Fc variants with mutant binding to FcRn

Abstract

Providing Fc variants with mutant binding to FcRn. [Solution] The present application relates to a mutant Fc region comprising at least one modification compared to a wild-type human Fc region, wherein the modification is selected from the group consisting of 434S, 252Y / 428L, 252Y / 434S, and 428L / 434S, numbered according to the EU index.

Description

[Technical Field]

[0001] This application is a division of U.S. Patent No. 61 / 016,793, filed December 26, 2007; U.S. Patent No. 61 / 031,353, filed February 25, 2008; U.S. Patent No. 61 / 046,353, filed April 18, 2008; U.S. Patent No. 61 / 050,172, filed May 2, 2008; U.S. Patent No. 61 / 079,779, filed July 10, 2008; and U.S. Patent No. 61 / 089,779, filed September 22, 2008, all of which are hereby incorporated by reference under 35 U.S.C. § 119(e). 11 / 932,151, filed October 31, 2007, which claims the benefit of USSN 61 / 099,178, filed May 17, 2006, which claims the benefit under 35 U.S.C. § 119(e) to USSN 60 / 951,536, filed July 24, 2007, which claims the benefit of USSN 60 / 951,536, filed November 14, 2005, which claims the benefit of USSN 60 / 951,536, filed July 24, 2007, under 35 U.S.C. § 119(e). 11 / 274,065, which is a continuation-in-part application of USSN 60 / 627,763, filed November 12, 2004; USSN 60 / 642,886, filed January 11, 2005; USSN 60 / 649,508, filed February 2, 2005; USSN 60 / 662,468, filed March 15, 2005; USSN 60 / 669, filed April 6, 2005; 311, filed May 16, 2005, USSN 60 / 681,607, filed June 13, 2005, USSN 60 / 690,200, filed July 5, 2005, USSN 60 / 696,609, filed July 5, 2005, USSN 60 / 703,018, filed July 27, 2005, and USSN 60 / 726,453, filed October 12, 2005, all of which are incorporated by reference in their entireties.

[0002] The present application relates to optimized IgG immunoglobulin variants, engineering methods for their production, and their applications, particularly for therapeutic purposes. [Background technology]

[0003] Antibodies are immune proteins that bind to specific antigens. In most mammals, including humans and mice, antibodies are constructed from paired heavy and light polypeptide chains. Each chain is formed by an individual immunoglobulin (Ig) domain, hence the general term immunoglobulin. Each chain consists of two distinct regions, termed the variable and constant regions. The light and heavy chain variable regions exhibit significant sequence diversity among antibodies and are responsible for target antigen binding. The constant regions exhibit less sequence diversity and are responsible for binding numerous natural proteins that trigger important biochemical events. In humans, there are five distinct classes of antibodies: IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. While subtle differences may exist in the V regions, the distinguishing features between these antibody classes are their constant regions. IgG antibodies are tetrameric proteins composed of two heavy chains and two light chains. The IgG heavy chain is composed of four immunoglobulin domains connected in the order VH-CH1-CH2-CH3 from N-terminus to C-terminus, which refer to the heavy chain variable domain, heavy chain constant domain 1, heavy chain constant domain 2, and heavy chain constant domain 3, respectively (also referred to as VH-Cγ1-Cγ2-Cγ3, which refer to the heavy chain variable domain, constant gamma 1 domain, constant gamma 2 domain, and constant gamma 3 domain, respectively). The IgG light chain is composed of two immunoglobulin domains connected in the order VL-CL, which refer to the light chain variable domain and light chain constant domain, respectively, from N-terminus to C-terminus.

[0004] In IgG, a site between the Cγ2 and Cγ3 domains on Fc mediates interaction with the neonatal receptor FcRn. Binding to FcRn allows endocytosed antibodies to be recycled from endosomes to the bloodstream (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766, both incorporated by reference in their entireties). This process, coupled with exclusion of full-length antibodies from renal filtration due to their large size, results in favorable antibody serum half-lives ranging from 1 to 3 weeks. Binding of Fc to FcRn also plays a key role in antibody transport. The FcRn-binding site on Fc is also the site of bacterial protein A and G binding. The tight binding of these proteins is typically exploited as a means of antibody purification, typically by using protein A or protein G affinity chromatography during protein purification. Therefore, the fidelity of this region on Fc is important for both the clinical properties of antibodies and their purification. The available structures of the rat Fc / FcRn complex (Burmeister et al., 1994, Nature, 372:379-383; Martin et al., 2001, Mol Cell 7:867-877, both incorporated by reference in their entireties) and the complex of Fc with proteins A and G (Deisenhofer, 1981, Biochemistry 20:2361-2370; Sauer-Eriksson et al., 1995, Structure 3:265-278; Tashiro et al., 1995, Curr Opin Struct Biol 5:471-481, all incorporated by reference in their entireties) provide insight into the interaction of Fc with these proteins.The FcRn receptor is also involved in the transport of IgG into the lumen of the neonatal viscera and adult intestinal epithelium (Ghetie and Ward, Annu. Rev. Immunol., 2000, 18:739-766; Yoshida et al., Immunity, 2004, 20(6):769-783, both of which are incorporated by reference in their entireties).

[0005] Studies of rat and human Fc domains have demonstrated the importance of certain Fc residues for FcRn binding. The rat and human sequences share approximately 64% sequence identity in the Fc region (residues 237-443 in the EU index numbering). See Figures 3, 4, and 5 for rat / human alignments of the Fc, FcRn heavy chain, and FcRn light chain (beta-2-microglobulin). A model of the human Fc / FcRn complex was constructed from the existing structure of the rat Fc / FcRn complex (Martin et al., 2001, Mol Cell 7:867-877, incorporated by reference in its entirety). The rat and human sequences share several residues critical for FcRn binding, such as H310 and H435 (Medesan et al., 1997 J. Immunol. 158(5):221-7; Shields et al., 2001 J. Biol. Chem. 276(9):6591-6604, both incorporated by reference in their entireties). However, at many positions, the human and rat proteins have different amino acids, thereby providing residues in the human sequence with a different environment, and likely a different identity, than the rat sequence. This variability limits the ability to transfer properties from one homolog to another.

[0006] In mouse Fc, random mutation and phage display selection at positions T252, T254, and T256 results in a triple mutant, T252L / T254S / T256F, with a 3.5-fold increase in FcRn affinity and a 1.5-fold increase in serum half-life (Ghetie et al., 1997, Nat. Biotech. 15(7):637-640, incorporated by reference in its entirety). Also, disruption of the Fc / FcRn interaction by mutations at positions 253, 310, and 435 leads to a decrease in in vivo half-life (Medesan et al. J. Immunol. 1997 158(5):2211-7, incorporated by reference in its entirety).

[0007] Mutational studies in human Fcγ have been performed on some residues important for binding to FcRn, demonstrating increased serum half-life. In human Fcγ1, Hinton et al. mutated three residues individually to 19 other common amino acids. They found that double mutations of several point mutations increased FcRn-binding affinity (Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216; Hinton et al. Journal of Immunology 2006, 176:346-356, both incorporated by reference in their entireties). Two mutations increased half-life in monkeys. Shields et al. mutated residues almost exclusively to Ala and studied their binding to FcRn and FcγR (Shields et al., 2001, J. Biol. Chem., 276(9):6591-6604, incorporated by reference in its entirety).

[0008] Dall'Acqua et al. used phage display to select Fc mutants that bind to FcRn with high affinity (Dall'Acqua et al. 2002, J. Immunol. 169:5171-5180, incorporated by reference in its entirety). The selected DNA sequences were primarily double and triple mutants. The reference expressed proteins encoded by many of these selected sequences and found that some bound to FcRn more strongly than wild-type Fc.

[0009] Administration of antibodies and Fc fusion proteins as therapeutics requires injections at a frequency determined by the protein's clearance and half-life characteristics. A longer in vivo half-life would allow for fewer injections or the administration of lower doses, a clear advantage. Previous mutations in the Fc domain have resulted in several proteins with increased FcRn-binding affinity and in vivo half-life, but these mutations did not identify the optimal mutation and increased in vivo half-life.

