ANTIBODY VARIANTS AND FRAGMENTS THEREOF

Abstract: A variant of a polypetide comprising a human IgG Fc region is described, which variant comprises an amino acid substitution at one or more of amino acid positions 270, 322, 326, 327, 329, 331, 333 or 334 of the human IgG Fc region. Such variants display altered effector function. For example, C1q binding and/or complement dependent cytotoxicity (CDC) activity may be altered in the variant polypeptide. The application also discloses a variant of a parent polypeptide comprising a human IgG Fc region, which variant has a better binding affinity for human C1q than the parent polypeptide.

Description: ANTIBODY VARIANTS AND FRAGMENTS THEREOF BACKGROUND OF THE INVENTION Field of the Invention The present invention concerns variants of polypeptides comprising an Fc region More particularly, the present invention concerns Fc region-containing polypeptides that have altered effector function as a consequence of one or more ammo acid substitutions in the Fc region of the nonvanant polypeptide The invention also relates to novel immune complexes and an assay for determining binding of an analyte, such as an Fc region-containing polypeptide, to a receptor Description of Related Art Antibodies are proteins, which exhibit binding specificity to a specific antigen Native antibodies are usually heterotetrameπcglycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobuiin isotypes Each heavy and light chain also has regularly spaced intrachain disulfide bridges Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains Each light chain has a variable domain at one end (V L ) and a constant domain at its other end, the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain Particular ammo acid residues are believed to form an interface between the light and heavy chain variable domains The term "variable" refers to the fact that certain portions of the vanable domains differ extensively in sequence among antibodies and are responsible for the binding specificity of each particular antibody for its particular antigen However, the variability is not evenly distributed through the variable domains of antibodies It is concentrated in three segments called complementarity determining regions (CDRs) both in the light chain and the heavy chain variable domains The more highly conserved portions of the variable domains are called the framework regions (FR) The variable domains of native heavy and light chains each comprise four FR regions largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat ef al , Sequences of Proteins of Immunological Interest, 5th Ed Public Health Service, National Institutes of Health, Bethesda, MD (1991)) The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions Depending on the ammo acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins can be assigned to different classes There are five major classes of immunoglobulins IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e g lgG1 , lgG2, lgG3, and lgG4, lgA1 and lgA2 The heavy chain constant regions that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, -1- respectively Of the various human immunoglobulinclasses only human lgG1 , lgG2, lgG3 and IgM are known to activate complement A schematic representation of the native lgG1 structure is shown in Fig 1 , where the various portions of the native antibody molecule are indicated Papain digestion of antibodies produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily The crystal structure of the human IgG Fc region has been determined (Deisenhofer, Biochemistry 20 2361-2370 (1981 )) In human IgG molecules the Fc region is generated by papain cleavage N-terminal to Cys 226 The Fc region is central to the effector functions of antibodies The effector functions mediated by the antibody Fc region can be divided into two categories (1 ) effector functions that operate after the binding of antibody to an antigen (these functions involve the participation of the complement cascade or Fc receptor (FcR)-bearιng cells), and (2) effector functions that operate independently of antigen binding (these functions confer persistence in the circulation and the ability to be transferred across cellular barriers by transcytosis) Ward and Ghetie, Therapeutic Immunology 2 77-94 (1995) While binding of an antibody to the requisite antigen has a neutralizing effect that might prevent the binding of a foreign antigen to its endogenous target (e g receptor or ligand), binding alone may not remove the foreign antigen To be efficient in removing and/or destructing foreign antigens, an antibody should be endowed with both high affinity binding to its antigen, and efficient effector functions C1α binding C1q and two serine proteases, Cl r and C1s, form the complex C1 , the first component of the complement dependent cytotoxicity (CDC) pathway C1q is a hexavalent molecule with a molecular weight of approximately 460,000 and a structure likened to a bouquet of tulips in which six collagenous "stalks" are connected to six globular head regions Burton and Woof, Advances in Immunol 51 1-84 (1992) To activate the complement cascade, it is necessary for C1q to bind to at least two molecules of lgG1 , lgG2, or lgG3 (the consensus is that lgG4 does not activate complement), but only one molecule of IgM, attached to the antigenic target Ward and Ghetie, Therapeutic Immunology 2 77-94 (1995) at page 80 Based upon the results of chemical modifications and crystallographic studies, Burton ef al (Nature, 288 338-344 (1980)) proposed that the binding site for the complement subcomponent C1q on IgG involves the last two (C-terminal) β-strands of the CH2 domain Burton later suggested (Molec Immunol , 22(3) 161-206 (1985)) that the region comprising ammo acid residues 318 to 337 might be involved in complement fixation Duncan and Winter (Nature 332 738-40 (1988)), using site directed mutagenesis, reported that Glu318, Lys320 and Lys322 form the binding site to C1q The data of Duncan and Winter were generated by testing the binding of a mouse lgG2b isotype to guinea pig C1q The role of Glu318, Lys320 and Lys322 residues in the binding of C1q was confirmed by the ability of a short synthetic peptide containing these residues to inhibit complement mediated lysis Similar results are disclosed -2- in U S Patent No 5,648,260 issued on July 15, 1997, and U S Patent No 5,624 821 issued on April 29, 1997 The residue Pro331 has been implicated in C1 q binding by analysis of the ability of human IgG subclasses to carry out complement mediated cell lysis Mutation of Ser331 to Pro331 in lgG4 conferred the ability to activate complement (Tao et al , J Exp Med , 178 661-667 (1993), Brekke ef al , Eur J Immunol , 24 2542-47 (1994)) From the comparison of the data of the Winter group, and the Tao ef al and Brekke et al papers, Ward and Ghetie concluded in their review article that there are at least two different regions involved in the binding of C1 q one on the β-strand of the CH2 domain bearing the Glu318, Lys320 and Lys322 residues, and the other on a turn located in close proximity to the same β-strand, and containing a key ammo acid residue at position 331 Other reports suggested that human lgG1 residues Lys235, and Gly237 located in the lower hinge region, play a critical role in complement fixation and activation Xu ef al ,J Immunol 150 152A (Abstract) (1993) W094/29351 published December 22, 1994 reports that ammo acid residues necessary for C1q and FcR binding of human lgG1 are located in the N-terminal region of the CH2 domain, i e residues 231 to 238 It has further been proposed that the ability of IgG to bind C1q and activate the complement cascade also depends on the presence, absence or modification of the carbohydrate moiety pos' oned between the two CH2 domains (which is normally anchored at Asn297) Ward and Ghetie, Therapeutic Immunology 2 77-94 ( 1995) at page 81 Fc receptor binding Effector functions can also be mediated by the interaction of the Fc region of an antibody with Fc receptors (FcRs), which are specialized cell surface receptors on hematopoietic cells Fc receptors belong in the immunoglobulin superfamily, and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes, and the lysing of erythrocytes and various other cellular targets (e g tumor cells) coated with the corresponding antibody, via antibody dependent cell mediated cytotoxicity (ADCC) Van de Winkel and Anderson, J Leuk Biol 49 511-24 (1991 ) FcRs are defined by their specificity for immunoglobulin isotypes, Fc receptors for IgG antibodies are referred to as FcγR, for IgE as FcεR, for IgA as FcαR and so on Three subclasses of gamma receptors have been identified FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) Because each FcγR subclass is encoded by two or three genes, and alternative RNA spicing leads to multiple transcripts, a broad diversity in FcγR isoforms exists The three genes encoding the FcγRI subclass (FcγRIA, FcγRIB and FcγRIC) are clustered in region 1q21 1 of the long arm of chromosome 1 , the genes encoding FcγRII isoforms (FcγRIIA, FcγRIIB and FcγRIIC) and the two genes encoding FcγRIII (FcγRIIIAand FcγRIIIB) are all clustered in region 1q22 FcRs are reviewed in Ravetch and Kmet, Annu Rev Immunol 9457-92 (1991 ), Capel ef al , Immunomethods 4 25-34 (1994), and de Haas et al , J Lab Clin Med 126 330-41 (1995) -3- While FcγRI binds monomeπc IgG with a high affinity, FcγRII and FcγRIII are low-affinity receptors, interacting with complexed or aggregated IgG The classical method for detecting these low- affinity receptors is by "rosettmg" using antibody-coated erythrocytes (EA) sensitized with IgGs Bredius ef al evaluated rosette formation between IgG-sensitized red blood cells and polymorphonuclear leukocytes (PMN) which express FcγRlla and FcγRlllb at their cell-surface Rosette was defined as three of more EA bound per PMN (Bredius ef al Immunology 83 624-630 (1994)) See, also, Tax ef al J Immunol 133(3) 1185-1189 (1984), Nagarajan ef al J Biol Chem 270(43) 25762-25770(1995), and Warmerdam ef al J Immunol 147(4) 1338-1343 (1991 ) concerning rosette assays However, binding of these EA "immune complexes" to FcR is not easily quantified Accordingly, more defined complexes with detectable affinity for these FcRs have been developed For example, IgG dimers have been formed using anti-light chain monoclonal antibodies (Nagarajan ef al , supra and Warmerdam ef al , supra) or chemical cross-linking agents (Hogarth ef al Immunomethods 4 17-24 (1994), and Tamm ef al J Biol Chem 271(7) 3659-3666(1996)) Heat-aggregated immune complexes have also been evaluated for binding to cells expressing FcRs (Tax ef al , supra and Tarn ef al , supra) The binding site for the FcγRs on human IgG was found to reside in the lower hinge region, primarily involving residues at ammo acid positions 233-238, all of which were found to be necessary for full FcγR binding activity Canfield and Morrison, J Exp Med 173 1483-91 (1991), Chappel ef al , Proc Natl Acad Sci USA, 88 9036-40 (1991), Lund et al , J Immunol , 147 2657-62 ( 391), Lund ef al , Molec Immunol , 29 53-59 (1992), Jeffensef a/ , Molec Immunol , 27 1237-40 (1990) and Sarmay ef al , Molec Immunol , 29 633-639 (1992) Pro331 in lgG3 was changed to Ser, and the affinity of this mutant to target cells analyzed The affinity was found to be six-fold lower than that of unmutated lgG3, indicating the involvement of Pro331 in FcγRI binding Morrison ef al , Immunologist, 2 119-124 (1994), and Canfield and Morrison, J Exp Med 173 1483-91 (1991) In addition, Glu318 was identified as being involved in binding to FcγRII Ward and Ghetie, Therapeutic Immunology 2 77-94 (1995) SUMMARY OF THE INVENTION The present invention provides a variant of a polypeptide comprising a human IgG Fc region, which variant comprises an ammo acid substitution at ammo acid position 270 or 329, or at two or more of ammo acid positions 270, 322, 329, and 331 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat The invention further relates to a variant of a polypeptide comprising a human IgG Fc region, which variant binds FcγRI, FcγRII, FcγRIII and FcRn but does not activate complement and comprises an ammo acid substitution at ammo acid position 322 or ammo acid position 329, or both ammo acid positions of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat The invention also pertains to a variant of a parent polypeptide comprising a human IgG Fc region, which variant has a better binding affinity for human C1q than the parent polypeptide and -4- comprises an ammo acid substitution in the IgG Fc region For example, the binding affinity of the variant for human C1q may be about two-fold or more improved compared to the binding affinity of the parent polypeptide for human C1q Preferably the parent polypeptide binds C1q and mediates CDC (for example, the parent polypeptide may comprise a human lgG1 , lgG2 or lgG3 Fc region) The variant with improved C1q binding preferably comprises an ammo acid substitution at one or more of am o acid positions 326 327, 333 and 334 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat In another aspect, the invention provides a variant of a polypeptide comprising a human IgG Fc region, which variant comprises an ammo acid substitution at ammo acid position 326, 327, 333 or 334 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat In yet a further aspect, the invention provides a method for modifying a polypeptide compπsing a human IgG Fc region comprising substituting an ammo acid residue at ammo acid position 270 or 329, or at two or more of ammo acid positions 270, 322, 329, and 331 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat The invention further provides a method for modifying a polypeptide comprising a human IgG Fc region comprising substituting an ammo acid residue at ammo acid position 326, 327, 333 or 334 of the human IgG Fc region, where the numbering of the residues in the IgG F - region is that of the EU index as in Kabat The method optionally further comprises a step wherein a variant with improved binding affinity for human C1q is identified The invention also provides a composition comprising the polypeptide variant and a physiologically acceptable carrier or diluent This composition for potential therapeutic use is sterile and may be lyophilized Diagnosticand therapeutic uses for the polypeptide variant are contemplated In one diagnostic application, the invention provides a method for determining the presence of a protein of interest comprising exposing a sample suspected of containing the protein to the polypeptide variant and determining binding of the polypeptide variant to the sample In one therapeutic application, the invention provides a method of treating a mammal suffering from a disorder comprising administering to the mammal a therapeutically effective amount of a variant of a polypeptide comprising a human IgG Fc region, which variant binds FcγRI, FcγRII, FcγRIII and FcRn but does not activate complement and comprises an ammo acid substitution at ammo acid position 270, 322, 329 or 331 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat The invention further provides isolated nucleic acid encoding the polypeptide variant, a vector comprising the nucleic acid, optionally, operably linked to control sequences recognized by a host cell transformed with the vector, a host cell comprising the vector, a process for producing the polypeptide variant comprising cultuπng this host cell so that the nucleic acid is expressed and, optionally, recovering the polypeptide variant from the host cell culture (e g from the host cell culture medium) The invention also pertains to an immune complex comprising (a) an Fc region-containing polypeptide, (b) a first target molecule which comprises at least two binding sites for the Fc region- -5- containing polypeptide, and (c) a second target molecule comprises at least two binding sites for the first target molecule The immune complex may be used in an FcR-bindmg assay, particularly where the FcR has a low affinity for the Fc region-containing polypeptide Other uses for the immune complex are disclosed herein Moreover, the invention provides a method for determining binding of an analyte, such as an Fc