This application claims benefit of provisional application serial number 60/603,057, filed on August 19, 2004, which application is incorporated herein by reference in their entirety.

FIELD OF THE INVENTION The present invention concerns polypeptides comprising a variant Fc region. More particularly, the present invention concerns Fc region-containing polypeptides that have altered effector function as a consequence of one or more amino acid modifications in the Fc region thereof.

BACKGROUND OF THE INVENTION

Antibodies are proteins that exhibit binding specificity to a specific antigen. Native antibodies are usually heterotetrameric glycoproteins 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 immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) 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 amino 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 variable 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 (FRs). The variable domains of native heavy and light chains each comprise four FRs, 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 FRs and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et ai, 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 amino 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. IgGl , IgG2, IgG3, and IgG4; IgAl and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called α, 5, ε, γ, and μ, respectively. Of the human immunoglobulin classes, only human IgGl , IgG2, IgG3 and IgM are known to activate complement, and human IgGl and IgG3 mediate ADCC more effectively than IgG2 and IgG4. A schematic representation of the native IgGl structure is shown in Fig. IA, 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.

Antibody Effector Functions 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)-bearing 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. The interaction of antibodies and antibody-antigen complexes with cells of the immune system effects a variety of responses, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) (reviewed in Daeron, Annu. Rev. Immunol. 15:203-234 (1997); Ward and Ghetie, Therapeutic Immunol. 2:77-94 (1995); as well as Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991)). Several antibody effector functions are mediated by Fc receptors (FcRs), which bind the Fc region of an antibody. FcRs are defined by their specificity for immunoglobulin isotypes; Fc receptors for IgG antibodies are referred to as Fc)R, for IgE as FcεR, for IgA as FcαR and so on. Three subclasses of FcγR have been identified: FcγRI (CD64), FcγRII (CD32) and Fc)RIII (CD 16). Because each FcγR subclass is encoded by two or three genes, and alternative RNA splicing 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 Iq21.1 of the long arm of chromosome 1; the genes encoding FcγRII isoforms (FcγRIIA, FcγRIIB and FcγRIIC) and the two genes encoding FcγRIII (FcγRIIIA and FcγRIIIB) are all clustered in region 1 q22. These different FcR subtypes are expressed on different cell types (reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991 )). For example, in humans, FcγRIIIB is found only on neutrophils, whereas FcγRIIIA is found on macrophages, monocytes, natural killer (NK) cells, and a subpopulation of T-cells. Structurally, the FcyR are all members of the immunoglobulin superfamily, having an IgG-binding α-chain with an extracellular portion comprised of either two (FcγRI and FcγRIII) or three (FcγRI ) Ig-like domains. In addition, FcγRI and FcγRIII have accessory protein chains (γ, ζ) associated with the α-chain which function in signal transduction. The receptors are also distinguished by their affinity for IgG. FcγRI exhibits a high affinity for IgG, Ka = 108-109M'' (Ravetch et al. Ann. Rev. Immunol. 19:275-290 (2001)) and can bind monomeric IgG. In contrast FcγRII and FcγRIII show a relatively weaker affinity for monomeric IgG Ka ≤ 107M"1 (Ravetch et al Ann. Rev. Immunol. 19:275-290 (2001)), and hence only interact effectively with multimeric immune complexes. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). NK cells carry only FcγRIIIA and binding of antibodies to FcγRIIIA leads to ADCC activity by the NK cells. Allelic variants of several of the human FcγR have been found in the human population. These allelic variant forms have been shown to exhibit differences in binding of human and murine IgG and a number of association studies have correlated clinical outcomes with the presence of specific allelic forms (reviewed in Lehrnbecher et al. Blood 94(12):4220-4232 (1999)). Several studies have investigated two forms of FcγRIIA, R131 and H131, and their association with clinical outcomes (Hatta et al. Genes and Immunity 1:53-60 (1999); Yap et al. Lupus 8:305-310 (1999); and Lorenz et al. European J. Immuno genetics 22:397-401 (1995)). Two allelic forms of FcγRIIIA, F158 and V158, are only now being investigated (Lehrnbecher et al., supra; and Wu et al. J. Clin. Invest. 100(5): 1059-1070 (1997)). The FcγRIIIA(Vall58) allotype interacts with human IgG better than the FcγRIIIA(Phel58) allotype (Shields et al. J. Biol. Chem. 276: 6591-6604 (2001); Koene et al. Blood 90:1109-1114 (1997); and Wu et al. J.Clin. Invest. 100: 1059-1070 (1997)). The binding site on human and murine antibodies for FcγR have been previously mapped to the so- called "lower hinge region" consisting of residues 233-239 (EU index numbering as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Woof et al. Molec. Immunol. 23:319-330 (1986); Duncan et al. Nature 332:563 (1988); Canfield and Morrison, J. Exp. Med. 173:1483-1491 (1991); Chappel et al, Proc. Natl. Acad. Sci USA 88:9036-9040 (1991). Of residues 233-239, P238 and S239 have been cited as possibly being involved in binding. Other previously cited areas possibly involved in binding to FcγR are: G316-K338 (human IgG) for r human FcγRI (by sequence comparison only; no substitution mutants were evaluated) (Woof er al. Molec. Immunol. 23:319-330 (1986)); K274-R301 (human IgGl) for human FcγRIII (based on peptides) (Sarmay et al Molec. Immunol. 21:43-51 (1984)); Y407-R416 (human IgG) for human FcγRIII (based on peptides) (Gergely et al. Biochem. Soc. Trans. 12:739-743 (1984)); as well as N297 and E318 (murine IgG2b) for murine FcγRII (Lund et al, Molec. Immunol. 29:53-59 (1992)). See also Armour et al Eur. J. Immunol. 29: 2613-2624 (1999). Presta in U.S. 6,737,056 describes polypeptide variants with improved or diminished binding to FcRs. See, also, Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001). Variant Fes that bind FcγR are also described in WO 2004/063351. CIq and two serine proteases, CIr and CIs, form the complex Cl, the first component of the complement dependent cytotoxicity (CDC) pathway. CIq 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 CIq to bind to at least two molecules of IgGl, IgG2, or IgG3 (the consensus is that IgG4 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 et al. Nature, 288:338-344 (1980) proposed that the binding site for the complement subcomponent CIq 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 amino 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 CIq. The data of Duncan and Winter were generated by testing the binding of a mouse IgG2b isotype to guinea pig CIq. The role of Glu318, Lys320 and Lys322 residues in the binding of CIq was confirmed by the ability of a short synthetic peptide containing these residues to inhibit complement mediated lysis. Similar results are disclosed 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 CIq binding by analysis of the ability of human IgG subclasses to carry out complement mediated cell lysis. Mutation of Ser331 to Pro331 in IgG4 conferred the ability to activate complement. (Tao et al., J. Exp. Med., 178:661-667 (1993); Brekke et al, Eur. J. Immunol, 24:2542-47 (1994)). From the comparison of the data of the Winter group, and the Tao et 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 CIq: 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 amino acid residue at position 331. Other reports suggested that human IgGl residues Lys235, and Gly237, located in the lower hinge region, play a critical role in complement fixation and activation. Xu et al.,J. Immunol. 150:152A (Abstract) (1993). WO94/29351 published December 22, 1994 reports that amino acid residues necessary for CIq and FcR binding of human IgGl 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 CIq and activate the complement cascade also depends on the presence, absence or modification of the carbohydrate moiety positioned between the two CH2 domains (which is normally anchored at Asn297). Ward and Ghetie, Therapeutic Immunology 2:77-94 (1995) at page 81. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased CIq binding capability are described in US patent No. 6,194,551B l and WO99/51642. The contents of those patent publications are specifically incorporated herein by reference. See, also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000). Another type of Fc receptor is the neonatal Fc receptor (FcRn). FcRn is structurally similar to major histocompatibility complex (MHC) and consists of an α-chain noncovalently bound to β2- microglobulin. The multiple functions of the neonatal Fc receptor FcRn are reviewed in Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. The FcRn plays a key role in IgG homeostasis based on a pH- dependent interaction with the antibody Fc region (Ghetie and Ward (2000) Annu Rev Immunol 18, 739-766; Ghetie and Ward (1997) Immunol Today 18, 592-598). Increasing the affinity of the Fc-FcRn complex at pH 6 while retaining low affinity at pH 7.4 has been shown to increase antibody half-life (Hinton et al. (2004) J Biol Cfiem 279, 6213-6216). FcRn plays a role in the passive delivery of immunoglobulin IgGs from mother to young and the regulation of serum IgG levels. FcRn acts as a salvage receptor, binding and transporting pinocytosed IgGs in intact form both within and across cells, and rescuing them from a default degradative pathway, as illustrated in Fig. 6. Although the mechanisms responsible for salvaging IgGs are still unclear, it is thought that unbound IgGs are directed toward proteolysis in lysosomes, whereas bound IgGs are recycled to the surface of the cells and released. This control takes place within the endothelial cells located throughout adult tissues. FcRn is expressed in at least the liver, mammary gland, and adult intestine. FcRn binds to IgG; the FcRn-IgG interaction has been studied extensively and appears to involve residues at the CH2, CH3 domain interface of the Fc region of IgG. These residues interact with residues primarily located in the α2 domain of FcRn Ghetie et al. in Nature Biotechnology 15: 637-640 (1997) reported random mutagenesis of Thr 252, Thr254, and Thr 256 in murine Fcγl, residues that are in proximity to the FcRn-IgG interaction site, to study the effect on the serum half-lives of these variant hinge-Fc fragments. The mutant with the highest affinity for murine FcRn has a longer half-life than the wild-type fragment despite its lower off-rate from FcRn at pH 7.4. In previous studies, extensive alanine-scanning by Presta and colleagues (Shields et al., J. Biol. Chem. 276: 6591-6604 (2001); Presta US patent 6,737,056) identified three Fc variants, N434A, E38OA, and T307A, that increase the affinity of Fc:FcRn by 3.5-fold, 2.2-fold, and 1.8-fold, respectively. The triple mutant has an affinity increase for FcRn at pH 6 of 12-fold relative to wild-type. Assuming structural homology between human Fc:FcRn and rat Fc-FcRn, for which an x-ray structure was known (Burnmeister et al., Nature 372: 336-343 (1994); Burnmeister et al., Nature 372: 379- 383 (1994)), Dall'Acqua et al. {Journal of Immunology. 169: 5171-5180 (2002); US2003/019031 1) pursued higher affinity improvements by phage display. They constructed four randomized libraries of Fc, each library having 4 or 5 residues completely randomized (i.e., having all possible amino acids substituted, resulting in two libraries of 204 diversity, and two libraries of 205 diversity) and selected for binding to murine FcRn. They reported that efforts to use human FcRn for screening the libraries were unsuccessful. Although the binding-affinity improvements identified from phage selections using murine FcRn also improved binding to human FcRn, direct phage selections using human FcRn were reportedly unsuccessful using the methods described (Dall'Acqua et al., 2002). From these libraries, they identified variants with mutations at H433, N434, and Y436 and at M252, S254, and T256. Two of their library-derived variants, H433K+N434F+Y436H and M252Y+S254T+T256E were found to have 10- to 20-fold increased affinity for both murine and human FcRn, at pH 6.0. The combination of these mutations led to a 30-fold increase in binding to murine FcRn and a 57-fold increase in binding to human FcRn. However, these variants also had increased affinity at pH 7.4, and do not have prolonged half-life in mice. This supports the conclusions that efficient IgG recycling is related to pH dependent affinity. No results were reported for these variants in primate species or in human FcRn transgenic animals. Ward et al, US 6,277,375, US 6,821,505 and US 6,165,745 describe immunoglobulin-like domains with increased half-lives and mutations at Fc positon 434. A resultant mutant N434Q actually showed reduced half-life. Israel and Simister in WO 98/23289 discuss altering residue 434 generally by addition, substitution or deletion of the residue to affect binding to FcRn but does not mention what that residue should be substituted with or what was to be added. Also assuming structural homology to the rat Fc-FcRn complex (Burnmeister et al., 1997) to model the human Fc-FcRn interface, Hinton et al., ( J. Biol. Chem. 279: 6213-6216 (2004)) identified residues T250, L314, and M428 in human IgG2 as residues that could be important for binding huFcRn. They identified mutations T250Q and M428L as having about 3-fold and 7-fold higher affinity, respectively, for human FcRn at pH 6.0, with no significant binding at pH 7.5. The combination variant T250Q+M428L was reported to have 28-fold increased binding. Similar binding was observed for rhesus monkey FcRn. Pharmacokinetic studies indicated that an IgG2 antibody with these two mutations has about a 1.9-fold longer elimination half-life (t 1/2 beta) in rhesus monkeys. There is a continuing need in the art to produce antibodies, in particular therapeutic antibodies having improved or modulated effector function. One of the goals of antibody engineering is to increase the half-life of antibodies in vivo. This can be achieved by modulating the interaction of the antibody with the neonatal Fc receptor (FcRn). The present invention satisfies these and other needs.