[0010] One feature of the Fc region is the conserved N-linked glycosylation that occurs at N297. This carbohydrate, or oligosaccharide (as it is sometimes called), plays a critical structural and functional role in antibodies and is one of the primary reasons why antibodies must be produced using mammalian expression systems. Umana et al., 1999, Nat Biotechnol 17:176-180, Davies et al., 2001, Biotechnol Bioeng 74:288-294, Mimura et al., 2001, J Biol Chem 276:45539-45547, Radaev et al., 2001, J Biol Chem 276:16478-16483, Shields et al.,2001,J Biol Chem 276:6591-6604,Shields et al.,2002,J Biol Chem 277:26733-26740,Simmons et al.,2002,J Immunol Methods 263:133-147, Radaev et al., 2001, J Biol Chem 276:16469-16477, and Krapp et al., 2003, J Mol Biol 325:979-989, all incorporated by reference in their entireties).

[0011] Antibodies for therapeutic use are being developed. Representative publications related to such therapies include Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200; Cragg et al., 1999, Curr Opin Immunol 11:541-547; Glennie et al., 2000, Immunol Today 21:403-410; McLaughlin et al., 1998, J Clin Oncol 16:2825-2833; and Cobleigh et al., 1999, J Clin Oncol 17:2639-2648, all of which are incorporated by reference in their entireties. Currently, for anticancer therapies, any small improvement in mortality rate is defined as success. Certain IgG variants disclosed herein enhance the ability of the antibody to limit further growth or at least partially destroy the targeted cancer cells.

[0012] The anti-tumor effects of antibodies are mediated by their enhanced ability to mediate cytotoxic effector functions such as ADCC, ADCP, and CDC. Examples include Clynes et al., 1998, Proc Natl Acad Sci USA 95:652-656; Clynes et al., 2000, Nat Med 6:443-446; and Cartron et al., 2002, Blood 99:754-758, all of which are incorporated by reference in their entireties.

[0013] Human IgG1 is the most commonly used antibody for therapeutic purposes, and the majority of antibody engineering research has been conducted in this context. However, these different isotypes of the IgG class, including IgG1, IgG2, IgG3, and IgG4, have unique physical, biological, and clinical properties. There is a need in the art to design improved IgG1, IgG2, IgG3, and IgG4 variants. Furthermore, there is a need to design such variants to enhance binding to FcRn and / or increase in vivo half-life compared to native IgG polypeptides. Furthermore, there is a need to combine variants with pharmacokinetic properties and variants with modifications to improve efficacy through altered FcγR binding. The present application addresses these and other needs. Summary of the Invention

[0014] The present application is directed to Fc variants of a parent polypeptide comprising at least one modification in the Fc region of the polypeptide. In various embodiments, the variant polypeptide exhibits altered binding to FcRn compared to the parent polypeptide. In particular variants, the modification is selected from the group consisting of 428L, 434M, and 434S, numbered according to the EU index of Kabat et al.

[0015] In another embodiment, the Fc variant comprises at least two modifications selected from the group consisting of 252Y / 428L, 428L / 434H, 428L / 434F, 428L / 434Y, 428L / 434A, 428L / 434M, and 428L / 434S.

[0016] In another embodiment, the Fc variant comprises at least one modification selected from the group consisting of: M428L / N434S, V308F / M428L / N434S.

[0017] In another embodiment, the Fc variant comprises at least one modification selected from the group consisting of 259I / 434S, 308F / 434S, 308F / 428L / 434S, 259I / 308F / 434S, 307Q / 308F / 434S, 250I / 308F / 434S, and 308F / 319L / 434S.

[0018] In another embodiment, the Fc variant comprises at least one modification.

[0019] In another embodiment, the invention includes a method of treating a patient in need of such treatment comprising administering an effective amount of an Fc variant described in the present application.

[0020] In another embodiment, the invention includes a method of modifying the Fc to increase the half-life of an antibody or immunoadhesin according to the modifications described in this application.

[0021] In another variant, the invention includes Fc variants with enhanced FcRn binding with additional Fc variants that modulate effector function. [Brief explanation of the drawings]

[0022] [Figure 1a] Sequence alignment of human IgG constant heavy chains. Grey indicates differences from IgG1, and boxed residues indicate common allotypic variants in the human population. [Figure 1b] As explained in Figure 1a. [Figure 2] (SEQ ID NOs: 1 to 6) Amino acid sequences of the constant regions used in the present invention. [Figure 3] (SEQ ID NOs: 7-12) Amino acid sequences of exemplary mutant constant regions. [Figure 4] (SEQ ID NOs: 13 to 22) Amino acid sequences of the VH and VL variable regions used in the present invention. [Figure 5a] (SEQ ID NOs: 23-29) Amino acid sequences of exemplary mutant antibodies. [Figure 5b] As explained in Figure 5a. [Figure 6]Relative VEGF binding by WT and selected mutant IgG1 anti-VEGF antibodies. The plot shows Biacore response units (RU) at the end of the association phase, when the antibody analyte binds to the immobilized VEGF antigen. An anti-Her2 IgG1 antibody was used as a negative control. [Figure 7] Biacore sensorgrams of WT and mutant IgG1 antibodies against immobilized human FcRn at low (6.0) and high (7.4) pH. [Figure 8] FcRn binding affinity of WT and selected mutant IgG1 antibodies to human FcRn at pH 6.0 as determined by Biacore. The graph shows a plot of the pseudo-affinity constant (Ka*) on a logarithmic scale. [Figure 9a] Relative binding of mutant IgG1 anti-VEGF antibodies to human FcRn as determined by Biacore. The table shows the fold Ka* of each mutant relative to human WT (native) IgG1. n indicates the number of times each mutant was tested, and mean and SD indicate the mean and standard deviation, respectively, for each mutant across n binding experiments. Within each binding experiment, FcRn fold values ​​relative to WT IgG1 were calculated for all mutants. NB indicates no binding was detected. ND indicates no binding was determined for that particular mutant. NF indicates no approximation could be made from the binding data. [Figure 9b] As explained in Figure 9a. [Figure 9c] As explained in Figure 9a. [Figure 9d] As explained in Figure 9a. [Figure 10a] Relative binding of mutant IgG2 and IgG1 / 2 anti-VEGF antibodies to human FcRn as determined by Biacore. Table as described for Figure 9. [Figure 10b] As explained in Figure 10a. [Figure 10c] As explained in Figure 10a. [Figure 10d] As explained in Figure 10a. [Figure 11a]Analysis of additive and synergistic substitution combinations. Figure 11a shows a plot of the experimentally determined binding fold to human FcRn by each mutant against the predicted FcRn binding fold determined by the products of the single mutants. The data points for the mutants are displayed, and the line represents perfect additivity. Figure 11b shows the difference between the experimental and predicted fold for each mutant combination. Figure 11c shows the synergy of each mutant combination. Percent synergy is calculated as 100 x [(experimental fold / predicted fold)-1]. [Figure 11b] As explained in Figure 11a. [Figure 11c] As explained in Figure 11a. [Figure 12a] Relative binding of mutant anti-TNF, -CD25, -EGFR, and -IgE antibodies to human FcRn as determined by Biacore. Table as described for Figure 9. [Figure 12b] As explained in Figure 12a. [Figure 12c] As explained in Figure 12a. [Figure 12d] As explained in Figure 12a. [Figure 13a] In vivo pharmacokinetics of WT and mutant antibodies in mFcRn- / - hFcRn+ mice. The graphs plot serum antibody concentration versus time after a single intravenous administration. Figure 13a shows data from one of four studies conducted with an IgG1 antibody (Study 3), and Figure 13b shows data from a study conducted with an IgG2 antibody (Study 5). [Figure 13b] As explained in Figure 13a. [Figure 14a]Figure 1 shows approximate PK parameters from all in vivo PK studies performed in mFcRn- / -hFcRn+ mice using mutant and WT antibodies. N represents the number of mice per group, and mean and standard deviation (SD) data are provided for PK parameters. Half-life represents the beta phase characterized by antibody elimination from serum. Cmax is the maximum observed serum concentration, AUC is the area under the concentration-time curve, and clearance is the clearance of antibody from serum. Half-life multiples are calculated as the half-life of the mutant antibody relative to that of the WT IgG1 or IgG2 parent within each study. [Figure 14b] As explained in Figure 14a. [Figure 15a] Correlation of half-life of IgG1 (Figure 15a) and IgG2 (Figure 15b) mutant antibodies with fold FcRn binding relative to WT IgG1 in mFcRn- / -hFcRn+ mice. Data on the y-axis are from Figure 14, and data on the x-axis are from Figures 9 and 10. Selected mutants are indicated, and mutant data from replicate experiments are circled. Figure 15c shows the correlation data for both IgG1 and IgG2, with the black and gray lines representing fits to the IgG1 and IgG2 data, respectively. [Figure 15b] As explained in Figure 15a. [Figure 15c] As explained in Figure 15a. [Figure 16a] (SEQ ID NOs: 30-35) Amino acid sequences of mutant and parent anti-TNF Fc immunoadhesins used in the present invention. [Figure 16b] As explained in Figure 16a. [Figure 17] Binding of anti-TNF immunoadhesins to TNF antigen as determined by Biacore. [Figure 18]Relative binding of mutant Fc immunoadhesins to human FcRn as determined by Biacore. The table shows the fold Ka* of each mutant relative to human WT (native) IgG1. n indicates the number of times each mutant was tested, and mean and SD indicate the mean and standard deviation, respectively, of each mutant across n binding experiments. Within each binding experiment, FcRn folds were calculated for all mutants relative to their respective parent IgG. [Figure 19] In vivo pharmacokinetics of parental and mutant Fc immunoadhesins in mFcRn- / -hFcRn+ mice. The graph plots serum concentrations of Fc fusions against time after a single intravenous administration. [Figure 20] Approximate PK parameters from in vivo PK studies of Fc fusions in mFcRn- / -hFcRn+ mice. Parameters are as described for Figure 14. The % increase in half-life is calculated as 100 x the half-life of the mutant Fc fusion divided by the parental half-life of WT IgG1 or IgG2. [Figure 21a] Relative binding of mutant IgG1 anti-VEGF antibodies to cynomolgus monkey and human FcRn as determined by Biacore. Figure 21a shows the data in tabular format. Figure legend is as in Figure 9, and data for binding to human FcRn is taken from Figure 9. Figure 21b shows a plot of the data. [Figure 21b] As explained in Figure 21a. [Figure 22] In vivo pharmacokinetics of WT and mutant antibodies in cynomolgus monkeys. The graph plots serum concentration of antibody against time after a single intravenous dose. [Figure 23] Approximate PK parameters from in vivo PK studies in cynomolgus monkeys with mutant and WT antibodies. Parameters are as described for Figure 14. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention discloses the generation of novel variants of Fc domains, including those found in antibodies, Fc fusions, and immunoadhesions, that have increased binding to the FcRn receptor. As described in this application, binding to FcRn results in longer serum retention in vivo.

