrosine kinase inhibitor ” means a molecule that inhibits to some extent the tyrosine kinase activity of a tyrosine kinase. In another embodiment, the Fc variants of the present invention are administered with a cytokine. As used herein, “cytokine” refers to the generic name of a protein released by a cell population that acts on another cell as an intercellular mediator.

  By pharmaceutical composition is intended that the Fc variants of the invention and one or more therapeutically active agents are formulated. The Fc variant formulations of the present invention may comprise IgG of desired purity, optionally pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980). , Which is incorporated herein by reference in its entirety) and is prepared for storage in the form of a lyophilized formulation or an aqueous solution. The formulation used for in vivo administration is preferably sterilized. This is easily accomplished by filtration through sterile filtration membranes or other methods. The Fc variants and other therapeutically active agents described herein can also be formulated as immunoliposomes and / or microencapsulations.

The concentration of therapeutically active Fc variant in the formulation can vary from about 0.001 to 100% by weight. In a preferred embodiment, the concentration of IgG is in the range of 0.003 to 1.0 mole. In order to treat a patient, a therapeutically effective dose of the Fc variants of the present invention can be administered. As used herein, “ therapeutically effective dose ” means the dose with which it is administered to produce an effect. The exact dose will vary depending on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. The dosage is in the range of 0.001 to 100 mg or more per kg body weight, for example 0.1, 1, 10, or 50 mg per kg body weight, with 1 to 10 mg / kg being preferred. As is known in the art, the rate of proteolysis, systemic versus local delivery, and new protein synthesis, as well as age, weight, general health, sex, diet, time of administration, severity of drug interactions and conditions Adjustment for the degree is necessary and can be ascertained by one skilled in the art through routine experimentation.

  Administration of a pharmaceutical composition comprising an Fc variant of the present invention, preferably in the form of a sterile aqueous solution, can be administered orally, subcutaneously, intravenously, nasally, intraotically, transdermally, topically (eg, gel , Ointments, lotions, creams, etc.), intraperitoneal, intramuscular, intrapulmonary (eg AERx® inhalable technology marketed by Aradigm, or Inance® pulmonary delivery system marketed by Inhale Therapeutics, etc. ), Vaginal, parenteral, rectal, or intraocularly, can be performed in a variety of ways. The therapeutic agents described herein can be administered in combination with other therapeutic agents.

Examples The following examples are provided to illustrate the present invention. These examples are not meant to limit the invention to any particular application or theory of operation.

Example 1. Fc variants USSN 10 / 672,280, USSN 10 / 822,231, USSN 11 / 124,620 and USSN 11 / 256,060 with effector function mediated by increased FcγR (all herein incorporated by reference in their entirety) Are used to increase Fc ligand binding, optimize effector function, and to reduce or decrease FcγR binding and effector function. . The mutant is an anti-CD20 antibody, PRO70769 (PCT / US2003 / 040426, known to mediate measurable CDC and ADCC in cell-based assays, which is hereby incorporated by reference in its entirety. ) Built in light of. The previously characterized variants were also constructed in PRO70769 to further characterize their properties and provide a comparator for the current series of new variants. FIG. 5 provides a list of these Fc variants. Among other things, this mutant set includes many insertions. For example, “insert L> 235-236 / I332E” refers to a double mutant comprising an I332E substitution and an insertion of leucine between residues 235 and 236.

  The variable region gene of PRO70769 (FIGS. 24a and 24b) was constructed using recursive PCR and pcDNA3.1Zeo, a mammalian expression vector containing a full length light chain kappa (Cκ) and heavy chain IgG1 constant region. Invitrogen). Mutants were constructed into variable regions of antibodies in pcDNA3.1Zeo vector using quick-change mutagenesis technology (Stratagene) and expressed in 293T cells. DNA was sequenced to confirm sequence fidelity. A plasmid containing the heavy chain gene (VH-CH1-CH2-CH3) (wild type or mutant) was co-transfected into a 293T cell with a plasmid containing the light chain gene (VL-Cκ). The medium was collected 5 days after transfection and the antibody was purified from the supernatant using protein A affinity chromatography (Pierce). Selected Fc variants were also expressed in alemtuzumab.

Binding affinity for human FcγR by IgG antibodies was measured using AlphaScreen ^™ competition analysis. AlphaScreen is a bead-based luminescent proximity analysis. Laser excitation of the donor beads can excite oxygen and, when sufficient access to the acceptor beads, results in a cascade of chemiluminescent events, ultimately leading to fluorescence emission at 520-620 nm. AlphaScreen was used as a competition analysis to screen for antibodies. Wild-type IgG1 antibody was biotinylated by standard binding methods to streptavidin donor beads and tagged FcγR was bound to glutathione chelate acceptor beads. In the absence of competing Fc polypeptides, wild-type antibody and FcγR interact to produce a signal at 520-620 nm. The addition of untagged antibody competes with the wild type Fc / FcγR interaction and quantitatively reduces fluorescence to allow determination of relative binding affinity.

  FIG. 6 provides AlphaScreen competition data for binding of selected PRO70769Fc variants to human activated receptors V158 FcγRIIIa (FIG. 6a) and F158 FcγRIIIa (FIG. 6b). The data was fitted to partial competition models using nonlinear regression and these fits are shown as curves in the figure. These fits provide a 50% inhibitory concentration (IC50) for each antibody (ie, the concentration required for 50% inhibition), and therefore the relative binding affinity compared to WT can be determined. FIG. 5 provides IC50 and IC50 multiples compared to WT for fitting to these binding curves.

  Selected Fc variants were re-expressed and retested using the AlphaScreen competition assay for binding to human V158 FcγRIIIa and F158 FcγRIIIa (FIG. 7). FIG. 7a shows the binding data for these variants and FIG. 7b provides the IC50 and IC50 fold compared to WT for fitting to these binding curves.