SUMMARY OF THE INVENTION The present invention provides polypeptides, in particular antibodies which demonstrate higher binding affinity for FcRn and FcγRIII than polypeptides having native sequence / wild type sequence Fc region. These Fc variant polypeptides and antibodies have the advantage of being salvaged and recycled rather than degraded. Increased serum half life will be beneficial to increase exposure to antibody and reduce the frequency of administration of Fc containing polypeptides such as Abs and other antibody fusion proteins such as immunoadhesins. The invention provides an isolated polypeptide comprising a variant IgG Fc region comprising at least an amino acid substitution at Asn 434 to Trp (N434W). A second isolated polypeptide is one comprising a variant IgG Fc region comprising at least an amino acid substitution at Asn 434 to His (N434H). Another isolated polypeptide provided by the invention is a polypeptide comprising a variant IgG Fc region comprising at least an amino acid substitution at Asn 434 to Tyr (N434Y) wherein the polypeptide does not further have an amino acid substitution selected from the group consisting of H433R, H433S, Y436H, Y436R, Y436T. Yet another polypeptide is an isolated polypeptide comprising a variant IgG Fc region comprising at least an amino acid substitution at Asn 434 to Phe (N434F) wherein the polypeptide does not further have an amino acid substitution of H433K,Y436H, M252Y, S254T, or T256E. The invention provides a polypeptide having a variant IgG Fc region wherein the variant IgG Fc region has an amino acid substitution consisting essentially of or consisting of Asn 434 to Tyr (N434Y). Also provided is a polypeptide having a variant IgG Fc wherein the variant IgG Fc has an amino acid substitution consisting essentially of or consisting of Asn 434 to Phe (N434F). In one embodiment, the isolated polypeptide of any of the preceding embodiments is an antibody. In another embodiment, the polypeptide is an immunoadhesin. In preferred embodiments, the IgG antibody of any of the preceding embodiments is murine or human, preferably human. Human IgG encompasses any of the human IgG isotypes of IgGl, IgG2, IgG3, IgG4. Murine IgG encompasses the isotypes of IgGl, 2a, 2b, 3. Preferably the therapeutic antibodies for human use are humanized, human or chimeric. In the preceding polypeptides which include antibodies, the polypeptide comprising the variant Fc region binds human FcRn at pH 6.0 with higher affinity than a polypeptide comprising native sequence IgG Fc region, and binds human FcRn with weaker binding affinity at pH 7.4 or pH 7.5 than at pH 6.0. In a preferred embodiment, the binding affinity of the Fc variant polypeptide for human FcRn at pH 6.0 is at least 4-, preferably at least 7-, 9-, or even more preferably at least 20-fold higher than native sequence/native sequence Fc. The polypeptides of the preceding embodiments have a longer serum half life in primate serum, particularly human or cynomolgus monkey serum, than a polypeptide with native sequence Fc region. Yet another aspect of the invention is an isolated polypeptide comprising a variant IgG Fc region comprising at least an amino acid substitution at Lys 334 to Leucine (K334L). In one embodiment this polypeptide binds human FcγRIII with higher affinity than a polypeptide having native sequence IgG Fc region, greater than 3-fold higher. This polypeptide also preferably exhibits increased ADCC over a polypeptide with native sequence IgG Fc region. Also provided is an isolated polypeptide comprising a variant IgG Fc region that exhibits improvement in binding to human FcRn at pH 6, but without increased binding at pH 7.4, which comprise at least an amino acid substitution at G385H, D312H, or N315H. In one embodiment, the isolated polypeptide of any of the preceding embodiments is an antibody. In another embodiment, the polypeptide is an immunoadhesin. In preferred embodiments, the IgG antibody of any of the preceding embodiments is murine or human, preferably human. Human IgG encompasses any of the human IgG isotypes of IgGl , IgG2, IgG3, IgG4. Murine IgG encompasses the isotypes of IgGl , 2a, 2b, 3. Preferably the therapeutic antibodies for human use are humanized, human or chimeric. The invention specifically provides antibodies of the preceding embodiments that bind the group of antigens consisting of CD20, Her2, BR3, TNF, VEGF, IgE, CD l Ia. In specific embodiments, the recombinantly produced, humanized antibodies that bind specific antigens comprise the sequences as disclosed in the SEQ ID NOs under the section subtitled antibody composition below. In a preferred embodiment the CD20 is a primate CD20. Human and cynomolgus monkey CD20 are specific embodiments. Where the antibody binds human CD20, in more specific embodiments, the antibody will comprise a VH sequence of SEQ ID NO. 2 and a L chain that comprises the VL sequence of SEQ ID NO. 1 or the full length L chain sequence of SEQ ID NO. 26. In another embodiment, the CD20 binding antibody comprises the C2B8 VL sequence from SEQ ID NO. 24 and the VH sequence from SEQ ID NO. 25 as shown in Fig. 10. In yet another embodiments, the isolated humanized antibody that binds human CD20 will comprise the VH and VL sequences disclosed below under humanized 2H7 variants. Where the antibody binds HER2, in more specific embodiments, the antibody will comprise VL and VH sequences selected from VL sequence of SEQ ID NO.3 paired with VH sequence of SEQ ID NO. 4; and VL sequence of SEQ ID NO. 5 paired with VH sequence of SEQ ID NO. 6. One specific anti-HER2 antibody comprises a variant IgG Fc region comprising at least an amino acid substitution at Asn 434 to His (N434H). Additionally, the invention provides an isolated anti-HER2 antibody comprising VL sequence of SEQ ID NO. 5, VH sequence of SEQ ID NO. 6, and a variant IgG Fc region comprising at least an amino acid substitution at Asn 434 to Ala (N434A). In preferred embodiments, the VH and VL sequences provided are joined to human IgGl constant region, the sequence of which is shown in FIG. 4 and FIG. 5. In one aspect, the antibodies of the preceding embodiments further comprise one or more amino acid substitutions in the Fc region that result in the antibody exhibiting one or more of the properties selected from increased FcγR binding, increased ADCC, increased CDC, decreased CDC, increased ADCC and CDC, increased ADCC but decreased CDC function, increased FcRn binding and serum half life, as compared to the antibody having native sequence Fc region. An antibody of the preceding embodiments may further comprise one or more amino acid substitutions in the IgG Fc region at a residue position selected from the group consisting of D265A, S298A/E333A/K334A, K334L, K322A, K326A, K326W, E380A and E380A/T307A, wherein the numbering of the residues is that of the EU index as in Kabat. Wherein the polypeptide comprises an amino acid substitution of K334L, it may further comprise one or more amino acid substitutions in the IgG Fc region at a residue position selected from the group consisting of D265 A, S298A/E333A, K322A, K326A, K326W, E380A and E380A/T307A. The invention also provides a composition comprising the polypeptide or antibody of any of the preceding embodiments and a carrier, such as a pharmaceutically acceptable carrier. Another aspect of the invention is an isolated nucleic acid encoding a polypeptide of any one of the preceding embodiments. Expression vectors encoding the polypeptides including antibodies of the invention are also provided. Also provided is a host cell comprising a nucleic acid encoding a polypeptide or antibody of the invention. Host cells that express and produce the polypeptide include CHO cell or E. coli bacterial cell. A method is also provided for producing the polypeptides, antibodies and immunoadhesins of the invention, comprising culturing a host cell comprising a nucleic acid encoding the polypeptide which host cell produces the polypeptide, and recovering the polypeptide from the cell culture. Still another aspect of the invention is an article of manufacture comprising a container and a composition contained therein, wherein the composition comprises a polypeptide or antibody of any of the preceding embodiments. The article of manufacture can further comprise a package insert indicating that the composition can be used to treat the indication the antibody as intended for. The invention provides a method of treating a B cell neoplasm or malignancy characterized by B cells expressing CD20, comprising administering to a patient suffering from the neoplasm or malignancy, a therapeutically effective amount of a CD20 binding antibody, in particular, a humanized CD20 binding antibody of the above embodiments. In specific embodiments, the B cell neoplasm is non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, lymphocyte predominant Hodgkin's disease (LPHD), follicular center cell (FCC) lymphomas, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) and Hairy cell leukemia. One embodiment provides for a method of treating chronic lymphocytic leukemia, comprising administering to a patient suffering from the leukemia, a therapeutically effective amount of an antibody of comprising a variant IgG Fc of the above embodiments, which antibody binds human CD20, wherein the antibody further comprises amino acid substitution K326A or K326W. A further aspect is a method of alleviating a B-cell regulated autoimmune disorder comprising administering to a patient suffering from the disorder, a therapeutically effective amount of a CD20 binding antibody comprising a variant IgG Fc of the above embodiments. In specific embodiments, the autoimmune disorder is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic throbocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus, Reynaud's syndrome, Sjorgen's syndrome and glomerulonephritis. Other treatment methods provided are as follows: A method of treating an angiogenesis related disorder is provided which comprises administering to a patient suffering from the disorder, a therapeutically effective amount of a VEGF binding antibody comprising a variant IgG Fc of the above embodiments. A method of treating a HER2 expressing cancer, comprising administering to a patient suffering from the cancer, a therapeutically effective amount of a HER2 binding antibody that comprises a variant IgG Fc of the above embodiments. A method of treating a LFA-I mediated disorder comprising administering to a patient suffering from the disorder, a therapeutically effective amount of an antibody that binds human anti-CD 11 a comprising a variant IgG Fc of the above embodiments. A method of treating an IgE-mediated disorder, comprising administering to a patient suffering from the disorder, a therapeutically effective amount of an antibody that binds human IgE comprising a variant IgG Fc of the above embodiments. Yet another aspect of the invention is a method of screening for a polypeptide with higher affinity binding to FcRn at pH 6.0 and with weaker binding affinity at pH 7.4 than at pH 6.0. Preferably the polypeptide has higher affinity binding to human FcRn at pH 6.0 than a polypeptide or antibody having native sequence IgG Fc. The method comprises expressing a candidate polypeptide on phage, providing huFcRn immobilized on a solid matrix, allow phage particles to bind to the FcRn on the matrix, removing unbound phage particles by multiple rounds of washes each round with increasing stringency; and eluting the remaining bound phage at pH 7.4.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a schematic representation of a native IgG and enzymatic digestion thereof to generate various antibody fragments. Disulfide bonds are represented by S-S between CHl and CL domains and the two CH2 domains. V is variable domain; C is constant domain; L stands for light chain and H stands for heavy chain. Figures 2A and 2B show the VL (FIG. 2A; SEQ ID No. 5) and VH (FIG. 2B; SEQ ID No. 6) amino acid sequences of an anti-Her2 antibody (Trastuzumab). Figures 3 A and 3B show the sequences of the light and heavy chains of specific anti-IgE antibodies E25, E26, E27 and Hu-901. Figure 4 depicts alignments of native sequence human IgG Fc region sequences, humlgGl (non-A and A allotypes; SEQ ID NOs:29 and 30, respectively), humIgG2 (SEQ ID NO:31), humIgG3 (SEQ ID NO:32) and humIgG4 (SEQ ID NO:33) with differences between the sequences marked with asterisks. Figure 5 depicts alignments of native sequence IgG Fc regions. Native sequence human IgG Fc region sequences, humlgGl (non-A and A allotypes) (SEQ ID NOs: 29 and 30, respectively), humIgG2 (SEQ ID NO:31), humIgG3 (SEQ ID NO:32) and humIgG4 (SEQ ID NO:33), are shown. The human IgGl sequence is the non-A allotype, and differences between this sequence and the A allotype (at positions 356 and 358; EU numbering system) are shown below the human IgGl sequence. Native sequence murine IgG Fc region sequences, murlgGl (SEQ ID NO:34), murIgG2A (SEQ ID NO:35), murIgG2B (SEQ ID NO:36) and murIgG3 (SEQ ID NO:37), are also shown. Figure 6 depicts the role of FcRn in IgG homeostasis. The ovals within the vesicles are FcRn. Figure 7 shows the sequence of the human IgGl Fc protein variant (W0437) used for phage- display. The mature protein sequence (SEQ ID NO. 38) of the soluble Fc is shown; the portion of the M13 g3p used for phage display is not shown. The first residue in the mature protein sequence, Ser, corresponds to a mutation of the second Cys of the hinge region (C229), and the last residue (Leu) is the site of fusion to M13 g3p. The underlined residue corresponds to N434. Figure 8 shows equilibrium analysis of E. coli-produced wild-type and variant Fc binding to huFcRn at pH 6.0 by SPR (BIAcore). Figure 9 shows ELISA analysis of 2H7 IgGl variants binding to human FcRn. Human IgGl variants were produced by transient transfection in mammalian cells, and compared to humanized 4D5 (Herceptin®) for binding FcRn at pH 6.0 or pH 7.4. NeutrAvidin coat/FcRn-biotinylated/antibody/goat anti- hu-IgG-F(ab)'2-HRP association (pH 6.0) and dissociation (pH 7.4). Figure 10 shows the C2B8 light (SEQ ID NO. 24) and heavy chain (SEQ ID NO. 25) sequences. The constant and Fc regions are boxed and the variable regions are outside of the box. Figure 11 shows binding affinity of 2H7 variants to human FcγRIII (V158) in an ELISA. Figure 12 shows the serum concentration-time profile of PRO145234, PRO145181, and PRO145182 following a single IV Dose of 20 mg/kg in Cynomolgus monkeys. Figure 13 shows the binding of Herceptin and hu4D5(N434H) to human FcRn at pH 6.0 and pH 7.4, as assayed by ELISA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An important component of the homeostasis of IgG is the recycling pathway mediated by the pH dependent interaction of the Fc region with the cell-surface neonatal receptor, FcRn. An important goal for the field of antibody engineering has been to identify mutations in the Fc that increase the affinity of the Fc- FcRn complex at pH 6.0, while retaining low affinity at pH 7.4 (Ghetie et al., 1997). Furthermore, it is highly desirable to minimize the number of mutations introduced to the Fc to avoid potential anti-drug immune responses in patients treated with therapeutic antibodies that include mutations to the highly conserved constant domains. In the present invention we identified single amino acid mutations (N434W, N434Y, and N434F; the numbering system used here for the IgG Fc region is the EU notation as described in Kabat, Sequences of Proteins of Immunological Interest (1991)) that increase the affinity of Fc for human FcRn, the N434W mutant increased Fc binding affinity by about 170-fold at pH 6.0 and retain low affinity for huFcRn at pH 7.4, through the use of phage-display and a novel method for constructing libraries of randomized amino acids. Methods of measuring binding to FcRn are known (see, e.g., Ghetie 1997, Hinton 2004) as well as described in the examples. Binding to human FcRn in vivo and serum half life of human FcRn high affinity binding polypeptides can be assayed, e.g, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with the Fc variant polypeptides. In separate embodiments, the polypeptide and specifically the antibody of the invention having a variant IgG Fc exhibits increased binding affinity for human FcRn over a polypeptide having wild-type IgG Fc, by at least 7 fold, at least 9 fold, more preferably at least 20 fold, preferably at least 40 fold, even more preferably at least 70 to 100 fold. In a specific embodiment, the binding affinity for human FcRn is increased about 70 fold. The invention also provides an isolated polypeptide comprising a variant IgG Fc region comprising at least an amino acid substitution at Lys 334 to Leucine (K334L). This polypeptide binds human FcγRIII with higher affinity than native sequence IgG Fc, greater than 3-fold higher. These polypeptides preferably exhibit increased ADCC in the presence of human effector cells over a polypeptide with native sequence IgG Fc. Where the antibody is a CD20 binding antibody, ADCC activity can be tested in transgenic mice expressing human CD20 plus CDl 6 (hCD20+/hCDl 6+ Tg mice). Assays for ADCC have been described, see, e.g., Presta U.S. Patent No. 6,737,056. For binding affinity to FcRn, in one embodiment, the EC50 or apparent Kd (at pH 6.0) of the polypeptide is <= 100 nM, more preferably <= 10 nM. For increased binding affinity to FcγRIII (F158; i.e. low-affinity isotype), in one embodiment the EC50 or apparent Kd <= 10 nM, and for FcgRIII (V158; high- affinity) the EC50 or apparent Kd <= 3 nM. Throughout the present specification and claims, the numbering of the residues in an immunoglobulin 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 IgGl EU antibody. A "parent polypeptide" is a polypeptide comprising an amino acid sequence which lacks one or more of the Fc region modifications disclosed herein and 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 pre-existing amino acid sequence modifications (such as additions, deletions and/or substitutions). The term "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain, e.g., as shown in Figure 1. The "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3, as shown, for example, in Fig. 1. The last residue, lysine, in the heavy chain of IgGl can but does not have to be present as the terminal residue in the Fc in the mature protein. The "CH2 domain" of a human IgG Fc region (also referred to as "C72" domain) usually extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG 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.22:16l-206 (1985). The "CH3 domain" comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e. from about amino acid residue 341 to about amino acid residue 447 of an IgG) A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include CIq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g. an antibody variable domain) and can be assessed using various assays as herein disclosed, for example. A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions are shown in Figures 4 and 5. and include a native sequence human IgGl Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof. Native sequence murine Fc regions are shown in Fig. 5. A "variant Fc region" comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one "amino acid modification" as herein defined. Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g. from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith. "Homology" is defined as the percentage of residues in the amino acid sequence variant that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. One such computer program is "Align 2", authored by Genentech, Inc., which was filed with user documentation in the United States Copyright Office, Washington, DC 20559, on December 10, 1991. 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 terms "Fc receptor" or "FcR" are used to describe a receptor that binds to the Fc region of an IgG antibody. The preferred FcR is a native sequence human FcR. In one embodiment, the FcR is a FcγR which includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITEM) in its cytoplasmic domain, (see review M. in Dae'ron, Annu. Rev. Immunol. 15:203-234 (1997)). The term includes allotypes, such as FcγRIIIA allotypes: FcγRIIIA-Phel58, FcγRIIIA-Vall58, FcγRIIA-R131 and/or FcγRIIA-H131. FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et 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 et al., J. Immunol. 117:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)). "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells {e.g. Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen- bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies "arm" the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998). "Human effector cells" are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred. The effector cells may be isolated from a native source thereof, e.g. from blood or PBMCs as described herein. "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (CIq) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed. A polypeptide with a variant IgG Fc with "altered" FcR binding affinity or ADCC activity is one which has either enhanced or diminished FcR binding activity (FcγR or FcRn) and/or ADCC activity compared to a parent polypeptide or to a polypeptide comprising a native sequence Fc region. The variant Fc which "exhibits increased binding" to an FcR binds at least one FcR with better affinity than the parent polypeptide. The improvement in binding compared to a parent polypeptide may be about 3 fold, preferably about 5, 10, 25, 50, 60, 100, 150, 200, up to 500 fold, or about 25% to 1000% improvement in binding. The polypeptide variant which "exhibits decreased binding" to an FcR, binds at least one FcR with worse affinity than a parent polypeptide. The decrease in binding compared to a parent polypeptide may be about 40% or more decrease in binding. Such Fc variants which display decreased binding to an FcR may possess little or no appreciable binding to an FcR, e.g., 0-20% binding to the FcR compared to a native sequence IgG Fc region, e.g. as determined in the Examples herein. The polypeptide having a variant Fc which binds an FcR with "better affinity" of "higher affinity" than a polypeptide or parent polypeptide having wild type or native sequence IgG Fc is one which binds any one or more of the above identified FcRs with substantially better binding affinity than the parent polypeptide with native sequence Fc, when the amounts of polypeptide with variant Fc and parent polypeptide in the binding assay are essentially the same. For example, the variant Fc polypeptide with improved FcR binding affinity may display from about 2 fold to about 300 fold, e.g. from about 3 fold to about 170 fold improvement in FcR binding affinity compared to the parent polypeptide, where FcR binding affinity is determined, for example, as disclosed in the Examples herein. The polypeptide comprising a variant Fc region which "exhibits increased ADCC" or mediates antibody-dependent cell-mediated cytotoxicity (ADCC) in the presence of human effector cells more effectively than a polypeptide having wild type IgG Fc is one which in vitro or in vivo is substantially more effective at mediating ADCC, when the amounts of polypeptide with variant Fc region and the polypeptide with wild type Fc region sed in the assay are essentially the same. Generally, such variants will be identified using the in vitro ADCC assay as herein disclosed, but other assays or methods for determining ADCC activity, e.g. in an animal model etc, are contemplated. The preferred variant is from about 5 fold to about 100 fold, e.g. from about 25 to about 50 fold, more effective at mediating ADCC than the wild type Fc. An "amino acid modification" refers to a change in the amino acid sequence of a predetermined amino acid sequence. Exemplary modifications include an amino acid substitution, insertion and/or deletion. The preferred amino acid modification herein is a substitution. An "amino acid modification at" a specified position, e.g. of the Fc region, refers to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent the specified residue. By insertion "adjacent" a specified residue is meant insertion within one to two residues thereof. The insertion may be N-terminal or C-terminal to the specified residue. An "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence with another different "replacement" amino acid residue. The replacement residue or residues may be "naturally occurring amino acid residues" {i.e. encoded by the genetic code) and selected from the group consisting of: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (GIn); glutamic acid (GIu); glycine (GIy); histidine (His); isoleucine (He): leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (VaI). Preferably, the replacement residue is not cysteine. Substitution with one or more non-naturally occurring amino acid residues is also encompassed by the definition of an amino acid substitution herein. A "non-naturally occurring amino acid residue" refers to a residue, other than those naturally occurring amino acid residues listed above, which is able to covalently bind adjacent amino acid residues(s) in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine and other amino acid residue analogues such as those described in Ellman et al. Meth. Enzym. 202:301-336 (1991). To generate such non-naturally occurring amino acid residues, the procedures of Noren et al. Science 244:182 (1989) and Ellman et al., supra, can be used. Briefly, these procedures involve chemically activating a suppressor tRNA with a non-naturally occurring amino acid residue followed by in vitro transcription and translation of the RNA. The term "conservative" amino acid substitution as used within this invention is meant to refer to amino acid substitutions which substitute functionally equivalent amino acids. Conservative amino acid changes result in silent changes in the amino acid sequence of the resulting peptide. For example, one or more amino acids of a similar polarity act as functional equivalents and result in a silent alteration within the amino acid sequence of the peptide. In general, substitutions within a group may be considered conservative with respect to structure and function. However, the skilled artisan will recognize that the role of a particular residue is determined by its context within the three-dimensional structure of the molecule in which it occurs. For example, Cys residues may occur in the oxidized (disulfide) form, which is less polar than the reduced (thiol) form. The long aliphatic portion of the Arg side chain may constitute a critical feature of its structural or functional role, and this may be best conserved by substitution of a nonpolar, rather than another basic residue. Also, it will be recognized that side chains containing aromatic groups (Tφ, Tyr, and Phe) can participate in ionic-aromatic or "cation-pi" interactions. In these cases, substitution of one of these side chains with a member of the acidic or uncharged polar group may be conservative with respect to structure and function. Residues such as Pro, GIy, and Cys (disulfide form) can have direct effects on the main chain conformation, and often may not be substituted without structural distortions. An "amino acid insertion" refers to the incorporation of at least one amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, the present application contemplates larger "peptide insertions", e.g. insertion of about three to about five or even up to about ten amino acid residues. The inserted residue(s) may be naturally occurring or non-naturally occurring as disclosed above. An "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence. Amino acids may be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), VaI (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M) (2) uncharged polar: GIy (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), GIn (Q) (3) acidic: Asp (D), GIu (E) (4) basic: Lys (K), Arg (R), His(H) Alternatively, naturally occurring residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, VaI, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, GIn; (3) acidic: Asp, GIu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: GIy, Pro; (6) aromatic: Trp, Tyr, Phe.