region-containing polypeptide, to a receptor (e g a low affinity FcR) comprising the following steps performed sequentially (a) forming a molecular complex between the analyte and a first target molecule, wherein the first target molecule comprises at least two binding sites for the analyte and (b) determining binding of the molecular complex of step (a) to the receptor (e g to a binding domain of the receptor coated on an assay plate) Optionally, the molecular complex of step (a) further comprises a second target molecule which comprises at least two binding sites for the first target molecule The invention also relates to an assay kit, such as a kit useful for determining binding of an analyte to a receptor comprising (a) a first target molecule which comprises at least two binding sites for the analyte, and (b) a second target molecule which comprises at least two binding sites for the first target molecule Brief Description of the Drawings Figure 1 is a schematic representation of a native IgG Disulfide bonds are represented by heavy lines between CH1 and CL domains and the two CH2 d mains V is variable domain, C is constant domain, L stands for light chain and H stands for heavy chain Figure 2 shows C1q binding of wild type (wt) C2B8 antibody, C2B8 antibody with a human lgG2 constant region (lgG2), and mutants K322A, K320A and E318A Figure 3 depicts C1q binding of mutants P331A, P329A and K322A Figures 4A (SEQ ID NO 1) and 4B (SEQ ID NO 2) depict the ammo acid sequences of E27 anti- IgE antibody light chain (Fig 4A) and heavy chain (Fig 4B) Figure 5 is a schematic diagram of the "immune complex" prepared for use in the FcR assay described in Example 1 The hexamer comprising three anti-lgE antibody molecules (the "Fc region- containing polypeptide") and three IgE molecules (the "first target molecule") is shown IgE has two "binding sites" for the anti-lgE antibody (E27) in the Fc region thereof Each IgE molecule in the complex is further able to bind two VEGF molecules ("the second target polypeptide") VEGF has two "binding sites" for IgE Figure 6 shows C1q binding results obtained for mutants D270K and D270V compared to wild type C2B8 Figure 7 depicts complement dependent cytotoxicity (CDC) of mutants D270K and D270V, compared to wild type C2B8 Figure 8 shows C1q binding ELISA results for 293 cell-produced wild type C2B8 antibody (293- Wt-C2B8), CHO-produced wild type C2B8 antibody (CHO-Wt-C2B8) and various mutant antibodies Figure 9 shows C1q binding ELISA results obtained for wild type (wt) C2B8 and various mutant antibodies as determined in Example 3 -6- Figure 10 depicts the three-dimensional structure of a human IgG Fc highlighting residues Asp270, Lys326, Pro329, Pro331 , Lys322 and Glu333 Figure 11 shows C1q binding ELISA results obtained for wild type C2B8 and various mutant antibodies as determined in Example 3 Figure 12 shows C1q binding ELISA results obtained for wild type C2B8 and double mutants, K326M-E333S and K326A-E333A Figure 13 shows CDC of wild type C2B8 and double mutants, K326M-E333S and K326A-E333A Figure 14 depicts C1q binding ELISA results obtained for C2B8 with a human lgG4 heavy chain constant region (lgG4), wild type C2B8 (Wt-C2B8), C2B8 with a human lgG2 heavy chain constant region (lgG2), and mutant antibodies as described in Example 3 Detailed Description of the Preferred Embodiments I. Definitions Throughoutthe present specification and claims, the numbering of the residues in an IgG heavy chain is that of the EU index as in Kabat et al , Sequences of Proteins of Immunological Interest 5th Ed Public Health Service, National Institutes of Health, Bethesda, MD (1991), expressly incorporated herein by reference The "EU index as in Kabat" refers to the residue numbering of the human lgG1 EU antibody The term "Fc region" is used to define a C-terminal region of an IgG heavy chain as shown in Figure 1 Although the boundaries of the Fc region of an igG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to stretch from ammo acid residue at position Cys226 to the carboxyl-terminus The term "Fc region-containing polypeptide" refers to a polypeptide, such as an antibody or immunoadhesin (see definitions below), which comprises an Fc region The Fc region of an IgG comprises two constant domains, CH2 and CH3, as shown in Figure 1 The "CH2" domain of a human IgG Fc region (also referred to as "Cγ2" domain) usually extends from am o acid 231 to ammo acid 340 The CH2 domain is unique in that it is not closely paired with another domain Rather, two N-lmked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain Burton, Molec Immunol 22 161-206 (1985) "Hinge region" is generally defined as stretching from Glu216 to Pro230 of human lgG1 (Burton, Molec lmmunol 22 161-206 (1985)) Hinge regions of other IgG isotypes may be aligned with the lgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S-S bonds in the same positions "C1q" is a polypeptide that includes a binding site for the Fc region of an immunoglobulin C1q together with two serine proteases, C1r and C1s, forms the complex C1 , the first component of the complement dependent cytotoxicity (CDC) pathway Human C1q can be purchased commercially from, e g Quidel, San Diego, CA The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody The preferred FcR is one, which binds an IgG antibody (a gamma receptor) and includes -7- receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors FcRs are reviewed in Ravetch and Kinet, Annu Rev Immunol 9 457-92 (1991 ), Capel ef al , Immunomethods 4 25-34 (1994), and de Haas et al . J Lab Clin Med 126 330-41 (1995) Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer ef al J Immunol 117 587 (1976) and Kim ef al , J Immunol 24249 (1994)) The term "binding domain" refers to the region of a polypeptide that binds to another molecule In the case of an FcR, the binding domain can comprise a portion of a polypeptide chain thereof (e g the α chain thereof) which is responsible for binding an Fc region One useful binding domain is the extracellular domain of an FcR α chain The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e g , bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity "Antibody fragments", as defined for the purpose of the present invention, comprise a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody, so long as they retain at least the CH2 region of an IgG immunoglobulin heavy chain constant domain, compπsing ammo acid residues 322, 329 ε> α 331, and have the ability, alone or in combination with anotner antibody fragment, to specifically bind a selected antigen Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments The antibody fragments preferably retain at least part of the hinge and optionally the CH1 region of an IgG heavy chain More preferably, the antibody fragments retain the entire constant region of an IgG heavy chain, and include an IgG light chain The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, / e , the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts Monoclonal antibodies are highly specific, being directed against a single antigenic site Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybπdoma method first described by Kohleref al , Nature 256 495 (1975), or may be made by recombinant DNA methods (see, e g , U S Patent No 4,816,567) The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson ef al , Nature 352 624-628 (1991 ) and Marks ef al , J Mol Biol 222 581-597 (1991), for example The monoclonal antibodies herein specifically include "chimeπc" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding -8- sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass while the remainder of the chaιn(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U S Patent No 4,816,567, and Morrison et al , Proc Natl Acad Sci USA 81 6851-6855 (1984)) "Humanized" forms of non-human (e g , muπne) antibodies are chimeπc antibodies that contain minimal sequence derived from non-human immunoglobulin For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervaπable region of the recipient are replaced by residues from a hypervaπable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody These modifications are made to further refine antibody performance In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervaπable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin For further details, see Jones ef al , Nature 321.522-525 (1986), Riechmann et al , Nature 332 323-329 (1988), and Presta, Curr Op Struct Biol 2 593-596 (1992) The term "hypervaπable region" when used herein refers to the ammo acid residues of an antibody which are responsible for antigen-binding The hypervaπable region comprises ammo acid residues from a "complementaπty determining region" or "CDR" (/ e residues 24-34 (L1 ), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain, Kabat ef al , Sequences of Proteins of Immunological Interest, 5th Ed Public Health Service, National Institutes of Health, Bethesda, MD (1991 )) and/or those residues from a "hypervaπable loop" (i e. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1 ), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain, Chothia and Lesk J Mol Biol 196 901-917 (1987)) "Framework" or "FR" residues are those variable domain residues other than the hypervanable region residues as herein defined As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the "binding domain" of a heterologous "adhesin" protein (e g a receptor, ligand or enzyme) with an immunoglobulin constant domain Structurally, the immunoadhesins comprise a fusion of the adhesin ammo acid sequence with the desired binding specificity which is other than the antigen recognition and binding site (antigen combining site) of an antibody (/ e is "heterologous") and an immunoglobulin constant domain sequence The term "ligand binding domain" as used herein refers to any native cell-surface receptor or any region or derivative thereof retaining at least a qualitative ligand binding ability of a corresponding native receptor In a specific embodiment, the receptor is from a cell-surface polypeptide having an -9- extracellular domain that is homologous to a member of the immunoglobulin supergenefamily Other receptors, which are not members of the immunoglobulin supergenefamily but are nonetheless specifically covered by this definition, are receptors for cytokines, and in particular receptors with tyrosme kmase activity (receptor tyrosine kinases), members of the hematopoietin and nerve growth factor receptor superfamilies, and cell adhesion molecules, e g (E-, L- and P-) selectins The term "receptor binding domain" is used to designate any native ligand for a receptor, including ceil adhesion molecules, or any region or derivative of such native ligand retaining at least a qualitative receptor binding ability of a corresponding native ligand This definition, among others, specifically includes binding sequences from ligands for the above-mentioned receptors An "antibody-immunoadhesin chimera" comprises a molecule that combines at least one binding domain of an antibody (as herein defined) with at least one immunoadhesin (as defined in this application) Exemplary antibody-immunoadhesin chimeras are the bispecific CD4-lgG chimeras described in Berg ef a/ , PNAS (USA) 88 4723-4727 (1991 ) and Chamow ef al , J Immunol 153 4268 (1994) An "isolated" polypeptide is one that has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other protemaceousor nonprote aceous solutes In preferred embodiments, the polypeptide will be purified (1) to greater than 95% by weight of polypeptide as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficientto obtain at least 15 residues of N-terminal or internal ammo acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducmg conditions using Coomassie blue or, preferably, silver stain Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present Ordinarily however isolated polypeptide will be prepared by at least one purification step "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented A "disorder" is any condition that would benefit from treatment with the polypeptide variant This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question The word "label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the polypeptide The label may be itself be detectable (e g , radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide nucleic acid An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature Isolated nucleic acid molecules therefore are distinguished from -10- the nucleic acid molecule as it exists in natural cells However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells The expression "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a πbosome binding site Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprote that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a πbosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase However, enhancers do not have to be contiguous Linking is accomplished by ligation at convenient restriction sites If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventiria! practice As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included Where distinct designations are intended, it will be clear from the context The term "molecular complex" when used herein refers to the relatively stable structure which forms when two or more heterologous molecules (e.g polypeptides) bind (preferably noncovalently) to one another The preferred molecular complex herein is an immune complex "Immune complex" refers to the relatively stable structure which forms when at least one target molecule and at least one heterologous Fc region-containing polypeptide bind to one another forming a larger molecular weight complex Examples of immune complexes are antigen-antibody aggregates and target molecule-immunoadhesin aggregates The term "immune complex" as used herein, unless indicated otherwise, refers to an ex vivo complex (/ e other than the form or setting in which it may be found in nature) However, the immune complex may be administered to a mammal, e g to evaluate clearance of the immune complex in the mammal The term "target molecule" refers to a molecule, usually a polypeptide, which is capable of being bound by a heterologous molecule and has one or more binding sites for the heterologous molecule The term "binding site" refers to a region of a molecule to which another molecule can bind The "first target molecule" herein comprises at least two distinct binding sites (for example, two to five separate binding -1 1 - sites) for an analyte (e g an Fc region-containing polypeptide) such that at least two analyte molecules can bind to the first target molecule In the preferred embodiment of the invention, the two or more binding sites are identical (e g having the same ammo acid sequence, where the target molecule is a polypeptide) In Example 1 below, the first target molecule was IgE and had two separate binding sites in the Fc region thereof to which the Fc region-containing polypeptide (an anti-lgE antibody, E27) could bind Other first target molecules include dimers of substantially identical monomors (e g neurotrophms, IL8 and VEGF) or are polypeptides comprising two or more substantially identical polypeptide chains (e g antibodies or immunoadhesins) The "second target molecule" comprises at least two distinct binding sites (for example, two to five separate binding sites) for the first target molecule such that at least two first target molecules can bind to the second target molecule Preferably, the two or more binding sites are identical (e g having the same ammo acid sequence, where the target molecule is a polypeptide) In Example 2, the second target molecule was VEGF, which has a pair of distinct binding sites to which the variable domain of the IgE antibody could bind Other second target molecules are contemplated, e g other dimers of substantially identical monomers (e g neurotrophms or IL8) or polypeptides comprising two or more substantially identical domains (e g antibodies or immunoadhesins) An "analyte" is a substance that is to be analyzed The preferred analyte is an Fc region- con^inmg polypeptide that is to be analyzed for its ability to bind to an Fc receptor A "receptor" is a polypeptide