  Based on these data, a number of additional Fc variants were constructed against PRO70769 IgG1. In addition, some Fc variants have been identified as IgG (1/1), a novel IgG molecule described in USSN 11 / 256,060, filed October 21, 2005, which is hereby incorporated by reference in its entirety. 2) Constructed against ELLGG. These variants were constructed as described above and expressed and purified along with a number of previously characterized Fc variants. These variants are listed in FIG. 8a. Binding of the mutant to human activated receptors V158 FcγRIIIa and F158 FcγRIIIa, and the inhibitory receptor FcγRIIb was measured using an AlphaScreen competition assay. FIG. 8b shows the binding data of selected variants with these receptors, and FIG. 8a provides the IC50 and IC50 fold compared to WT PRO70769 IgG1 for all of this series of Fc variants.

  Due to the highly reactive nature of the assay, AlphaScreen provides only relative affinity. Accurate binding constants were determined by competing SPR devices (Nieba et al., 1996, Anal Biochem 234: 155-65, which are incorporated herein by reference in their entirety), with unbound antibody in the antibody / FcγR equilibrium captured on the FcγRIIIa surface. Included). This experiment was performed with the I332E and S239D / I332E mutants (USSN 10 / 672,280, USSN 10 / 822,231, and USSN 11 / 124,620, in the light of the trastuzumab IgG1 constructed and characterized above. All of which are incorporated herein by reference in their entirety). WT and mutant trastuzumab antibodies were expressed and purified as described above. For this experiment, data was obtained on a BIAcore 3000 instrument (BIAcore). V158 FcγRIIIa-His-GST was captured using immobilized anti-GST antibody, blocked using recombinant GST, and binding competition analyte to antibody / receptor was measured. Anti-GST antibody was covalently attached to the CM5 sensor using the BIAcore GST capture kit. One flow cell for each sensor chip was coupled with ethanolamine as a non-specific binding control and the bulk refractive index change was subtracted online. The electrophoresis buffer is HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v surfactant P20, BIAcore), and the chip regeneration buffer is glycine 1.5. (10 mM glycine-HCl, pH 1.5, BIAcore). 1 μM V158 FcγRIIIa-His-GST was bound to the anti-GST CM5 chip in HBS-EP for 5 minutes at 1 μl / min. The surface was blocked by injecting 5 μM recombinant GST (Sigma) at 1 μl / min for 2 minutes. 100 nM wild type or mutant trastuzumab antibody was combined with between 4 and 1000 nM serial dilutions of V158 FcγRIIIa-His-GST and incubated at room temperature for at least 2 hours. The competitive binders were injected at 50 μl / min onto the V158 FcγRIIIa-His-GST / recombinant GST surface for 30 seconds of binding in HBS-EP. One cycle with antibody but no competitive receptor provided a baseline response.

Using the “competitive BIAcore” method described above, the kinetic curve was adapted to derive binding rates (Nieba et al., 1996, Anal Biochem 234: 155-65, which is hereby incorporated by reference in its entirety. Included). The inventors have found that this method is not very reliable because the binding rate obtained from the kinetic curve showed a non-linear correlation with the antibody concentration used. The analysis used in this study is based on the proportion of initial binding rate R to free antibody concentration (Holwill et al., 1996, Process Control and Quality 8: 133-145; Edwards & Leatherbarrow, 1997, Anal Biochem 246: 1 -6, all incorporated herein by reference in their entirety). Response unit data was output using BIA evaluation software (BIAcore) and analyzed using Microsoft Excel (IDBS) with Xlfit version 3.0.5. Initial binding values (for signal increase) were determined from the raw data for each sensorgram using the Excel formula of the slope. The equilibrium dissociation binding constant (K _D ) was determined by plotting the log value of the FcγRIIIa concentration against the initial binding rate obtained at each concentration. Using GraphPad Prism (GraphPad Software), the data was fit to the following formula:

〔式中、
［Ａ_０］＝抗体濃度
Ｒ_０＝競合する受容体の不存在下での、抗体濃度Ａ_０での初期結合率
Ｘ＝ｌｏｇ［Ｌ_０］（ここで、［Ｌ_０］＝インプット受容体濃度である。）
Ｋ_Ｄ＝平衡解離定数である。〕。
Ｒ_０は、抗体と固定化受容体との結合率を示し（競合する受容体の不存在下での）、それらの異なる受容体親和性のため、それを、ＷＴ、Ｉ３３２Ｅ、およびＳ２３９Ｄ／Ｉ３３２Ｅ抗体について別個に計算した。

[Where,
[A ₀ ] = antibody concentration R ₀ = initial binding rate at antibody concentration A ₀ in the absence of competing receptors X = log [L ₀ ] (where [L ₀ ] = input receptor concentration .)
K _D = equilibrium dissociation constant. ].
R ₀ indicates the binding rate of the antibody to the immobilized receptor (in the absence of competing receptors) and due to their different receptor affinities it is expressed as WT, I332E, and S239D / I332E. Calculated separately for antibodies.

で示される定義、および質量保存式

〔式中：
［Ｌ］＝遊離受容体の濃度である。〕
から導かれる。 The formula for the initial binding rate R, K _D for a single binding site:

Definitions indicated by and mass conservation formulas

[In the formula:
[L] = concentration of free receptor. ]
Derived from.