"Hinge region" is generally defined as stretching from Glu216 to Pro230 of human IgGl (Burton, Molec. Immunol.22Λ6l-2Q6 (1985)). Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain S-S bonds in the same positions. The "lower hinge region" of an Fc region is normally defined as the stretch of residues immediately C-terminal to the hinge region, i.e. residues 233 to 239 of the Fc region. Prior to the present invention, FcγR binding was generally attributed to amino acid residues in the lower hinge region of an IgG Fc region. "CIq" is a polypeptide that includes a binding site for the Fc region of an immunoglobulin. CIq together with two serine proteases, CIr and CIs, forms the complex Cl , the first component of the complement dependent cytotoxicity (CDC) pathway. Human CIq can be purchased commercially from, e.g. Quidel, San Diego, CA. 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. "Functional fragments", of the antibodies of the invention comprise a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody or the Fc region of an antibody which retains FcR binding capability. Examples of antibody fragments include linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can 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 which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. 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 hybridoma method first described by Kohler et 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 et al., Nature, 352:624-628 (1991) and Marks et al., J. MoI. Biol., 222:581-597 (1991), for example. The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(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; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Methods of making chimeric antibodies are known in the art. "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit 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 which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize 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 hypervariable 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 although the FR regions may include one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR are typically no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321 :522- 525 (1986); Reichmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992). The humanized antibody includes a PRIMATIZED® antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest. Methods of making humanized antibodies are known in the art. Human antibodies can also be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. MoI. Biol., 227:381 (1991); Marks et al., J. MoI. Biol., 222:581 (1991). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies. Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(l):86-95 (1991). As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin can be obtained from any immunoglobulin, such as IgG-I, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-I and IgA-2), IgE, IgD or IgM. For example, useful immunoadhesins according to this invention are polypeptides that comprise the BLyS binding portions of a BLyS receptor without the transmembrane or cytoplasmic sequences of the BLyS receptor. In one embodiment, the extracellular domain of BR3, TACI or BCMA is fused to a constant domain of an immunoglobulin sequence. A "fusion protein" and a "fusion polypeptide" refer to a polypeptide having two portions covalently linked together, where each of the portions is a polypeptide having a different property. The property may be a biological property, such as activity in vitro or in vivo. The property may also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, etc. The two portions may be linked directly by a single peptide bond or through a peptide linker containing one or more amino acid residues. Generally, the two portions and the linker will be in reading frame with each other. An "isolated" polypeptide or antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the polypeptide or antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. The biological activity of the CD20 binding and humanized CD20 binding antibodies of the invention will include at least binding of the antibody to human CD20, more preferably binding to human and other primate CD20 (including cynomolgus monkey, rhesus monkey, chimpanzees). The antibodies would bind CD20 with a Kj value of no higher than 1 x 10"8, preferably a K<j value no higher than about 1 x 10"9, and be able to kill or deplete B cells in vivo, preferably by at least 20% when compared to the appropriate negative control which is not treated with such an antibody. B cell depletion can be a result of one or more of ADCC, CDC, or other mechanism. In some embodiments of disease treatment herein, specific effector functions or mechanisms may be desired over others and certain variants of the humanized 2H7 are preferred to achieve those biological functions, such as ADCC. "Treating" or "treatment" or "alleviation" refers to therapeutic treatment wherein the object is to lessen or slow down the targeted pathologic condition or disorder. A subject is successfully "treated" for example, a CD20 positive cancer or an autoimmune disease if, after receiving a therapeutic amount of a CD20 binding antibody of the invention according to the methods of the present invention, the subject shows observable and/or measurable reduction in or absence of one or more signs and symptoms of the particular disease. For example, for cancer, reduction in the number of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition (i.e., slow to some extent and preferably stop) of tumor metastasis; inhibition, to some extent, of tumor growth; increase in length of remission, and/or relief to some extent, one or more of the symptoms associated with the specific cancer; reduced morbidity and mortality, and improvement in quality of life issues. Reduction of the signs or symptoms of a disease may also be felt by the patient. Treatment can achieve a complete response, defined as disappearance of all signs of cancer, or a partial response, wherein the size of the tumor is decreased, preferably by more than 50 percent, more preferably by 75%. A patient is also considered treated if the patient experiences stable disease. In a preferred embodiment, the cancer patients are still progression-free in the cancer after one year, preferably after 15 months. These parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician of appropriate skill in the art. A "therapeutically effective amount" refers to an amount of an antibody or a drug effective to "treat" a disease or disorder in a subject. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. See preceding definition of "treating". To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. "Chronic" administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g. At2", I131, 1125, Y90, Re'86, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. A "growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially a CD20 expressing cancer cell, either in vitro or in vivo. Thus, the growth inhibitory agent may be one which significantly reduces the percentage of PSCA expressing cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest Gl also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells. A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE®, Rhδne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. "Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. The term "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal herein is human.

Compositions In specific embodiments, the antibodies will comprise the V domain sequences or full length sequences shown below but will have the Fc mutations of the present invention that improve one or more of the Fc effector functions. The polypeptides and antibodies of the present invention may further comprise other amino acid substitutions that, e.g., improve or reduce other Fc function or further improve the same Fc function, increase antigen binding affinity, increase stability, alter glycosylation, or include allotypic variants. The antibodies may further comprise one or more amino acid substitutions in the Fc region that result in the antibody exhibiting one or more of the properties selected from increased FcγR binding, increased ADCC, increased CDC, decreased CDC, increased ADCC and CDC function, increased ADCC but decreased CDC function (e.g., to minimize infusion reaction), increased FcRn binding and serum half life, as compared to the polypeptide and antibodies that have wild type Fc. These activities can be measured by the methods described herein. For additional amino acid alterations that improve Fc function, see US 6,737,056, incorporated herein by reference. Any of the antibodies of the present invention may further comprise at least one amino acid substitution in the Fc region that decreases CDC activity, for example, comprising at least the substitution K322A. See US Patent No. 6,528,624Bl (Idusogie et al.). Mutations that improve ADCC and CDC include S298A/E333A/K334A also referred to herein as the triple Ala mutant. K334L increases binding to CDl 6. K322A results in reduced CDC activity; K326A or K326W enhances CDC activity D265A results in reduced ADCC activity. Glycosylation variants that increase ADCC function are described in WO 03/035835 incorporated herein by reference. Stability variants are variants that show improved stability with respect to e.g., oxidation, deamidation. A recombinant humanized version of the murine HER2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2, Trastuzumab or HERCEPTIN®; U.S. Patent No. 5,821 ,337) is clinically active in patients with HER2-overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy (Baselga et al., J. Clin. Oncol. \A:12>1-1AA (1996)). Trastuzumab received marketing approval from the Food and Drug Administration September 25, 1998 for the treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 protein. Other HER2 antibodies with various properties have been described in Tagliabue et al. Int. J. Cancer 47:933-937 (1991); McKenzie et al. Oncogene 4:543-548 (1989); Maier et al. Cancer Res. 51 :5361- 5369 (1991); Bacus et al. Molecular Carcinogenesis 3:350-362 (1990); Stancovski et al. PNAS (USA) 88:8691-8695 (1991); Bacus et al. Cancer Research 52:2580-2589 (1992); Xu et al. Int. J. Cancer 53:401- 408 (1993); WO94/00136; Kasprzyk et al. Cancer Research 52:2771-2776 (1992);Hancock et al. Cancer Res. 51:4575-4580 (1991); Shawver et al. Cancer Res. 54: 1367-1373 (1994); Arteaga et al. Cancer Res. 54:3758-3765 (1994); Harwerth et al. J. Biol. Chem. 267:15160-15167 (1992); U.S. Patent No. 5,783,186; and Klapper et al. Oncogene 14:2099-2109 (1997). In one embodiment, the anti-HER2 antibody comprises the following VL and VH domain sequences (the CDRs are indicated in bold): humanized 2C4 version 574 antibody VL (SEQ ID NO: 3 ) 1 10 20 30 40 50 60

D IIQMT IQSPSS ILSASV IGDRVT IITCKA ISQDVS IIGVAW IYQQKP IGKAPK ILLIYS IASYRY ITGVPS I

RFSGSGSG

and humanized 2C4 version 574 antibody VH (SEQ ID NO: 4 )

1 10 20 30 40 50 60 E IVQLV IESGGG ILVQPG IGSLRL ISCAAS IGFTFT IDYTMD IWVRQA IPGKGL IEWVAD IVNPNS IGGSIY I 70 80 90 100 110 120 I I I I I I I I I I I I NQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS

In another embodiment, the anti-HER2 antibody comprises the VL (SEQ ID NO. 5) and VH (SEQ ID NO. 6) domain sequences of Trastuzumab as shown in Figure 2A and Figure 2B. In specific embodiments, the anti-VEGF antibodies of the invention comprise the following sequences: In one embodiment, the anti-VEGF antibody comprises VL sequence of : (SEQ ID NO: 7) DIQMTQTTSS LSASLGDRVI ISCSASQDIS NYLNWYQQKP DGTVKVLIYF TSSLHSGVPS RFSGSGSGTD YSLTISNLEP EDIATYYCQQ YSTVPWTFGG GTKLEIKR; and VH sequence of: (SEQ ID NO: 8) EIQLVQSGPE LKQPGETVRI SCKASGYTFT NYGMNWVKQA PGKGLKWMGW INTYTGEPTY AADFKRRFTF SLETSASTAY LQISNLKNDD TATYFCAKYP HYYGSSHWYF DVWGAGTTVT VSS;

In another embodiment, the anti-VEGF antibody comprises VL sequence of: (SEQ ID NO: 9) DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKR; and

VH sequence of: (SEQ ID NO: 10) EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS.

In a third embodiment, the anti-VEGF antibody comprises VL sequence of : (SEQ ID NO: 1 1) DIQLTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKR; and

VH sequence of: (SEQ ID NO: 12)

EVQLVESGGG LVQPGGSLRL SCAASGYDFT HYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP YYYGTSHWYF DVWGQGTLVT VSS

The humanized anti-CDl Ia antibody efalizumab or Raptiva® (U.S. Patent No. 6,037,454) received marketing approval from the Food and Drug Administration on October 27, 2003 for the treatment for the treatment of psoriasis. One embodiment provides for an anti-human CDl 1 a antibody comprising the Fc mutations of the present invention that improve one or more of the Fc effector functions, the antibody comprising the VL and VH sequences of HuMHM24 below:

Variable Light (SEQ ID NO: 13)

HUMHM24 DIQMTQSPSSLSASVGDRVTITCRASKTISKYLAWYQQKPGKAPKLLIY 1 10 20 30 40 HUMHM24 SGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPLTFGQ 60 70 80 90 100

HUMHM24 GTKVEIKR

Variable Heavy (SEQ ID NO: 14)

HUMHM24 EVQLVESGGGLVQPGGSLRLSCAASGYSFTGHWMNWVRQAPGKGLEWV 1 10 20 30 40

HUMHM24 GMIHPSDSETRYNQKFKDRFTISVDKSKNTLYLQMNSLRAEDTAVYYCAR 50 a 60 70 80 abc 90

HuMHM24 GIYFYGTTYFDYWGQGTLVTVSS 100 110

The anti-human CDl Ia antibody may comprise the VH of SEQ ID NO: 14 and the full length L chain of HuMHM24 having the sequence of:

DIQMTQSPSSLSASVGDRVTITCRASKTISKYLAWYQQKPGKAPKLLIYS GSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPLTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQIDNO: 15)

In specific embodiments, the anti-IgE antibodies having the Fc mutations of the present invention that improve one or more of the Fc effector functions comprise at least the V region sequences of the anti- IgE antibodies E25, E26, E27 and Hu-901, the L and H chain sequences of which are shown in Figures 3A and 3B. The light chain sequences are as follows: E25 L chain (SEQ ID NO. 16); E26 L chain (SEQ ID NO. 17); E27 L chain (SEQ ID NO. 18); and Hu-901 L chain (SEQ ID NO. 19). The heavy chains sequences are as follows: E25 H chain (SEQ ID NO. 20 ); E26 H chain (SEQ ID NO. 21); E27 H chain (SEQ ID NO. 22); and Hu-901 H chain (SEQ ID NO. 23 ). For the anti-IgE antibodies shown in Figures 3 A and 3B, the VL ends at VEIK (residue 1 1 1 in Figure 3A) and the VH ends at VTVSS [around residue#121 in Figure 3B). The VL sequences of E25, E26, E27 and Hu-901 antibodies are as SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51 and SEQ ID NO. 53, respectively. The VH sequences of E25, E26, E27 and Hu- 901 antibodies are as SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52 and SEQ ID NO. 54, respectively. In another embodiment, the anti-IgE antibodies having the Fc mutations of the present invention will comprise a L chain selected from any one of the antibodies whose sequences are shown in Figure 3A: E25 L chain (SEQ ID NO. 16); E26 L chain (SEQ ID NO. 17); E27 L chain (SEQ ID NO. 18); and Hu-901 L chain (SEQ ID NO. 19).

Examples of antibodies which bind the CD20 antigen include: "C2B8" which is now called "Rituximab" ("RITUXAN®") (US Patent No. 5,736,137, expressly incorporated herein by reference); the yttrium-[90]-labeled 2B8 murine antibody designated "Y2B8" or "Ibritumomab Tiuxetan" ZEVALIN® (US Patent No. 5,736,137, expressly incorporated herein by reference); murine IgG2a "Bl," also called "Tositumomab," optionally labeled with 131I to generate the "13 H-B 1 " antibody (iodine 1131 tositumomab, BEXXAR™) (US Patent No. 5,595,721, expressly incorporated herein by reference); murine monoclonal antibody "1F5" (Press et al. Blood 69(2):584-591 (1987) and variants thereof including "framework patched" or humanized 1F5 (WO03/002607, Leung, S.); ATCC deposit HB-96450); murine 2H7 and chimeric 2H7 antibody (Clark et al. PNAS 82: 1766-1770 (1985); US Patent No. 5,500,362, expressly incorporated herein by reference); humanized 2H7; huMax-CD20 (WO 04/035607, Genmab, Denmark); AME-133 (Applied Molecular Evolution); A20 antibody or variants thereof such as chimeric or humanized A20 antibody (cA20, hA20, respectively) (US 2003/0219433, Immunomedics); and monoclonal antibodies L27, G28-2, 93-1B3, B-Cl or NU-B2 available from the International Leukocyte Typing Workshop (Valentine et al., In: Leukocyte Typing III (McMichael, Ed., p. 440, Oxford University Press (1987)). The terms "rituximab" or "RITUXAN®" herein refer to the genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen and designated "C2B8" in US Patent No. 5,736,137, expressly incorporated herein by reference, including fragments thereof which retain the ability to bind CD20. The C2B8 light (SEQ ID NO. 24) and heavy chain (SEQ ID NO. 25) sequences are shown in Figure 10. The VL and VH are delineated. In specific embodiments, antibodies which bind the CD20 antigen include the humanized 2H7 vl6 antibody and variants thereof described below. Humanized 2H7 v.16 refers to an intact antibody or antibody fragment comprising the variable light sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIY APSNLASGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKR (SEQ ID NO: 1); and

variable heavy sequence:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGD TSYNQKF KGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSS (SEQ ID NO: 2)

Where the humanized 2H7v.l6 antibody is an intact antibody, preferably it comprises the vl6 full length light chain amino acid sequence: 2H7.vl6 Light Chain DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIY APSNLASGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 26); and full length heavy chain amino acid sequence:

2H7.vl6 Heavy Chain EVQLVESGGGLVQPGGSLRLSCAASG YTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSL RAEDTAVYYCARVVYYSNSYWYFDVWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKD YFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALP APIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 27).

The V region of all other variants based on version 16 will have the amino acid sequences of vl 6 except at the positions of amino acid substitutions which are indicated in the table below. Unless otherwise indicated below, the 2H7 variants will have the same L chain as that of vl6.

Each of versions 114, 115, 1 16,138 ,477, 511 comprises the VL sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIY APSNLASGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKR (SEQ ID NO: 41)

Each of versions 96, 114, 115, 116,138, 477 comprises the VH sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSY NQKF KGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSASYWYFDVWGQGTLVTVSS (SEQ ID NO: 42)

A variant of the preceding humanized 2H7 mAb is 2H7v.31 having the same VL (SEQ ID NO. 1) and VH (SEQ ID NO. 2) and L chain (SEQ ID NO: 26) sequences as vl6 above, with the full length H chain amino acid sequence: 2H7v.3 I H chain: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSY NQKF KGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSAS TKGP SVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYR VVSVLTVLHQDWLNGKEYKC KVSNKALP APIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 28).

Another variant of the humanized 2H7 antibody is 2H7v.l 38 having the L chain sequence of SEQ ID NO. 39 and the H chain sequence of SEQ ID NO. 40 shown below:

2H7.vl38 Light chain (SEQ ID NO. 39) DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIY APSNLASGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQW AFNPPTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

2H7.vl38 Heavy Chain (SEQ ID NO. 40) EVQLVESGGGLVQPGGSLRLSCAASG YTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSL RAEDTAVYYCARVVYYSASYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKD YFPEPVTVSWNSGALTSGVHTFP A VLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSOTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKC KVSNAALP APIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK

Variant humanized 2H7 v.477 has the L chain sequence of SEQ ID NO. 39 and the H chain sequence of SEQ ID NO. 43 (with the amino acid substitution of N434W): EVQLVESGGGLVQPGGSLRLSCAASG YTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSL RAEDTAVYYCAR WYYSASYWYFDVWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRWSVLTVLHQDWLNGKEYKC KVSNAALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHWHYTQKSL SLSPGK (SEQ ID NO: 43). Other variants of vl38 have amino acid substitution of N434Y or N434F.