capable of binding at least one ligand The prefe r red receptor is a cell-surface receptor having an extracellularligand-bindingdomain and, optionally, other domains (e transmembrane domain, intracellular domain and/or membrane anchor) The receptor to be evaluated in the assay described herein may be an intact receptor or a fragment or derivative thereof (e g a fusion protein comprising the binding domain of the receptor fused to one or more heterologous polypeptides) Moreover, the receptor to be evaluated for its binding properties may be present in a cell or isolated and optionally coated on an assay plate or some other solid phase The phrase "low affinity receptor" denotes a receptorthat has a weak binding affinity for a ligand of interest, e g having a binding constant of about 50nM or worse affinity Exemplary low affinity receptors include FcγRII and FcγRIII as well as adhesion molecules, such as integnns A "parent polypeptide" is a polypeptide comprising an ammo acid sequence which lacks one or more of the Fc region substitutions disclosed herein and/or which differs in effector function compared to a polypeptide variant as herein disclosed The parent polypeptide may comprise a native sequence Fc region or an Fc region with existing ammo acid sequence modifications (such as additions, deletions and/or substitutions) II. Modes for Carrying Out the Invention The invention herein relates to a method for making a polypeptide variant The "parent", "starting" or "nonvaπant" polypeptide is prepared using techniques available in the art for generating polypeptides comprising an Fc region In the preferred embodiment of the invention, the polypeptide is an antibody and exemplary methods for generating antibodies are described in more detail in the following sections The polypeptide may, however, be any other polypeptide comprising an Fc region, -12- e g an immunoadhesin Methods for making immunoadhesins are elaborated in more detail herembelow The starting polypeptide of particular interest herein is usually one that binds to C1q and displays complement dependent cytotoxicity (CDC) The ammo acid substitutions described herein will generally serve to alter the ability of the starting polypeptide to bind to C1 q and/or modify its complement dependent cytotoxicity function, e g to reduce and preferably abolish these effector functions However, polypeptides comprising substitutions at one or more of the described positions with improved effector functions are contemplated herein For example, the starting polypeptide may be unable to bind C1q and/or mediate CDC and may be modified according to the teachings herein such that it acquires these effector functions Moreover, polypeptides with pre-existing C1q binding activity, optionally further having the ability to mediate CDC may be modified such that one or both of these activities are enhanced To generate the polypeptide variant, one or more ammo acid alterations (e g substitutions) are introduced in the Fc region of the starting polypeptide The ammo acid positions to be modified are generally selected from heavy chain positions 270, 322, 326, 327, 329, 331 333, and 334, where the numbering of the residues in an IgG heavy chain is that of the EU index as in Kabat ef al , Sequences of Proteins of Immunological Interest, 5th Ed Public Health Service, National Institutes of Health, Bethesda, MD (1991 ) The Fc region is preferably from a human IgG and mcst preferably a human lgG1 or human lgG3 The human lgG1 Fc region may be a human A or non-A allotype Prolme is conserved at position 329 in human IgG's This residue is preferably replaced with alanine, however substitution with any other am o acid is contemplated, e g , serine, threonme, asparagine, glycine or valine Prolme is conserved at position 331 in human lgG1, lgG2 and lgG3, but not lgG4 (which has a serine residue at position 331 ) Residue 331 is preferably replaced by alanine or another ammo acid, e g serine (for IgG regions other than lgG4), glycine or valine Lysine 322 is conserved in human IgGs, and this residue is preferably replaced by an alanine residue, but substitution with any other ammo acid residue is contemplated, e g serine, threonme, glycine or valine D270 is conserved in human IgGs, and this residue may be replaced by another ammo acid residue, e g alanine, serine, threonme, glycine, valine, or lysine K326 is also conserved in human IgGs This residue may be substituted with another residue including, but not limited to, valine, glutamic acid, alanine, glycine, aspartic acid, methionine or tryptophan, with tryptophan being preferred Likewise, E333 is also conserved in human IgGs E333 is preferably replaced by an ammo acid residue with a smaller side chain volume, such as valine, glycine, alanine or serine, with serine being preferred K334 is conserved in human IgGs and may be substituted with another residue such as alanine or other residue -13- In human lgG1 and lgG3, residue 327 is an alanine In order to generate a variant with improved C1q binding, this alanine may be substituted with another residue such as glycine In lgG2 and lgG4, residue 327 is a glycine and this may be replaced by alanine (or another residue) to diminish C1q binding Preferably, substitutions at two, three or all of positions 326, 327, 333 or 334 are combined, optionally with other Fc region substitutions, to generate a polypeptide with improved human C1 q binding and preferably improved CDC activity in vitro or in vivo In one embodiment, only one of the eight above-identified positions is altered in order to generate the polypeptide variant Preferably only residue 270, 329 or 322 is altered if this is the case Alternatively, two or more of the above-identified positions are modified If substitutions are to be combined, generally substitutions which enhance human C1 q binding (e g at residue positions 326, 327, 333 and 334) or those which dimmish human C1 q binding (e g , at residue positions 270, 322, 329 and 331 ) are combined In the latter embodiment, all four positions (; e , 270, 322, 329 and 331) may be substituted A variant may be generated in which the native ammo acid residue at position 329 of the human heavy chain constant region is substituted with another ammo acid, optionally in combination with a substitution of the amino acid residue at position 331 and/or substitution of the ammo acid residue at position 322 Otherwise, the native am o acid residue at position 331 and the native ammo acid residue at position 322 of the human IgG Fc region may both be substituted with another ammo acid residue One may also combine an ammo acid substitution at position 270 with further substιtutιon(s) at posιtιon(s) 322, 329 and/or 331 DNA encoding am o acid sequence variant of the starting polypeptide is prepared by a variety of methods known in the art These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding the polypeptide Site-directed mutagenesis is a preferred method for preparing substitution variants This technique is well known in the art (see, e g , Carter ef al Nucleic Acids Res 13 4431-4443 (1985) and Kunkel etal , Proc Natl Acad Sci USA 82 488 (1987)) Briefly, in carrying out site-directed mutagenesis of DNA, the starting DNA is altered by first hybridizing an oligonucleotideencodmg the desired mutation to a single strand of such starting DNA After hybridization, a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and using the single strand of the starting DNA as a template Thus, the oligonucleotide encoding the desired mutation is incorporated in the resulting double-stranded DNA PCR mutagenesis is also suitable for making ammo acid sequence variants of the starting polypeptide See Htguchi, in PCR Protocols, pp 177-183 (Academic Press, 1990), and Vallette et al , Nuc Acids Res 17 723-733 (1989) Briefly, when small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template -14- Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells ef al , Gene 34 315-323 (1985) The starting material is the plasmid (or other vector) comprising the starting polypeptide DNA to be mutated The codon(s) in the starting DNA to be mutated are identified There must be a unique restriction endonuclease site on each side of the identified mutation sιte(s) If no such restriction sites exist, they may be generated using the above-described o gonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the starting polypeptide DNA The plasmid DNA is cut at these sites to linearize it A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutatιon(s) is synthesized using standard procedures, wherein the two strands of the oligonucleotide are synthesized separately and then hybridized together using standard techniques This double-stranded oligonucleotide is referred to as the cassette This cassette is designed to have 5' and 3' ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid This plasmid now contains the mutated DNA sequence Alternatively, or additionally, the desired ammo acid sequence encoding a polypeptide variant can be determined, and a nucleic acid sequence encoding such ammo acid sequence variant can be generated synthetically The polypeptide varιant(s) so prepared may be subjected to further modifications, oftentimes depending on the intended use of the polypeptida Such modifications may involve further alteration of the am o acid sequence, fusion to heterologous polypeptιde(s) and/or covalent modifications For example, it may be useful to combine the above am o acid substitutions with one or more further ammo acid substitutions that reduce or ablate FcR binding For example, the native am o acid residues at any one or more of heavy chain positions 233-238, 318 or 331 (where the numbering of the residues in an IgG heavy chain is that of the EU index as in Kabat ef al , supra) may be replaced with non-native resιdue(s), e g alanine With respect to further ammo acid sequence alterations, any cysteine residue not involved in maintaining the proper conformation of the polypeptide variant also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross linking Another type of ammo acid substitution serves to alter the glycosylation pattern of the polypeptide This may be achieved by deleting one or more carbohydrate moieties found in the polypeptide, and/or adding one or more glycosylation sites that are not present in the polypeptide Glycosylation of polypeptides is typically either N-lmked or O-lmked N-lmked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue The tnpeptide sequences asparagine-X-seπne and asparagme-X-threonine, where X is any ammo acid except prolme, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain Thus, the presence of either of these tnpeptide sequences in a polypeptide creates a potential glycosylation site O-lmked glycosylation refers to the attachment of one of the sugars N- aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonme, although 5-hydroxyprolιne or 5-hydroxylysιne may also be used Addition of glycosylation sites to the polypeptide is conveniently accomplished by altering the am o acid sequence such that it contains one -15- or more of the above-described tnpeptide sequences (for N-lmked glycosylation sites) The alteration may also be made by the addition of, or substitution by, one or more serine or threonme residues to the sequence of the original polypeptide (for 0-lιnked glycosylation sites) An exemplary glycosylation variant has an ammo acid substitution of residue Asn 297 of the heavy chain The polypeptide variant may be subjected to one or more assays to evaluate any change in biological activity compared to the starting polypeptide For example, the ability of the variant to bind C1q and mediate complement dependent cytotoxicity (CDC) may be assessed To determine C1q binding, a C1q binding ELISA may be performed Briefly, assay plates may be coated overnight at 4°C with polypeptide variant or starting polypeptide (control) in coating buffer The plates may then be washed and blocked Following washing, an aliquot of human C1q may be added to each well and incubated for 2 hrs at room temperature Following a further wash, 100 μl of a sheep antι-complementC1q peroxidase conjugated antibody may be added to each well and incubated for 1 hour at room temperature The plate may again be washed with wash buffer and 100 μl of substrate buffer containing OPD (0-phenylenedιamιnedιhydrochloπde (Sigma)) may be added to each well The oxidation reaction, observed by the appearance of a yellow color, may be allowed to proceed for 30 minutes and stopped by the addition of 100 μl of 4 5 N H 2 S0 4 The absorbance may then read at (492-405) nm An exemplary polypeptide variant is one that displays a "significant reduction in C1q binding' in this assay This means that about 10Oμg/ml of the polypeptide variant displays about 50 fold or more reduction in C1q binding compared to 100μg/ml of a control antibody having a nonmutated lgG1 Fc region In the most preferred embodiment, the polypeptide variant "does not bind C1q", / e 100μg/ml of the polypeptide variant displays about 100 fold or more reduction in C1q binding compared to 100μg/ml of the control antibody Another exemplary variant is one which "has a better binding affinity for human C1q than the parent polypeptide" Such a molecule may display, for example, about two-fold or more, and preferably about five-fold or more, improvement in human C1q binding compared to the parent polypeptide (e g at the IC 50 values for these two molecules) For example, human C1q binding may be about two-fold to about 500-fold, and preferably from about two-fold or from about five-fold to about 1000-fold improved compared to the parent polypeptide To assess complement activation, a complement dependent cytotoxicity (CDC) assay may be performed, e g as described in Gazzano-Santoro et al , J Immunol Methods 202 163 (1996) Briefly, various concentrations of the polypeptide variant and human complement may be diluted with buffer Cells which express the antigen to which the polypeptide variant binds may be diluted to a density of ~1 x 10 6 cells /ml Mixtures of polypeptide variant, diluted human complement and cells expressing the antigen may be added to a flat bottom tissue culture 96 well plate and allowed to incubate for 2 hrs at 37°C and 5% C0 2 to facilitate complement mediated cell lysis 50 μl of alamar blue (Accumed International) may then be added to each well and incubated overnight at 37°C The absorbance is measured using a 96-well fluorometer with excitation at 530 nm and emission at 590 nm The results -16- may be expressed in relative fluorescence units (RFU) The sample concentrations may be computed from a standard curve and the percent activity as compared to nonvaπant polypeptide is reported for the polypeptide variant of interest Yet another exemplary variant ' does not activate complement" For example, 0 6 μg/ml of the polypeptide variant displays about 0-10% CDC activity in this assay compared to a 0 6 μg/ml of a control antibody having a nonmutated lgG1 Fc region Preferably the variant does not appear to have any CDC activity in the above CDC assay The invention also pertains to a polypeptide variant with enhanced CDC compared to a parent polypeptide, e g , displaying about two-fold to about 100-fold improvement in CDC activity in vitro or in vivo (e g at the IC 50 values for each molecule being compared) Preferably the polypeptide variant essentially retains the ability to bind antigen compared to the nonvaπant polypeptide, ; e the binding capability is no worse than about 20 fold, e g no worse than about 5 fold of that of the nonvanant polypeptide The binding capability of the polypeptide variant may be determined using techniques such as fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA), for example The ability of the polypeptide variant to bind an FcR may also be evaluated Where the FcR is a high affinity Fc receptor, such as FcγRI or FcRn, binding can be measured by titrating monomenc polypeptide variant and measuring bound polypeptide variant using an antibody which specifical'y binds to the polypeptide variant in a standard ELISA format (see Example 2 below) Another FcR binding assay for low affinity FcRs is elaborated in more detail in the following section