The initial binding rate was determined from the raw data of sensorgrams (Fig. 9a), the K _D values were calculated by plotting the log value of the receptor concentration for the initial binding rate obtained at each concentration (Fig. 9b, 9c (Edwards & Leatherbarrow, 1997, Anal Biochem 246: 1-6, which is hereby incorporated by reference in its entirety). WT K _D (252 nM) is in good agreement with published data (208 nM from SPR, 535 nM from calorimetry) (Okazaki et al., 2004 J Mol Biol 336: 1239-49, which is entirely incorporated by reference. Included in the specification). I332E (30 nM) and _{the K D} value of S239D / I332E (2nM) mutants show a strong affinity respectively about 10 times and 100 times values for V158 Fc [gamma] RIIIa (1-and 2-logs).

In order to investigate the ability of antibodies comprising Fc variants of the present invention to exert FcγR-mediated effector functions, an in vitro cell-based ADCC assay was performed using human PBMC as effector cells. . ADCC was measured by the release of lactose dehydrogenase using an LDH cytotoxicity detection kit (Roche Diagnostic). Human PBMC was purified from leukopack using the ficoll gradient method and the CD20 + target lymphoma cell line WIL2-S was obtained from ATCC. Target cells were seeded at 10,000 cells / well in 96 well plates and opsonized with Fc variants or WT antibodies at the final concentrations indicated. Only Triton X100 and PBMC were used as controls. Effector cells were added PBMC: target cells at 25: 1 and the plates were incubated at 37 ° C. for 4 hours. Cells were incubated with the LDH reaction mixture and fluorescence was measured using Fusion ^™ Alpha-FP (Perkin Elmer). Data were normalized to maximal lysis (Triton) and minimal lysis (PBMC only) and fit to a sigmoidal dose response model. FIG. 10 provides these data for variable region PRO70769 and selected Fc variant antibodies in either IgG1 or IgG (1/2) ELLGG. Fc variants provide a distinct increase in CD20 + target cell lysis mediated by FcγR compared to WT PRO70769 IgG1 antibody.

  These in vitro analyzes suggest that the Fc variants of the present invention may provide enhanced efficacy and / or efficacy in a clinical setting. In vivo performance can be affected by many factors, including several factors not considered in these in vitro experiments. One such parameter is the high concentration of non-specific IgG in serum that has been shown to affect the clinical efficacy of antibodies (Vugmeyster & Howell, 2004, Int Immunopharmacol 4: 1117-24; Preithner et al., 2005, Mol Immunol, 43 (8): 1183-93, all of which are hereby incorporated by reference in their entirety). To investigate how the Fc mutations of the present invention occur in solutions that more closely resemble in vivo biology, ADCC assays were performed using IgG purified from biologically relevant concentrations (1 mg / ml) of human serum (Jackson In the presence of (commercially available from Immunoresearch Lab, Inc.). These data are shown in FIG. The efficacy of the WT anti-CD20 antibody is not only reduced in the presence of serum levels of IgG, but is completely eliminated. In contrast, Fc variant antibodies are significantly reduced but still show substantial ability to mediate killing of the target cell line.

Example 2 Fc variants with effector function mediated by increased complement A number of variants were designed with the goal of enhancing complement-dependent cytotoxicity (CDC). In the same way that Fc / FcγR binding mediates ADCC, Fc / C1q binding mediates complement dependent cytotoxicity (CDC). There is currently no structure available for the Fc / C1q complex; however, mutagenesis studies have mapped the binding site on human IgG for C1q to a region centered on residues D270, K322, P329 and P331 (Idusogie et al., 2000, J Immunol 164: 4178-4184; Idusogie et al., 2001, J Immunol 166: 2571-2575, both of which are hereby incorporated by reference in their entirety). FIG. 12 shows the structure of the human IgG1 Fc region containing this mapped center point. USSN 10 / 672,280, USSN 10 / 822,231, USSN 11 / 124,620, and USSN 11 / 256,060, which are structurally close to these four residues (all incorporated herein by reference in their entirety). The selected amino acid modifications disclosed in (1) were investigated to search for variants that could mediate increased affinity for C1q and / or provide increased CDC. Variants that already showed increased FcγR affinity and effector function mediated by FcγR included this series of variants and characterized their complementary properties. This mutant library is shown in FIG.

  The mutants were constructed as described above in the light of anti-CD20 antibody PRO70769 (variable region) and either IgG1 or IgG (1/2) ELLGG as the heavy chain constant region. Mutants were expressed and purified as described above. Cell based analysis was used to determine the ability of Fc variants to mediate CDC. Lysis was measured using Alamar Blue release to monitor lysis of WIL2-S lymphoma cells opsonized with Fc variants and WT PRO70769 by human serum complement. Target cells were washed 3 times by centrifugation and resuspension in 10% FBS medium and WT or mutant rituximab antibody was added at the indicated final concentrations. Human serum complement (Quidel) was diluted to 50% using media and added to target cells opsonized with antibody. The final concentration of complement was 1/6 of the stock solution. Plates were incubated for 2 hours at 37 ° C., alamar blue was added, cells were incubated for 2 days, and fluorescence was measured. Representative data for this analysis is shown in FIG. Binding data was normalized to maximum and minimum luminescence signals for each particular curve by baseline at low and high concentrations of antibody, respectively. The data was fitted to a sigmoidal dose response including a variable slope model using non-linear regression, and these fits are shown as curves in the figure. These fits provide a 50% effective concentration (EC50) for each antibody (ie, the concentration required for a 50% response) that allows quantitative determination of the relative binding affinity of the Fc variants. To do. Dividing the EC50 of each mutant by the EC50 of WT PRO70769 yielded an fold increase or decrease fold compared to WT PRO70769 (WT fold). These values are shown in FIG. Here, multiples greater than 1 indicate an increase in CDC EC50 compared to WT PRO70769, and multiples less than 1 indicate a decrease in CDC EC50.