Variant 2H7v.l 14 has the complete L chain sequence of SEQ ID NO. 39 and the complete H chain amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASG YTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSL RAEDTAVYYCARWYYSASYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRWSVLTVLHQDWLNGKEYKC KVSNKALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO : 44 ) .

Variant 2H7v.51 1 comprises VL of SEQ ID NO. 41 above and VH of SEQ ID NO. 45: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSY NQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARWYYSYRYWYFDVWGQGTLVTV SS (SEQ ID NO. 45) and VH of SEQ ID NO. 45: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSY NQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARWYYSYRYWYFDVWGQGTLVTV SS (SEQIDNO.45)

2H7.v51 1 has the Light Chain sequence is SEQ ID NO. 39 above and the Heavy Chain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGA TSY NQKFKGRFTI SVDKSKNTLYLQMNSLRAEDTAVYYCARWYYSYRYWYFDVWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSWTVPSSSLGTQTYICNWHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNATYRWSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ( SEQ ID NO. 46). Anti-BR3 antibodies are also provided that contain a substitution of N434 to aromatic residues F, Y, H or S.

Methods of the Invention The invention also provides a method of screening for a polypeptide with high affinity binding to FcRn at pH 6.0 and with weaker binding affinity at pH 7.4 than at pH 6.0, as described in the examples. The method comprises expressing a candidate polypeptide on phage, providing huFcRn immobilized on a solid matrix, allow phage particles to bind to the FcRn on the matrix, removing unbound phage particles by multiple rounds of washes each round with increasing stringency; and eluting the remaining bound phage at pH 7.4. As in Example 1, increasing stringency means increasing no and/or length of washes. The eluted phage can be propagated and the polypeptide isolated from the phage. In one embodiment the polypeptide is an IgG Fc containing polypeptide such as an antibody or an immunoadhesin.

Antibody production Monoclonal antibodies Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Patent No. 4,816,567). In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above 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. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which medium preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. Preferred fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells. Preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the SaIk Institute Cell Distribution Center, San Diego, California USA, and SP-2 and derivatives e.g., 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 described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al, Anal. Biochem., 107:220 (1980). Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, 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 hybridoma cells may be grown in vivo as ascites tumors in an animal e.g, by i.p. injection of the cells into mice. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc. 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 murine antibodies). The hybridoma 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 E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra e/ α/., Curr. Opinion in Immunol, 5:256-262 (1993) and Plϋckthun, Immunol. Revs., 130:151-188 (1992). In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et ai, Nature, 348:552-554 (1990). Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. MoI. Biol, 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al, Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al, Nuc. Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies. The DNA that encodes the antibody may be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy chain and light chain constant domain (CH and CL) sequences for the homologous murine sequences (U.S. Patent No. 4,816,567; and Morrison, et al, Proc. Natl Acad. ScL USA, 81 :6851 (1984)), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence for a non-immunoglobulin polypeptide (heterologous polypeptide). The non- immunoglobulin polypeptide sequences can substitute 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 chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.

Humanized antibodies Methods for humanizing non-human antibodies have been described in the art. Preferably, a humanized antibody has one or more amino acid residues introduced into it from a source which is non- human. These non-human amino 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 et aL, Nature, 321:522-525 (1986); Reichmann et al. Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric 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 a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region 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 and HAMA response (human anti-mouse antibody) when the antibody is intended for human therapeutic use. 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 V domain sequence which is closest to that of the rodent is identified and the human framework region (FR) within it accepted for the humanized antibody (Sims et ai, J. Immunol., 151:2296 (1993); Chothia et al, J. MoL Biol, 196:901 (1987)). Another method uses a particular framework region 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 et al., Proc. Natl. Acad. ScL USA, 89:4285 (1992); Presta et al., J. Immunol., 151 :2623 (1993)). It is further important that antibodies be humanized with retention of high binding 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, i.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 antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding. The humanized antibody may be an antibody fragment, such as a Fab, which is optionally conjugated with one or more cytotoxic agent(s) in order to generate an immunoconjugate. Alternatively, the humanized antibody may be an full length antibody, such as an full length IgG 1 antibody.

Human antibodies and phage display methodology As an alternative to humanization, human antibodies can be generated. For example, 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 (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc. Natl. Acad. ScL USA, 90:2551 (1993); Jakobovits et al. Nature, 362:255-258 (1993); Bruggemann et al, Year in Immuno., 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669 (all of GenPharm); 5,545,807; and WO 97/17852. Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 [1990]) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. MoI. Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S. Patent Nos. 5,565,332 and 5,573,905. As discussed above, human antibodies may also be generated by in vitro activated B cells (see U.S. Patents 5,567,610 and 5,229,275).

A ntibody fragments In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to solid tumors. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al. , Journal of Biochemical and Biophysical Methods 24:107 -1 17 (1992); and Brennan et at, Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et ai, Bio/Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. Fv and sFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a "linear antibody", e.g., as described in U.S. Patent 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.

Bispecific antibodies Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the CD20 protein. Other such antibodies may combine a CD20 binding site with a binding site for another protein. Alternatively, an anti-CD20 arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), or NKG2D or other NK cell activating ligand, so as to focus and localize cellular defense mechanisms to the CD20-expressing cell. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express CD20. These antibodies possess a CD20-binding arm and an arm which binds the cytotoxic agent (e.g. saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIII antibody and U.S. Patent No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcγRI antibody. A bispecific anti-ErbB2/Fcα antibody is shown in WO98/02463. U.S. Patent No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody. Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et ai, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (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 et 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. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light chain bonding, 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 cell. This provides for greater 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 yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant affect on the yield of the desired chain combination. 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 et al, Methods in Enzymology, 121:210 (1986). According to another approach described in U.S. Patent No. 5,731,168, 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 CH3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. 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 (U.S. 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 U.S. Patent No. 4,676,980, along with a number of cross-linking techniques. Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. Recent progress has facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al, J. Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al, J. Immunol, 148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. ScL USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a VH connected to a VL by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al, J. Immunol, 152:5368 (1994). Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. TvAt et al. J. Immunol. 147: 60 (1991).

Multivalent Antibodies A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present invention can be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g. tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VDl- (Xl)n-VD2-(X2)n-Fc, wherein VDl is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, Xl and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH-CHl -flexible linker- VH-CHl -Fc region chain; or VH- CHl-VH-CHl -Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.

Vectors, Host Cells and Recombinant Methods Selection and transformation of host cells Suitable host cells for cloning or expressing the recombinant mAbs, immunoadhesins and other polypeptide antagonists described herein are prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marc' escans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P. aeruginosa, 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. Full length antibody, antibody fragments, and antibody fusion proteins can be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, such as when the therapeutic antibody is conjugated to a cytotoxic agent (e.g., a toxin) and the immunoconjugate by itself shows effectiveness in tumor cell destruction. Full length antibodies have greater half life in circulation. Production in E. coli is faster and more cost efficient. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. 5,648,237 (Carter et. al.), U.S. 5,789,199 (JoIy et al.), and U.S. 5,840,523 (Simmons et al.) which describes translation initiation region (TIR) and signal sequences for optimizing expression and secretion, these patents incorporated herein by reference. After expression, the antibody is isolated from the E. coli cell paste in a soluble fraction and can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out similar to the process for purifying antibody expressed e.g,, in CHO cells. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding, such as CD20 antibody-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. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K . thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger. Suitable host cells for the expression of, e.g., glycosylated CD20 binding antibody 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 mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-I variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori 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. 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 CVl 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 ai, J. Gen Virol. 36:59 (1977)) ; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cellsΛDHFR (CHO, Urlaub et ai, Proc. Natl. Acad. ScL USA 77:4216 (1980)) ; mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980) ); monkey kidney cells (CVl 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 (W 138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et ai. Annals N. Y. Acad. ScL 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). Host cells are transformed with expression or cloning vectors for a B cell depleting antibody such as CD20 binding antibody, or an integrin anagonist antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Culturing the host cells The host cells used to produce an antibody of this invention may be cultured in a variety of media. Commercially available media such as Ham's FlO (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et ai, Anal. Biochem.\02:255 (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, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine 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. Purification of antibody When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al, Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenyl methylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants 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 antibody 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 domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γl, γ2, or γ4 heavy chains (Lindmark et al, J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et 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(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 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 Phase HPLC, chromatography on silica, chromatography on heparin 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 antibody to be recovered. Following any preliminary purification step(s), the mixture comprising the antibody 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). Antibody conjugates The antibody may be conjugated to a cytotoxic agent such as a toxin or a radioactive isotope. In certain embodiments, the toxin is calicheamicin, a maytansinoid, a dolastatin, auristatin E and analogs or derivatives thereof, are preferable.

Therapeutic Uses of the Antibody compositions The CD20 binding antibodies of the invention are useful to treat a number of malignant and non- malignant diseases including autoimmune diseases and related conditions, and B cell neoplasm or malignancy characterized by B cells expressing CD20, including B cell lymphomas and leukemias. Stem cells (B-cell progenitors) in bone marrow lack the CD20 antigen, allowing healthy B-cells to regenerate after treatment and return to normal levels within several months. An "autoimmune disease" herein is a disease or disorder arising from and directed against an individual's own tissues or a co-segregate or manifestation thereof or resulting condition therefrom. Examples of autoimmune diseases or disorders include, but are not limited to arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and ankylosing spondylitis), psoriasis, dermatitis including atopic dermatitis; chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis/dermatomyositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, responses associated with inflammatory bowel disease (IBD) (Crohn's disease, ulcerative colitis), and IBD with co- segregate of pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, and/or episcleritis), respiratory distress syndrome, including adult respiratory distress syndrome (ARDS), meningitis, IgE-mediated diseases such as anaphylaxis and allergic rhinitis, encephalitis such as Rasmussen's encephalitis, uveitis, colitis such as microscopic colitis and collagenous colitis, glomerulonephritis (GN) such as membranous GN, idiopathic membranous GN, membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN, allergic conditions, eczema, asthma, conditions involving infiltration of T cells and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) such as cutaneous SLE, lupus (including nephritis, cerebritis, pediatric, non-renal, discoid, alopecia), juvenile onset diabetes, multiple sclerosis (MS) such as spino-optical MS, allergic encephalomyelitis, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis including Wegener's granulomatosis, agranulocytosis, vasculitis (including Large Vessel vasculitis (including Polymyalgia Rheumatica and Giant Cell (Takayasu's) Arteritis), Medium Vessel vasculitis (including Kawasaki's Disease and Polyarteritis Nodosa), CNS vasculitis, and ANCA-associated vasculitis , such as Churg-Strauss vasculitis or syndrome (CSS)), aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Bechet disease, Castleman's syndrome, Goodpasture's Syndrome, Lambert-Eaton Myasthenic Syndrome, Reynaud's syndrome, Sjorgen's syndrome, Stevens-Johnson syndrome, solid organ transplant rejection (including pretreatment for high panel reactive antibody titers, IgA deposit in tissues, and rejection arising from renal transplantation, liver transplantation, intestinal transplantation, cardiac transplantation, etc.), graft versus host disease (GVHD), pemphigoid bullous, pemphigus (including vulgaris, foliaceus, and pemphigus mucus- membrane pemphigoid), autoimmune polyendocrinopathies, Reiter's disease, stiff-man syndrome, immune complex nephritis, IgM polyneuropathies or IgM mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic throbocytopenic purpura (TTP), thrombocytopenia (as developed by myocardial infarction patients, for example), including autoimmune thrombocytopenia, autoimmune disease of the testis and ovary including autoimune orchitis and oophoritis, primary hypothyroidism; autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's Thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Grave's disease, autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), Type I diabetes also referred to as insulin-dependent diabetes mellitus (IDDM), including pediatric IDDM, and Sheehan's syndrome; autoimmune hepatitis, Lymphoid interstitial pneumonitis (HIV), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre Syndrome, Berger's Disease (IgA nephropathy), primary biliary cirrhosis, celiac sprue (gluten enteropathy), refractory sprue with co-segregate dermatitis herpetiformis, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune inner ear disease (AIED), autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS), polychondritis such as refractory polychondritis, pulmonary alveolar proteinosis, amyloidosis, giant cell hepatitis, scleritis, monoclonal gammopathy of uncertain/unknown significance (MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS; autism, inflammatory myopathy, and focal segmental glomerulosclerosis (FSGS). A B cell neoplasm or malignancy characterized by B cells expressing CD20 is one comprising abnormal proliferation of cells that express CD20 on the cell surface. The B cell neoplasms having CD20+ B cells include CD20-positive Hodgkin's disease- including lymphocyte predominant Hodgkin's disease (LPHD); non-Hodgkin's lymphoma (NHL); follicular center cell (FCC) lymphomas; acute lymphocytic leukemia (ALL); chronic lymphocytic leukemia (CLL); Hairy cell leukemia. The non-Hodgkins lymphoma include low grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic lymphoma (SLL), intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, plasmacytoid lymphocytic lymphoma, mantle cell lymphoma, AIDS- related lymphoma and Waldenstrom's macroglobulinemia. Treatment of relapses of these cancers are also contemplated. LPHD is a type of Hodgkin's disease that tends to relapse frequently despite radiation or chemotherapy treatment and is characterized by CD20-positive malignant cells. CLL is one of four major types of leukemia. A cancer of mature B-cells called lymphocytes, CLL is manifested by progressive accumulation of cells in blood, bone marrow and lymphatic tissues. Indolent lymphoma is a slow-growing, incurable disease in which the average patient survives between six and 10 years following numerous periods of remission and relapse. The term "non-Hodgkin's lymphoma" or "NHL", as used herein, refers to a cancer of the lymphatic system other than Hodgkin's lymphomas. Hodgkin's lymphomas can generally be distinguished from non-Hodgkin's lymphomas by the presence of Reed-Sternberg cells in Hodgkin's lymphomas and the absence of said cells in non-Hodgkin's lymphomas. Examples of non-Hodgkin's lymphomas encompassed by the term as used herein include any that would be identified as such by one skilled in the art (e.g., an oncologist or pathologist) in accordance with classification schemes known in the art, such as the Revised European-American Lymphoma (REAL) scheme as described in Color Atlas of Clinical Hematology (3rd edition), A. Victor Hoffbrand and John E. Pettit (eds.) (Harcourt Publishers Ltd., 2000). See, in particular, the lists in Fig. 1 1.57, 11.58 and 11.59. More specific examples include, but are not limited to, relapsed or refractory NHL, front line low grade NHL, Stage III/IV NHL, chemotherapy resistant NHL, precursor B lymphoblastic leukemia and/or lymphoma, small lymphocytic lymphoma, B cell chronic lymphacytic leukemia and/or prolymphocyte leukemia and/or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and/or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone - MALT lymphoma, nodal marginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasma cell myeloma, low grade/follicular lymphoma, intermediate grade/follicular NHL, mantle cell lymphoma, follicle center lymphoma (follicular), intermediate grade diffuse NHL, diffuse large B-cell lymphoma, aggressive NHL (including aggressive front-line NHL and aggressive relapsed NHL), NHL relapsing after or refractory to autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, Burkitt's lymphoma, precursor (peripheral) T-cell lymphoblastic leukemia and/or lymphoma, adult T-cell lymphoma and/or leukemia, T cell chronic lymphocytic leukemia and/or prolymphacytic leukemia, large granular lymphocytic leukemia, mycosis fungoides and/or Sezary syndrome, extranodal natural killer/T-cell (nasal type) lymphoma, enteropathy type T-cell lymphoma, hepatosplenic T-cell lymphoma, subcutaneous panniculitis like T-cell lymphoma, skin (cutaneous) lymphomas, anaplastic large cell lymphoma, angiocentric lymphoma, intestinal T cell lymphoma, peripheral T-cell (not otherwise specified) lymphoma and angioimmunoblastic T-cell lymphoma. In specific embodiments, the methods of treatment of B cell cancers having CD20+ B cells of the invention are used to treat non-Hodgkin's lymphoma (NHL), lymphocyte predominant Hodgkin's disease (LPHD), small lymphocytic lymphoma (SLL), chronic lymphocytic leukemia (CLL). In specific embodiments, the methods of treating autoimmune disease or of depleting B cells are used to treat rheumatoid arthritis and juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE) including lupus nephritis, Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic throbocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus, Reynaud's syndrome, Sjorgen's syndrome and glomerulonephritis. The desired level of B cell depletion will depend on the disease. For the treatment of a CD20 positive cancer, it may be desirable to maximize the depletion of the B cells which are the target of the anti- CD20 antibodies of the invention. Thus, for the treatment of a CD20 positive B cell neoplasm, it is desirable that the B cell depletion be sufficient to at least prevent progression of the disease which can be assessed by the physician of skill in the art, e.g., by monitoring tumor growth (size), proliferation of the cancerous cell type, metastasis, other signs and symptoms of the particular cancer. Preferably, the B cell depletion is sufficient to prevent progression of disease for at least 2 months, more preferably 3 months, even more preferably 4 months, more preferably 5 months, even more preferably 6 or more months. In even more preferred embodiments, the B cell depletion is sufficient to increase the time in remission by at least 6 months, more preferably 9 months, more preferably one year, more preferably 2 years, more preferably 3 years, even more preferably 5 or more years. In a most preferred embodiment, the B cell depletion is sufficient to cure the disease. In preferred embodiments, the B cell depletion in a cancer patient is at least about 75% and more preferably, 80%, 85%, 90%, 95% , 99% and even 100% of the baseline level before treatment. For treatment of an autoimmune disease, it may be desirable to modulate the extent of B cell depletion depending on the disease and/or the severity of the condition in the individual patient, by adjusting the dosage of CD20 binding antibody. Thus, B cell depletion can but does not have to be complete. Or, total B cell depletion may be desired in initial treatment but in subsequent treatments, the dosage may be adjusted to achieve only partial depletion. In one embodiment, the B cell depletion is at least 20%, i.e., 80% or less of CD20 positive B cells remain as compared to the baseline level before treatment. In other embodiments, B cell depletion is 25%, 30%, 40%, 50%, 60%, 70% or greater. Preferably, the B cell depletion is sufficient to halt progression of the disease, more preferably to alleviate the signs and symptoms of the particular disease under treatment, even more preferably to cure the disease. The parameters for assessing efficacy or success of treatment of the neoplasm will be known to the physician of skill in the appropriate disease. Generally, the physician of skill will look for reduction in the signs and symptoms of the specific disease. Parameters can include median time to disease progression, time in remission and stable disease. The following references describe lymphomas and CLL, their diagnoses, treatment and standard medical procedures for measuring treatment efficacy. Canellos GP, Lister, TA, Sklar JL: The Lymphomas. W.B.Saunders Company, Philadelphia, 1998; van Besien K and Cabanillas, F: Clinical Manifestations, Staging and Treatment of Non-Hodgkin's Lymphoma, Chap. 70, pp 1293-1338, in: Hematology , Basic Principles and Practice, 3rd ed. Hoffman et al. (editors). Churchill Livingstone, Philadelphia, 2000; and Rai, K and Patel, D: Chronic Lymphocytic Leukemia, Chap. 72, pp 1350-1362, in: Hematology , Basic Principles and Practice, 3rd ed. Hoffman et al. (editors). Churchill Livingstone, Philadelphia, 2000. The parameters for assessing efficacy or success of treatment of an autoimmune or autoimmune related disease will be known to the physician of skill in the appropriate disease. Generally, the physician of skill will look for reduction in the signs and symptoms of the specific disease. The following are by way of examples. In one embodiment, the methods and compositions of the invention are useful to treat rheumatoid arthritis. RA is characterized by inflammation of multiple joints, cartilage loss and bone erosion that leads to joint destruction and ultimately reduced joint function. Additionally, since RA is a systemic disease, it can have effects in other tissues such as the lungs, eyes and bone marrow. The CD20 binding antibodies can be used as first-line therapy in patients with early RA (i.e., methotrexate (MTX) naive), or in combination with, e.g., MTX or cyclophosphamide. Or, the antibodies can be used in treatment as second-line therapy for patients who were DMARD and/or MTX refractory, in combination with, e.g., MTX. The CD20 binding antibodies can also be administered in combination with B cell mobilizing agents such as integrin antibodies that mobilize B cells into the bloodstream for more effective killing. The CD20 binding antibodies are useful to prevent and control joint damage, delay structural damage, decrease pain associated with inflammation in RA, and generally reduce the signs and symptoms in moderate to severe RA. The RA patient can be treated with the CD20 antibody prior to, after or together with treatment with other drugs used in treating RA (see combination therapy below). In one embodiment, patients who had previously failed disease-modifying antirheumatic drugs and/or had an inadequate response to methotrexate alone are treated with a CD20 binding antibody. In another embodiment, the patients are administered humanized CD20 binding antibody plus cyclophosphamide or CD20 binding antibody plus methotrexate. One method of evaluating treatment efficacy in RA is based on American College of Rheumatology (ACR) criteria, which measures the percentage of improvement in tender and swollen joints, among other things. The RA patient can be scored at for example, ACR 20 (20 percent improvement) compared with no antibody treatment (e.g., baseline before treatment) or treatment with placebo. Other ways of evaluating the efficacy of antibody treatment include X-ray scoring such as the Sharp X-ray score used to score structural damage such as bone erosion and joint space narrowing. Patients can also be evaluated for the prevention of or improvement in disability based on Health Assessment Questionnaire [HAQ] score, AIMS score, SF-36 at time periods during or after treatment. The ACR 20 criteria may include 20% improvement in both tender (painful) joint count and swollen joint count plus a 20% improvement in at least 3 of 5 additional measures: 1. patient's pain assessment by visual analog scale (VAS), 2. patient's global assessment of disease activity (VAS), 3. physician's global assessment of disease activity (VAS), 4. patient's self-assessed disability measured by the Health Assessment Questionnaire, and 5. acute phase reactants, CRP or ESR.