[0024] To increase the retention of Fc proteins in vivo, there must be an increase in binding affinity at approximately pH 6 while maintaining low affinity at approximately pH 7.4. Although still experimental, it is believed that Fc regions have a longer in vivo half-life because binding to FcRn at pH 6 in endosomes sequesters the Fc (Ghetie and Ward, 1997 Immunol Today. 18(12):592-598, incorporated by reference in its entirety). The endosomal compartment then recycles the Fc to the cell surface. Once the compartment is open to the extracellular space, a higher pH (approximately 7.4) induces the release of Fc back into the blood. In mice, Dall'Acqua et al. indeed demonstrated that Fc mutations with increased FcRn binding at pH 6 and pH 7.4 had reduced serum concentrations and half-lives similar to those of wild-type Fc (Dall'Acqua et al. 2002, J. Immunol. 169:5171-5180, incorporated by reference in their entirety). The increased affinity of Fc for FcRn at pH 7.4 prevents Fc release back into the blood. Therefore, Fc mutations that increase the in vivo half-life of Fc ideally increase FcRn binding at low pH while allowing Fc release at high pH. The amino acid histidine changes its charge state at pHs ranging from 6.0 to 7.4. Therefore, it is not uncommon to find His residues at key positions within the Fc / FcRn complex (Figure 6).

[0025] An additional aspect of the present invention is an increase in FcRn binding over wild-type, particularly at low pH (approximately pH 6.0), to facilitate intraendosomal Fc / FcRn binding. Fc variants with altered FcRn binding and altered binding to other classes of Fc receptors are also disclosed, and differential binding to FcγR (sometimes referred to as Fc gamma R) has been shown to result in decreased efficiency, specifically increased binding to FcγRIIIb and decreased binding to FcγRIIb.

[0026] definition

[0027] In order that this application may be more fully understood, certain definitions are set forth below. Such definitions are intended to include grammatical equivalents.

[0028] As used in this application, ADCC "or" Antibody-dependent cell-mediated cytotoxicity " refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize bound antibody on a target cell, resulting in subsequent lysis of the target cell.

[0029] As used in this application, ADCP "or" Antibody-dependent cell-mediated phagocytosis " refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize bound antibody on target cells, resulting in phagocytosis of the target cells.

[0030] As used in this application, qualification " refers to amino acid substitutions, insertions and / or deletions in the polypeptide sequence, or alterations to the moiety chemically attached to the protein. For example, modifications can be altered carbohydrates or PEG structures attached to the protein. As used in this application, " Amino acid modifications " refers to amino acid substitutions, insertions, and / or deletions in a polypeptide sequence.

[0031] As used in this application, Amino acid substitutions "or" replacement" refers to the substitution of an amino acid at a particular position in a parent polypeptide with another amino acid. For example, the substitution E272Y refers to a mutant polypeptide, in this case an Fc variant, in which glutamic acid at position 272 has been substituted with tyrosine.

[0032] As used in this application, Amino acid insertion "or" Insert " refers to the addition of an amino acid sequence at a particular position within a parent polypeptide sequence. For example, -233E or ^233E refers to the insertion of a glutamic acid after position 233 and before position 234. Furthermore, -233ADE or ^233ADE refers to the insertion of AlaAspGlu after position 233 and before position 234.

[0033] As used in this application, Amino acid deletion "or" deletion " refers to the deletion of an amino acid sequence at a particular position within a parent polypeptide sequence. For example, E233- or E233# refers to the deletion of a glutamic acid at position 233. Furthermore, EDA233- or EDA233# refers to the deletion of the sequence GluAspAla starting at position 233.

[0034] As used in this application, Mutant proteins "or" Protein variants ",or" Mutants" means a protein that differs from the parent protein by at least one amino acid modification. Protein variant may refer to the protein itself, a composition comprising the protein, or the amino acid sequence encoding it. Preferably, the protein variant has at least one amino acid modification compared to the parent protein, for example, about 1 to about 70 amino acid modifications, and preferably about 1 to about 5 amino acid modifications compared to the parent protein. Preferably, the protein variant sequences of the present application have at least about 80% homology, most preferably at least about 90% homology, and even more preferably at least about 95% homology with the parent protein sequence. Mutant protein may refer to the mutant protein itself, a composition comprising the protein variant, or the DNA sequence encoding it. Thus, as used in this application, " Antibody variants "or" Mutant antibodies " refers to an antibody that differs from a parent antibody by at least one amino acid modification, and is used in this application as " IgG variants "or" Mutant IgG " refers to an antibody that differs from a parent IgG by at least one amino acid modification, and is used in this application as " Immunoglobulin variants "or" Mutant immunoglobulins " refers to an immunoglobulin sequence that differs from a parent immunoglobulin sequence by at least one amino acid modification. Fc variants "or" Mutant Fc" refers to a protein comprising a modification within the Fc domain. The Fc variants of the present invention are defined according to the amino acid modifications that comprise them. Thus, for example, N434S or 434S is an Fc variant having a substituted serine at position 434 relative to the parent Fc polypeptide, where the numbering is according to the EU index. Similarly, M428L / N434S defines an Fc variant having the substitutions M428L and N434S.A relative to the parent Fc polypeptide. The identity of the WT amino acids may not be specified, in which case the variant is referred to as 428L / 434S. Note that the order in which the substitutions are provided is arbitrary; i.e., for example, 428L / 434S is the same Fc variant as M428L / N434S. The numbering of all positions described herein is according to the EU index. The EU index or Kabat EU index or EU numbering scheme refers to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, incorporated herein by reference in its entirety). Modifications may be additions, deletions, or substitutions. Substitutions may include naturally occurring amino acids, non-naturally occurring amino acids. Variants may include non-naturally occurring amino acids. Examples include U.S. Pat. No. 6,586,207, International Publication Nos. WO 98 / 48032, WO 03 / 073238, U.S. Pat. No. 2004-0214988 A1, WO 05 / 35727 A2, WO 05 / 74524 A2, J.W. Hin et al., (2002), Journal of the American Chemical Society 124:9026-9027, J.W. Hin, & P.G. Schultz, (2002), ChemBioChem 11:1135-1137, J.W. Hin, et al., (2002), PICAS United States of America 99:11020-11024, and L. Wang, & P.G. Schultz, (2002), Chem. 1-10, all of which are incorporated by reference in their entireties.