  The data in FIGS. 13 and 14 show that it provides increased CDC compared to WT PRO70769 IgG1. For example, a CDC increase of 2 or more is indicated by modification 239D, 267D, 267Q, 268D, 268E, 268F, 268G, 272I, 276D, 276L, 276S, 278R, 282G, 284T, 285Y, 293R, 300T, 324I, 324T, Observed at 324V, 326E, 326T, 326W, 327D, 330H, 330S, 332E, 333F, 334T and 335D (FIG. 15). Furthermore, the data show that many modifications provide reduced CDC compared to WT PRO70769 IgG1. For example, modifications that show a relative CDC of 0.5 times or less include 235D, 239D, 284D, 322H, 322T, 322Y, 327R, 330E, 330I, 330L, 330N, 330V, 331D, 331L, and 332E ( FIG. 15). These modifications provide additional valuable structure activity relationship (SAR) information that can be used to guide further design of variants for increased CDC. At the same time, the data are 235, 239, 267, 268, 272, 276, 278, 282, 284, 285, 293, 300, 322, 324, 326, It suggests that modifications at positions 327, 330, 331, 332, 333, 334 and 335 (FIG. 15) can provide increased CDC compared to the parent Fc polypeptide.

Example 3 Fc variants with reduced FcγR and complement-mediated effector functions As described above, one type of antibody and clinical application, as opposed to antibody therapy and indications where effector function contributes to clinical efficacy Reduce or eliminate the above binding to FcγR, or reduce or eliminate effector function mediated by one or more FcγR or complement, including but not limited to ADCC, ADCP and / or CDC Is preferred. This is often the case for therapeutic antibodies whose mechanism of action is not the death of cells with the target antigen, but with its blocking or antagonism. In these cases, target cell reduction is undesirable and can be considered a side effect. Effector function may also be a challenge for radiolabeled antibodies called radioconjugates and antibodies complexed with toxins called immunotoxins. Although these drugs can be used to destroy cancer cells, the capture of immune cells by the interaction of Fc and FcγR can result in healthy immune cells in close proximity to harmful payloads (radioactive substances or toxins). Resulting in a decrease in normal lymphoid tissue with the target cancer cells.

  A previously unconsidered advantage of reduced FcγR binding and complement binding is that FcγR and complement binding can effectively reduce the active concentration of the drug when effector function is not required. Binding to the Fc ligand can be achieved by binding the antibody or Fc fusion, or a complex with a serum protein that is less active or inactive compared to when the serum protein is free (not complexed). Can be localized to the surface. This is because the antibody is at a reduced effective concentration at the desired binding site, or perhaps because Fc ligand binding can render the Fc polypeptide a less active conformation than if unbound. . A further consideration is that FcγR receptors may be one mechanism of antibody turnover and mediate uptake and processing by antigen presenting cells such as dendritic cells and macrophages. This can affect the pharmacokinetics (or in vivo half-life) of the antibody or Fc fusion and its immunogenicity (both are important parameters of clinical performance).

  Appearance of the Fc / FcγR structure (FIG. 2) and the Fc / C1q junction (FIG. 12), and the above and USSN 10 / 672,280, USSN 10 / 822,231, USSN 11 / 124,620, and USSN 11/256, 060 (all incorporated herein by reference in its entirety) was used to guide the design of libraries to screen for variants with reduced affinity for FcγR and reduced CDC. . This mutant library is shown in FIG. Mutants were constructed against PRO70769 IgG1, expressed and purified as described above. Relative FcγR affinity was measured using the AlphaScreen competition assay as described above. FIG. 17 shows AlphaScreen data for binding of selected Fc variants to human V158 FcγRIIIa, and FIG. 16 shows its IC50 fold compared to WT PRO70769 IgG1. Mutants were also examined for their ability to mediate complement-mediated lysis against CD20 + WIL2-S lymphoma target cells using the CDC assay described above. FIG. 18 shows CDC data for selected Fc variants, and FIG. 16 shows EC50 fold compared to WT PRO70769 IgG1. Based on the results of these experiments, selected Fc variants were characterized for their ability to mediate effector functions mediated by FcγR. ADCC assays using human PBMC as effector cells and WIL2-S lymphoma cells as target cells were performed as described above. FIG. 19 shows these ADCC data for the selected mutants.

  The data indicate that modifications at many positions provide reduced or reduced FcγR affinity, reduced FcγR-mediated effector function, and reduced complement-mediated effector function. Furthermore, modifications at several positions, including but not limited to positions 235 and 330, provide reduced CDC, but FcγR affinity may be at WT levels. For example, 235D, 330L, 330N, and 330R exhibit such action. Alternatively, modifications at several positions including but not limited to positions 236 and 299 provide reduced FcγR affinity, but CDC may be at WT levels. For example, 236I and 299A exhibit these properties.

  Based on the results of these experiments, many modifications that simultaneously reduce FcγR affinity and CDC can be achieved by mutations with fully reduced FcγR affinity, effector function mediated by FcγR, and effector function mediated by complement. Combined with variants with multiple mutations in a new library of Fc variants designed to screen the body. These variants include modifications at positions 234, 235, 236, 267, 269, 325 and 328 and are shown in FIG. WT IgG1 antibody, and IgG2 and IgG4 antibody variants, glycosylation variant N297S, and two variants already characterized as having reduced effector function: L234A / L235A (Xu et al., 2000, Cellular Immunology 200: 16-26; USSN 10 / 267,286, which is hereby incorporated by reference in its entirety), and E233P / L234V / L235A / G236- (Armour et al., 1999, Eur J Immunol 29: 2613) -2624, which is incorporated herein by reference in its entirety).