The ACR 50 and 70 are defined analogously. Preferably, the patient is administered an amount of a CD20 binding antibody of the invention effective to achieve at least a score of ACR 20, preferably at least ACR 30, more preferably at least ACR50, even more preferably at least ACR70, most preferably at least ACR 75 and higher. Psoriatic arthritis has unique and distinct radiographic features. For psoriatic arthritis, joint erosion and joint space narrowing can be evaluated by the Sharp score as well. The humanized CD20 binding antibodies disclosed herein can be used to prevent the joint damage as well as reduce disease signs and symptoms of the disorder. Yet another aspect of the invention is a method of treating Lupus or SLE by administering to the patient suffering from SLE, a therapeutically effective amount of a humanized CD20 binding antibody of the invention. SLEDAI scores provide a numerical quantitation of disease activity. The SLEDAI is a weighted index of 24 clinical and laboratory parameters known to correlate with disease activity, with a numerical range of 0-103. see Bryan Gescuk & John Davis, "Novel therapeutic agent for systemic lupus erythematosus" in Current Opinion in Rheumatology 2002, 14:515-521. Antibodies to double-stranded DNA are believed to cause renal flares and other manifestations of lupus. Patients undergoing antibody treatment can be monitored for time to renal flare, which is defined as a significant, reproducible increase in serum creatinine, urine protein or blood in the urine. Alternatively or in addition, patients can be monitored for levels of antinuclear antibodies and antibodies to double-stranded DNA. Treatments for SLE include high- dose corticosteroids and/or cyclophosphamide (HDCC). Spondyloarthropathies are a group of disorders of the joints, including ankylosing spondylitis, psoriatic arthritis and Crohn's disease. Treatment success can be determined by validated patient and physician global assessment measuring tools. Various medications are used to treat psoriasis; treatment differs directly in relation to disease severity. Patients with a more mild form of psoriasis typically utilize topical treatments, such as topical steroids, anthralin, calcipotriene, clobetasol, and tazarotene, to manage the disease while patients with moderate and severe psoriasis are more likely to employ systemic (methotrexate, retinoids, cyclosporine, PUVA and UVB) therapies. Tars are also used. These therapies have a combination of safety concerns, time consuming regimens, or inconvenient processes of treatment. Furthermore, some require expensive equipment and dedicated space in the office setting. Systemic medications can produce serious side effects, including hypertension, hyperlipidemia, bone marrow suppression, liver disease, kidney disease and gastrointestinal upset. Also, the use of phototherapy can increase the incidence of skin cancers. In addition to the inconvenience and discomfort associated with the use of topical therapies, phototherapy and systemic treatments require cycling patients on and off therapy and monitoring lifetime exposure due to their side effects. Treatment efficacy for psoriasis is assessed by monitoring changes in clinical signs and symptoms of the disease including Physician's Global Assessment (PGA) changes and Psoriasis Area and Severity Index (PASI) scores, Psoriasis Symptom Assessment (PSA), compared with the baseline condition. The patient can be measured periodically throughout treatment on the Visual analog scale used to indicate the degree of itching experienced at specific time points. Anti-HER2 antibodies having the Fc mutations of the present invention are useful to treat a HER2- expressing or over-expressing cancer. A "HER2-expressing cancer" is one comprising cells which have HER2 protein present at their cell surface. A cancer which "overexpresses" a HER receptor is one which has significantly higher levels of a HER receptor, such as HER2, at the cell surface thereof, compared to a noncancerous cell of the same tissue type. Such overexpression may be caused by gene amplification or by increased transcription or translation. HER receptor overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the HER protein present on the surface of a cell (e.g. via an immunohistochemistry assay; IHC). Alternatively, or additionally, one may measure levels of HER- encoding nucleic acid in the cell, e.g. via fluorescent in situ hybridization (FISH; see WO98/45479 published October, 1998), southern blotting, or polymerase chain reaction (PCR) techniques, such as real time quantitative PCR (RT-PCR). One may also study HER receptor overexpression by measuring shed antigen (e.g., HER extracellular domain) in a biological fluid such as serum (see, e.g., U.S. Patent No. 4,933,294 issued June 12, 1990; WO91/05264 published April 18, 1991 ; U.S. Patent 5,401,638 issued March 28, 1995; and Sias et al. J. Immunol. Methods 132: 73-80 (1990)). Aside from the above assays, various in vivo assays are available to the skilled practitioner. For example, one may expose cells within the body of the patient to an antibody which is optionally labeled with a detectable label, e.g. a radioactive isotope, and binding of the antibody to cells in the patient can be evaluated, e.g. by external scanning for radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody. Various cancers that can be treated with the Her-2 antibody compositions are listed below. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma,and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophagael cancer, tumors of the biliary tract, as well as head and neck cancer. It is also contemplated that the HER2 antibody may be used to treat various non-malignant diseases or disorders, such as autoimmune disease (e.g. psoriasis); endometriosis; scleroderma; restenosis; polyps such as colon polyps, nasal polyps or gastrointestinal polyps; fibroadenoma; respiratory disease; cholecystitis; neurofibromatosis; polycystic kidney disease; inflammatory diseases; skin disorders including psoriasis and dermatitis; vascular disease; conditions involving abnormal proliferation of vascular epithelial cells; gastrointestinal ulcers; Menetrier's disease, secreting adenomas or protein loss syndrome; renal disorders; angiogenic disorders; ocular disease such as age related macular degeneration, presumed ocular histoplasmosis syndrome, retinal neovascularization from proliferative diabetic retinopathy, retinal vascularization, diabetic retinopathy, or age related macular degeneration; bone associated pathologies such as osteoarthritis, rickets and osteoporosis; damage following a cerebral ischemic event; fibrotic or edemia diseases such as hepatic cirrhosis, lung fibrosis, carcoidosis, throiditis, hyperviscosity syndrome systemic, Osier Weber-Rendu disease, chronic occlusive pulmonary disease, or edema following burns, trauma, radiation, stroke, hypoxia or ischemia; hypersensitivity reaction of the skin; diabetic retinopathy and diabetic nephropathy; Guillain-Barre syndrome; graft versus host disease or transplant rejection; Paget's disease; bone or joint inflammation; photoaging (e.g. caused by UV radiation of human skin); benign prostatic hypertrophy; certain microbial infections including microbial pathogens selected from adenovirus, hantaviruses, Borrelia burgdorferi, Yersinia spp. and Bordetella pertussis; thrombus caused by platelet aggregation; reproductive conditions such as endometriosis, ovarian hyperstimulation syndrome, preeclampsia, dysfunctional uterine bleeding, or menometrorrhagia; synovitis; atheroma; acute and chronic nephropathies (including proliferative glomerulonephritis and diabetes-induced renal disease); eczema; hypertrophic scar formation; endotoxic shock and fungal infection; familial adenomatosis polyposis; neurodedenerative diseases (e.g. Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration); myelodysplastic syndromes; aplastic anemia; ischemic injury; fibrosis of the lung, kidney or liver; T-cell mediated hypersensitivity disease; infantile hypertrophic pyloric stenosis; urinary obstructive syndrome; psoriatic arthritis; and Hasimoto's thyroiditis. Preferred non-malignant indications for therapy herein include psoriasis, endometriosis, scleroderma, vascular disease (e.g. restenosis, artherosclerosis, coronary artery disease, or hypertension), colon polyps, fibroadenoma or respiratory disease (e.g. asthma, chronic bronchitis, bronchieactasis or cystic fibrosis). The anti-angiogenesis and anti-VEGF antibodies of the invention are useful to treat angiogenesis related disorders which include the following neoplastic and non-neoplastic disorders. The neoplastic disorders include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include kidney or renal cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, lung cancer including small-cell lung cancer, non- small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, squamous cell cancer (e.g. epithelial squamous cell cancer), cervical cancer, ovarian cancer, prostate cancer, liver cancer, bladder cancer, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, head and neck cancer, glioblastoma, retinoblastoma, astrocytoma, thecomas, arrhenoblastomas, hepatoma, hematologic malignancies including non-Hodgkins lymphoma (NHL), multiple myeloma and acute hematologic malignancies, endometrial or uterine carcinoma, endometriosis, fibrosarcomas, choriocarcinoma, salivary gland carcinoma, vulval cancer, thyroid cancer, esophageal carcinomas, hepatic carcinoma, anal carcinoma, penile carcinoma, nasopharyngeal carcinoma, laryngeal carcinomas, Kaposi's sarcoma, melanoma, skin carcinomas, Schwannoma, oligodendroglioma, neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome. More particularly, cancers that are amenable to treatment by the antagonists of the invention include renal cancer, breast cancer, colorectal cancer, non-small cell lung cancer, non-Hodgkins lymphoma (NHL), prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma. Non-neoplastic conditions that are amenable to treatment with anti-angiogenesis antibodies including anti-VEGF antibodies include, but are not limited to, e.g., undesired or aberrant hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriatic plaques, sarcoidosis, atherosclerosis, atherosclerotic plaques, edema from myocardial infarction, diabetic and other proliferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, diabetic macular edema, corneal neovascularization, corneal graft neovascularization, corneal graft rejection, retinal/choroidal neovascularization, neovascularization of the angle (rubeosis), ocular neovascular disease, vascular restenosis, arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, lung inflammation, acute lung injury/ ARDS, sepsis, primary pulmonary hypertension, malignant pulmonary effusions, cerebral edema (e.g., associated with acute stroke/ closed head injury/ trauma), synovial inflammation, pannus formation in RA, myositis ossificans, hypertropic bone formation, osteoarthritis (OA), refractory ascites, polycystic ovarian disease, endometriosis, 3rd spacing of fluid diseases (pancreatitis, compartment syndrome, burns, bowel disease), uterine fibroids, premature labor, chronic inflammation such as IBD (Crohn's disease and ulcerative colitis), renal allograft rejection, inflammatory bowel disease, nephrotic syndrome, undesired or aberrant tissue mass growth (non-cancer), hemophilic joints, hypertrophic scars, inhibition of hair growth, Osier-Weber syndrome, pyogenic granuloma retrolental fibroplasias, scleroderma, trachoma, vascular adhesions, synovitis, dermatitis, preeclampsia, ascites, pericardial effusion (such as that associated with pericarditis), and pleural effusion. Anti-CDl Ia antibodies having the Fc amino acid alterations of the present are useful to treat LFA-I mediated disorders. The term "LFA-I -mediated disorders" refers to pathological states caused by cell adherence interactions involving the LFA-I receptor on lymphocytes. Examples of such disorders include T cell inflammatory responses such as inflammatory skin diseases including psoriasis; responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); adult respiratory distress syndrome; dermatitis; meningitis; encephalitis; uveitic; allergic conditions such as eczema and asthma and other conditions involving infiltration of T cells and chronic inflammatory responses; skin hypersensitivity reactions (including poison ivy and poison oak); atherosclerosis; leukocyte adhesion deficiency; autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE), diabetes mellitus, multiple sclerosis, Reynaud's syndrome, autoimmune thyroiditis, experimental autoimmune encephalomyelitis, Sjorgen's syndrome, juvenile onset diabetes, and immune responses associated with delayed hypersensitivity mediated by cytokines and T-lymphocytes typically found in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; pernicious anemia; diseases involving leukocyte diapedesis; CNS inflammatory disorder, multiple organ injury syndrome secondary to septicaemia or trauma; autoimmune haemolytic anemia; myethemia gravis; antigen-antibody complex mediated diseases; all types of transplantation rejection, including graft vs. host or host vs. graft disease. Anti-IgE antibodies having the Fc amino acid alterations of the present that increase binding to FcRn and increase serum half life are useful to treat IgE mediated disorders. IgE mediated disorders includes atopic disorders, which are characterized by an inherited propensity to respond immunologically to many common naturally occurring inhaled and ingested antigens and the continual production of IgE antibodies. Specific atopic disorders includes allergic asthma, allergic rhinitis, atopic dermatitis and allergic gastroenteropathy. Atopic patients often have multiple allergies, meaning that they have IgE antibodies to, and symptoms from, many environmental allergens, including seasonal, perennial and occupational allergens. Example seasonal allergens include pollens (e.g., grass, tree, rye, timothy, ragweed), while example perennial allergens include fungi (e.g., molds, mold spores), feathers, animal and insect (e.g., dust mite) debris. Example occupational allergens include detergents, metals and isocyanates. Non-antigen specific stimuli that can result in an IgE-mediated reaction include infection, irritants such as smoke, combustion fumes, diesel exhaust particles and sulphur dioxide, exercise, cold and emotional stress. Hypersensitivity reactions may result from exposure to proteins in foods, venom, vaccines, hormones, antiserum, enzymes and latex, antibiotics, muscle relaxants, vitamins, cytotoxins, opiates, and polysaccharides such as dextrin, iron dextran and polygeline. However disorders associated with elevated IgE levels are not limited to those with an inherited (atopic) etiology. Other disorders associated with elevated IgE levels, that appear to be IgE-mediated and are treatable with the formulations of this present invention include hypersensitivity (e.g., anaphylactic hypersensitivity), eczema, urticaria, allergic bronchopulmonary aspergillosis, parasitic diseases, hyper-IgE syndrome, ataxia-telangiectasia, Wiskott-Aldrich syndrome, Churg-Strauss Syndrome, systemic vasculitis, thymic alymphoplasia, IgE myeloma, graft-versus-host reaction, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, indeterminate colitis and infectious colitis), gastroenteropathy, enteritis, mucositis (e.g., oral mucositis, gastrointestinal mucositis, nasal mucositis and proctitis), necrotizing enterocolitis and esophagitis.