[0035] As used in this application, protein" refers to at least two covalently attached amino acids, including proteins, polypeptides, oligopeptides, and peptides. Peptidyl groups may include naturally occurring amino acids and peptide bonds, or synthetic peptide mimetic structures, i.e., "analogs," such as peptoids (see Simon et al., PNAS USA 89(20):9367 (1992), incorporated by reference in its entirety). Amino acids may be either naturally occurring or non-naturally occurring, as would be understood by one of skill in the art. For example, homo-phenylalanine, citrulline, and norleucine are contemplated amino acids for purposes of the present invention, and amino acids in the D- and L- (R or S) configurations may be used. Variants of the invention can include modifications, including, but not limited to, the use of unnatural amino acids introduced using techniques developed by Schultz and coworkers, including, but not limited to, those described in Cropp & Shultz, 2004, Trends Genet. 20(12):625-30; Anderson et al., 2004, Proc Natl Acad Sci USA 101(2):7566-71; Zhang et al., 2003, 303(5656):371-3; and Chin et al., 2003, Science 301(5635):964-7, all of which are incorporated by reference in their entireties. Polypeptides can also include synthetic derivatization of one or more chains or termini, glycosylation, PEGylation, circular permutation, cyclization, linkers to other molecules, fusion to proteins or protein domains, and the addition of peptide tags or labels.

[0036] As used in this application, residue " refers to a position in a protein and its associated amino acid identity. For example, asparagine 297 (also called Asn297 or N297) is the residue at position 297 in the human antibody IgG1.

[0037] As used in this application, Fab "or" Fab area" refers to a polypeptide comprising VH, CH1, VL, and CL immunoglobulin domains. Fab can refer to this region in isolation or to this region in the context of a full-length antibody, antibody fragment, or Fab fusion protein. As used in this application, " Fv "or" Fv fragment "or" Fv area " refers to a polypeptide comprising the VL and VH domains of a single antibody.

[0038] As used in this application, IgG subclass modification " refers to an amino acid modification that converts a different amino acid from one IgG isotype to the corresponding amino acid of the aligned IgG isotype. For example, IgG1 has a tyrosine at EU position 296 and an IgG2a phenylalanine, so the F296Y substitution in IgG2 is considered an IgG subclass modification.

[0039] As used in this application, Non-naturally occurring modifications " refers to an amino acid modification that is not isomorphic. For example, the substitution 434S in IgG1, IgG2, IgG3, or IgG4 is considered a non-naturally occurring modification because none of the IgGs contain a serine at position 434.

[0040] As used in this application, amino acid " and " Amino acid identity " refers to one of the 20 naturally occurring amino acids or any non-natural analogue that may occur at a specifically defined position.

[0041] As used in this application, Effector function " refers to the biochemical events that result from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC.

[0042] As used in this application, IgG Fc ligand" refers to a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an IgG antibody to form an Fc / Fc ligand complex. Fc ligands include, but are not limited to, FcγR, FcγR, FcγR, FcRn, C1q, C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), a group of Fc receptors that are homologous to FcγR (Davis et al., 2002, Immunological Reviews 190:123-136, incorporated by reference in its entirety). Fc ligands may also include undiscovered molecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gamma receptors. As used in this application, " Fc ligand " refers to a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc / Fc ligand complex.

[0043] As used in this application, Fcγ receptor "," FcγR ",or" Fc Gamma R" refers to any member of the family of proteins that binds the IgG antibody Fc region and is encoded by the FcγR genes. In humans, this group includes, but is not limited to, FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, incorporated by reference in its entirety), and any undiscovered human FcγR or FcγR isoform or allotype. FcγRs may be from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any unidentified mouse FcγR or FcγR isoform or allotype.

[0044] As used in this application, FcRn "or" Neonatal Fc receptor" refers to a protein that binds the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. FcRn may be from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey. As is well known in the art, a functional FcRn protein comprises two polypeptides, often referred to as a heavy chain and a light chain. The light chain is beta-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise stated in this application, FcRn or FcRn protein refers to the complex of the FcRn heavy chain with beta-2-microglobulin. Sequences of FcRn species of particular interest, particularly the human species, are shown in the figures.

[0045] As used in this application, Parent Polypeptide " refers to an unmodified polypeptide that is subsequently modified to generate a variant. A parent polypeptide may be a naturally occurring polypeptide, or a variant or engineered version of a naturally occurring polypeptide. A parent polypeptide may refer to the polypeptide itself, a composition comprising the parent polypeptide, or the amino acid sequence that encodes it. Additionally, as used in this application, " Parent immunoglobulin " refers to an unmodified immunoglobulin polypeptide that is modified to produce a variant, and is used in this application as " Parent antibody " refers to an unmodified antibody that is modified to generate a mutant antibody. Note that, as described below, a "parent antibody" is a commercially known, recombinantly produced antibody.

[0046] As used in this application, position " refers to a location within the sequence of a protein. Positions may be numbered consecutively or according to an established format, for example, the EU index for antibody numbering.

[0047] As used in this application, target antigen " refers to a molecule that is specifically bound by the variable region of a given antibody. The target antigen may be a protein, carbohydrate, lipid, or other chemical.

[0048] As used in this application, target cell The term "cells" refers to cells that express a target antigen.

[0049] As used in this application, Variable region " refers to a region of an immunoglobulin that includes one or more Ig domains substantially encoded by either the Vκ, Vλ, and / or VH genes that make up the κ, λ, and heavy chain immunoglobulin loci, respectively.

[0050] As used in this application, Wild type or WT " refers to an amino acid or nucleotide sequence that occurs in the wild, including allelic variations. A WT protein has an amino acid or nucleotide sequence that has not been intentionally modified.

[0051] The present invention is directed to antibodies that exhibit increased binding to FcRn relative to wild-type antibodies. For example, in some instances, increased binding results in cellular recycling of the antibody, thereby increasing half-life. Antibodies that exhibit increased binding to FcRn and altered binding to other Fc receptors (e.g., FcγR) are also useful in the present invention. antibody

[0052] The present application is directed to antibodies containing amino acid modifications that modulate binding to FcRn. Of particular interest are antibodies that minimally contain an Fc region or functional variants thereof that exhibit reduced binding affinity to FcRn at low pH and do not exhibit substantial changes in binding at high pH.

[0053] Typically, a conventional antibody structural unit comprises a tetramer. Typically, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, which define antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. Therefore, as used herein, "isotype" refers to any of the immunoglobulin subclasses defined by the chemical and antigenic properties of the constant regions. The well-known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE.

[0054] The amino-terminal portion of each chain contains approximately 100-110 variable regions, or additional amino acids primarily responsible for antigen recognition. In the variable regions, three loops gather in each of the heavy and light chain V domains to form the antigen-binding site. Each of the loops is called a complementarity-determining region (hereafter referred to as "CDR"), which is the area of ​​most significant variation in amino acid sequence.

[0055] The carboxy-terminal portions of each chain define a constant region that is primarily responsible for effector function. Kabat et al. collected many primary sequences of heavy and light chain variable regions. Based on the degree of sequence conservation, they classified the individual primary sequences into CDRs and frameworks and compiled a list (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, E.A. Kabat et al., incorporated by reference in its entirety).

[0056] In the IgG subclass of immunoglobulins, there are various immunoglobulin domains within the heavy chain. Immunoglobulin (Ig) domain " refers to regions of immunoglobulins that have different tertiary structures. Of interest in this invention are the heavy chain domains, including the constant heavy (CH) domain and the hinge domain. In IgG antibodies, each of the IgG isotypes has three CH regions. Thus, the "CH" domains in IgG are as follows: "CH1" refers to positions 118-220 according to the EU index of Kabat; "CH2" refers to positions 237-340 according to the EU index of Kabat; and "CH3" refers to positions 341-447 according to the EU index of Kabat.