  These variants contained IgG1, which is a heavy chain constant region other than IgG2 and IgG4 antibodies, and were constructed against the anti-CD20 antibody PRO70769. The antibody was expressed and purified as described above. The AlphaScreen competition assay was used as described above to determine the relative FcγR affinity of Fc variants. FIG. 21 shows AlphaScreen data for binding of selected variants to the low affinity human activated receptor V158 FcγRIIIa as well as the high affinity human activated receptor FcγRI. FIG. 20 shows the IC multiple of 50 compared with WT. Due to its greater binding affinity for the Fc region, FcγRI provides a more stringent test of variants. The data in FIGS. 20 and 21 support this and show that the mutant can substantially reduce or completely reduce the affinity for FcγRIIIa but has little effect on FcγRI binding. Fc variants were also tested for their ability to mediate complement-mediated lysis against CD20 + WIL2-S cells using the CDC assay described above. FIG. 22 shows CDC data for selected Fc variants and FIG. 20 shows EC50 fold compared to WT PRO70769 IgG1.

  To examine the ability of Fc variants to mediate ADCC, selected variants were subcloned into trastuzumab, an anti-Her2 / neu antibody (variable region sequences are shown in FIGS. 24c and 24d). Trastuzumab reliably provides a substantial signal in the ADCC assay to the Her2 + expressing cell line and thus provides a rigorous examination of Fc variants that reduce / reduce effector function. Fc variants L235G, G236R, G237K, N325L, N325A, L328R, L235G / G236R, G236R / G237K, G236R / N325L, G236R / L328R, G237K / N325L, L235G / G236R / G236K / G236R / G236K Constructed in the light of WT IgG1, WT IgG2 and WT IgG4 antibody variants were similarly constructed. The ADCC assay was performed as described above except that Her2 + breast cancer cell line SkBr-3 was used as the target cell. FIG. 23 shows the results of the ADCC experiment. The data shows that some variants completely reduce ADCC. Furthermore, IgG2 also appears to not mediate ADCC, whereas IgG4 exhibits a significant level of ADCC.

  Results are 232, 234, 235, 236, 237, 238, 239, 265, 267, 269, 270, 297, 299, 325, 327, 328 Amino acid modifications at a number of positions, including but not limited to positions 329, 330, and 331, are not desirable for FcγR binding, FcγR-mediated effector functions, and / or complement-mediated effector functions; We show that it provides promising candidates for improving the clinical properties of antibodies and Fc fusions. For example, 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 331L provide significantly reduced Fc ligand binding properties and / or effector functions. Particularly effective in reducing Fc ligand binding and effector function are variants 236R / 237K, 236R / 325L, 236R / 328R, 237K / 325L, 237K / 328R, 325L / 328R, 235G / 236R, 267R / 269R. 234G / 235G, 236R / 237K / 325L, 236R / 325L / 328R, 235G / 236R / 237K, and 237K / 325L / 328R. In particular, amino acid modifications including these variants, including 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R, can reduce binding to both FcγRIIIa and FcγRI and reduce CDC by a factor of 10 or more. . Furthermore, the data show that human IgG2 has significantly reduced FcγR affinity, FcγR-mediated effector function, and complement-mediated effector function compared to human IgG4.

As noted above, decreased FcγR affinity and / or effector function may be optimal for Fc polypeptides where Fc ligand binding or effector function results in toxic and / or reduced effects. For example, antibodies targeting CTLA4 are effective in preventing inhibition of T cell activation and promoting an anti-tumor immune response, but destruction of T cells by effector functions mediated by antibodies is And / or adverse effects on potential toxicity. Actual toxicity was observed in clinical application of ipilimumab, an anti-CTLA4 antibody (Maker et al., 2005, Ann Surg Oncol 12: 1005-16, which is hereby incorporated by reference in its entirety). Ipilimumab, an anti-CTLA4 antibody, extracted from SEQ ID NO: 7 (VL, FIG. 24e) and SEQ ID NO: 17 (VH, FIG. 24f) of US 6,984,720 (incorporated herein in its entirety by reference) The sequence of (Mab 10D.1, MDX010) is shown in FIG. For purposes of illustration, many Fc variants of the present invention are incorporated into the sequence of antibodies that target CTLA4. Since the combination of Fc variants of the present invention typically resulted in additive or synergistic binding modulation and thereby additive or synergistic modulation of effector function, the Fc variants provided in the present invention are still It is expected that combinations that have not been elucidated or in combination with other previously reported modifications may also provide favorable results. A possible Fc variant is shown in Figure 26a. The optimized antibody sequence includes at least one non-WT amino acid selected from the group consisting of X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ , X ₇ , and X ₈ . For example, improved anti-CTLA-4 antibody sequences comprising L235G and G236R modifications in the IgG1 constant region are shown in FIGS. 26b and 26c. Alternatively, as the present invention shows, IgG2 and IgG4 can also be used to reduce Fc ligand binding and Fc-mediated effector functions. Figures 26b and 26d provide the sequence of the improved anti-CTLA4 IgG2 antibody sequence. Herein, the use of anti-CTLA4 is only an example and does not imply that the application of the Fc variant is limited to this antibody or any other specific Fc polypeptide. Other exemplary applications of reduced Fc ligand binding and / or effector function include anti-TNFα antibodies including, for example, infliximab and adalimumab, anti-VEGF antibodies including, for example, bevacizumab, anti-α4-integrin antibodies including, for example, natalizumab, and USSN10 Anti-CD32b antibodies, including but not limited to those described in US Pat. No. 6,643,857, which is hereby incorporated by reference in its entirety.

  This list of preferred Fc variants is not meant to limit the invention. Indeed, all combinations of any Fc variants provided provide aspects of the invention. Furthermore, combinations of any Fc variants of the present invention with other discovered or undiscovered Fc variants can also provide favorable properties, and these combinations are also contemplated as aspects of the present invention. Is done. Finally, from these results, other substitutions at mutated positions in the present invention may also provide favorable binding enhancement and specificity, and therefore substitutions at all positions described herein are considered. Is expected.