Dosage Depending on the indication to be treated and factors relevant to the dosing that a physician of skill in the field would be familiar with, the antagonists and antibodies of the invention will be administered at a dosage that is efficacious for the treatment of that indication while minimizing toxicity and side effects. For the treatment of a CD20 positive cancer or an autoimmune disease, the therapeutically effective dosage will be in the range of about 250mg/m2 to about 400 mg/m2 or 500 mg/m2 , preferably about 250-375mg/m2. In one embodiment, the dosage range is 275-375 mg/m2. In one embodiment of the treatment of a CD20 positive B cell neoplasm, the antibody is administered at a range of 300-375 mg/m2. For the treatment of patients suffering from B-cell lymphoma such as non-Hodgkins lymphoma, in a specific embodiment, the anti-CD20 antibodies and humanized anti-CD20 antibodies of the invention will be administered to a human patient at a dosage of 10mg/kg or 375mg/m2. In one embodiment, Rituximab can be administered at a dosage range of 7-15mg/kg. For treating NHL, one dosing regimen would be to administer one dose of the antibody composition a dosage of 10mg/kg in the first week of treatment, followed by a 2 week interval, then a second dose of the same amount of antibody is administered. Generally, NHL patients receive such treatment once during a year but upon recurrence of the lymphoma, such treatment can be repeated. In another dosing regimen, patients treated with low-grade NHL receive four weeks of a version of humanized 2H7, preferably vl6 (375 mg/m2 weekly) followed at week five by three additional courses of the antibody plus standard CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) or CVP (cyclophosphamide, vincristine, prednisone) chemotherapy, which was given every three weeks for three cycles. For treating rheumatoid arthritis, in one embodiment, the dosage range for the humanized antibody is 125mg/m2 (equivalent to about 200mg/dose) to 600mg/m2, given in two doses, e.g., the first dose of 200mg is administered on day one followed by a second dose of 200mg on day 15. In different embodiments, the dosage is 250mg/dose, 275mg, 300mg, 325mg, 350mg, 375mg, 400mg, 425mg, 450mg, 475mg, 500mg, 525mg, 550mg, 575mg, 600mg. In treating disease, the B cell targeting antibodies such as the CD20 binding antibodies of the invention can be administered to the patient chronically or intermittently, as determined by the physician of skill in the disease. A patient administered a drug by intravenous infusion or subcutaneously may experience adverse events such as fever, chills, burning sensation, asthenia and headache. To alleviate or minimize such adverse events, the patient may receive an initial conditioning dose(s) of the antibody followed by a therapeutic dose. The conditioning dose(s) will be lower than the therapeutic dose to condition the patient to tolerate higher dosages. Route of administration The antibodies used in the methods of the invention are administered to a human patient in accord with methods known to medical practitioners, such as by intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by subcutaneous, intramuscular, intra-arterial, intraperitoneal, intrapulmonary, intracerobrospinal, intra-articular, intrasynovial, intrathecal, intralesional, or inhalation routes (e.g., intranasal), generally by intravenous or subcutaneous administration. In on embodiment, the humanized 2H7 antibody is administered by intravenous infusion with 0.9% sodium chloride solution as an infusion vehicle.

Combination Therapy In treating the B cell neoplasms described above, the patient can be treated with the CD20 binding antibodies of the present invention in conjunction with one or more therapeutic agents such as a chemotherapeutic agent in a multidrug regimen. The CD20 binding antibody can be administered concurrently, sequentially, or alternating with the chemotherapeutic agent, or after non-responsiveness with other therapy. Standard chemotherapy for lymphoma treatment may include cyclophosphamide, cytarabine, melphalan and mitoxantrone plus melphalan. CHOP is one of the most common chemotherapy regimens for treating Non-Hodgkin's lymphoma. The following are the drugs used in the CHOP regimen: cyclophosphamide (brand names Cytoxan, neosar); adriamycin (doxorubicin / hydroxydoxorubicin); vincristine (Oncovin); and prednisolone (sometimes called Deltasone or Orasone). In particular embodiments, the CD20 binding antibody is administered to a patient in need thereof in combination with one or more of the following chemotherapeutic agents of doxorubicin, cyclophosphamide, vincristine and prednisolone. In a specific embodiment, a patient suffering from a lymphoma (such as a non-Hodgkin's lymphoma) is treated with an anti-CD20 antibody of the present invention in conjunction with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) therapy. In another embodiment, the cancer patient can be treated with a humanized CD20 binding antibody of the invention in combination with CVP (cyclophosphamide, vincristine, and prednisone) chemotherapy. In a specific embodiment, the patient suffering from CD20-positive NHL is treated with humanized 2H7.vl6 or a variant thereof disclosed above, in conjunction with CVP. In a specific embodiment of the treatment of CLL, the CD20 binding antibody is administered in conjunction with chemotherapy with one or both of fludarabine and Cytoxan. In treating the autoimmune diseases or autoimmune related conditions described above, the patient can be treated with the B cell depleting agent such as the CD20 binding antibodies of the present invention in conjunction with a second therapeutic agent, such as an immunosuppressive agent, such as in a multi drug regimen. The B cell depleting agent can be administered concurrently, sequentially or alternating with the immunosuppressive agent or upon non-responsiveness with other therapy. The immunosuppressive agent can be administered at the same or lesser dosages than as set forth in the art. The preferred adjunct immunosuppressive agent will depend on many factors, including the type of disorder being treated as well as the patient's history. "Immunosuppressive agent" as used herein for adjunct therapy refers to substances that act to suppress or mask the immune system of a patient. Such agents would include substances that suppress cytokine production, down regulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include steroids such as glucocorticosteroids, e.g., prednisone, methylprednisolone, and dexamethasone; 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077), azathioprine (or cyclophosphamide, if there is an adverse reaction to azathioprine); bromocryptine; glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat. No. 4,120,649); anti-idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A; cytokine or cytokine receptor antagonists including anti- interferon-γ, -β, or -α antibodies; anti-tumor necrosis factor-α antibodies; anti-tumor necrosis factor-β antibodies; anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3 binding domain (WO 90/08187 published 7/26/90); streptokinase; TGF-β; streptodornase; RNA or DNA from the host; FK506; RS-61443; deoxyspergualin; rapamycin; T-cell receptor (U.S. Pat. No. 5,114,721); T-cell receptor fragments (Offner et at, Science 251:430-432 (1991); WO 90/11294; and WO 91/01133); and T cell receptor antibodies (EP 340,109) such as T10B9. For the treatment of rheumatoid arthritis, the patient can be treated with a CD20 antibody of the invention in conjunction with any one or more of the following drugs: DMARDS (disease-modifying anti- rheumatic drugs (e.g., methotrexate), NSAI or NSAID (non-steroidal anti-inflammatory drugs), HUMIRA™ (adalimumab; Abbott Laboratories), ARAVA® (lefiunomide), REMICADE® (infliximab; Centocor Inc., of Malvern, Pa), ENBREL (etanercept; Immunex, WA), COX-2 inhibitors. DMARDs commonly used in RA are hydroxycloroquine, sulfasalazine, methotrexate, lefiunomide, etanercept, infliximab, azathioprine, D-penicillamine, Gold (oral), Gold (intramuscular), minocycline, cyclosporine, Staphylococcal protein A immunoadsorption. Adalimumab is a human monoclonal antibody that binds to TNFα. Infliximab is a chimeric monoclonal antibody that binds to TNFα. Etanercept is an "immunoadhesin" fusion protein consisting of the extracellular ligand binding portion of the human 75 kD (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of a human IgGl. For conventional treatment of RA, see, e.g., "Guidelines for the management of rheumatoid arthritis" Arthritis & Rheumatism 46(2): 328-346 (February, 2002). In a specific embodiment, the RA patient is treated with a CD20 antibody of the invention in conjunction with methotrexate (MTX). An exemplary dosage of MTX is about 7.5— 25 mg/kg/wk. MTX can be administered orally and subcutaneously. For the treatment of ankylosing spondylitis, psoriatic arthritis and Crohn's disease, the patient can be treated with a CD20 binding antibody of the invention in conjunction with, for example, Remicade® (infliximab; from Centocor Inc., of Malvern, Pa.), ENBREL (etanercept; Immunex, WA). Treatments for SLE include high-dose corticosteroids and/or cyclophosphamide (HDCC). For the treatment of psoriasis, patients can be administered a CD20 binding antibody in conjunction with topical treatments, such as topical steroids, anthralin, calcipotriene, clobetasol, and tazarotene, or with methotrexate, retinoids, cyclosporine, PUVA and UVB therapies. In one embodiment, the psoriasis patient is treated with the CD20 binding antibody sequentially or concurrently with cyclosporine.

Pharmaceutical Formulations Therapeutic formulations of the antibodies used in accordance with the present invention are prepared for storage by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, 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; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as olyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; 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). Exemplary anti-CD20 antibody formulations are described in WO98/56418, expressly incorporated herein by reference. Another formulation is a liquid multidose formulation comprising the anti-CD20 antibody at 40 mg/mL, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 at pH 5.0 that has a minimum shelf life of two years storage at 2-8°C. Another anti-CD20 formulation of interest comprises lOmg/mL antibody in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7mg/mL polysorbate 80, and Sterile Water for Injection, pH 6.5. Yet another aqueous pharmaceutical formulation comprises 10-30 mM sodium acetate from about pH 4.8 to about pH 5.5, preferably at pH5.5, polysorbate as a surfactant in a an amount of about 0.01-0.1% v/v, trehalose at an amount of about 2-10% w/v, and benzyl alcohol as a preservative (U.S. 6,171,586). Lyophilized formulations adapted for subcutaneous administration are described in WO97/04801. Such lyophilized formulations may be reconstituted with a suitable diluent to a high protein concentration and the reconstituted formulation may be administered subcutaneously to the mammal to be treated herein. One formulation for the humanized 2H7 variants is antibody at 12-14 mg/mL in 10 mM histidine, 6% sucrose, 0.02% polysorbate 20, pH 5.8. In a specific embodiment, 2H7 variants and in particular 2H7.vl6 is formulated at 20mg/mL antibody in 1OmM histidine sulfate, 60mg/ml sucrose., 0.2 mg/ml polysorbate 20, and Sterile Water for Injection, at pH5.8. 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. For example, it may be desirable to further provide a cytotoxic agent, chemotherapeutic agent, cytokine or immunosuppressive agent (e.g. one which acts on T cells, such as cyclosporin or an antibody that binds T cells, e.g. one which binds LFA-I). The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein or about from 1 to 99% of the heretofore employed dosages. The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-) release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. J

Articles of Manufacture and Kits Another embodiment of the invention is an article of manufacture containing materials useful for the treatment of the aforementioned disorders, e.g., an autoimmune disease or a cancer such as CLL. The article of manufacture comprises at least one container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. At least one container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Two therapeutic compositions may be provided in the article of manufacture. At least one active agent in the first composition is an Fc variant antibody of the invention. The label or package insert indicates that the composition is used for treating the particular condition. The label or package insert will further comprise instructions for administering the compositions to the patient. Package insert refers to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. Additionally, the article of manufacture may further comprise a container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

Experimental Examples These experimental examples are by way of illustration and not intended to be a limitation on the scope of the invention. Example 1 Phage display of human IgGl-Fc for selection of single-site mutants with optimized pH-dependent binding to human FcRn For phage-display, we used a "hingeless" variant of the human IgGl Fc in which the disulfides of the hinge region have been removed (C226 is deleted and C229 is replaced by Ser) and the C-terminus is fused to the C-terminal region of the Ml 3 gill protein (g3p) in a phagemid construct (pW0437). The protein sequence (SEQ ID. NO. 38) is shown in Fig.7. We used as the phage-selection target, huFcRn that was biotinylated and captured on neutravidin-coated immunosorbent (Maxisorp™) plates in pH 6.0 buffer, washed extensively with pH 6.0 buffer to remove lower affinity variants, and eluted higher affinity, pH sensitive variants with pH 7.4 buffer. By the third round of selection, N434W was the dominant clone (44 of 48 sequenced clones corresponded to this variant), with N434Y (3/48) and N434F (1/48) also present. With the goal of minimizing the number of mutations to the Fc (and therefore minimizing the risk of immunogenicity in therapeutic antibodies), only single amino acid substitutions comprised the phage- display libraries. The most common methods to create randomized libraries of an antibody fragment for selection by phage display are oligonucleotide-directed mutagenesis to mutate multiple residues simultaneously and random mutagenesis via error prone PCR or a similar method (reviewed in Clackson and Lowman (eds.) in Phage Display: A Practical Approach, Oxford University Press (2004)); however, these methods generally lead to multiple mutations per molecule, which must then be deconvoluted to determine the minimal number of mutations needed to achieve the desired molecular properties. As an alternative, we constructed a randomized library by systematically mutating each residue within 10 A of FcRn (in the structure when FcRn is bound to IgG) individually (by homology to the rat FcRn structure (Burnmeister, 1997)), by oligonucleotide-directed mutagenesis. Thus 43 "mini-libraries," each consisting of 20 variants at a particular amino acid position (Table 1 ), comprise the complete library of 8600 variants used for selection of single-site Fc variants with enhanced pH-dependent binding to FcRn. Table 1. Residues randomized in human IgGl Fc hingeless variant.

Previous attempts to select for Fc variants that bind human FcRn with higher affinity have been unsuccessful (Dall'Acqua 2002; US2003/0190311). In those studies huFcRn was coated directly on a Maxisorp plate. We have found that huFcRn is very sensitive to coating conditions, and must be biotinylated and captured on neutravidin coated Maxisorp plates in order to retain its ability to bind Fc. Maxisorp plates were coated with 2 μg/mL neutravidin in 50 mM carbonate buffer, pH 9.6 overnight at 4°C, and then blocked with Assay Diluent (PBS + 0.5 % BSA, pH 6.0). 50 μL huFcRn (-0.2 mg/mL) was biotinylated by mixing with lOμL freshly prepared 10 mM biotin-LC-NHS (Pierce) at room temperature for 30 minutes. Unreacted biotin-LC-NHS was inactivated by the addition of 10 μL 1 M Tris. huFcRn-biotin was purified from excess biotin-Tris by gel filtration on a PBS pH 6.0 equilibrated PD-10 column (Amersham-Pharmacia). 2 mL was collected (huFcRn-biotin, 5 μg/mL). huFcRn-biotin was captured in 8 neutravidin coated and blocked wells for 1 hour (1 10 μL/well). The plate was rinsed quickly three times with Assay Diluent before adding phage. Phage particles from a dense overnight growth were harvested by precipitation with 2.5M NaCl/20 % PEG and resuspended in Assay Diluent, typically at a concentration of 1013 phage/mL. Phage were then diluted 1 :10 in Assay Diluent and allowed to bind huFcRn-biotin coated wells (or uncoated wells, as a negative control) for ~ 1 hour (100 μL/well). The plates were washed with PBS + 0.05% Tween, pH 6.0. Washings were performed with increasing stringency for each round of selections (first round: 10 quick washes; second round: 20 quick washes; third round: 40 quick washes; fourth round: 16 quick washes and 20 washes of 15 sec; fifth round: 4 quick washes and one 3 min. wash, repeated 5 times (20 washes total)). The remaining bound phage were eluted with 110 μL PBS pH 7.4 and added to 10 mL log-phase E. coli (XLl- Blue; Stratagene, Inc.) culture for propagation. Example 2 Characterization of soluble Fc variant proteins Soluble Fc variants with these mutations were expressed, purified, and assayed in a BIAcore binding assay for their affinity for human, cynomolgus monkey, rat, and murine FcRn. The Fc variants were also analyzed by size exclusion chromatography to determine their aggregation tendencies. Variant Fc fragments were expressed by transforming 34B8 E. coli cells with the mutant pW0437 phagemids, growing for 24 hours at 30°C in phosphate-free media to induce expression of the Fc genes, and harvesting the cells. Cell paste was frozen overnight, and lysed by osmotic shock in 10 mM Tris, 1 mM EDTA. Lysate was cleared by centrifugation and applied to a Protein A column. The column was washed with PBS, soluble Fc eluted with Protein A Citrate Elution Buffer (0.1 M citrate, pH 3.0), and neutralized with Tris pH 7.5. Soluble Fc was concentrated in an Amicon Centriprep. FcRn from human, cynomolgus monkey, rat, or mouse was immobilized by NHS chemistry on Biacore CM5 chips at varying densities (100-3000 RU). Fc variants were serially diluted from 10 μM to 1 nM in PBS at pH 6.0, and binding was monitored over time. For the parental hingeless Fc (i.e., wild-type), equilibrium binding was reached almost immediately for huFcRn, indicating that it has very fast on- and off- rates, and an approximate Kd of 700 nM as determined by equilibrium analysis (Fig. 8). For the N434W variant, the on-rate is noticeably slower, and the off-rate is extremely slow. By injecting N434W at a slower flow rate and for a longer period of time, equilibrium analysis was possible, and the Kd is approximately 4 nM. The variants N434W, N434F, N434A, and the triple mutant N434A+E380A+T307A had apparent increases in affinity over wild-type Fc of -170-fold, ~9-foId, ~2.7-fold, and ~14-fold, respectively, at pH 6.0. In contrast, at pH 7.4, affinity of the N434 variants for huFcRn is effectively too low to be measured in this assay. The improvement of N434W relative to wild-type is not as significant for binding to cyno FcRn, showing only about 10-fold better for binding to rat FcRn, and virtually the same as WT for binding to murine FcRn (data not shown). Thus the improvement achieved by this mutation is a specific for human FcRn. Aggregated Fc may appear to have higher affinity for FcRn from a valency effect. We analyzed the Fc variants (WT, N434W, N434Y, N434F, N434A, and N434A+E380A+T307A) by quantitative size exclusion chromatography. All the variants appeared to be at least 90% monomeric, except N434W, which had significant populations of dimeric, tetrameric, and octameric forms. Monomeric N434W variant was purified from the oligomeric forms by preparative size exclusion chromatography on an S-200 column. The purified monomeric material retained its high affinity for huFcRn in an SPR binding assay, indicating that the increase in affinity observed for N434W is in fact due to changes in its intrinsic affinity for huFcRn, and not an artifact of aggregation. Purified oligomeric N434W in fact showed reduced affinity for FcRn (Fig. 8).

Example 3 Characterization of humanized anti-CD20 IgGl variants with FcRn mutations Mutations identified by phage display of human Fc were also tested for their effects in the background of an intact antibody, 2H7.vl38. 2H7.vl38 is a humanized anti-CD20 antibody in which the Fc has been modified for increased ADCC and CDC activities through the following mutations: S298A, K326A, E333A, K334A. Mutations at position N434 were introduced into this background, and IgG (Table 2) prepared by transient transfection of 293 cells as previously described. In each case, purified IgG variants were shown to have low levels of protein aggregation by size-exclusion chromatography as described above.