[0057] Another type of Ig domain in a heavy chain is the hinge region. As used herein, "hinge" or "hinge region," or "antibody hinge region," or "immunoglobulin hinge region," refers to a flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the CH1 domain of an IgG ends at EU position 220, and the CH2 domain of an IgG begins at EU position 237. Thus, in an IgG, the antibody hinge is defined herein to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1), numbering according to the EU index of Kabat. In some embodiments, for example, in the Fc region, the lower hinge is included, and "lower hinge" typically refers to positions 226 or 230.

[0058] Of interest in the present invention is the Fc region. As used herein, "Fc" or "Fc region" refers to a polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain and, in some instances, a portion of the hinge. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, plus the flexible hinge N-terminal to these domains. In IgA and IgM, Fc may also include the J chain. As illustrated in Figure 1, in IgG, Fc may also include immunoglobulin domains Cγ2 and Cγ3 (Cg2 and Cg3) and the lower hinge region between Cγ1 (Cg1) and Cγ2 (Cg2). Although the boundaries of the Fc region may vary, the heavy chain Fc region of human IgG is typically defined to include residues C226 or P230 at its carboxyl terminus, as numbered according to the EU index of Kabat. As described below, Fc can refer to this region in isolation or to this region in an Fc polypeptide. As used herein, "Fc polypeptide" refers to a polypeptide that includes all or part of an Fc region. Fc polypeptides include antibodies, Fc fusions, isolated Fcs, and Fc fragments.

[0059] In some embodiments, the antibody is full-length. As used herein, "full-length antibody" refers to the structure that constitutes the natural biological form of an antibody, including mutations and constant regions, including one or more modifications, as described herein.

[0060] Alternatively, antibodies may be of various structures, including, but not limited to, antibody fragments, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments of each.

[0061] antibody fragments

[0062] In one embodiment, the antibody is an antibody fragment. Of particular interest are antibodies that include an Fc region, an Fc fusion, and the constant region of the heavy chain (CH1-hinge-CH2-CH3), and also include a constant heavy region fusion.

[0063] Specific antibody fragments include (i) Fab fragments consisting of the VL, VH, CL, and CH1 domains; (ii) Fd fragments consisting of the VH and CH1 domains; (iii) Fv fragments consisting of the V1 and VH domains of a single antibody; (iv) dAb fragments consisting of a single mutation (Ward et al., 1989, Nature 341:544-546, incorporated by reference in its entirety); (v) isolated CDR regions; (vi) F(ab')2 fragments, bivalent fragments comprising two linked Fab fragments; and (vii) single-chain Fv molecules (scFv) in which the VH and VL domains are joined by a peptide linker which enables the two domains to combine to form the antibody binding site (Bird et al., 1988, Science 242:423-426; Huston et al., 1989, Science 242:423-426). (viii) bispecific single-chain Fvs (International Publication No. WO 03 / 11161, incorporated by reference in its entirety), and (ix) "diabodies" or "triabodies," multivalent or multispecific fragments constructed by gene fusion (Tomlinson et al., 2000, Methods Enzymol. 326:461-479; International Publication No. WO 94 / 13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448, all of which are incorporated by reference in their entirety). Antibody fragments may be modified. For example, the molecules may be stabilized by the incorporation of disulfide bridges linking the VH and VL domains (Reiter et al., 1996, Nature Biotech. 14:1239-1245, incorporated by reference in its entirety).

[0064] Chimeric and Humanized Antibodies

[0065] In some embodiments, the scaffold component may be a mixture of different species. In that case, if the protein is an antibody, such an antibody may be a chimeric and / or humanized antibody. Generally, both "chimeric antibody" and "humanized antibody" refer to antibodies that combine regions from more than one species. For example, traditionally, a "chimeric antibody" contains variable regions from a mouse (or, in some cases, a rat) and constant regions from a human. Generally, a "humanized antibody" refers to a non-human antibody with framework regions of variant domains that replace sequences found in human antibodies. Generally, in a humanized antibody, all of the antibody except for the CDRs is encoded by a polynucleotide of human origin and is identical to such an antibody except within its CDRs. Some or all of the CDRs encoded by nucleic acid from a non-human organism are grafted onto the beta-sheet framework of a human antibody variable region to form an antibody whose specificity is determined by the grafted CDRs. The generation of such antibodies is described, for example, in International Publication No. WO 92 / 11018; Jones, 1986, Nature 321:522-525; Verhoeyen et al., 1988, Science 239:1534-1536, all of which are incorporated by reference in their entireties. "Backmutation" of selected acceptor framework residues to the corresponding donor residues is often necessary to restore affinity lost in the original grafted construct (U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,762, 6,180,370, 5,859,205, 5,821,337, 6,054,297, 6,407,213, all of which are incorporated by reference in their entireties). Optimally, a humanized antibody also comprises at least a portion of an immunoglobulin constant region, typically a human immunoglobulin, and thus typically comprises a human Fc region. Humanized antibodies can also be generated using mice with genetically engineered immune systems (Roque et al., 2004, Biotechnol. Prog. 20:639-654, incorporated by reference in its entirety).Various techniques and methods for humanizing and reshaping non-human antibodies are well known in the art (see Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and references cited in this application, all of which are incorporated by reference in their entirety). Humanization methods are described in Jones et al., 1986, Nature 321:522-525, Riechmann et al., 1988, Nature 332:323-329, Verhoeyen et al., 1988, Science, 239:1534-1536, Queen et al., 1989, Proc Natl Acad Sci, USA. 86:10029-33, He et al.,1998, J.Immunol.160:1029-1035, Carter et al.,1992, Proc Natl Acad Sci USA 89:4285-9, Presta et al.,1997, Cancer Res.57(20):4593-9, Gorman et al. al.,1991,Proc.Natl.Acad.Sci.USA 88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8, which are incorporated by reference in their entireties. Other methods for humanizing or reducing the immunogenicity of non-human antibody variable regions may include resurfacing methods, for example, as described in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973, which are incorporated by reference. In one embodiment, the parent antibody is affinity matured, as is well known in the art. Structure-based methods may be used for humanization and affinity maturation, for example, as described in USSN 11 / 004,590.Selection-based methods may be used to humanize and / or affinity mature antibody variable regions, including, but not limited to, those described in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37):22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95:8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759. Other humanization methods involve grafting only a portion of the CDRs, and include, but are not limited to, those described in USSN 09 / 810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol. 169:3076-3084 (incorporated by reference in their entireties).

[0066] bispecific antibody

[0067] In one embodiment, the antibodies of the present invention are multispecific antibodies, and in particular bispecific antibodies, sometimes referred to as "diabodies." Some antibodies bind to two (or more) different antigens. Diabodies can be produced by a variety of methods well known in the art (e.g., hybridomas) (Holliger and Winter, 1993, Current Opinion Biotechnol. 4:446-449, incorporated by reference in its entirety).

[0068] Mini Body

[0069] In one embodiment, the antibody is a minibody, which is a minimized antibody-like protein comprising an scFv linked to a CH3 domain (Hu et al., 1996, Cancer Res. 56:3055-3061, incorporated by reference in its entirety). In some cases, the scFv can be linked to the Fc region and may include some or all of the hinge region.

[0070] antibody fusion

[0071] In one embodiment, the antibody of the present invention is an antibody fusion protein (sometimes referred to herein as an "antibody conjugate"). One type of antibody fusion comprises an Fc fusion, which links the Fc region to a conjugate partner. As used in this application, "antibody conjugate" refers to an antibody fusion protein. Fc fusion" refers to a protein in which one or more polypeptides are operably linked to an Fc region. In this application, Fc fusion is synonymous with the terms "immunoadhesin," "Ig fusion," "Ig chimera," and "receptor globulin" (sometimes preceded by a dash) as used in the art (Chamow et al., 1996, Trends Biotechnol. 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol. 9:195-200, both of which are incorporated by reference in their entireties). Generally, an Fc fusion combines the Fc region of an immunoglobulin with a fusion partner, which may be any protein or small molecule. Virtually any protein or small molecule may be linked to the Fc to create an Fc fusion. Protein fusion partners include, but are not limited to, the variable region of an antibody, the target-binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or another protein or protein domain. The small molecule fusion partner may include any therapeutic agent that directs the Fc fusion to a therapeutic target. Such a target may be any molecule, preferably an extracellular receptor responsible for a disease. Thus, the IgG variants can bind to one or more fusion partners. In an alternative embodiment, the IgG variants are conjugated or operably linked to another therapeutic compound. The therapeutic compound may be a cytotoxic agent, a chemotherapeutic agent, a toxin, a radioisotope, a cytokine, or other therapeutically active agent. The IgG may also be conjugated to one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol.