  All cited references are expressly incorporated herein in their entirety by reference.

  While particular embodiments of the present invention have been described above for purposes of illustration, those skilled in the art will recognize that numerous modifications can be made without departing from the present invention as set forth in the appended claims. Will be.

Antibody structure and function. Humanized Fab structure from pdb accession code 1CE1 (James et al., 1999, J Mol Biol 289: 293-301, incorporated herein by reference in its entirety) and human IgG1Fc from pdb accession code 1DN2 1 shows a model of a full-length human IgG1 antibody modeled using the structure (DeLano et al., 2000, Science 287: 1279-1283, which is hereby incorporated by reference in its entirety). The flexible hinge connecting the Fab and Fc regions is not shown. IgG1 is a heterodimeric homodimer consisting of two light chains and two heavy chains. The Ig domains containing antibodies, including _VL and C _L of the light chain, and V _{H of} the heavy chain, C gamma 1 (Cγ1), C gamma 2 (Cγ2), and C gamma 3 (Cγ3) are shown. The Fc region is indicated. The binding sites of related proteins are shown, including antigen binding sites in the variable region and binding sites for FcγR, FcRn, C1q and proteins A and G in the Fc region. Fc / FcγRIIIb complex structure 1IIS. Fc is shown as a gray ribbon and FcγRIIIb is shown as a black ribbon. N297 carbohydrate is shown as a black bar. Alignment of amino acid sequences of human IgG immunoglobulins IgG1, IgG2, IgG3 and IgG4. FIG. 3a provides the sequence and hinge domain of CH1 (Cγ1), and FIG. 3b provides the sequence of CH2 (Cγ2) and CH3 (Cγ3) domains. The positions are numbered according to the EU index of the IgG1 sequence and the differences between IgG1 and other immunoglobulins IgG2, IgG3 and IgG4 are shown in gray. Polymorphisms are present at multiple positions (Kim et al., 2001, J. Mol. Evol. 54: 1-9, which is hereby incorporated by reference in its entirety), and thus the sequence shown There may be slight differences from conventional sequences. Possible starting points for the Fc region are indicated and are described herein as either EU positions 226 or 230. The allotype and isoallotype positions of the gamma 1 chain of human IgG1 and the associated amino acid substitutions are shown (Gorman & Clark, 1990, Semin Immunol 2 (6): 457-66, which is hereby incorporated by reference in its entirety. ) For comparison, amino acids found at equivalent positions in human IgG2, IgG3 and IgG4 gamma chains are also shown. Fc variant and FcγR binding data. All Fc variants were constructed against the antibody PRO70769 IgG1. Fold indicates IC50 fold compared to WT PRO70769 IgG1 for binding to human V158 and F158 FcγRIIIa as measured by AlphaScreen competition assay. Binding of human V158 FcγRIIIa (FIG. 6a) and F158 FcγRIIIa (FIG. 6b) by selected PRO70769 Fc variants as determined by AlphaScreen competition assay. In the presence of a competing antibody (Fc variant or WT), a characteristic inhibition curve is observed as a decrease in luminescent signal. Binding data was normalized to the maximum and minimum luminescence signals for each particular curve provided by the baseline at low and high concentrations of antibody, respectively. The curve shows the fit of the data to a partial competition model using nonlinear regression. Binding of human V158 FcγRIIIa and F158 FcγRIIIa by PRO70769 Fc variants as measured by AlphaScreen competition assay. FIG. 7a shows data for selected mutants and FIG. 7b shows IC50 values and folds compared to WT PRO70769 IgG1. Fc variant and FcγR binding data. All Fc variants were constructed against the variable region PRO70769 and either human IgG1 or IgG (1/2) ELLGG. FIG. 8a shows IC50 values and IC50 fold compared to WT PRO70769 IgG1 for binding to human activated receptors V158 and F158 FcγRIIIa, and inhibitory receptor FcγRIIb as measured by AlphaScreen competition assay. FIG. 8b shows AlphaScreen data for selected mutants. Competition surface plasmon resonance (SPR) experiment measuring the binding affinity of I332E and S239D / I332E mutants in trastuzumab against human V158 FcγRIIIa. FIG. 9a shows sensorgram raw data, FIG. 9b shows a plot of the log value (logarithmic value) of the receptor concentration against the initial binding rate obtained at each concentration, and FIG. The affinities obtained from the fit to these data as described in Example 1 are shown. Cell-based ADCC assay of selected Fc variants in anti-CD20 antibody PRO70769. ADCC was measured by the release of lactose dehydrogenase using the LDH cytotoxicity detection kit (Roche Diagnostic). CD20 + lymphoma WIL2-S cells were used as target cells and human PBMC was used as effector cells. FIG. 5 shows the dose dependence of ADCC at the antibody concentration of a given antibody, normalized to the minimum and maximum fluorescence signals for each particular curve provided by the baseline at low and high concentrations of antibody, respectively. The curve shows the