Preferably the variant retains the ability to bind one or more FcRs, e g the ability of the polypeptide variant to bind FcγRI, FcγRII, FcγRIII and/or FcRn is no more than about 20 fold reduced, preferably no more than about 10 fold reduced, and most preferably no more than about two fold reduced compared to the starting polypeptide as determined in the FcγRI or FcRn assays of Example 2 or the FcγRII or FcγRIII assays described in the following section A. Receptor Binding Assay and Immune Complex A receptor binding assay has been developed herein which is particularly useful for determining binding of an analyte of interest to a receptor where the affinity of the analyte for the receptor is relatively weak, e g in the micromolar range as is the case for FcγRlla, FcγRllb, FcγRllla and FcγRlllb The method involves the formation of a molecular complex that has an improved avidity for the receptor of interest compared to the noncomplexed analyte The preferred molecular complex is an immune complex comprising (a) an Fc region-containing polypeptide (such as an antibody or an immunoadhesin), (b) a first target molecule which comprises at least two binding sites for the Fc region- containing polypeptide, and (c) a second target molecule which comprises at least two binding sites for the first target molecule In Example 1 below, the Fc region-containing polypeptide is an anti-lgE antibody, such as the E27 antibody (Figs 4A-4B) E27, when mixed with human IgE at an 1 1 molar ratio, forms a stable hexamer consisting of three E27 molecules and three IgE molecules In Example 1 below the "first -17- target molecule is a chimeπc form of IgE in which the Fab portion of an anti-VEGF antibody is fused to the human IgE Fc portion and the "second target molecule" is the antigen to which the Fab binds (/ e VEGF) Each molecule of IgE binds two molecules of VEGF VEGF also binds two molecules of IgE per molecule of VEGF When recombinant human VEGF was added at a 2 1 molar ratio to IgE E27 hexamers, the hexamers were linked into larger molecular weight complexes via the IgE VEGF interaction (Fig 5) The Fc region of the anti-lgE antibody of the resultant immune complex binds to FcR with higher avidity than either uncomplexed anti-lgE or anti-lgE IgE hexamers Other forms of molecular complexes for use in the receptor assay are contemplated Examples comprising only an Fc region-containing polypeptide first target molecule combination include an immunoadhesin ligand combination such as VEGF receptor (KDR)-ιmmunoadhesιn VEGF and a full- length bispecific antibody (bsAb) first target molecule A further example of an Fc region-containing polypeptide first target molecule second target molecule combination include a nonlocking antibody soluble receptor ligand combination such as anti-Trk antibody soluble Trk receptor neurotrophin (Urfer ef a/ J Biol Chem 273(10) 5829-5840 (1998)) Aside from use in a receptor binding assay, the immune complexes described above have further uses including evaluation of Fc region-containing polypeptide function and immune complex clearance in vivo Hence, the immune complex may be administered to a mammal (e g in a pre-clinical animal study) and evaluated for its half-life etc To determine receptor binding, a polypeptide comprising at least the binding domain of the receptor of interest (e g the extracellular domain of an α subunit of an FcR) may be coated on solid phase, such as an assay plate The binding domain of the receptor alone or a receptor-fusion protein may be coated on the plate using standard procedures Examples of receptor-fusion proteins include receptor-glutathione S-transferase (GST) fusion protein, receptor-chitin binding domain fusion protein, receptor-hexaHis tag fusion protein (coated on glutathione, chitin, and nickel coated plates, respectively) Alternatively, a capture molecule may be coated on the assay plate and used to bind the receptor-fusion protein via the non-receptor portion of the fusion protein Examples include anti-hexaHis F(ab') 2 coated on the assay plate used to capture receptor-hexaHis tail fusion or anti-GST antibody coated on the assay plate used to capture a receptor-GST fusion In other embodiments, binding to cells expressing at least the binding domain of the receptor may be evaluated The cells may be naturally occurring hematopoietic cells that express the FcR of interest or may be transformed with nucleic acid encoding the FcR or a binding domain thereof such that the binding domain is expressed at the surface of the cell to be tested The immune complex described heremabove is added to the receptor-coated plates and incubated for a sufficient period of time such that the analyte binds to the receptor Plates may then be washed to remove unbound complexes, and binding of the analyte may be detected according to known methods For example, binding may be detected using a reagent (e g an antibody or fragment thereof) which binds specifically to the analyte, and which is optionally conjugated with a detectable label (detectable labels and methods for conjugating them to polypeptides are described below in the section entitled "Non-Therapeutic Uses for the Polypeptide Variant") -18- As a matter of convenience, the reagents can be provided in an assay kit, / e , a packaged combination of reagents, for combination with the analyte in assaying the ability of the analyte to bind to a receptor of interest The components of the kit will generally be provided in predetermined ratios The kit may provide the first target molecule and/or the second target molecule, optionally complexed together The kit may further include assay plates coated with the receptor or a binding domain thereof (e the extracellular domain of the α subunit of an FcR) Usually, other reagents, such as an antibody that binds specifically to the analyte to be assayed, labeled directly or indirectly with an enzymatic label, will also be provided in the kit Where the detectable label is an enzyme, the kit will include substrates and cofactors required by the enzyme (e g a substrate precursor which provides the detectable chromophore or fluorophore) In addition, other additives may be included such as stabilizers, buffers (e g assay and/or wash lysis buffer) and the like The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents that substantially optimize the sensitivity of the assay Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients that on dissolution will provide a reagent solution having the appropriate concentration The kit also suitably includes instructions for carrying out the assay B. Antibody Preparation In the preferred embodiment of the invention, the Fc region-containing polypeptide which is modified according to the teachings herein is an antibody Techniques for producing antibodies follow (i) Antigen selection and preparation Where the polypeptide is an antibody, it is directed against an antigen of interest Preferably, the antigen is a biologically important polypeptide and administration of the antibody to a mammal suffering from a disease or disorder can result in a therapeutic benefit in that mammal However, antibodies directed against nonpoly peptide antigens (such as tumor-associated glycolipid antigens, see US Patent 5,091,178) are also contemplated Where the antigen is a polypeptide, it may be a transmembrane molecule (e g receptor) or ligand such as a growth factor Exemplary antigens include molecules such as renin, a growth hormone, including human growth hormone and bovine growth hormone, growth hormone releasing factor, parathyroid hormone, thyroid stimulating hormone, lipoprotems, alpha-1-antιtrypsιn, insulin A-cham, insulin B-cham, promsulin, follicle stimulating hormone, calcitonm, luteinizing hormone, glucagon, clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor, anti-clotting factors such as Protein C, atnal natnuretic factor, lung surfactant, a plasminogen activator, such as urokmase or human urine or tissue-type plasminogen activator (t-P A), bombesm, thrombm, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkepha nase, RANTES (regulated on activation normally T-cell expressed and secreted), human macrophage inflammatory protein (MIP-1-alpha), a serum albumin such as human serum albumin, Muelleπan-inhibiting substance, relaxin A-cham, relaxin B-cham, prorelaxin, mouse gonadotropm-associated peptide, a microbial protein, such as beta-lactamase, DNase, IgE, a cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4, mhibin, activin, vascular endothehal growth factor (VEGF), receptors for hormones or growth factors, protein A or D, rheumatoid factors, a neurotrophic factor such as bone-derived neurotrophic factor (BDNF) -19- neurotrophιn-3 -4, -5, or -6 (NT-3 NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-β, platelet-derived growth factor (PDGF), fibroblast growth factor such as aFGF and bFGF, epidermal growth factor (EGF), transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF- β1 , TGF-β2, TGF-β3, TGF-β4, orTGF-β5, insulin-like growth factor-l and -II (IGF-I and IGF-II), des(1-3)- IGF-I (brain IGF-I), insulin-like growth factor binding proteins, CD proteins such as CD3, CD4, CD8, CD19 and CD20 erythropoietm, osteoinductive factors, immunotoxins, a bone morphogenetic protein (BMP), an mterferon such as terferon-alpha, -beta, and -gamma, colony stimulating factors (CSFs), e g , M-CSF, GM-CSF, and G-CSF, interleukms (ILs), e g , IL-1 to IL-10, superoxide dismutase, T-cell receptors, surface membrane proteins, decay accelerating factor, viral antigen such as, for example, a portion of the AIDS envelope, transport proteins, homing receptors, address s, regulatory proteins, integπns such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and VCAM, a tumor associated antigen such as HER2, HER3 or HER4 receptor, and fragments of any of the above-listed polypeptides Preferred molecular targets for antibodies encompassed by the present invention include CD proteins such as CD3, CD4, CD8, CD19, CD20 and CD34, members of the ErbB receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor, cell adhesion molecules such as LFA-1, Mad , p150 95, VLA-4, ICAM-1 , VCAM and αv/β3 integπn including either α or β subunits thereof (e g anti- CD11 a, antι-CD18 or antι-CD11 b antibodies), growth factors such as VEGF, IgE, blood group antigens, flk2/flt3 receptor, obesity (OB) receptor, mpl receptor, CTLA-4, protein C etc Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens for generating antibodies For transmembrane molecules, such as receptors, fragments of these (e g the extracellular domain of a receptor) can be used as the immunogen Alternatively, cells expressing the transmembrane molecule can be used as the immunogen Such cells can be derived from a natural source (e cancer cell lines) or may be cells which have been transformed by recombinant techniques to express the transmembrane molecule Other antigens and forms thereof useful for preparing antibodies will be apparent to those in the art (II) Polyclonal antibodies Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e g , keyhole limpet hemocyanin, serum albumin, bovine thyroglobulm, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succmic anhydride, SOCI 2 , or R 1 N=C=NR, where R and R 1 are different alkyl groups Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites One month later the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites Seven to 14 days later the -20- animals are bled and the serum is assayed for antibody titer Animals are boosted until the titer plateaus Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent Conjugates also can be made in recombinant cell culture as protein fusions Also, aggregating agents such as alum are suitably used to enhance the immune response (in) Monoclonal antibodies Monoclonal antibodies may be made using the hybπdoma method first described by Kohler ef al , Nature, 256495 (1975), or may be made by recombinant DNA methods (U S Patent No 4,816,567) In the hybπdoma method, a mouse or other appropriate host animal, such as a hamster or macaque monkey, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization Alternatively, lymphocytes may be immunized in vitro Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene gly col, to form a hybπdoma cell (Goding, Monoclonal Antibodies Principles and Practice, pp 59-103 (Academic Press, 1986)) The hybπdoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells For example, if the parental myeloma cells lack the enzyme hypoxanthine guanme phosphonbosyl transferase (HGPRT or HPRT), the culture medium for the hybπdomas typically will include hypoxanthine, aminopteπn, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producmgcells, and are sensitive to a medium such as HAT medium Among these, preferred myeloma cell lines are muπne myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland USA Human myeloma and mouse-human heteromyeloma cell lines also have been descnbed for the production of human monoclonal antibodies (Kozbor, J Immunol , 133 3001 (1984), Brodeur ef al , Monoclonal Antibody Production Techniques and Applications, pp 51-63 (Marcel Dekker, Inc , New York, 1987)) Culture medium in which hybπdoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen Preferably, the binding specificity of monoclonal antibodies produced by hybπdoma cells is determined by immunoprecipitationor by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA) After hybndoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies Principles and Practice, pp 59-103 (Academic Press, 1986)) Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium In addition, the hybndoma cells may be grown in vivo as ascites tumors in an animal -21- The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis dialysis, or affinity chromatography DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies) The hybndoma cells serve as a preferred source of such DNA Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as £ coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells Recombinant production of antibodies will be described in more detail below In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al , Nature 348 552-554 (1990) Clackson ef a/ , Nature, 352 624-628 (1991) and Marks ef a/ , J Mol Biol , 222 581 -597 (1991 ) descπbe the isolation of muπne and human antibodies, respectively, using phage libraries Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks ef al , Bio/Technology, 10 779-783 (1992)), as well as combinatorial infection and in v /o recombination as a strategy for constructing very large phage libraries (Waterhouse ef al , Nuc Acids Res , 21 2265-2266 (1993)) Thus, these techniques are viable alternatives to traditional monoclonal antibody hybndoma techniques for isolation of monoclonal antibodies The DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous muπne sequences (U S Patent No 4,816,567, Morrison, et al , Proc Natl Acad Sci USA, 81 6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeπc bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen (iv) Humanized and human antibodies A humanized antibody has one or more ammo acid residues introduced into it from a source which is non-human These non-human ammo acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain Humanization can be essentially performed following the method of Winter and co-workers (Jones ef al , Nature, 321.522-525 (1986), Riechmann et al , Nature, 332 323-327 (1988), Verhoeyen et al , Science, 239 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody Accordingly, such "humanized" antibodies are chimeπc antibodies (U S Patent No 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from -22- a non-human species In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al , J Immunol , 151 2296 (1993), Chothia et al , J Mol Biol , 196 901 (1987)) Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains The same framework may be used for several different humanized antibodies (Carter ef al , Proc Natl Acad Sci USA, 89 4285 (1992), Presta e a/ , J Immnol , 151 2623 (1993)) It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties To achieve this goal, according to a preferred method humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art Computer programs