[0072] In addition to Fc fusions, antibody fusions include fusions of the heavy chain constant region with one or more fusion partners (again, including any antibody variable region); other antibody fusions are substantially or entirely full-length antibodies with the fusion partner. In one embodiment, the role of the fusion partner is to mediate target binding and, therefore, is (and may be) functionally similar to the variable region of an antibody. Virtually any protein or small molecule may be bound to Fc to create an Fc fusion (or antibody fusion). Protein fusion partners include, but are not limited to, the target-binding region of a receptor, adhesion molecule, ligand, enzyme, cytokine, or chemokine; some other protein or protein domains. Small molecule fusion partners may include any therapeutic agent that directs the Fc fusion to a therapeutic target. Such a target may be any molecule, preferably an extracellular receptor responsible for a disease.

[0073] The conjugate partner may be of proteinaceous or non-proteinaceous nature, the latter typically being generated using functional groups on the antibody and the conjugate partner. For example, linkers are well known in the art, e.g., homo-hetero-bifunctional linkers are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference).

[0074] Suitable conjugates include, but are not limited to, drugs and cytotoxic agents, including but not limited to, the labels described below, cytotoxic agents (e.g., chemotherapeutic agents), or toxins or active fragments of such toxins. Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, and the like. Cytotoxic agents also include radioactive chemistries produced by conjugating a radioisotope to an antibody or by binding a radionuclide to a chelator that is covalently attached to an antibody. Additional embodiments utilize calicheamicin, auristatin, geldanamycin, maytansine, and duocarmycin, and analogs, the latter of which are described in U.S. Patent Publication No. 2003 / 0050331A1, incorporated by reference in its entirety.

[0075] Covalent modification of antibodies

[0076] Covalent modifications of antibodies are included within the scope of the present invention and are generally, but not always, carried out post-translationally. For example, some types of covalent modifications of antibodies are introduced into the molecule by reacting specific amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or N- or C-terminal residues.

[0077] Cysteinyl residues are most commonly reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues may also be derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetylphosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuric benzoate, 2-chloromercuric-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0078] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5 to 7.0 because this agent is relatively specific for the histidyl side chain. Parabromophenacyl bromide is also useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

[0079] Lysinyl and amino-terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residue. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborate, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactions with glyoxylic acid.

[0080] Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues must be performed under alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with lysine groups as well as the arginine epsilon-amino group.

[0081] The specific modification of tyrosyl residues has been accomplished by reaction with aromatic diazonium compounds or tetranitromethane, with particular interest in introducing spectroscopic labels into tyrosyl residues. N-acetylimidizole and tetranitromethane are typically used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are also labeled with 125I or 131I to prepare labeled proteins for use in radioimmunoassay, preferably the chloramine T method described above.

[0082] Carboxyl side groups (aspartyl or glutaminyl) are selectively modified by reaction with carbodiimides (R'-N=C=N--R'), where R and R' are optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutaminyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0083] Derivatization with bifunctional agents is useful for crosslinking antibodies to water-insoluble support matrices or surfaces for use in a variety of methods, including those described below. Commonly used crosslinkers include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters (e.g., esters with 4-azidosalicylic acid, homobifunctional imidoesters including disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. methyl-3-[(p-azidophenyl)dithio] Derivatizing agents such as propioimidates produce photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and reactive substrates, as described in U.S. Patent Nos. 3,969,287, 3,691,016, 4,195,128, 4,247,642, 4,229,537, and 4,330,440, which are incorporated by reference in their entireties, are used for protein immobilization.

[0084] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamine and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues is within the scope of the present invention. Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (Tecreighton, Protein: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86

[1983] , incorporated by reference in its entirety), acetylation of the N-terminal amine, and amidation of either the C-terminal carboxyl group.

[0085] Glycosylation

[0086] Another type of covalent modification is glycosylation. In another embodiment, the IgG variants disclosed herein can be modified to contain one or more engineered glycans. As used herein, "engineered glycan" refers to a carbohydrate composition that is covalently attached to an IgG, which carbohydrate composition is chemically different from that of the parent IgG. Engineered glycans can be useful for a variety of purposes, including, but not limited to, enhancing or reducing effector function. Engineered glycans may be generated by a variety of methods well known in the art (Umana et al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473, US 6,602,684, USSN 10 / 277,370, USSN 10 / 113,929, PCT WO 00 / 61739 A1, PCT WO 01 / 29246 A1, PCT WO 02 / 31140 A1, PCT WO 02 / 30954 A1, all of which are incorporated by reference in their entireties) (Potelligent® technology [Biowa, Inc., Princeton, NJ], GlycoMAb® glycosylation engineering technology [Glycart Biotechnology AG, Zurich, Switzerland]. Many of these techniques are based on controlling the level of fucosylation and / or splitting oligosaccharides covalently attached to the Fc region, for example, by expressing or otherwise manipulating IgG in various organisms or cell lines (e.g., Lee-13 CHO cells or rat hybridoma YB2 / 0 cells), by inhibiting enzymes involved in the glycosylation pathway (e.g., FUT8 [α1,6-fucosyltransferase] and / or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by modifying the carbohydrates after IgG has been expressed.Typically, an altered glycosylation refers to a different carbohydrate or oligosaccharide, and thus an IgG variant, e.g., an antibody or Fc fusion, can comprise an altered glycosylation. Alternatively, an altered glycosylation can refer to an IgG variant comprising a different carbohydrate or oligosaccharide. As is well known in the art, glycosylation patterns can vary depending on both the protein sequence (e.g., the presence or absence of specific glycosylated amino acid residues, as described below), or the host cell or organism in which the protein is produced. Specific expression systems are described below.

[0087] Typically, glycosylation of polypeptides is either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tri-peptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tri-peptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of the sugars N-acetylgalactosamine, galactose, or xylose to one hydroxyamino acid, usually serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0088] Addition of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence to include one or more of the above-described tri-peptide sequences (N-linked glycosylation sites). Alterations may also be made by adding or substituting one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites). To facilitate this, antibody amino acid sequences are suitably altered through alterations at the DNA level, particularly by altering the DNA encoding the target polypeptide with bases that are preselected to generate codons and convert them into the desired amino acids.

[0089] Another method for increasing the number of carbohydrate moieties on an antibody is by chemical or enzymatic conjugation to the protein. These approaches are advantageous because they do not require production of the protein in a host cell capable of N- and O-linked glycosylation. Depending on the mode of attachment used, saccharides may be attached to (a) adenine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those of cysteine, (d) free hydroxyl groups, such as those of serine, threonine, or hydroxyproline, (e) aromatic residues, such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in International Publication No. WO 87 / 05330 and in Alpin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306, both of which are incorporated by reference in their entireties.

[0090] Removal of carbohydrate moieties present on the starting antibody may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact. Chemical deglycosylation is described in Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52, and Edge et al., 1981, Anal. Biochem. 118:131, both of which are incorporated by reference in their entireties. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved using a variety of endo- and exo-glucosidases, as described in Thokakura et al., 1987, Meth. Enzymol. 138:350, which are incorporated by reference in their entireties. Glycosylation at potential glycosylation sites can be prevented by the use of the compound tunicamycin, as described in Duskin et al., 1982, J. Biol. Chem. 257:3105, which is incorporated by reference in its entirety. Tunicamycin inhibits the formation of protein-N-glycosidic linkages.

[0091] Another type of covalent modification of antibodies includes various polymers of nonproteinaceous nature, including, but not limited to, various polyols (such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene), in a manner described, for example, in Nektar Therapeutics' 2005-2006 PEG Catalog (available on the Nektar website), U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337 (all of which are incorporated by reference in their entireties). Also, as is well known in the art, amino acid substitutions may be made at various positions within the antibody to facilitate the addition of polymers such as PEG. See, for example, U.S. Publication No. 2005 / 0114037A1, incorporated by reference in its entirety.