fit of the data to a sigmoidal dose response model using non-linear regression. Cell-based ADCC assay of selected Fc variants in PRO70769 IgG1 in the absence and presence of serum levels of human IgG. ADCC was measured by the release of lactose dehydrogenase using the LDH cytotoxicity detection kit (Roche Diagnostic). CD20 + lymphoma WIL2-S cells were used as target cells and human PBMC was used as effector cells. Residues mutated in Fc variants designed to enhance CDC. The structure of the human IgG1 Fc region is shown (pdb accession code 1E4K, Sondermann et al., 2000, Nature 406: 267-273, which is hereby incorporated by reference in its entirety). Black spheres and bars indicate residues D270, K322, P329 and P331 which have been shown to be important in mediating binding to complement protein C1q, gray bars affect CDC The residues mutated in the present invention in order to examine the mutants are shown. Fc variants and CDC data screened for complement-mediated cytotoxicity (CDC). The variable region is that of the anti-CD20 antibody PRO70769, the heavy chain constant region is IgG1, and unless otherwise stated, is IgG (1/2) ELLGG. CDC fold indicates relative CDC activity compared to WT PRO70769 IgG1. Fc variant CDC assay of anti-CD20 antibody. The dose dependence of antibody concentration of complement-mediated lysis is shown for the indicated PRO70769 antibody against CD20 + WIL2-S lymphoma target cells. Lysis was measured using the release of Alamar Blue, and the data is relative to the minimum and maximum fluorescence signals for each specific curve provided by the baseline at low and high concentrations of antibody, respectively. And standardized. The curve shows the fit of the data to a sigmoidal dose response model with a slope change using non-linear regression. Amino acid modifications that provide increased and decreased CDC, and positions that can be modified to provide increased / regulated CDC. The positions are numbered according to the EU index. Fc variants screened for reduced FcγR affinity, effector function mediated by FcγR, and effector function mediated by complement. The variable region is that of the anti-CD20 antibody PRO70769, and the heavy chain constant region is IgG1. The figure shows the IC50 fold for binding to human V158 FcγRIIIa and the EC50 fold for CDC activity compared to WT PRO70769 IgG1. Binding to human V158 FcγRIIIa by selection of PRO70769 Fc variants as determined by AlphaScreen competition assay. CDC assay of selected anti-CD20 antibody Fc variants against CD20 + WIL2-S lymphoma target cells. Dissolution was measured by Alamar Blue release. Cell-based ADCC activity of selected anti-CD20 antibody Fc variants against CD20 + lymphoma WIL2-S cells. Human PBMC were used as effector cells and lysis was measured by LDH release. Fc variants screened for reduced FcγR affinity, FcγR-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 IgG1. The figure shows the IC50 fold for binding to human V158 FcγRIIIa compared to WT, the IC50 fold for binding to human FcγRI compared to WT, and the EC50 fold for CDC activity compared to WT by two separate tests. Indicates. Binding of the low affinity human activated receptor V158 FcγRIIIa and the high affinity human activated receptor FcγRI by selected PRO70769 Fc variants as determined by AlphaScreen competition assay. CDC activity of selected PRO70769Fc variants against CD20 + WIL2-S lymphoma target cells. Dissolution was measured by the release of alamar blue. Cell-based ADCC activity of anti-Her2 Fc variants and WT IgG antibodies against Her2 / neu + SkBr-3 breast cancer target cells. Human PBMC were used as effector cells and lysis was measured by LDH release. Amino acid sequences of variable light chain (VL) and variable heavy chain (VH) used in the present invention, including PRO70769 (Figures 24a and 24b), trastuzumab (Figures 24c and 24d), and ipilimumab (Figures 24e and 24f). Amino acid sequences of human constant light chain kappa (FIG. 25a) and constant heavy chain (FIGS. 25b-25f) used in the present invention. A sequence showing a constant heavy chain sequence that may have reduced Fc ligand binding and effector function properties (FIG. 26a) and an improved anti-CTLA-4 antibody sequence (FIGS. 26b-26d). FIG. 26a shows a possible Fc variant constant heavy chain sequence with variable positions shown highlighted as X1, X2, X3, X4, X5, X6, X7 and X8. The table below the sequence shows WT amino acids and possible substituents at these positions. Improved antibody sequences can include one or more non-WT amino acids selected from this potential modifying group. FIG. 26b shows the anti-CTLA-4 antibody light chain sequence, and FIGS. 26c and 26d show the anti-CTLA-4 antibody heavy chain sequence with reduced Fc ligand binding and Fc-mediated effector function. These include the L235G / G236R IgG1 heavy chain (FIG. 26c) and the IgG2 heavy chain (FIG. 26d). The positions are numbered according to the EU index described in Kabat and therefore do not correspond to the sequential order in the sequence.