Table 2. Variants of humanized anti-CD20 antibody 2H7.vl 38

Human IgGl variants of 2H7.vl38 were analyzed for pH-dependent binding to human FcRn in an ELISA using biotinylated FcRn. MaxiSorp 96-well microwell plates (Nunc, Roskilde, Denmark) were coated with 2μg/ml NeutrAvidin (Pierce, Rockford, IL), at 100 μl/well in 50 mM carbonate buffer, pH 9.6, at 4°C overnight. Plates were washed with PBS containing 0.05% polysorbate (wash buffer), pH 7.4, and blocked with PBS containing 0.5% BSA, pH 7.4, at 150 μl/well. After a one-hour incubation at room temperature, plates were washed with wash buffer, pH 7.4. Human FcRn was biotinylated using biotin-X-NHS (Research Organics, Cleveland, OH). Biotinylated FcRn was added to the plates at 2 μg/ml, 100 μl/well, in PBS containing 0.5% BSA, 0.05% polysorbate 20 (sample buffer), pH 7.4. The plates were incubated for one hour and washed with wash buffer, pH 6.0. Seven twofold serial dilutions of IgG antibodies (3.1-200 ng/ml) in sample buffer, pH 6.0, were added to the plates. After a two-hour incubation, plates were washed with wash buffer, pH 6.0. Bound IgG was detected by adding peroxidase labeled goat F(ab')2 anti-human IgG F(ab')2 (Jackson ImmunoResearch, West Grove, PA) at 100 μl/well in sample buffer, pH 6.0. After a one- hour incubation, plates were washed with wash buffer, pH 6.0, and the substrate 3,3',5,5'-tetramethyl benzidine (TMB) (Kirkegaard & Perry Laboratories) was added at 100 μl/well. The reaction was stopped by adding 1 M phosphoric acid at 100 μl/well. Absorbance was read at 450 nm on a multiskan Ascent reader (Thermo Labsystems, Helsinki, Finland). The absorbance at the midpoint of the standard curve (mid-OD) was calculated. The corresponding concentrations of standard and samples at this mid-OD were determined from the titration curves using a four-parameter nonlinear regression curve-fitting program (KaleidaGraph, Synergy software, Reading, PA). The relative activity was calculated by dividing the mid-OD concentration of standard by that of sample. For evaluation of dissociation of bound IgG from FcRn at pH 6.0 or pH 7.4, the assay was carried out similarly except that after the sample incubation step and when the plates were washed, sample buffer at pH 6.0 or 7.4 was added at 100 μl/well. Plates were incubated for 45 min and washed. The assay was then continued as described above. The results (Fig. 9) indicate relative binding affinities similar to those observed for the Fc variants. At pH 6.0, the relative binding affinities are v477 > v478 = v479 > v364 > vl38. At pH 7.4, the relative binding affinities are consistently weaker than at pH 6.0, with the same relative binding: v477 > v478 = v479 > v364 > vl38. These Fc mutations are broadly applicable to human IgG antibodies.

Example 4 In vivo studies of FcRn binding effects on pharmacokinetics

To determine'the effects of improved FcRn binding on the pharmacokinetics of these Fc variant antibodies in vivo, cynomolgus (Macaca fascicularis) monkeys or other primate species are injected intravenously with each antibody variant, and blood samples collected over time to monitor the clearance of the antibody. Several animals are injected at one or more dose levels. In one experiment, a single i.v. dose of 1-20 mg/kg is injected at time 0 on day 1. Blood (serum) samples are collected from each animal prior to dosing and at 6h, 24h, and 72h after dosing. Additional samples are collected on day 8, day 10, day 30, and day 60. The concentrations of antibody in the serum samples are determined using an ELISA. The time- dependent decrease in antibody concentration in the serum is modeled using standard pharmacology techniques (Shargel and Yu, Applied Pharmaceutics and Pharmacokinetics, Fourth edition, pp. 67-98, Appleton and Lange, Stamford, CT (1999)). A two-compartment model is used to account for the initial distribution of antibody to the tissues (alpha phase), followed by a terminal or elimination phase (beta phase). The elimination half-life (ti/2p) so calculated reveals effects of improved FcRn binding because FcRn functions to maintain IgG in the circulation. In one example of such a study, the pharmacokinetics of three humanized monoclonal anti-BR3 antibodies (PRO145234, PRO145181, and PRO145182) with different binding affinities to FcRn were compared in cynomolgus monkeys. BR3 (also known as BAFF-R) is a 184-residue type III transmembrane protein expressed on the surface of B cells (Thompson, J. S., et al., (2001) Science 293, 2108-211 1 ; Yan, M., et al., (2001) Curr. Biol. 11, 1547-1552). PRO145234 is the wild type anti-BR3 antibody while PRO145181 and PRO145182 are N434A and N434W variants, respectively, that have increased binding affinity to human and cyno FcRn. Seventeen male and 17 female cynomolgus monkeys (Macaca fascicularis) were obtained from SNBL USA stock. The monkeys were 45 years old and weighed 24 kg at the time of the physical examination at the start of the study {e.g., initiation of acclimation). Only animals that appeared to be healthy and that were free of obvious abnormalities were used for the study. Thirty animals were randomized by weight into one of three groups. Animals in Groups 1, 2, and 3 received a single IV dose of 20 mg/kg of PRO145234 (wild type), PRO145181 (N434A), or PRO145182 (N434W), respectively. The study design is summarized in Table 1 1. Table 11. Study Design

Dose Level Dose Cone. Dose Volume Group No ./Sex Test Material Route (mg/kg) (mg/mL) (mL/kg)a 1 5/M. 5/F PRO 145234 IV 20 20 1 (wild type) 2 5/M. 5/F PRO145181 IV 20 20 1 (N434A) 3 5/M. 5/F PRO145182 IV 20 20 1 (N434W) Cone. = concentration. a Total dose volume (mL) was calculated based on the most recent body weight. Dose volumes were interpolated to the nearest 0.1 mL.

Approximately 1.0 mL of blood for pharmacokinetic analysis was collected from a peripheral vein of each animal at the following timepoints:

• Predose • 30 minutes, and 6 hours post-dose on Study Day 1. • Once on Study Days 2, 3, 4, 5, 8, 11, 15, 18, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 92, 99, 106, 113, 120, 127, and 134 Approximately 1.0 mL of blood for anti-therapeutic antibody analysis was collected from a peripheral vein of each animal at the following timepoints: • Predose • Once on Study Days 15, 29, 43, 57, 71, 85, 99, 113, 127 and 134 Blood samples for pharmacokinetic (PK) and anti -therapeutic antibody (ATA) analysis were collected into serum separator tubes and allowed to clot at room temperature for approximately 30- 80 minutes. Serum (approximately 0.5 mL) was obtained by centrifugation (2000 x g for 15 minutes at room temperature). Serum samples were transferred into prelabeled 1.5-mL Eppendorf tubes and stored in a freezer set to maintain a temperature of -600C to -800C until packed on dry ice and shipped overnight to Genentech for determination of PRO145234, PRO145181 , and PRO145182 concentrations. The concentrations of PRO145234, PRO145181, or PRO145182 in each serum sample were determined using an ELISA assay. The assay lower limit of quantification (LLOQ) in serum is 0.05 μg/mL. Values below this limit were recorded as less than reportable (LTR). Anti -therapeutic antibodies in each sample were determined using a bridging ECLA assay. Nominal dose and sample collection times with minimal deviation from the schedule were used in the data analysis. Mean and SD of serum PRO145234, PRO145181 , and PRO145182 concentrations in male and female cynomolgus monkeys were calculated using Excel (version 2000, Microsoft Corporation, Redmond, WA,) and plotted using SigmaPlot (version 9.0; Systat Software, Inc., Point Richmond, CA). Serum concentrations that were less than reportable were excluded from all data analysis. The SD was not calculated when n < 2. Results are presented to three significant figures. PK parameters for each animal were estimated using a Gauss-Newton (Levenberg and Hartley) two-compartmental model with a 1 over y hat weighting scheme (WinNonlin Version 3.2; Pharsight Corporation; Mountain View, CA). Eight out of ten cynos in Group 1 (wild type; PRO 145234) and five out of 10 cynos in Group 3 (N434W; PRO145182) developed ATA's by day 57. In general, detection of ATA's at a particular time correlated with a sharp drop in serum concentrations during or after that time, resulting in a shorter terminal half-life and decreased drug exposure. To understand the magnitude of the effect of the ATA response on PK, mean PK parameters for each group were calculated using two methods. In method 1, PK parameters (mean ± standard deviation) were calculated using data from all 10 cynos in each group. In method 2, PK parameters were calculated using data only from cynos that did not develop anti-therapeutic antibodies by day 57 (n=2 for group 1, n=10 for group 2, and n=5 for group 3). For groups 1 and 3, method 1 resulted in lower estimates of terminal half-life (t m, β) and exposure (AUC; measure of overall drug exposure) compared to method 2. However, the overall conclusions using the two methods were similar. Therefore, the mean PK parameters reported here were calculated using method 1 {e.g., including data from all cynos).

Results Following a single IV bolus administration of 20 mg/kg of PRO 145234 (wild type antibody), PRO 145181 (N434A variant), and PRO 145182 (N434W variant), serum concentrations exhibited biphasic disposition, with a rapid initial distribution phase followed by a slower elimination phase (Figure 12). Estimated PK parameters for each group are shown in Table 12 and include data from all ten cynos in each group. The terminal half-life (mean ± SD) of PRO145234 (wild type antibody) was 6.15 ± 2.01 days and ranged from 4.24 to 11.0 days in ten cynos. The mean terminal half-life (t \a, β) of PRO145234 in the two cynos that did not develop ATA's by day 57 was 8.95 days. For PRO 145181 (N434A variant), the mean terminal half-life was 14.1 ± 1.55 days which is 1.6-2.3 fold greater than that of PRO145234 (p<0.05). For PRO145182 (N434W variant), the mean + SD terminal half-life in ten cynos was 9.55 ± 2.49 days. This value is significantly greater than the overall mean 1 1/2, p of PROl 45234 (wild type antibody) in ten cynos (p<0.05), but it is very similar to the mean t m, β of PRO145234 in the two cynos that did not develop detectable ATA's (8.95 days). It is likely that the observed difference in t u2, β between PRO145234 (wild type antibody) and PRO145182 (N434W variant) is confounded by the ATA response in these two groups. The area under the concentration-time curve extrapolated to infinity (AUC) of PRO145234 (wild type antibody) was 2440 ± 398 day*ug/mL and ranged from 1740 to 3140 day*ug/mL for the ten cynos. The mean AUC of PRO145234 in the two cynos that did not develop ATA's by day 57 was 2850 day*ug/mL. For PRO145181 (N434A variant), the mean AUC was 4450 ± 685 day*ug/mL which is 1.6-1.8 fold greater than that of PRO145234 (wild type antibody) (p<0.05). There was no difference in the AUC of PRO145234 (wild type antibody) and PRO145182 (N434W variant). Table 12: PK Parameters (Mean ± SD) of PRO145234, PRO145181, and PRO145182

* Presence of anti-drug antibodies in 8/10 and 5/10 cynos in PRO145234 & PRO145182 groups may confound PK parameters of PRO145234 & PRO145182 (e.g., decrease AUC and decrease 1 1/2 p) ** Different from PROl 45234 with p<0.05

In summary, the pharmacokinetics of PRO1451234, PRO145181, and PRO145182 were examined following a single IV dose of 20 mg/kg to cynomologus monkeys. Eight out of 10 cynos developed anti- therapeutic antibodies (ATA's) to PRO145234 by day 56 while 5 out of 10 cynos developed ATA's to PRO145182 by day 56. No cynos developed ATA's to PRO145181 by day 56. PRO145181 (N343A variant) exhibited an increased terminal half-life and increased AUC compared to PRO 145234 (wild-type antibody) (p<0.05). PRO145182 exhibited a slight increase in terminal half-life compared to PRO145234; however, it is likely that this observed difference is confounded by the anti-therapeutic antibody response to both PRO145234 and PRO145182.

Example 5 Human IgGl variants with enhanced binding to FcγRIII Mutations have been previously described (Shields et al., J. Biol. Chem. 276:6591-6604 (2001); Presta et al., Biochem. Soc. Trans. 30:487-490 (2002)) for improving antibody-dependent cell-mediated cytotoxicity (ADCC) through enhanced IgG binding to activating Fcγ receptors and reduced IgG binding to inhibitory Fcγ receptors. Mutations that increase ADCC activity while maintaining or increasing complement dependent cytotoxicity (CDC) are of particular interest because both mechanisms may be important for lysis of CD20-positive cells in vivo. In particular, three Ala mutations in combination have been previously identified for improving CDC activity through improved CIq binding (Idusogie et al., J. Immunol. 164:4178-4184 (2000)); Idusogie et al., J. Immunol. 166:2571-2575 (2001 )), and ADCC activity through improvement in FcγRIII binding and reduction in FcγRII binding: S298A+E333A+K334A (Shields et al., 2001). These mutations, along with a further substitution that enhances ADCC and CDC activity, K326A, have been incorporated into a humanized anti-CD20 antibody variant known as 2H7.vl 38 (Table 3). Here we describe additional amino acid substitutions at positions 298, 333, and 334. Each substitution was made in the background of 2H7.vl6 and compared to vl6 in an ELISA for relative binding to the high-affinity or low-affinity isotype of human FcγRIII (Fig. 11). The results indicate that several substitutions at these positions, such as S298T, S298L, E333L, and K334G, are well tolerated, having little effect on binding affinity to FcγRIII (Table 1). Other substitutions such as S298G, E333G, and K334R are deleterious to binding, either because of unfavorable interactions of these side chains with the receptor or because of destabilizing effects on the Fc structure. One mutation, K334L, was identified with >3-fold increased binding affinity to FcγRIII. These results indicate that substitutions other than Ala at selected positions in the Fc that show effects with Ala substitution can improve binding to Fcγ receptors. In particular, K334L improves binding to FcγRIII and may further improve binding in combination with other Fc mutations such as those in 2H7.vl38. Such antibody variants are predicted to have improved ADCC activity and to be more potent in depleting target cells in vivo.

Table 3. Effects of substitutions in the Fc region on CD20 binding. Fc mutations are indicated by EU numbering (Kabat, supra) and are relative to the 2H7.vl6 parent. The relative binding is expressed as the concentration of 2H7.vl6 divided by the concentration of the variant required for equivalent binding; hence a ratio <1 indicates weaker affinity for the variant and a ratio >1 indicates higher affinity. Results for the F158 (low affinity) and V158 (high affinity) isotypes of FcγRIII are shown.

Example 6 Histidine mutants In this example, point mutations were investigated through a histidine-scanning (His-scan) of the residues in the interface between Fc and FcRn as deduced from the published structure of rat IgG in complex with rat FcRn (Burnmeister, 1997). We reasoned that substitutions of His could be advantageous to pH- dependent effects on the binding of IgG to FcRn because the side chain of His is often titratable between pH 6 and 7. Substitution of His at a site in the Fc where the protonated form is advantageous to FcRn binding, but the unprotonated form is not advantageous, should yield the desired characteristics for an enhanced half- life molecule in vivo. We describe the further characterisation of previously described point mutants in the context of a full-length antibody (Herceptin®) and investigate the characteristics of several new variants, including combinations of point mutations. CONSTRUCTION OF TRASTUZUMAB FcRn VARIANTS Previous examples described some mutations in the Fc that improve binding to FcRn and were studied as Fc fragments, or on a background of an intact antibody, 2H7.vl38. To study the effect of these mutations on a full length human IgGl, but in the absence of other Fc changes, the mutations N434W, N434Y, N434F, and N434A were introduced into the background of rhuMab 4D5 (trastuzumab, Herceptin®) using oligonucleotide site-directed mutagenenesis as previously described. Because three of the four aromatic amino acids were found substituting at position N434 using Fc-phage point-mutation libraries, we concluded that aromatic substitutions in general enhance FcRn (low-pH, and possible high-pH) binding. Therefore, an additional mutation, N434H was also constructed. These 5 variants of Herceptin were purified from large scale transient CHO cell cultures, using Protein A affinity chromatography followed by size exclusion chromatography to remove aggregates. Additionally, mutations E380A, E380A + T307A, +/- N434A, and +/- N434H were constructed on Herceptin. Antibodies were expressed in 293 cells, purified by Protein A chromatography, and assayed for binding to FcRn. To identify additional residues important in FcRn binding, and mutations that would improve binding, a histidine scanning approach was used. These experiments substituted histidine for residues on the interface between Fc and FcRn (by homology to the rat FcRn structure (Burnmeister, 1997)). Antibodies with these mutations were expressed in 293 cells and purified and assayed for binding as described above. Histidine scanning mutations that gave improved binding to FcRn were combined with mutations at N434 to give an additional set of variants.

FcRn ELISA Soluble FcRn was prepared as described previously (Shields et. al., 2001). MaxiSorp 96-well microwell plates (Nunc, Roskilde, Denmark) were coated with 2 μg/ml NeutrAvidin (Pierce, Rockford, IL), at 100 μl/well in 50 mM carbonate buffer, pH 9.6, at 4°C overnight. Plates were washed with PBS containing 0.05% polysorbate (wash buffer), pH 7.4, and blocked with PBS containing 0.5% BSA, pH 7.4, at 150 μl/well. After an one-hour incubation at room temperature, plates were washed with wash buffer, pH 7.4. Human FcRn was biotinylated using biotin-X-NHS (Research Organics, Cleveland, OH). Biotinylated FcRn was added to the plates at 2 μg/ml, 100 μl/well, in PBS containing 0.5% BSA, 0.05% polysorbate 20 (assay buffer), pH 7.4. The plates were incubated for one hour and washed with wash buffer, pH 6.0. Seven twofold serial dilutions of IgG antibodies (3.1-200 ng/ml) in assay buffer, pH 6.0, were added to the plates. Herceptin was used as the standard. After a two-hour incubation, plates were washed with wash buffer, pH 6.0. A dissociation step was carried out by adding assay buffer at pH 6.0 or pH 7.4, 100 μl/well. Plates were incubated for 45 min and washed with wash buffer, pH 6.0. Bound IgG was detected by adding peroxidase labeled goat F(ab')2 anti-human IgG F(ab')2 (Jackson ImmunoResearch, West Grove, PA) at 100 μl/well in assay buffer, pH 6.0. After a one-hour incubation, plates were washed with wash buffer, pH 6.0, and the substrate 3,3',5,5'-tetramethyl benzidine (TMB) (Kirkegaard & Perry Laboratories) was added at 100 μl/well. The reaction was stopped by adding 1 M phosphoric acid at 100 μl/well. Absorbance was read at 450 nm on a multiskan Ascent reader (Thermo Labsystems, Helsinki, Finland). The absorbance at the midpoint of the Herceptin curve at pH 6.0 (mid-OD) was calculated. The corresponding concentrations of Herceptin and samples at this mid-OD were determined from the titration curves using a four-parameter nonlinear regression curve-fitting program (KaleidaGraph, Synergy software, Reading, PA). The relative activity was calculated by dividing the mid-OD concentration of standard by that of sample.