[0092] Labeled antibody

[0093] In some embodiments, covalent modifications of the antibodies of the present invention include the addition of one or more labels. In some cases, these are considered antibody fusions. The term "labeling group" refers to any detectable label. In some embodiments, the labeling group is attached to the antibody via a spacer arm of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are well known in the art and may be used in practicing the present invention.

[0094] Generally, labels are divided into various classes depending on the assay in which they are detected: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox-active moieties; d) optical dyes; enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylation groups; and f) predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metals, binding domains, epitope tags, etc.). In some embodiments, the labeling group is attached to the antibody via a spacer arm of various lengths to reduce potential steric hindrance. A variety of methods for labeling proteins are well known in the art and may be used in practicing the present invention.

[0095] Specific labels include optical dyes, including but not limited to chromophores, luminophores, and fluorophores, the latter being specific in many instances. Fluorophores may be either "small molecule" fluorescers or fluorescers of a protein nature.

[0096] "Fluorescent label" means any molecule that may be detected via its unique fluorescent properties. Suitable fluorescent labels include fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue J, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy5, Cy5.5, LC Red 705, Oregon Green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-Phycoerythrin (PE) (Molecular Suitable optical dyes include, but are not limited to, fluorescein ...

[0097] Suitable fluorescent labels of protein nature also include green fluorescent protein (GFP) from Renilla, Sea Pansy, or Aequorea species (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., Genbank accession number U55762), blue fluorescent protein (BEP, Quantum Biotechnologies, Inc., 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al. al., 1993, J. Immunol. 150:5408-5417), β-galactosidase (Nolan et al. al., 1988, Proc. Natl. Acad. Sci. USA 85:2603-2607), and Renilla reniformis (International Publication Nos. WO 92 / 15673, WO 95 / 07463, WO 98 / 14605, WO 98 / 26277, WO 99 / 49019, U.S. Pat. Nos. 5,292,658, 5,418,155, 5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995, 5,925,558). All of the above-mentioned references cited in this section are expressly incorporated by reference.

[0098] IgG variants

[0099] In one embodiment, the present invention provides mutant IgG proteins. At a minimum, IgG mutants comprise antibody fragments containing the CH2-CH3 regions of the heavy chain. Suitable IgG mutants also include the Fc domain (e.g., including the lower hinge region), as well as IgG mutants containing the heavy chain constant region (CH1-hinge-CH2-CH3) that are also useful in the present invention, all of which can be fused to a fusion partner.

[0100] IgG variants contain one or more amino acid modifications relative to the parent IgG polypeptide, and in some cases, relative to the wild-type IgG. The IgG variants can have one or more optimized properties. The IgG variants differ in amino acid sequence from their parent IgG due to at least one amino acid modification. Thus, the IgG variants have at least one amino acid modification compared to the parent IgG. Alternatively, the IgG variants may have one or more amino acid modifications compared to the parent IgG, for example, about 1 to 50 amino acid modifications, preferably about 1 to 10 amino acid modifications, and most preferably about 1 to about 5 amino acid modifications compared to the parent IgG.

[0101] Thus, the sequence of the IgG variant and the sequence of the parent Fc polypeptide are substantially homologous. For example, the variant sequence of the mutated IgG of the present application has about 80% homology with the variant sequence of the parent IgG, preferably at least about 90%, and most preferably at least about 95% homology. Modifications may be made genetically, using molecular biology, or enzymatically or chemically.

[0102] Antibody target antigen

[0103] Virtually any antigen may be targeted by the IgG variants, including, but not limited to, proteins, subunits, domains, motifs, and / or epitopes belonging to the following list of target antigens (including both soluble factors such as cytokines and membrane-bound factors such as transmembrane receptors): 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, AlK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM , BLK, BMP, BMP-2, BMP-2a, BMP-3 osteogenin, BMP-4BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7(OP-1), BMP- 8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP , b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin CDPPI, cathepsin D, cathepsin E, cathepsin H,Cathepsin L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Botulinum toxin, Verotoxin, Ckb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cytokeratin tumor-associated antigen, DAN, DCCDcR3, DC-SIGN, Degradation promoting factor, Des(1-3)-IGF-I (Brain IGF-1), Dhh, Digoxin, DNAM-1, D Nase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIc, factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF 3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-α1, GFR-α2, GFR-α3, GITR, glucagon, Glut 4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone-releasing factor, hapatene (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMVgH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), HepB gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein,IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-α, INF-β, INF-γ, inhibin, iNOS, insulin A-chain, insulin B-chain, insulin-like growth factor 1, integrin α2, integrin α3, integrin α4, integrin α4 / β1, integrin Integrin α4 / β7, integrin α5 (αV), integrin α5 / β1, integrin α5 / β3, integrin α6, integrin β1, integrin β2, interferon γ, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1 , latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin β receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metal, LOPROTEASES, MGDF receptor, MGMT, MHC ( HLA-DR), MIF, MIGMIP, MIP-1-α, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, nutlin,Nerve growth factor (NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prolactin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A-chain, relaxin B-chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptor (e.g., T-cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β pan-specificity, TGF-βRI (ALK-5), TGF-βRII, TGF-βRIIb, TGF-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRESS2, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD)TNFRSF11A(RANK ODF R、TRANCE R)、TNFRSF11B(OPG OCIF、TR1)、TNFRSF12(TWEAK R FN14)、TNFRSF13B(TACI)、TNFRSF13D(BAFF R)、TNFRSF14(HVEM、 ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR AITR)、TNFRSF19(TROY TAJ、TRADE)、TNFRSF19L(RELT)、TNFRSF1A(TNF). RI CD120a, p55–60, TNFRSF1B(TNF RII CD120b, p75–80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1). R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB). CD137、ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2 TNFRH2), TNFRSF23(DcTRAIL R1TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL). Apo-2 polymer TL2) TNFSF11(TRANCE / RANK polymer ODF) TNFSF12(TWEAK). Apo-3リンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LIGHT, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM LTg, TNFSF15(TL1A / VEGI), TNFSF18 (GITR AITR ligand TL6), TNFSF1A(TNF-linker, DIF, TNFSF2), TNFSF1B(TNF-b LTa, TNFSF1), TNFSF3(LTb).TNFSF, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transduction receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expression Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA, VLA-1, VLA-4, VLR integrins, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, and hormone and growth factor receptors.

[0104] Those skilled in the art will understand that the targets in the foregoing list refer not only to specific proteins and biomolecules but also to biochemical pathways or pathways that include them. For example, reference to CTLA-4 as a target antigen means that the ligands and receptors that make up the T cell costimulatory pathway (including CTLA-4, B7-1, B7-2, CD28, and any other yet-to-be-discovered ligands or receptors that bind these proteins) are also targets. Thus, as used herein, target refers not only to a specific biomolecule but also to a set of proteins that interact with the target and members of the biochemical pathway to which the target belongs. Those skilled in the art will further understand that any of the aforementioned target antigens, ligands, or receptors that bind them, or other members of their corresponding biochemical pathways, may be operably linked to the Fc variants of the present invention to generate Fc fusions. Thus, for example, Fc variants targeting EGFR can be constructed by operably linking an EGFR-binding Fc variant, discovered or yet-to-be discovered, to EGF, TGF-β, or any other ligand. Alternatively, the Fc variants of the invention, discovered or yet to be discovered, may be operably linked to EGFR to generate Fc fusions that bind EGF, TGF-β, or other ligands that bind EGFR. Thus, virtually any polypeptide, whether a ligand, receptor, or other protein or protein domain (including, but not limited to, the aforementioned targets and proteins, including their corresponding biochemical pathways), may be operably linked to an Fc variant of the invention to develop an Fc fusion.

[0105] The selection of an appropriate antigen depends on the desired application. For anti-cancer therapy, it is desirable to have a target whose expression is restricted to cancer cells. Some targets that have proven particularly suitable for antibody therapy have signaling functions. Other therapeutic antibodies work by inhibiting receptor signaling, inhibiting the binding between a receptor and its cognate ligand. Another mechanism of action of therapeutic antibodies is receptor downregulation. Other antibodies do not function by signaling through their target antigen. In some cases, antibodies directed to infectious disease drugs are used.