Claims (18)

  An Fc variant of a parent Fc polypeptide, wherein the Fc variant exhibits altered binding or altered antibody-dependent cell-mediated cytotoxicity with at least one FcγR compared to the parent Fc polypeptide. And includes at least one amino acid modification in the Fc region of the parent Fc polypeptide, wherein the variants are 227G / 332E, 234D / 332E, 234E / 332E, 234Y / 332E, 234I / 332E, 234G / 332E, 235I / 332E, 235S / 332E, 235D / 332E, 235E / 332E, 246H / 332E, 255Y / 332E, 258H / 332E, 260H / 332E, 267E / 332E, 267D / 332E, 268E / 330Y, 268D / 330Y, 272R / 332E, 272H / 332E, 28 H / 332E, 293R / 332E, 295E / 332E, 304T / 332E, 324I / 332E, 324G / 332E, 324I / 332D, 324G / 332D, 327D / 332E, 328F / 332E, 328Y / 332E, 328D / 332E, 328A / 332D, 328T / 332D, 328V / 332D, 328I / 332D, 328F / 332D, 328Y / 332D, 328M / 332D, 328D / 332D, 328E / 332D, 328N / 332D, 328Q / 332D, 335D / 332E, 239D / 268E / 330Y, 239D / 332E / 268E / 330Y, 239D / 332E / 327A, 239D / 332E / 268E / 327A, 239D / 332E / 330Y / 327A, 332E / 3 An Fc variant selected from the group consisting of 30Y / 268E / 327A and 239D / 332E / 268E / 330Y / 327A, where the numbers are according to EU Index.   The Fc variant of claim 1, wherein the Fc polypeptide comprises a human IgG1 antibody.   The Fc variant of claim 2, wherein the Fc polypeptide comprises a human IgG1 antibody, and the IgG1 antibody has allotype residues 356D and 358L.   The Fc variant of claim 2, wherein the Fc polypeptide comprises a human IgG1 antibody, and the IgG1 antibody has allotype residues 356E and 358M.   The Fc variant of claim 1, wherein the Fc polypeptide constitutes an IgG (1/2) ELLGG antibody as defined in SEQ ID NO: 12.   An IgG1 variant of a parent IgG1 polypeptide, wherein the IgG1 variant exhibits improved binding to at least one FcγR, wherein the Fc variant is at least 1 in the Fc region of the parent IgG1 polypeptide. IgG1 variant containing a single amino acid insertion.   The insertion occurs between two positions selected from the group consisting of positions 235 and 236, positions 297 and 298, and positions 326 and 327, where the numbers follow the EU Index. Item 7. The IgG1 variant according to Item 6.   Insertion of G between positions 235 and 236, insertion of A between positions 235 and 236, insertion of S between positions 235 and 236, insertion of T between positions 235 and 236, 235 and N insertion between positions 236, D insertion between positions 235 and 236, V insertion between positions 235 and 236, L insertion between positions 235 and 236, G between positions 235 and 236 Insertion between positions 235 and 236, insertion of S between positions 235 and 236, insertion of T between positions 235 and 236, insertion of N between positions 235 and 236, 235 and 236 Insertion of D between positions, insertion of V between positions 235 and 236, insertion of L between positions 235 and 236, insertion of G between positions 297 and 298, insertion of A between positions 297 and 298 Insertion, 297 Insertion between positions 298 and 298, insertion of D between positions 297 and 298, insertion of G between positions 326 and 327, insertion of A between positions 326 and 327, between positions 326 and 327 8. The insertion selected from the group consisting of a T insertion, a D insertion between positions 326 and 327, and an E insertion between positions 326 and 327, where the number is according to EU Index. IgG1 variants of   An Fc variant of a parent Fc polypeptide, wherein the Fc variant exhibits altered complement dependent cytotoxicity compared to the parent Fc polypeptide and is at least in the Fc region of the parent Fc polypeptide. 1 amino acid modification, including 233I, 234Y, 235D, 235Y, 239D, 267D, 267E, 267Q, 268D, 268E, 268F, 268G, 271A, 271D, 271I, 272H, 272I, 272R, 274E, 274R, 274Y, 276D, 276L, 276S, 278E, 278H, 278Q, 278R, 281D, 282G, 284D, 284E, 284T, 285Y, 293R, 300D, 300T, 320I, 320T, 320Y, 322H, 322T, 322Y, 324D, 324H, 324I, 3 4L, 324T, 324V, 326L, 326T, 327A, 327D, 327H, 327R, 328Q, 330E, 330G, 330H, 330I, 330L, 330N, 330V, 330Y, 331D, 331L, 332E, 333F, 334T, 335D and 335Y ( Wherein the numbers are according to EU Index.) Fc variants selected from the group consisting of.   An Fc variant of a parent Fc polypeptide, wherein the Fc variant exhibits improved complement dependent cytotoxicity compared to the parent Fc polypeptide and is at least in the Fc region of the parent Fc polypeptide. It includes one amino acid modification, which is 235, 239, 267, 268, 272, 276, 278, 282, 284, 285, 293, 300, 322 An Fc variant that is a position selected from the group consisting of positions 324, 327, 330, 331, 332 and 335 (where the numbers are according to EU Index).   The Fc variants are 239D, 267D, 267Q, 268D, 268E, 268F, 268G, 272I, 276D, 276L, 276S, 278R, 282G, 284T, 285Y, 293R, 300T, 324I, 324T, 324V, 327D, 330H, 11. The Fc variant of claim 10, comprising one or more amino acid modifications selected from the group consisting of 330S, 332E, 335D.   An Fc variant of a parent Fc polypeptide, wherein the Fc variant has reduced binding to one or more FcγRs, reduced antibody-dependent cell-mediated cytotoxicity compared to the parent Fc polypeptide; Or exhibit reduced complement dependent cytotoxicity, comprising at least one amino acid modification in the Fc region of the parent Fc polypeptide, wherein the variants are 236R / 237K, 236R / 325L, 236R / 328R, 237K / 325L, 237K / 328R, 325L / 328R, 235G / 236R, 267R / 269R, 234G / 235G, 236R / 237L, 236R / 325L / 328R, 235G / 236R / 237K, and 237K / 325L / 328R An Fc variant selected from.   An Fc variant of a parent Fc polypeptide, wherein the Fc variant has an affinity for human FcγRIIIa, an affinity for human FcγRI, and complement dependent cytotoxicity compared to the parent Fc polypeptide Fc variants with a decrease of at least 10-fold.   The Fc polypeptide comprises at least one modification at a position selected from the group consisting of 234G, 235G, 236R, 237K, 267R, 269R, 325L, 328R (where the number is according to EU index). The Fc variant according to claim 13.   IgG antibody targeting CTLA-4, TNFα, VEGF, α4-integrin, or CD32b, with reduced FcγR affinity, reduced antibody-dependent cell-mediated cytotoxicity compared to WT antibody, or IgG antibody with reduced complement dependent cytotoxicity.   The antibody consists of the group consisting of positions 234, 235, 236, 237, 267, 269, 325, and 328 (numbers are according to EU Index) compared to the parent IgG antibody. 16. The IgG antibody of claim 15, comprising a single amino acid modification at a position selected from   16. The IgG antibody according to claim 15, wherein the antibody is a human IgG2 or IgG4 antibody.   An IgG antibody variant of a parent IgG antibody that exhibits improved antibody-dependent cell-mediated cytotoxicity in the presence of competing IgG.

Images