are available which illustrate and display probable three- dimensional conformational structures of selected candidate immunoglobulin sequences Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, / e , the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antιgen(s), is achieved In general, the CDR residues are directly and most substantially involved in influencing antigen binding Alternatively, it is now possible to produce transgenic animals (e g , mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (J H ) gene in chimeπc and germ-line mutant mice results in complete inhibition of endogenous antibody production Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge See, e g , Jakobovits ef al , Proc Natl Acad Sci USA, 90 2551 (1993), Jakobovits ef al , Nature, 362 255-258 (1993), Bruggermann ef al , Year in Immuno , 7 33 (1993), and Duchosal ef al Nature 355 258 (1992) Human antibodies can also be derived from phage-display libraries (Hoogenboom et al , J Mol Biol , 227 381 (1991), Marks et al , J Mol Biol , 222 581-597 (1991), Vaughan ef al Nature Biotech 14 309 (1996)) -23- (v) Multispecific antibodies Multispecific antibodies have binding specificities for at least two different antigens While such molecules normally will only bind two antigens (/ e bispecific antibodies, BsAbs), antibodies with additional specificities such as tπspecific antibodies are encompassed by this expression when used herein Examples of BsAbs include those with one arm directed against a tumor cell antigen and the other arm directed against a cytotoxic trigger molecule such as antι-FcγRI/antι-CD15, anti- p185 HER2 /FcγRIII (CD16), antι-CD3/antι-maiιgnantB-cell (1 D10), antι-CD3/antι-p185 HER2 , antι-CD3/antι- p97, antι-CD3/antι-renalcell carcinoma, antι-CD3/antι-OVCAR-3,antι-CD3/L-D1 (anti-colon carcinoma), antι-CD3/antι-melanocyte stimulating hormone analog, anti-EGF receptor/antι-CD3, antι-CD3/antι- CAMA1, antι-CD3/antι-CD19, antι-CD3/MoV18, anti-neural cell ahesion molecule (NCAM)/antι-CD3, anti- folate binding protein (FBP)/antι-CD3, anti-pan carcinoma associated antigen (AMOC-31 )/antι-CD3; BsAbs with one arm which binds specifically to a tumor antigen and one arm which binds to a toxin such as antι-sapoπn/antι-ld-1 , antι-CD22/antι-sapoπn, antι-CD7/antι-sapoπn, antι-CD38/antι-sapoπn, anti- CEA/anti-πcin A chain, anti-mterferon-α (IFN-α)/antι-hybπdoma idiotype, anti-CEA/anti-vinca alkaloid, BsAbs for converting enzyme activated prodrugs such as antι-CD30/antι-alkalιne phosphatase (which catalyzes conversion of mitomycin phosphate prodrug to mitomycin alcohol), BsAbs which can be used as fibπnolytic agents such as anti-fibπn/anti-tissueplasminogen activator (tPA), anti-fibnn/anti-urokinase- type plasminogen activator (uPA), BsAbs for targeting immune complexes to cell surface receptors such as anti-low density poprotein (LDL)/antι-Fc receptor (e g FcγRI, FcγRII or FcγRIII), BsAbs for use in therapy of infectious diseases such as antι-CD3/antι-herpes simplex virus (HSV), anti-T-cell receptor CD3 complex/anti-influenza, anti-FcγR/anti-HIV, BsAbs for tumor detection in vitro or in vivo such as anti-CEA/anti-EOTUBE, anti-CEA/anti-DPTA, antι-p185 HER2 /antι-hapten, BsAbs as vaccine adjuvants, and BsAbs as diagnostic tools such as anti-rabbit IgG/anti-ferπtin, anti-horse radish peroxιdase (HRP)/antι-hormone,antι-somatostatιn/antι-substanceP, antι-HRP/antι-FITC,antι-CEA/antι- β-galactosidase Examples of tπspecific antibodies include antι-CD3/antι-CD4/antι-CD37 antι-CD3/antι- CD5/antι-CD37 and antι-CD3/antι-CD8/antι-CD37 Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e g F(ab') 2 bispecific antibodies) Methods for making bispecific antibodies are known in the art Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein ef al , Nature, 305 537-539 (1983)) Because of the random assortment of immunoglobulin heavy and light chains, these hybπdomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low Similar procedures are disclosed in WO 93/08829, and in Traunecker ef al , EMBO J , 10 3655-3659 (1991 ) According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising -24- at least part of the hinge, CH2, and CH3 regions It is preferred to have the first heavy-chain constant region (CH1 ) containing the site necessary for light chain binding, present in at least one of the fusions DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation This approach is disclosed in WO 94/04690 For further details of generating bispecific antibodies see, for example, Suresh ef al , Methods in Enzymology, 121 210 (1986) According to another apDroach described in W096/27011 , the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture The preferred interface comprises at least a part of the C H 3 domain of an antibody constant domain In this method, one or more small ammo acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e g tyrosme or tryptophan) Compensatory "cavities" of identical or similar size to the large side chaιn(s) are created on the interface of the second antibody molecule by replacing large ammo acid side chains with smaller ones (e g alanine or threonme) This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers Bispecific antibodies include cross-linked or "heteroconjugate" antibodies For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (US Patent No 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089) Heteroconjugate antibodies may be made using any convenient cross-linking methods Suitable cross- linking agents are well known in the art, and are disclosed in US Patent No 4,676,980, along with a number of cross-linking techniques Antibodies with more than two valencies are contemplated For example, tπspecific antibodies can be prepared Tutt ef a/ J Immunol 147 60 (1991) While the polypeptide of interest herein is preferably an antibody, other Fc region-containing polypeptides which can be modified according to the methods described herein are contemplated An example of such a molecule is an immunoadhesin -25- C. Immunoadhesin Preparation The simplest and most straightforward immunoadhesin design combines the binding domaιn(s) of the adhesin (e g the extracellular domain (ECD) of a receptor) with the Fc region of an immunoglobulin heavy chain Ordinarily, when preparing the immunoadhesins of the present invention, nucleic acid encoding the binding domain of the adhesin will be fused C-terminaily to nucleic acid encoding the N-terminus of an immunoglobulin constant domain sequence, however N-terminal fusions are also possible Typically, in such fusions the encoded chimeπc polypeptide will retain at least functionally active hinge, C H 2 and C H 3 domains of the constant region of an immunoglobulin heavy chain Fusions are also made to the C-termmus of the Fc portion of a constant domain, or immediately N-termmal to the C H 1 of the heavy chain or the corresponding region of the light chain The precise site at which the fusion is made is not critical, particular sites are well known and may be selected in order to optimize the biological activity, secretion, or binding characteristics of the immunoadhesin In a preferred embodiment, the adhesin sequence is fused to the N-terminus of the Fc region of immunoglobulin G 1 (IgG^ It is possible to fuse the entire heavy chain constant region to the adhesin sequence However, more preferably, a sequence beginning in the hinge region just upstream of the papain cleavage site which defines IgG Fc chemically (/ e residue 216, taking the first residue of heavy chain constant region ιo be 114), or analogous sites of other immunoglobulins is used in the fusion In a particularly preferred embodiment, the adhesin ammo acid sequence is fused to (a) the hinge region and C H 2 and C H 3 or (b) the C H 1 , hinge, C H 2 and C H 3 domains, of an IgG heavy chain For bispecific immunoadhesins, the immunoadhesins are assembled as multimers, and particularly as heterodimersor heterotetramers Generally, these assembled immunoglobulins will have known unit structures A basic four chain structural unit is the form in which IgG, IgD, and IgE exist A four chain unit is repeated in the higher molecular weight immunoglobulins, IgM generally exists as a pentamer of four basic units held together by disulfide bonds IgA globulin, and occasionally IgG globulin may also exist in multimeπc form in serum In the case of multimer, each of the four units may be the same or different Various exemplary assembled immunoadhesins within the scope herein are schematically diagrammed below (a) AC L -AC L , (b) AC H -(AC H , AC L -AC H , AC L -V H C H , or V L C L -AC H ), (c) AC L -AC H -(AC L -AC H , AC L -V H C H , V L C L -AC H , or V L C L -V H C H ) (d) AC L -V H C H -(AC H , or AC L -V H C H , or V L C L -AC H ), (e) V L C L -AC H -(AC L -V H C H , or V L C L -AC H ), and (f) (A-Y) n -(V L C L -V H C H ) 2 , wherein each A represents identical or different adhesin ammo acid sequences, V L is an immunoglobulin light chain variable domain, -26- V H is an immunoglobulin heavy chain variable domain C L is an immunoglobulin light chain constant domain, C H is an immunoglobulin heavy chain constant domain n is an integer greater than 1 , Y designates the residue of a covalent cross-linking agent In the interests of brevity, the foregoing structures only show key features, they do not indicate joining (J) or other domains of the immunoglobulins, nor are disulfide bonds shown However, where such domains are required for binding activity, they shall be constructed to be present in the ordinary locations which they occupy in the immunoglobulin molecules Alternatively, the adhesin sequences can be inserted between immunoglobulin heavy chain and light chain sequences, such that an immunoglobulin comprising a chimeπc heavy chain is obtained In this embodiment, the adhesin sequences are fused to the 3' end of an immunoglobulin heavy chain in each arm of an immunoglobulin, either between the hinge and the C H 2 domain, or between the C H 2 and C H 3 domains Similar constructs have been reported by Hoogenboom, ef al , Mol Immunol 28 1027- 1037 (1991 ) Although the presence of an immunoglobulin light chain is not required in the immunoadhesins of the present invention, an immunoglobulin light chain might be present either covalently assr -ated to an adhesin-immunoglobulin heavy chain fusion polypeptide, or directly fused to the adhesin In the former case, DNA encoding an immunoglobulin light chain is typically coexpressed with the DNA encoding the adhesin-immunoglobulin heavy chain fusion protein Upon secretion, the hybrid heavy chain and the light chain will be covalently associated to provide an immunoglobulin-iike structure comprising two disulfide-linked immunoglobulin heavy chain-light chain pairs Methods suitable for the preparation of such structures are, for example, disclosed in U S Patent No 4,816,567, issued 28 March 1989 Immunoadhesins are most conveniently constructed by fusing the cDNA sequence encoding the adhesin portion in-frame to an immunoglobulin cDNA sequence However, fusion to genomic immunoglobulin fragments can also be used (see, e g Aruffo ef al , Cell 61 1303-1313 (1990), and Stamenkovic ef al , Cell 66 1133-1144 (1991)) The latter type of fusion requires the presence of Ig regulatory sequences for expression cDNAs encoding IgG heavy-chain constant regions can be isolated based on published sequences from cDNA libraries derived from spleen or peripheral blood lymphocytes, by hybridization or by polymerase chain reaction (PCR) techniques The cDNAs encoding the "adhesin" and the immunoglobulin parts of the immunoadhesin are inserted in tandem into a plasmid vector that directs efficient expression in the chosen host cells D. Vectors, Host Cells and Recombinant Methods The invention also provides isolated nucleic acid encoding a polypeptide variant as disclosed herein, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the polypeptide variant -27- For recombinant production of the polypeptide variant the nucleic acid encoding it is isolated and inserted into a rephcable vector for further cloning (amplification of the DNA) or for expression DNA encoding the polypeptide variant is readily isolated and sequenced using conventional procedures (e g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the polypeptide variant) Many vectors are available The vector components generally include, but are not limited to, one or more of the following a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence (i) Signal sequence component The polypeptide variant of this invention may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide The heterologous signal sequence selected preferably is one that is recognized and processed (/ e , cleaved by a signal peptidase) by the host cell For prokaryotic host cells that do not recognize and process the native polypeptide variant signal sequence, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicil nase, Ipp, or heat-stable enterotoxin II leaders For yeast secretion the native signal sequence may be substituted by, e g , the yeast invertase leader, α factor leader (including Saccharomyces and Kluyviromyces α-factor leaders), or acid phosphatase leader, the C albicans glucoamy'ase leader, or the signal described in WO 90/13646 In mammaπan cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available The DNA for such precursor region is gated in reading frame to DNA encoding the polypeptide variant (II) Origin of replication component Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences Such sequences are well known for a variety of bacteria, yeast, and viruses The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter) (in) Selection gene component Expression and cloning vectors may contain a selection gene, also termed a selectable marker Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e g , ampicillin, neomycm, methotrexate, or tetracyclme, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e , the gene encoding D-alanine racemase for Bacilli -28- One example of a selection scheme utilizes a drug to arrest growth of a host cell Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycm Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the polypeptide variant nucleic acid, such as DHFR, thymidine kinase, metallothιoneιn-1 and -II, preferably primate metallothionein genes, adenosme deaminase, ornithine decarboxylase, etc For example, cells transformed with the DHFR selection gene are first identified by cultuπng all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding polypeptide variant wild-type DHFR protein, and another selectable marker such as ammoglycoside 3'-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e g , kanamycin, neomycin, or G418 See U S Patent No 4,965,199 A suitable selection gene for use in yeast is the trpλ gene -esent in the yeast plasmid YRp7 (Stmchcomb et al , Nature, 282 39 (1979)) The frpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No 44076 or PEP4-1 Jones, Genetics, 85 12 ( 1977) The presence of the frpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan Similarly, etv2-deficιent yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene In addition, vectors derived from the 1 6 μm circular plasmid pKD1 can be used for transformation of Kluyveromyces yeasts Alternatively, an expression system for large-scale production of recombinant calf chymos was reported for K lactis Van den Berg, Bio/Technology, 8 135 (1990) Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed Fleer ef al , Bio/Technology, 9 968-975 (1991) (iv) Promoter component Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the polypeptide variant nucleic acid Promoters suitable for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter However, other known bacterial promoters are suitable Promoters for use in bacterial systems also will contain a Shine-Dalgamo (S D ) sequence operably linked to the DNA encoding the polypeptide variant Promoter sequences are known for eukaryotes Virtually ail eukaryotic genes have an AT-πch region located approximately 25 to 30 bases upstream from the site where transcription is initiated -29- Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence All of these sequences are suitably inserted into eukaryotic expression vectors Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kmase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructok ase, glucose-6-phosphate isomerase, 3-phosphoglyceratemutase, pyruvate k ase, tπosephosphate isomerase, phosphoglucose isomerase, and glucokmase Other yeast promoters, which are mducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and gaiactose utilization Suitable vectors and promoters for use in yeast expression are further described in EP 73,657 Yeast enhancers also are advantageously used with yeast promoters Polypeptide variant transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian s.-rcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV4U), from heterologous mammalian promoters, e g , the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided sucn promoters are compatible with the host cell systems The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hmdlll E restriction fragment A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U S Patent No 4,419,446 A modification of this system is described in U S Patent No 4,601 ,978 See also Reyes ef al , Nature 297 598-601 (1982) on expression of human β-mterferon cDNA in mouse cells under the control of a thymidine kmase promoter from herpes simplex virus Alternatively, the rous sarcoma virus long terminal repeat can be used as the promoter (v) Enhancer element component Transcription of a DNA encoding the polypeptide variant of this invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector Many enhancer sequences are now known from mammalian genes (globm, elastase, albumin, α-fetoprotein, and insulin) Typically, however, one will use an enhancer from a eukaryotic cell virus Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers See also Yaniv, Nature 297 17-18 (1982) on enhancing elements for activation of eukaryotic promoters The enhancer may be spliced into the vector at a position 5' or 3' to the polypeptide variant-encoding sequence, but is preferably located at a site 5' from the promoter -30- (vi) Transcription termination component Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the polypeptide variant One useful transcription termination component is the bovine growth hormone polyadenylation region See W094/11026 and the expression vector disclosed therein (VII) Selection and transformation of host cells Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above Suitable prokaryotes for this purpose include eubacteπa, such as Gram-negative or Gram-positive organisms, for example, Enterobacteπaceaesuch as Eschenchia, e g , E coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e g , Salmonella typhimunum Serratia, e g , Serratia marcescans, and Shigella, as well as Bacilli such as B subtilis and B lichemformis (e g , B lichemformis 41 P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P aerugmosa, and Streptomyces One preferred E coli cloning host is E coli 294 (ATCC 31 ,446), although other strains such as E coli B, E coli X1776 (ATCC 31 ,537), and E coli W3110 (ATCC 27,325) are suitable These examples are illustrative rather than limiting addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning o expression hosts for polypeptide variant-encoding vectors Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe, Kluyveromyces hosts such as, e g , K lactis, K fragilis (ATCC 12,424), K bulgaπcus (ATCC 16,045), K wickeramii (ATCC 24,178), K waltn (ATCC 56,500), K drosophilarum (ATCC 36,906), K thermotolerans, and K marxianus, yarrowia (EP 402,226), Pichia pastoπs (EP 183,070), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa, Schwanmomyces such as Schwa nniomyces occidentals, and filamentous fungi such as, e g , Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A nidulans and A mger Suitable host cells for the expression of glycosylated polypeptide variant are derived from multicellular organisms Examples of invertebrate cells include plant and insect cells Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mon have been identified A variety of viral strains for transfection are publicly available, e , the L-1 vaπant of Autographa califomica NPV and the Bm-5 strain of Bombyx mon NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts -31- However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al , J Gen Virol 36 59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells/-DHFR (CHO, Urlaub ef al , Proc Natl Acad Sci USA 77 4216 (1980)), mouse sertoli cells (TM4, Mather, Biol Reprod 23 243-251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather ef al , Annals N Y Acad Sci 383 44-68 (1982)), MRC 5 cells, FS4 cells, and a human hepatoma line (Hep G2) Host cells are transformed with the above-described expression or cloning vectors for polypeptide variant production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences (vm) Cultuπng the host cells The host cells used to produce the polypeptide variant of this invention may be cultured in a variety of media Commercially available media such as Ham's F10 (Sigma), Minima. Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for cultuπng the host cells In addition, any of the media described in Ham et al , Meth Enz 58 44 (1979), Barnes et al , Anal Biochem 102 255 (1980), U S Pat Nos 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90/03430, WO 87/00195, or U S Patent Re 30,985 may be used as culture media for the host cells Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferπn, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenos e and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan (ix) Polypeptide variant purification When using recombinant techniques, the polypeptide variant can be produced mtracellularly, in the peπplasmic space, or directly secreted into the medium If the polypeptide variant is produced mtracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration Carter ef al , Bio Technology 10 163-167 (1992) describe a procedure for isolating antibodies which are secreted to the peπplasmic space of E coli Briefly, cell paste is thawed in the presence of sodium acetate (pH 3 5), EDTA, and phenylmethylsulfonylfluoπde -32- (PMSF) over about 30 mm Cell debris can be removed by centrifugation Where the poly peptide variant is secreted into the medium, supematants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants The polypeptide variant composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc region that is present in the polypeptide variant Protein A can be used to purify polypeptide variants that are based on human γ1 , γ2, or γ4 heavy chains (Lindmark ef al , J Immunol Meth 62 1-13 (1983)) Protein G is recommended for all mouse isotypes and for human γ3 (Guss ef al , EMBO J 5 15671575 (1986)) The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available Mechanically stable matrices such as controlled pore glass or poly(styrenedιvιnyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose Where tne polypeptide variant comprises a C H 3 domain, the Bakerbond ABX™ resin (J T Baker, Phillipsburg, NJ) is useful for purification Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phac HPLC, chromatography on silica, chromatography on hepaπn SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the polypeptide variant to be recovered Following any preliminary purification step(s), the mixture comprising the polypeptide variant of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2 5-4 5, preferably performed at low salt concentrations (e g ,from about 0-0 25M salt) E. Pharmaceutical Formulations Therapeutic formulations of the polypeptide variant are prepared for storage by mixing the polypeptide variant having the desired degree of purity with optional physiologically acceptable earners, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A Ed (1980)), in the form of lyophilized formulations or aqueous solutions Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcmol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptide, proteins, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrohdone, am o acids such as glycine, glutamine, asparagine, histidme, argmine, or lysine, monosacchaπdes, disacchaπdes, and other -33- carbohydrates including glucose, mannose, or dextπns, chelating agents such as EDTA sugars such as sucrose, mannitol, trehalose or sorbitol, salt-forming counter-ions such as sodium metal complexes (e g , Zn-protein complexes), and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG) The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other Such molecules are suitably present in combination in amounts that are effective for the purpose intended The active ingredients may also be entrapped in microcapsule prepared for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylceliuloseor gelatin- microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, mtcroemulsions, nano-particles and nanocapsules) or in macroemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A Ed (1980) The formulations to be used for in vivo administration must be sterile This is readily accomplished by filtration through sterile filtration membranes Sustained-release preparations may be prepared Suitable examples of sustained-release preparations include serr permeable matrices of solid hydrophobic polymers containing the polypeptide variant, which matrices are in the form of shaped articles, e g , films, or microcapsule Examples of sustained-release matrices include polyesters, hydrogels (foi example, poiy(2-hydroxyethyl- methacrylate), or poly(vιnylalcohol)), polylactides (U S Pat No 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate,non-degradableethylene-vιnylacetate, degradable lactic acid-glycohcacid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyπc acid While polymers such as ethylene-vmyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity Rational strategies can be devised for stabilization depending on the mechanism involved For example, if the aggregation mechanism is discovered to be mtermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions F. Non-Therapeutic Uses for the Polypeptide Variant The polypeptide variant of the invention may be used as an affinity purification agent In this process, the polypeptide variant is immobilized on a solid phase such a Sephadex resin or filter paper, using methods well known in the art The immobilized polypeptide variant is contacted with a sample containing the antigen to be purified, and thereafter the support is washed with a suitable solvent that -34- will remove substantially all the material in the sample except the antigen to be purified, which is bound to the immobilized polypeptide variant Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5 0, that will release the antigen from the polypeptide variant The polypeptide variant may also be useful in diagnostic assays, e , for detecting expression of an antigen of interest in specific cells, tissues, or serum For diagnostic applications, the polypeptide variant typically will be labeled with a detectable moiety Numerous labels are available which can be generally grouped into the following categories (a) Radioisotopes, such as 35 S, 1 C, 125 l, 3 H, and 31 l The polypeptide variant can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen ef al , Ed Wiley-lnterscience, New York, New York, Pubs (1991 ) for example and radioactivity can be measured using scintillation counting (b) Fluorescent labels such as rare earth chelates (europium chelates) or fluorescem and its derivatives, rhodamme and its derivatives, dansyl, Lissamine, phycoerythπn and Texas Red are available The fluorescent labels can be conjugated to the polypeptide variant using the techniques disclosed in Current Protocols in Immunology, supra, for example Fluorescence can be quantified using a fluoπmeter (c) Various enzyme-substrate labels are available and U S Patent No 4,275,149 provides a review of some of these The enzyme generally catalvzes a chemical alteration of the chron qenic substrate that can be measured using various techniques For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometπcally Alternatively, the enzyme may alter the fluorescence or chemiluminescenceof the substrate Techniques for quantifying a change in fluorescence are described above The chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using a chemiluminometer, f nr example) or donates energy to a fluorescent acceptor Examples of enzymatic labels include luciferases (e g , firefly luciferase and bacterial luciferase, U S Patent No 4 737,456) lucifeπn, 2,3- dιhydrophthalazιnedιones,malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sacchaπde oxidases (e g , glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uncase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like Techniques for conjugating enzymes to antibodies are described in O'Sullivan ef al , Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym (ed J Langone & H Van Vunakis), Academic press, New York, 73 147-166 (1981) Examples of enzyme-substrate combinations include, for example (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e g .orthophenylene diamine (OPD) or 3, 3', 5,5'- tetramethyl benzidme hydrochloride (TMB)), (n) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate, and -35- (in) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e g , p-nitrophenyl-β-D- galactosidase) or fluorogenic substrate 4-methylumbellιferyl-β-D-galactosιdase Numerous other enzyme-substrate combinations are available to those skilled in the art For a general review of these, see U S Patent Nos 4,275,149 and 4,318,980 Sometimes, the label is indirectly conjugated with the polypeptide variant The skilled artisan will be aware of various techniques for achieving this For example, the polypeptide variant can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa Biotin binds selectively to avidin and thus, the label can be conjugated with the polypeptide variant in this indirect manner Alternatively, to achieve indirect conjugation of the label with the polypeptide variant, the polypeptide variant is conjugated with a small hapten (e , digoxin) and one of the different types of labels mentioned above is conjugated with an anti- hapten polypeptide variant (e g , anti-digoxm antibody) Thus, indirect conjugation of the label with the polypeptide variant can be achieved In another embodiment of the invention, the polypeptide variant need not be labeled and the presence thereof can be detected using a labeled antibody which binds to the polypeptide variant The poly peptide variant of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assay- Zola, Monoclonal Antibodies A Manual of Techniques, pp 147-158 (CRC Pre r »;, I nc 1987) The polypeptide variant may also be used for in vivo diagnostic assays Generally, the poly peptide variant is labeledwith a radιonuclιde(such as 111 ln, 99 Tc, 14 C, 131 l, 125 l, 3 H, 32 P or 35 S) so that the antigen or cells expressing it can be localized using immunoscintiography G. In Vivo Uses for the Polypeptide Variant It is contemplated that the polypeptide variant of the present invention may be used *o treat a mammal e g a patient suffering from a disease or disorder who could benefit from administration of the polypeptide variant The conditions which can be treated with the polypeptide variant are many and include cancer (e g where the polypeptide variant binds the HER2 receptor or CD20), allergic conditions such as asthma (with an anti-lgE antibody), and LFA-mediated disorders (e g where the polypeptide variant is an antι-LFA-1 or antι-ICAM-1 antibody) etc Where the polypeptide variant does not bind complement, but retains FcR binding capability, exemplary diseases or disorders to be treated include cancer (e g where ADCC function is desirable, but complement activation would lead to amplified side effects, such as vasculitis in the blood vessels at the tumor site), disorders treated with an agonist antibody, disorders wherein the polypeptide variant binds a soluble antigen and wherein stoichiometry leads to immune complexes which activate the complement cascade and result in unwanted side effects, conditions employing an antagonist antibody which downmoduiates receptor function without damaging tissue or organ function, intravenous immunoglobulin