BIACORE METHODS The apparent association rate (ka), apparent dissociation rate (Ig) and apparent (KD) of several 4D5 variants binding to human and cynomolgous monkey FcRn were measured using the BIAcore-3000 surface plasmon resonance system (6,7). The binding affinity of each antibody for antigen, as described by an apparent equilibrium dissociation constant, was both calculated (7) as KD= k^/kg, as well as directly measured in equilibrium binding experiments. The amine-coupling method was used for immobilization of human and cyno FcRn onto a carboxymethylated dextran biosensor chip (cat. no. CM5, BIAcore, Inc.) essentially as described in the manufacturer's instructions (6,8). Briefly, the biosensor chip was activated using N-ethyl-N'-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC) mixed with N-hydroxysuccinimide (NHS). FcRn, pre-equilibrated in 10 mM sodium acetate pH 4, was then injected over the activated chip to yield immobilized concentrations of 700 - 900 response units (RU) of cynoFcRn and approximately 2000 RU of human FcRn. Unreacted succinimide groups were blocked with an injection of 1 M ethanolamine. Kinetics measurements were conducted as follows. 3-fold serial dilutions (1 uM to 0.5 nM) of antibody were injected for 2 minutes in running buffer (phosphate-buffered saline, pH 6, containing 0.05% Tween-20) at 250C using a flow rate of 0.02 ml/min. Regeneration was achieved with a 0.01 ml injection of 10 mM Tris, pH 9, 150 mM NaCl. Data were fit to a simple 1 : 1 Langmuir binding model for each binding curve. Pseudo-first order rate constant (ks) were calculated for each association curve, and plotted as a function of protein concentration to obtain ka +/- s.e. (standard error of fit). Equilibrium binding measurements were performed both at pH 6 and pH 7.4. 3-fold serial dilutions (1 uM to 0.5 nM) of antibody were injected for 6 minutes in running buffer (phosphate-buffered saline containing 0.05% Tween-20) at 250C using a flow rate of 2 ul/min. Following injection, the flow rate was increased to 0.02 ml/min. Regeneration was achieved with a 0.01 ml injection of 10 mM Tris, pH 9, 150 mM NaCl. The amount of antibody bound at equilibrium (R«j) was plotted as a function of the antibody concentration and fit to a four parameter dose response curve in order to determine the KD.

ELISA RESULTS Binding to FcRn at pH 6.0 and 7.4 was measured. Relative binding affinities were calculated as described in Materials and Methods and are shown in Table 4. The results indicate improved pH 6 binding for mutations N434A, N434W, N434Y, N434F, or N434H. Increased binding relative to Herceptin is also observed at pH 7.4 for N434W, N434Y, and N434F. Table 4. FcRn ELISA results for N434 point mutants. Relative binding values >1 indicate increased , binding; values <1 indicate decreased binding, compared to the parental Herceptin molecule.

Ratio of mutant binding at pH 6.0 to Ratio of mutant binding at pH 7.4 to mutant Herceptin binding at pH 6.0 Herceptin binding at pH 6.0

Combination variants with N434H or N434A and previously described Ala substituions (Shields et al, 2000) also showed improved binding at pH 6.0, with little or no increase in pH 7.4 binding (Table 5).

Table 5. FcRn ELISA results for N434 combination mutants. Relative binding values >1 indicate increased binding; values <1 indicate decreased binding, compared to the parental Herceptin molecule.

Ratio of mutant binding at pH 6.0 to Ratio of mutant binding at pH 7.4 to mutant Herceptin binding at pH 6.0 Herceptin binding at pH 6.0

The His-scan of the Fc interface region identified several His substitutions that increased or decreased FcRn binding (Table 6). Some of these variants were shown to have pH-dependent binding, with little or no detectable interaction with FcRn at pH 7.4. Although none of these additional His mutations alone increased pH 6 binding more than N434H, the substitutions Q31 IH, D312H, N315H, and G385H each improved pH 6 binding >4-fold without significant increases in pH 7.4 binding.

Table 6. FcRn ELISA results for His-scan mutants. Relative binding values >1 indicate increased binding; values <1 indicate decreased binding, compared to the parental Herceptin molecule.

Ratio of mutant binding at pH 6.0 to Ratio of mutant binding at pH 7.4 to mutant Herceptin binding at pH 6.0 Herceptin binding at pH 6.0

Finally, several His-scan point mutations were combined pairwise with the mutations N434A or N434H. Among these variants, the double mutants T289H/N434H and N315H/N434H appeared most improved in pH 6 binding, with no apparent improvement in pH 7.4 binding. Table 7. FcRn ELISA results for His-scan combination mutants. Relative binding values >1 indicate increased binding; values <1 indicate decreased binding, compared to the parental Herceptin molecule.

Ratio of mutant binding at pH 6.0 to Ratio of mutant binding at pH 7.4 to mutant Herceptin binding at pH 6.0 Herceptin binding at pH 6.0

BIAcore RESULTS Several antibodies were selected based upon ELISA FcRn binding and divided into three groups for BIAcore analysis. Set 1 consisted primarily of mutants at Asn434, set 2 consisted of Histidine scan mutants, while set 3 consisted of previously published mutants (3). Results from kinetic and equilibrium binding analysis of antibodies from set 1 at pH 6 (Table 8), suggest that improvements in KD are the result of changes in kd and/or kj. Furthermore, mutants that are improved in binding to human FcRn appear to be likewise improved in binding to cynoFcRn. The mutants that have the most improved binding at pH 6 are N434W, N434Y and N434F. However, these mutants also show significantly increased binding at pH 7.4. In contrast, Herceptin, N434A, N434H and T250Q/M428L all show little to no binding at pH 7.4. Note that while the pH 7.4 binding of N434W, N434Y and N434F are noticeably increased over that of the wild-type Fc, the association and dissociation rates are so rapid that the equilibrium dissociation constants cannot be accurately determined. Binding at pH 7.4 has therefore been represented as + or - in Table 4. In particular, - denotes similar levels of binding as Herceptin, +/- denotes negligible binding, + denotes noticeable binding and ++ denotes significant binding.

TABLE 8. Comparison of kinetic and equilibrium binding of set 1 mutants.

Protein ka (xlO5 M-'s-1) kd (x 102 s-1) KD" KD* (nM) pH 7.4 binding (nM) huFcRn Herceptin 9.76 8.85 90.6 189 N434A 10.3 4.12 39.9 90.94 N434W 35.9 0.54 1.51 9.61 ++ N434Y 33 1.25 3.78 21.89 N434F 25.2 1.11 4.41 28.21 ++ N434H 20.2 2.92 14.5 49.63 +/- T250Q/M428Lc 12.5 1.9 15.2 44.38 +/-

cynoFcRn Herceptin 9.77 5.25 53.7 103.2 - N434A 22.5 1.62 7.19 41.1 - N434W 38 0.29 0.76 4.08 ++ N434Y 40.5 0.45 1.1 9.24 ++ N434F 29.7 0.59 1.98 12.14 ++ N434H 36.9 1.45 3.91 26.84 +/- T250Q/M428Lc 16.9 0.78 4.62 21.39 +/-

"K0 calculated from kinetic parameters. *KD calculated from equilibrium binding expts. cProteins were expressed in 293 cells.

There are some differences between the KDs determined by kinetic and equilibrium binding analysis. However, a comparison of these values shows a correlation coefficient of 0.994 for binding to human FcRn and 0.934 for binding to cynoFcRn, suggesting a systematic deviation. Kinetic and equilibrium binding analyses of antibodies from set 2 (Table 9), show similar results to those of antibodies from set 1. Mutants that are improved in binding to human FcRn appear to be also improved in binding to cynoFcRn. The mutants that have the most improved binding at pH 6 are N434H/T370A/E380A, N434H/T289H and N434H/N315H. However, these mutants also show significantly increased binding at pH 7.4. In contrast, the remaining antibodies all show little to no binding at pH 7.4.

TABLE 9. Comparison of kinetic and equilibrium binding of set 2 mutants.

Protein R3 (XlO5 M 1S 1) Mx IO-2 S 1) KD" (nM) KD* (nM) pH 7.4 binding huFcRn Herceptin 12.6 7.16 56.9 156.9 - N434H/T370A/E380Ac 54.2 0.93 1.71 23.43 ++ N434H/T289HC 44.9 1.11 2.46 27.48 + N434H/N315HC 51 1.34 2.63 25.33 +/- G385HC 20.9 3.67 17.5 286.6 - D312He 13.4 1.61 12 75.42 - N315HC 8.14 2.14 26.3 61.08 - N434H 35.2 1.83 5.19 37.29 -

cynoFcRn Herceptin 15.9 9.79 61.6 1 14.3 N434H/T370A/E380Ae 51.9 1.19 2.29 19.03 ++ N434H/T289HC 55.8 1.1 1 1.98 20.46 + N434H/N315HC 57 1.29 2.25 18.47 +/- G385HC 19.1 3.94 20.6 40.95 - D312HC 10.9 3.41 31.3 55.41 - N315HC 8.3 2.46 29.6 47.68 - N434H 33.6 2.02 6.02 26.29 _

"KQ calculated from kinetic parameters. *KD calculated from equilibrium binding expts. Troteins were expressed in 293 cells. An examination of the correlation coefficient between the KβS determined by kinetic and equilibrium binding analyses showed that the correlation coefficient for human FcRn binding was 0.246, while the correlation coefficient for cynoFcRn binding was 0.966. The cause of the low correlation coefficient for human FcRn binding was G385H, which experienced some aggregation problems during equilibrium binding. Excluding this data point gave a correlation coefficient of 0.916. Kinetic and equilibrium binding analyses of antibodies from set 3 (Table 10), show similar results to those of antibodies from set 1. Mutants that are improved in binding to human FcRn appear to be also improved in binding to cynoFcRn. The T250Q/M428L combination mutant has the most improved binding at pH 6. While it also has slightly increased binding at pH 7.4, this level is low compared to those of mutants from sets 1 and 2.

ABLE 10. Comparison of kinetic and equilibrium binding of set 3 mutants.

Protein ka (xlO5 M 1S"1) k,, (x 102 s 1) KD" (nM) KD* (nM) pH 7.4 binding huFcRn Herceptin 5.47 11.4 209 172.1 - T250Qc 7.16 6.27 87.6 59.9 - M428LC 5.92 5.43 91.7 56.4 T250Q/M428I/ 9.62 3.09 32.2 32.8 +/-

cynoFcRn Herceptin 12.8 7.63 59.8 49.5 - T250Qc 15.7 2.29 14.6 17.7 - M428LC 9.83 3.41 34.7 20.7 - T250Q/M428Lc 13.5 1.72 12.7 6.8 +/-

"KD calculated from kinetic parameters. 6KD calculated from equilibrium binding expts. Troteins were expressed in 293 cells.

An examination of the correlation coefficient between the KQS determined by kinetic and equilibrium binding analyses showed that the correlation coefficient for human FcRn binding was 0.966, while the correlation coefficient for cynoFcRn binding was 0.902.

Previously we reported that the affinities of hingeless Fes containing the mutations N434W, N434F and N434A were 170-, 9- and 2.7-fold improved in human FcRn binding, compared to wild-type at pH 6. In this example we have looked at these mutations, and others, in the context of the full-length 4D5 antibody. Comparing the results gained by equilibrium binding analysis, we now find that N434W, N434F and N434A show 20-, 7- and 2-fold improvements in binding affinity to human FcRn at pH 6. Similar results were seen with binding to cyno FcRn. Additional variants containing aromatic mutations such as N434Y and N434H showed 9- and 4-fold increases in pH 6 binding to human FcRn. However, the N434W, N434Y and N434F mutants all also exhibited large increases in binding to both human and cyno FcRn at pH 7.4. Given the promising results from N434H, and the importance of histidine residues in the pH- dependent Fc-FcRn interaction (9), we decided to investigate histidine mutations in other contexts. Using the structure solved for the rat Fc-FcRn complex (9,10) and assuming homology for the human proteins, a histidine-scan was conducted. Additionally, investigations were performed on the N434H mutation combined with the T370A and E380A mutations identified by Presta and colleagues (4). Variants that showed improvements in pH 6 binding, but without increased pH 7.4 binding, by BIAcore analysis included G385H, D312H, N315H, and N434H. None of the new histidine mutations, or histidine combination mutations, showed improvements in pH 6 FcRn binding that were much greater than that of the N434H mutation alone. However, the combination mutations all showed significantly increased FcRn binding at pH 7.4. Additional variants combining two or more histidine mutations may provide enhanced pH-dependent binding to FcRn. In addition, the identification of sites affected by His mutations suggests new sites for substitutions of additional amino acids. Finally, we also investigated the mutations reported by Hinton and colleagues (3) in the context of an IgGl molecule. Whereas they had reported increases in pH 6 binding of 3-, 7- and 28-fold for T250Q, M428L and T250Q/M428L, respectively, in the background of an IgG2 molecule, we found more modest increases of 3-, 3- and 5-fold, respectively, in the context of an IgG 1. Similar results were seen for binding to cyno FcRn. The single mutants showed no increase in binding to human or cyno FcRn at pH 7.4, while the double mutant showed only slightly increased binding. In this study we have quantitated the affinities of several Fc mutants to human and cyno FcRn at both pH 6 and pH 7.4. We have determined that overall affinities and affinity improvements are similar between human and cyno FcRn, for a given molecule. The rankings of improved variants based on pH 6 binding by ELISA and BIAcore assays were in general agreement; however, pH 7.4 binding assessments sometimes differed. These discrepancies may results from differences in behavior of FcRn or IgG under the different immobilization procedures used.

REFERENCES FOR EXAMPLE 6

1. Ghetie, V., and Ward, E. S. (2000) Annu Rev Immunol 18, 739-766

2. Ghetie, V., and Ward, E. S. (1997) Immunol Today 18, 592-598

3. Hinton, P. R., Johlfs, M. G., Xiong, J. M., Hanestad, K., Ong, K. C, Bullock, C, Keller, S., Tang, M. T., Tso, J. Y., Vasquez, M., and Tsurushita, N. (2004) J Biol Chem 279, 6213-6216

4. Shields, R. L., Namenuk, A. K., Hong, K., Meng, Y. G., Rae, J., Briggs, J., Xie, D., Lai, J., Stadlen, A., Li, B., Fox, J. A., and Presta, L. G. (2001) J Biol Chem 116, 6591-6604

5. Dall'Acqua, W. F., Woods, R. M., Ward, E. S., Palaszynski, S. R., Patel, N. K., Brewah, Y. A., Wu, H., Kiener, P. A., and Langermann, S. (2002) J Immunol 169, 5171-5180

6. Johnsson, B., Lofas, S., and Lindquist, G. (1991) Anal Biochem 198, 268-277

7. Karlsson, R., Michaelsson, A., and Mattsson, L. (1991) J Immunol Methods 145, 229-240 8. BIAcore, Inc. (1991) BIAcore Methods Manual, Piscataway, NJ

9. Martin, W. L., West, A. P., Jr., Gan, L., and Bjorkman, P. J. (2001 ) MoI Cell 7, 867-877

10. Burmeister, W. P., Huber, A. H., and Bjorkman, P. J. (1994) Nature 372, 379-383

References References cited within this application, including patents, published applications and other publications, are hereby incorporated by reference. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology and the like, which are within the skill of the art. Such techniques are explained fully in the literature. See e.g., Molecular Cloning: A Laboratory Manual, (J. Sambrook et al, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989); Current Protocols in Molecular Biology (F. Ausubel et al, eds., 1987 updated); Essential Molecular Biology (T. Brown ed., IRL Press 1991); Gene Expression Technology (Goeddel ed., Academic Press 1991 ); Methods for Cloning and Analysis of Eukarvotic Genes (A. Bothwell et al. eds., Bartlett Publ. 1990); Gene Transfer and Expression (M. Kriegler, Stockton Press 1990); Recombinant DNA Methodology II (R. Wu et al. eds., Academic Press 1995); PCR: A Practical Approach (M. McPherson et al., IRL Press at Oxford University Press 1991); Oligonucleotide Synthesis (M. Gait ed., 1984); Cell Culture for Biochemists (R. Adams ed., Elsevier Science Publishers 1990); Gene Transfer Vectors for Mammalian Cells (J. Miller & M. Calos eds., 1987); Mammalian Cell Biotechnology (M. Butler ed., 1991); Animal Cell Culture (J. Pollard et al. eds., Humana Press 1990); Culture of Animal Cells, 2nd Ed. (R. Freshney et al. eds., Alan R. Liss 1987); Flow Cytometry and Sorting (M. Melamed et al. eds., Wiley-Liss 1990); the series Methods in Enzvmology (Academic Press, Inc.);Wirth M. and Hauser H. (1993); Immunochemistry in Practice, 3rd edition, A. Johnstone & R. Thorpe, Blackwell Science, Cambridge, MA, 1996; Techniques in Immunocytochemistry, (G. Bullock & P. Petrusz eds., Academic Press 1982, 1983, 1985, 1989); Handbook of Experimental Immunology, (D. Weir & C. i Blackwell, eds.); Current Protocols in Immunology (J. Coligan et al. eds. 1991); Immunoassay (E. P. Diamandis & T.K. Christopoulos, eds., Academic Press, Inc., 1996); Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed) Academic Press, New York; Ed Harlow and David Lane, Antibodies A laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988; Antibody Engineering, 2nd edition (C. Borrebaeck, ed., Oxford University Press, 1995); and the series Annual Review of Immunology; the series Advances in Immunology.