treatment for, e g , immunodeficient individuals with autoimmune disorders The polypeptide variant is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and tranasal, and, if desired for local -36- immunosuppressive treatment, mtralesional administration Parenteral infusions include intramuscular, intravenous, intraarteπal, intraperitoneal, or subcutaneous administration In addition, the polypeptide variant is suitably administered by pulse infusion, particularly with declining doses of the polypeptide variant Preferably the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic For the prevention or treatment of disease, the appropriate dosage of polypeptide variant will depend on the type of disease to be treated, the severity and course of the disease, whether the polypeptide variant is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the polypeptide variant, and the discretion of the attending physician The polypeptide vanant is suitably administered to the patient at one time or over a series of treatments Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e g , 0 1- 20mg/kg) of polypeptide variant is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs However, other dosage regimens may be useful The progress of this therapy is easily monitored by conventional techniques and assays The polypeptide variant composition will be formulated, dosed, and admi istered in a fashion consistent with good medical practice Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners The "therapeutically effective amount" of the polypeptide variant to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat a disease or disorder The polypeptide variant need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question The effective amount of such other agents depends on the amount of polypeptide variant present in the formulation, the type of disorder or treatment, and other factors discussed above These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages The invention will be more fully understood by reference to the following examples They should not, however, be construed as limiting the scope of this invention All literature and patent citations mentioned herein are expressly incorporated by reference EXAMPLE 1 Low Affinity Receptor Binding Assay This assay determines binding of an IgG Fc region to recombinant FcγRlla, FcγRllb and FcγRllla α subunits expressed as Hιs6-glutathιone S transferase (GST)-tagged fusion proteins Since the affinity of the Fc region of lgG1 for the FcγRI is in the nanomolar range, the binding of lgG1 Fc mutants can be measured by titrating monomeπc IgG and measuring bound IgG with a polyclonal anti- -37- IgG m a standard ELISA format (Example 2 below) The affinity of the other members of the FcγR family, i e FcγRlla, FcγRlib and FcγRllla for lgG is however in the micromolar range and binding of monomenc lgG1 for these receptors can not be reliably measured in an ELISA format The following assay utilizes Fc mutants of recombinant anti-lgE E27 (Figures 4A and 4B) which, when mixed with human IgE at a 1 1 molar ratio, forms a stable hexamer consisting of three anti-lgE molecules and three IgE molecules A recombinant chimeric form of IgE (chimeric IgE) was engineered and consists of a human IgE Fc region and the Fab of an anti-VEGF antibody (Presta ef al Cancer Research 57 4593-4599 (1997)) which binds two VEGF molecules per mole of anti-VEGF When recombinant human VEGF is added at a 2 1 molar ratio to chimeric IgE E27 hexamers, the hexamers are linked into larger molecular weight complexes via the chimeric IgE Fab VEGF interaction The E27 component of this complex binds to the FcγRlla, FcγRllb and FcγRllla α subunits with higher avidity to permit detection in an ELISA format MATERIALS AND METHODS Receptor Coat Fcγ receptor α subunits were expressed as GST fusions of Hιs6 tagged extracellular domains (ECDs) in 293 cells resulting in an ECD-6Hιs-GST fusion protein (Graham ef al J Gen Virol 36 59-72 (1977) and Gorman ef al DNA Prot Eng Tech 2 3-10 (1990)) and purified by Ni-NTA column chromatography (Qiagen, Australia) and buffer exchanged into phosphate buffered saline (PBS) Concentrations were determined by absorption at 280nm u: n , extinction coefficients derived by ammo acid composition analysis Receptors were coated onto Nunc F96 maxisorb plates (cat no 439454) at 100ng per well by adding 100 μl of receptor-GST fusion at 1 μg/ml in PBS and incubated for 48 hours at 4°C Prior to assay, plates are washed 3x with 250 μl of wash buffer (PBS pH 74 containing 0 5% TWEEN 20™) and blocked with 250 μl of assay buffer (50mM Tris buffered saline, 0 05% TWEEN 20™, 0 5% RIA grade bovine albumin (Sigma A7888), and 2mM EDTA pH 7 4) Immune Complex Formation Equal molar amounts (1 1 ) of E27 and recombinant chimeric IgE which binds two moles recombinant human VEGF per mole of chimeric IgE are added to a 12 x 75mm polypropylene tube in PBS and mixed by rotation for 30 minutes at 25°C E27 (anti-lgE) /chimeric IgE (IgE) hexamers are formed during this incubation Recombinant human VEGF (165 form, MW 44,000) is added at a 2 1 molar ratio to the IgE concentration and mixed by rotation an additional 30 minutes at 25°C VEGF- chimeric IgE binding links E27 chimeric IgE hexamers into larger molecular weight complexes which bind FcγR α subunit ECD coated plates via the Fc region of the E27 antibody E27:chimeric lgE:VEGF (1 1 2 molar ratio; complexes are added to FcγR subunit coated plates at E27 concentrations of 5 μg and 1 μg total IgG in quadruplicate in assay buffer and incubated for 120 minutes at 25°C on an orbital shaker Complex Detection Plates are washed 5x with wash buffer to remove unbound complexes and IgG binding is detected by adding 100 μl horse radish peroxidase (HRP) conjugated goat anti- human IgG (γ) heavy chain specific (Boehπnger Mannheim 1814249) at 1 10,000 in assay buffer and incubated for 90mιn at 25°C on an orbital shaker Plates are washed 5x with wash buffer to remove unbound HRP goat anti-human IgG and bound anti-lgG is detected by adding 100 μl of substrate -38- solution (0 4mg/ml o-phenylenedaimme dihydrochlonde, Sigma P6912, 6 mM H 2 0 2 in PBS) and incubating for 8 m at 25°C Enzymatic reaction is stopped by the addition of 100 μl 4 5N H 2 S0 4 and coloπmetπc product is measured at 490 nm on a 96 well plate densitometer (Molecular Devices) Binding of E27 mutant complexes is expressed as a percent of the wild type E27 containing complex EXAMPLE 2 Identification of Unique C1q Binding Sites in a Human IgG Antibody In the present study, mutations were identified in the CH2 domain of a human lgG1 antibody, "C2B8" (Reff ef al , Blood 83 435 (1994)), that ablated binding of the antibody to C1 q but did not alter the conformation of the antibody nor affect binding to each of the FcγRs By alanine scanning mutagenesis, five mutants in human lgG1 were identified, D270K, D270V, K322A P329A, and P331, that were non-lytic and had decreased binding to C1q The data suggested that the core C1q binding sites in human lgG1 is differentfrom that of muπne lgG2b In addition, K322A, P329Aand P331 A were found to bind normally to the CD20 antigen, and to four Fc receptors, FcγRI, FcγRII, FcγRIII and FcRn MATERIALS AND METHODS Construction of C2B8 Mutants: The chimeric light and heavy chains of antι-CD20 antibody C2B8 (Reff ef al , Blood 83 435 (1994)) subcloned separately into previously described PRK vectors (Gorman ef al , DNA Protein Eng Tech 2 3 (1990)) were used By site directed mutagenesis (Kunkel ef al , Proc Natl Acad Sci USA 82 488 (1985)), alanine scan vaπanrε of the Fc region in the heavy chain were constructed The heavy and light chain plasmids were co-transfected into an adenovirus transformed human embryonic kidney cell line as previously described (Werther ef al J Immunol 157 4986 (1996)) The media was changed to serum-free 24 hours after transfection and the secreted antibody was harvested after five days The antibodies were purified using Protein A-SEPH AROSE CL- 4B™ (Pharmacia), buffer exchanged and concentrated to 0 5 ml wlh PBS using a Centπcon-30 (Amicon), and stored at 4°C The concentration of the antibody was determined using total Ig-bindmg ELISA C1q Binding ELISA: Costar 96 well plates were coat-d overnight at 4°C with the indicated concentrations of C2B8 in coating buffer (0 05 M sodium carbonate buffer), pH 9 The plates were then washed 3x with PBS/ 0 05% TWEEN 20™, pH 7 4 and blocked with 200μl of ELISA diluent without thimerosal (0 1 M NaP04 / 0 1M NaCl / 0 1% gelatin / 0 05% TWEEN 20™/ 0 05% ProClιn300) for 1hr at room temperature The plate was washed 3x with wash buffer, an aliquot of 100μl of 2 μg/ml C1q (Quidel, San Diego, CA) was added to each well and incubated for 2 hrs at room temperature The plate was then washed 6x with wash buffer 100 μl of a 1 1000 dilution of sheep anti-complement C1q peroxidase conjugated antibody (Biodesign) was added to each well and incubated for 1 hour at room temperature The plate was again washed 6x with wash buffer and 100 μl of substrate buffer (PBS/0 012% H 2 0 2 ) containing OPD (0-phenylenedιamιnedιhydrochloπde (Sigma)) was added to each well The oxidation reaction, observed by the appearance of a yellow color, was allowed to proceed for 30 minutes and stopped by the addition of 100 μl of 4 5 N H 2 S0 4 The absorbance was then read at (492-405) nm using a microplate reader (SPECTRA MAX 250™, Molecular Devices Corp ) The -39- appropriate controls were run in parallel (i e the ELISA was performed without C1q for each concentration of C2B8 used and also the ELISA was performed without C2B8) For each mutant, C1q binding was measured by plotting the absorbance (492-405) nm versus concentration of C2B8 in μg/ml using a 4-parameter curve fitting program (KALEIDAGRAPH™) and comparing EC 50 values Complement Dependent Cytotoxicity (CDC) Assay This assay was performed essentially as previously described (Gazzano-Santoro et al , J Immunol Methods 202 163 (1997)) Various concentrations of C2B8 (0 08-20 μg/ml) were diluted with RHB buffer (RPMI 1640/20mM HEPES (pH 7 2)/2mM Glutamιne/0 1% BSA/100 μg/ml Gentamicm) Human complement (Quidel) was diluted 1 3 in RHB buffer and WIL2-S cells (available from the ATCC, Manassas, VA) which express the CD20 antigen were diluted to a density of 1 x 10 6 cells /ml with RHB buffer Mixtures of 150 μl containing equal volumes of C2B8, diluted human complement and WIL2-S cells were added to a flat bottom tissue culture 96 well plate and allowed to incubate for 2 hrs at 37°C and 5% C0 2 to facilitate complement mediated cell lysis 50 μl of alamar blue (Accumed International) was then added to each well and incubated overnight at 37°C The absorbance was measured using a 96-well fluorometerwith excitation at 530 nm and emission at 590 nm As described by Gazzano-Santoro ef al , the results are expressed in relative fluorescence units (RFU) The sample concentrations were computed from a C2B8 standard curve and the percent activity as compared to wild type C2B8 is reported for each mutant CD20 Binding Potency of the C2B8 Mutants The binding of C2B8 and mutants to the CD20 antigen were assessed by a method previously described (Reff ef al , (1994), supra, reviewed in Gazzano-Santoro ef al , (1996), supra) WIL2-S cells were grown for 3-4 days to a cell density of 1 x 10 δ cells/ml The cells were washed and spun twice in FACS buffer (PBS/0 1 % BSA/0 02% NaN 3 ) and resuspended to a cell density of 5 x 10 6 cells/ml 200 μl of cells (5 x 10 6 cells/ml) and 20 μl of diluted C2B8 samples were added to a 5 ml tube and incubated at room temperature for 30 minutes with agitation The mixture was then washed with 2 ml of cold FACS buffer, spun down and resuspended in 200 μl of cold FACS buffer To the suspension, 10 μl of goat anti-human IgG-FITC (American Qualex Labs ) was added and the mixture was incubated in the dark at room temperature for 30 minutes with agitation After incubation, the mixture was washed with 2 ml of FACS buffer, spun down and resuspended in 1 ml of cold fixative buffer (1 % formaldehyde in PBS) The samples were analyzed by flow cytometry and the results expressed as relative fluorescence units (RFU) were plotted against antibody concentrations using a 4-parameter curve fitting program (KALEIDAGRAPH™) The EC 50 values are reported as a percentage of that of the C2B8 reference material FcyR Binding ELISAs FcγRI α subunit-GST fusion was coated onto Nunc F96 maxisorb plates (cat no 439454) by adding 100 μl of receptor-GST fusion at 1 μg/ml in PBS and incubated for 48 hours at 4°C Prior to assay, plates are washed 3x with 250 μl of wash buffer (PBS pH 7 4 containing 0 5% TWEEN 20™) and blocked with 250 μl of assay buffer (50mM Tris buffered saline, 0 05% TWEEN 20™, 0 5% RIA grade bovine albumin (Sigma A7888), and 2mM EDTA pH 7 4) Samples diluted to 10μg/ml in 1 ml of assay buffer are added to FcγRI subunit coated plates and incubated for 120 minutes at 25°C -40- on an orbital shaker Plates are washed 5x with wash buffer to remove unbound complexes and IgG binding is detected by adding 100 μl horse radish peroxidase (HRP) conjugated goat anti-human IgG (γ) heavy chain specific (Boehπnger Mannheim 1814249) at 1 10,000 in assay buffer and incubated for 90mιn at 25°C on an orbital shaker Plates are washed 5x with wash buffer to remove unbound HRP goat anti-human IgG and bound anti-IgG is detected by adding 100 μl of substrate solution (0 4mg/ml o-phenyienedaιmιne dihydrochloπde, Sigma P6912, 6 mM H 2 0 2 in PBS) and incubating for 8 mm at 25°C Enzymatic reaction is stopped by the addition of 100 μl 4 5N H 2 S0 4 and coloπmetπc product is measured at 490 nm on a 96 well plate densitometer (Molecular Devices) Binding of variant is expressed as a percent of the wild type molecule FcγRII and III binding ELISAs were performed as described in Example 1 above For measuring FcRn binding activity of IgG variants, ELISA plates were coated with 2 μg/ml streptavidin (Zymed, South San Francisco) in 50 mM carbonate buffer, pH 9 6, at 4°C overnight and blocked with PBS-0 5% BSA, pH 7 2 at room temperature for one hour Biotinylated FcRn (prepared using biotin-X-NHSfrom Research Organics, Cleveland, OH and used at 1-2 μg/ml) in PBS-0 5% BSA, 0 05% polysorbate 20, pH 7 2, was added to the plate and incubated for one hour Two fold serial dilutions of IgG standard (1 6-100 ng/ml) or variants in PBS-0 5% BSA, 0 05% polysorbate 20, pH 6 0, were added to the plate and incubated for two hours Bound IgG was detected using peroxidase labeled goat F(ab') 2 anti-human IgG F(ab') 2 in the above pH 6 0 buffer (Jackson ImmunoResearch, West Grove, PA) followed by 3,3',5,5'-tetramethylbenzιdιne (Kirgaard & Perry Laboratories) as the substrate Plates were washed between steps with PBS-0 05% polysorbate 20 at either pH 7 2 or 6 0 Absorbance was read at 450 nm on a Vmax plate reader (Molecular Devices, Menlo Park, CA) Titration curves were fit with a four-parameter nonlinear regression curve-fitting program (KaleidaGraph, Synergy software, Reading, PA) Concentrations of IgG variants corresponαirigto the mid-point absorbance of the titration curve of the standard were calculated and then divided by the concentration of the standard corresponding to the mid-point absorbance of the standard titration curve RESULTS AND DISCUSSION By alanine scanning mutagenesis, several single point mutations were constructed in the CH2 domain of C2B8 beginning with E318A, K320A and K322A All the mutants constructed bound normally to the CD20 antigen (Table 1) Table 1 wt E318A K320A K322A P329A P331A FcRn + + + + CD20 + + + + + + FcγRI + + + + + + FcγRII + + + + + + FcγRIII + + + + + + * C1q +++ ++ +++ - - - -41- CDC + + + - - - (+) indicates binding and (-) signifies binding abolished * Wιth respect to C1q binding, each + sign is equivalent to approximately 33% binding Where binding of human complement to an antibody with a human Fc was analyzed, the ability of E318A and K320A to activate complement was essentially identical to that of wild type C2B8 (Table 1 ) When compared to wild type C2B8, there appears to be little difference in the binding of E318A and K320A to C1 q There is only a 10% decrease in the binding of K320A and about a 30% decrease in the binding of E318A to C1q (Fig 2) The results indicate that the effect of the E318A and the K320A substitution on complement activation and C1q binding is minimal Also, the human lgG1 of C2B8 was substituted for human lgG2 and used as a negative control in the C1q binding studies The lgG2 mutant appears to have a much lower affinity for C1q than the E318A and K320A mutants (Fig 2) Thus, the results demonstrate that E318 and K320 do not constitute the core C1q binding sites for human lgG1 Conversely, the K322A substitution had a significant effect on both complement activity and C1q binding The K322A mutant did not have CDC activity when tested in the above CDC assay and was more than a 100 fold lower than wild type C2B8 m binding to C1q (Fig 2) In the human system, K322 is the only residue of the proposed core C1q binding sites that appeared to have a significant effect on complement activation and C1q binding Since the Duncan and Winter study was performed using mouse lgG2b ana the above results reveal that K320 and E318 in human lgG1 rve not involved in C1q binding, and without being bound to any one theory, the above data suggest that the C1q binding region in muπne IgGs is different from that of the human To investigate this further and also to identify additional mutants that do not bind to C1q and hence do not activate complement, several more point mutations in the vicinity of K322 were constructed as assessed from the three dimensional structure of the C2B8 Fc Mutants constructed, K274A, N276A, Y278A, S324A, P329A, P331.H K334A, and T335A, were assessed for their ability to bind C1q and also to activate complement Many of these substitutions had little or no effect on C1q binding or complement activation In the above assays, the P329A and the P331A mutants did not activate complement and had decreased binding to C1q The P331A mutant did not activate complement and was 60 fold lower in binding to C1q (Fig 3) when compared to wild type C2B8 (Fig 2) The concentration range of the antibody variants used in Fig 3 is expanded to 10Oμg/ml in order to observe saturation of C1q binding to the P331 A variant The mutation P329A results in an antibody that does not activate complement and is more than a 100 fold lower in binding to C1q (Fig 3) when compared to wild type C2B8 (Fig 2) Mutants that did not bind to C1q and hence did not activate complement were examined for their ability to bind to the Fc receptors FcγRI, FcγRlla, FcγRllb, FcγRllla and FcRn This particular study was performed using a humanized anti-lgE antibody, an lgG1 antibody with these mutations (see Example 1 above) The results revealed the mutants, K322A and P329A, bind to all the Fc receptors to the same extent as the wild type protein (Table 2) However, there was a slight decrease in the binding of P331 A to FcγRllb -42- In conclusion, two ammo acid substitutions in the COOH terminal region of the CH2 domain of human lgG1 , K322A and P329A were identified that result in more than 100 fold decrease in C1 q binding and do not activate the CDC pathway These two mutants, K322A and P329A, bind to all Fc receptors with the same affinity as the wild type antibody Based on the results, summarized in Table 2, and without being bound to any one theory, it is proposed that the C1q binding epicenter of human lgG1 is centered around K322, P329 and P331 and is different from the muπne lgG2b epicenter which constitutes E318, K320 and K322 Table 2 wt E318A K320A K322A P329A P331A CD20 100 89 102 86 112 103 a FcγRI 100 93 102 90 104 74 a FcγRlla 100 113 94 109 111 86 a FcγRllb 100 106 83 101 96 58 a FcγRIII 100 104 72 90 85 73 CDC 100 108 108 None none none 3 For binding to the FcγRs th e mutants were made in the E. IT background anti-lgE) The results are presented as a percentage of the wild type A further residue involved in binding human C1q was identified using the methods described in the present example The residue D270 was replaced with lysine and valine to generate mutants D270K and D270V, respectively These mutants both showed decreased binding to h^man C1q (Fig 6) and were non-lytic (Fig 7) The two mutants bound the CD20 antigen normally and recruited ADCC EXAMPLE 3 Mutants *vith Improved C1q Binding The following study shows that substitution of residues at positions K326, A327, E333 and K334 resulted in mutants with at least about a 30% increase in binding to C1 q when compared to the wild type antibody This indicated K326, A327, E333 and K334 are potential sites for improving the efficacy of antibodies by way of the CDC pathway The aim of this study was to improve CDC activity of an antibody by increasing binding to C1 q By site directed mutagenesis at K326 and E333, several mutants with increased binding to C1q were constructed The residues in order of increased binding at K326 are K<V<E<A<G<D<M<W, and the residues in order of increased binding at E333 are E<Q<D<V<G<A<S Four mutants, K326M, K326D, K326E and E333S were constructed with at least a two-fold increase in binding to C1q when compared to wild type Mutant K326W displayed about a five-fold increase in binding to C1q Mutants of the wild type C2B8 antibody were prepared as described above in Example 2 A further control antibody, wild type C2B8 produced in Chinese hamster ovary (CHO) cells essentially as described in US Patent 5,736,137, was included in a C1q binding ELISA to confirm that wt C2B8 produced in the 293 kidney cell line had the same C1q binding activity as the CHO-produced antibody -43- (see 'CHO-wt-C2B8"ιn Fig 8) The C1q binding ELISA, CDC assay, and CD20 binding potency assay in this example were performed as described in Example 2 above As shown in Fig 8, alanine substitution at K326 and E333 in C2B8 resulted in mutants with about a 30% increase in binding to C1q Several other single point mutants at K326 and E333 were constructed and assessed for their ability to bind C1q and activate complement All the mutants constructed bound normally to the CD20 antigen With respect to K326, the other single point mutants constructed were K326A, K326D, K326E, K326G, K326V, K326M and K326W As shown in Fig 9, these mutants all bound to C1q with a better affinity than the wild type antibody K326W, K326M, K326D and K326E showed at least a two-fold increase in binding to C1q (Table 3) Among the K326 mutants, K326W had the best affinity for C1q Table 3 Mutant EC 50 value Wild type 153 K326V 130 K326A 103 K326E 108 K326G 095 K326D 076 K326M 067 K326W 047 E333S 081 E333Λ 098 E333G 114 E333V 118 E333D 122 E333Q 152 K334A 107 Substitutions with hydrophobic as well as charged residues resulted in mutants with increased binding to C1q Even substitution with glycine which is known to impart flexibility to a chain and is well conserved in nature, resulted in a mutant with higher affinity for C1q when compared to the wild type It would appear that any ammo acid substitution at this site would result in a mutant with higher affinity forClq As assessed from the three-dimensional structure, K326 and E333 are in the vicinity of the core C1q binding sites (Fig 10) In addition to alanine, E333 was also substituted with other ammo acid residues These mutants, E333S, E333G, E333V, E333D, and E333Q, all had increased binding to C1q when compared to the wild type (Fig 11 ) As shown in Table 3, the order of binding affinity for C1q was as follows -44- E333S>E333A>E333G>E333V>E333D>E333Q Substitutions with ammo acid residues with small side chain volumes, t e serine, alanine and glycine, resulted in mutants with higher affinity for C1q in comparison to the other mutants, E333V, E333D and E333Q, with larger side chain volumes The mutant E333S had the highest affinity for C1q, showing a two-fold increase in binding when compared to the wild type Without being bound to any one theory, this indicates the effect on C1q binding at 333 may also be due in part to the polarity of the residue Double mutants were also generated As shown in Figs 12 and 13, double mutants K326M- E333S and K326A-E333Awere at least three-fold better at binding human C1q than wild type C2B8 (Fig 12) and at least two-fold better at mediating CDC compared to wild type C2B8 (Fig 13) Additivity indicates these are independently acting mutants An additional double mutant K326W-E333S was generated which was six-fold better at binding human C1q and three-fold better at mediating CDC compared to wild type C2B8 This double mutant was deficient in ADCC activity in a cell-based assay As shown in Fig 14, a further mutant with improved C1q binding (50% increase) was made by changing A327 in a human lgG1 constant region to glycine Conversely, in a human lgG2 constant region, changing G327 to alanine reduced C1q binding of the lgG2 antibody -45-

Claims: WHAT IS CLAIMED IS 1 A variant of a polypeptide comprising a human IgG Fc region, which variant compπses an ammo acid substitution at ammo acid position 270 or 329, or at two or more of ammo acid positions 270, 322, 329 and 331 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat 2 The variant of claim 1 having Pro329 of the human IgG Fc region substituted with another ammo acid 3 The variant of claim 1 having Asp270 of the human IgG Fc region substituted with another ammo acid 4 The variant of claim 1 wherein the polypeptide comprises an antibody 5 The variant of claim 1 wherein the polypeptide comprises a human igG1 Fc region 6 The variant of claim 1 which does not activate complement 7 The variant of claim 6 which binds an FcR 8 The variant of claim 7 which binds FcγRI, FcγRII, FcγRIII and FcRn 9 A vaπantof a polypeptide comprising a human IgG Fc region, which variant binds FcγRI, FcγRII, FcγRIII and FcRn but does not activate complement and comprises an ammo acid substitution at ammo acid position 322 or ammo acid position 329, or both ammo acid positions of the human IgG Fc region, w μ ere the numbering of the residues in the IgG Fc region is that of the EU index as r Kabat 10 A composition comprising the variant of claim 1 and a physiologically acceptable earner 11 A method for modifying a polypeptide comprising a human IgG Fc region comprising substituting an ammo acid residue at ammo acid position 270 or 329, or at two or more of ammo acid positions 270, 322, 329 and 331 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat 12 Isolated nucleic acid encoding the variant of claim 1 13 A vector comprising the nucleic acid of claim 12 14 A host cell comprising the vector of claim 13 15 A process of producing an polypeptide variant comprising culturing the host cell of claim 14 so that the nucleic acid is expressed 16 The process of claim 15 further comprising recovering the variant from the host cell culture 17 A method of treating a mammal suffering from a disorder comprising administering to the mammal a therapeutically effective amount of a variant of a polypeptide comprising a human IgG Fc region, which variant binds FcγRI, FcγRII, FcγRIII and FcRn but does not activate complement and comprises an ammo acid substitution at ammo acid position 270, 322, 329 or 331 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat 18 A variant of a parent polypeptide comprising a human IgG Fc region, which variant has a better binding affinity for human C1q than the parent polypeptide and comprises an ammo acid substitution in the IgG Fc region -46- 19 The variant of claim 18 wherein the binding affinity of the variant for human C1q is about two-fold or more improved compared to the binding affinity of the parent polypeptide for human C1q 20 The variant of claim 18 wherein the binding affinity of the variant for human C1q is about five-fold or more improved compared to the binding affinity of the parent polypeptide for human C1q 21 The variant of claim 18 wherein the parent polypeptide mediates complement dependent cell killing 22 The variant of claim 18 wherein the parent polypeptide comprises a human lgG1, lgG2 or lgG3 Fc region 23 The variant of claim 18 wherein the parent polypeptide comprises a human lgG1 Fc region 24 The variant of claim 18 which comprises an am o acid substitution at ammo acid position 326 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat 25 The variant of claim 24 wherein the am o acid residue at am o acid position 326 is substituted with tryptophan 26 The variant of claim 18 which comprises an ammo acid substitution at ammo acid position 333 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of *'-<? EU index as in Kabat 27 The variant of claim 18 which comprises an ammo acid substitution at am o acid position 334 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat 28 The variant of claim 18 which comprises an ammo acid substitution at ammo acid position 327 of the human IgG Fc region, where the numbering of residues in the IgG Fc region is that of the EU index as in Kabat 29 The variant of claim 28 wherein the human IgG Fc region is a human lgG1 or lgG3 Fc region 30 The variant of claim 18 which comprises ammo acid substitutions at two or more of ammo acid positions 326, 327, 333 and 334 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat 31 The variant of claim 18 wherein the parent polypeptide comprises an antibody 32 A composition comprising the variant of claim 18 and a physiologically acceptable carrier 33 A variant of a polypeptide comprising a human IgG Fc region, which variant compπses an ammo acid substitution at ammo acid position 326, 327, 333 or 334 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat 34 The variant of claim 33 which comprises ammo acid substitutions at two or more of ammo acid positions 326, 327, 333 or 334 35 The variant of claim 34 which compπses ammo acid substitutions at three or more am o acid positions 326, 327, 333 or 334 -47- 36 The variant of claim 35 which compπses ammo acid substitutions at ammo acid positions 326, 327, 333 and 334 37 A method for modifying a polypeptide comprising a human IgG Fc region comprising substituting an ammo acid residue at am o acid position 326, 327, 333 or 334 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat 38 Isolated nucleic acid encoding the variant of claim 18 39 A vector compnsmg the nucleic acid of claim 38 40 A host cell comprising the vector of claim 39 41 A process of producing an polypeptide variant comprising cultuπng the host cell of claim 40 so that the nucleic acid is expressed 42 The process of claim 41 further comprising recovering the variant from the host cell culture 43 A method of treating a mammal suffering from a disorder comprising administering to the mammal a therapeutically effective amount of the variant of claim 18 44 An immune complex comprising (a) an Fc region-containing polypeptide, (b) a first target molecule which comprises at least two binding sites for the Fc re > icn-contaιnιng polypeptide, and (c) a second target molecule which compnses at least two binding sites for the first target molecule 45 A method for determining binding of an analyte to a receptor comprising the following steps performed sequentially (a) forming a molecular complex between the analyte and a first target molecule, wherein the first target molecule comprises at least two binding sites for the analyte, and (b) determining binding of the molecular complex of step (a) to the receptor 46 The method of claim 45 wherein the analyte is an Fc region-containing polypeptide 47 The method of claim 45 wherein the receptor is a low affinity receptor 48 The method of claim 45 wherein the receptor is an FcR 49 The method of claim 45 wherein the molecular complex of step (a) further comprises a second target molecule which comprises at least two binding sites for the first target molecule 50 A kit useful for determining binding of an analyte to a receptor comprising (a) a first target molecule which compπses at least two binding sites for the analyte, and (b) a second target molecule which compπses at least two binding sites for the first target molecule -48-

Inventors: IDUSOGIE ESOHE EKINADUESE, MULKERRIN MICHAEL GEORGE, PRESTA LEONARD G, SHIELDS ROBERT LAIRD, IDUSOGIE, ESOHE, EKINADUESE, MULKERRIN, MICHAEL, GEORGE, PRESTA, LEONARD, G., SHIELDS, ROBERT, LAIRD
Applicants: GENENTECH INC, GENENTECH, INC.
Application year: 1999
Main classification: C07K16
IPC codes: A61K38, A61K47, C07K16, C07K19, C12N1, C12N5, C12N15, C12P21, G01N33