[0106] In one embodiment, the Fc variants of the invention are incorporated into antibodies against cytokines. Alternatively, the Fc variants are fused or conjugated to cytokines. As used herein, "cytokine" is a general term referring to a protein released from one cell population and acting on another cell as an intercellular mediator. For example, cytokines can be fused to antibodies to provide a range of desirable properties, as described in Penichet et al., 2001, J Immunol Methods 248:91-101 (expressly incorporated by reference). Examples of such cytokines include lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones (such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones (such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH), hepatocyte growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-alpha and -beta, Müllerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrins, thrombopoietin (TPO), nerve growth factors such as NGF-beta, platelet growth factor, transforming growth factors (TGs) such as TGF-alpha and TGF-beta. F), insulin-like growth factor-I and -II, erythropoietin (EPO), osteoinductive factors, interferons (such as interferon-alpha, beta, and gamma), colony-stimulating factors (CSF) (such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF)), interleukins (IL) (such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15), tumor necrosis factors such as TNF-alpha or TNF-beta, C5a, and other polypeptide factors including LIF and kit ligand (KL).The term cytokine, as used herein, includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0107] Cytokines and soluble targets, such as members of the TNF superfamily, are suitable targets for use with the variants of the present invention. For example, anti-VEGF, anti-CTLA-4, and anti-TNF antibodies, or fragments thereof, are particularly suitable for use with Fc variants that increase FcRn binding. Treatments for these targets often involve the treatment of autoimmune diseases, requiring multiple injections over a long period of time. Therefore, the longer serum half-life and less frequent treatments afforded by the variants of the present invention are particularly advantageous.

[0108] Many antibodies and Fc fusions approved for use in clinical trials or development may benefit from the Fc variants of the present invention. In this application, these antibodies and Fc fusions are referred to as "clinical products and candidates." Thus, in preferred embodiments, the Fc polypeptides of the present invention may find use in a range of clinical products and candidates. For example, many antibodies targeting CD20 may benefit from the Fc polypeptides of the present invention. For example, the Fc polypeptides of the present invention may be used in combination with other antibodies against rituximab (Rituxan®, IDEC / Genentech / Roche) (see, e.g., U.S. Pat. No. 5,736,137), a chimeric anti-CD20 antibody approved for treating non-Hodgkin's lymphoma, HuMax-CD20, an anti-CD20 antibody currently being developed by Genmab, the anti-CD20 antibody described in U.S. Pat. No. 5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel), and PRO70769 ("Immunoglobulin Variants and Uses").The Fc polypeptides of the present invention may find use in antibodies that are substantially similar to those disclosed in PCT Application No. PCT / US2003 / 040426, entitled "Thereof." Many antibodies that target members of the epidermal growth factor receptor family, including EGFR (ErbB-1), Her2 / neu (ErbB-2), Her3 / neu (ErbB-3), and Her4 / neu (ErbB-4), may benefit from the Fc polypeptides of the present invention. For example, the Fc polypeptides of the present invention may be used in combination with antibodies such as trastuzumab (Herceptin®, Genentech) (see, e.g., U.S. Pat. No. 5,677,171), a humanized anti-Her2 / neu antibody approved for treating breast cancer, pertuzumab (rhuMab-2C4, Omnitarg®), currently being developed by Genentech, the anti-Her2 antibody described in U.S. Pat. No. 4,753,894, cetuximab (Erbitux®, I), and the like. mclone) (US Pat. No. 4,943,533, PCT No. WO96 / 40210), chimeric anti-EGFR antibodies in clinical trials for various cancers, ABX-EGF (US Pat. No. 6,235,883) currently being developed by Abgenix-Immunex-Amgen, HuMax-EGFr (US Pat. No. 10 / 172,317) currently being developed by Genmab, 425, EMD55900, EMD62000, and EMD72000 (Merck KGaA) (US No. 5,558,864, Murthy et al., 1987, Arch Biochem Biophys. 252(2):549-60, Rodeck et al., 1987, J Cell Biochem. 35(4): 315-20, Kettleborough et al., 1991, Protein Eng.4(7):773-83), ICR62(Institute of Cancer Research)(PCT No. WO95 / 20045, Modjtahedi et al.,1993, J.Cell Biophys.1993,22(1-3):129-46, Modjtahedi et al.,1993,Br J Cancer.1993,67(2):247-53, Modjtahedi et al.,1996,Br J Cancer,73(2):228-35, Modjtahedi et al.al., 2003, Int J Cancer, 105(2):273-80), TheraCIM hR3 (YM Biosciences, Canada and Centro de Immunologia Molecular, Cuba (US No. 5,891,996, US No. 6,506,883, Mateo et al. al., 1997, Immunotechnology, 3(1): 71-81), mAb-806 (Ludwig Institue for Cancer Research, Memorial Sloan-Kettering) (Jungbluth et al., 2003, Proc Natl Acad Sci USA. 100(2): 639-44), KSB-102 (KS Biomedix), MR1-1 (IVAX, National Cancer In another preferred embodiment, the Fc polypeptides of the present invention may find use in antibodies substantially similar to Alemtuzumab (Campath®, Millennium), a humanized monoclonal antibody currently approved for the treatment of B-cell chronic lymphocytic leukemia. The Fc polypeptides of the present invention may find use in various antibodies or Fc fusions substantially similar to other clinical products and candidates, including muromonab-CD3 (Orthoclone OKT3®), an anti-CD3 antibody developed by Ortho Biotech / Johnson & Johnson, ibritumomab tiuxetan (Zevalin®), IDEC / ScheringGemtuzumab ozogamicin (Mylotarg®), an anti-CD20 antibody developed by AG; Alefacept (Amevive®), an anti-CD33 (p67 protein) antibody developed by Celltech / Wyeth; Anti-LFA-3 Fc fusion developed by Biogen; Abiciximab (ReoPro®), developed by Centocor / Lilly; Basiliximab (Simulect®), developed by Novartis; MediImmune Palivizumab (Synagis®), infliximab (Remicade®), an anti-TNF alpha antibody developed by Centocor, adalimumab (Humira®), an anti-TNF alpha antibody developed by Abbott, Humicade®, an anti-TNF alpha antibody developed by Celltech, etanercept (Enbrel®), an anti-TNF alpha Fc fusion developed by Immunex / Amgen, ABX- CBL, an anti-CD147 antibody being developed by Abgenix; ABX-IL8, an anti-IL8 antibody being developed by Abgenix; ABX-MA1, an anti-MUC18 antibody being developed by Abgenix; pemtumomab (R1549, 90Y-muHMFG1), an anti-MUC1 antibody being developed by Antisoma; Therex (R1550), an anti-MUC1 antibody being developed by Antisoma; AngioMab (AS1405), an anti-MUC1 antibody being developed by Antisoma; HuBC-1 being developed by a, thioplatin (AS1407) being developed by Antisoma, Antegren® (natalizumab), anti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7 antibodies being developed by Biogen, VLA-1 mAb, an anti-VLA-1 integrin antibody being developed by Biogen, LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibody being developed by Biogen, CAT-152, an anti-TGF-β2 antibody being developed by Cambridge Antibody Technology, J695, CambridgeCAT-192, an anti-IL-12 antibody being developed by Antibody Technology and Abbott; CAT-213, an anti-TGFβ1 antibody being developed by Cambridge Antibody Technology and Genzyme; CAT-213, an anti-eotaxin 1 antibody being developed by Cambridge Antibody Technology; Lympho Stat-B®, an anti-Blys antibody being developed by Cambridge Antibody Technology and Human Genome Sciences Inc.; TRAIL-R1 mAb, Cambridge Antibody Technology and Human Genome Sciences anti-TRAIL-R1 antibody being developed by Inc., Avastin® (bevacizumab, rhuMAb-VEGF), anti-VEGF antibody being developed by Genentech, anti-HER receptor group antibody being developed by Genentech, anti-tissue factor (ATF), anti-tissue factor antibody being developed by Genentech, Xolair® (omalizumab), anti-IgE antibody being developed by Genentech, Raptiva® (efalizumab), anti-CD11a antibody being developed by Genentech and Xoma, MLN-02 antibody (formerly LDP-02) being developed by Genentech and Millennium Pharmaceuticals, HuMaxCD4, anti-CD4 antibody being developed by Genmab, HuMax-IL15, anti-IL15 antibody being developed by Genmab and Amgen, HuMax-inflammation, HuMax-cancer, Genmab and Medarex, and Oxford an anti-heparanase I antibody being developed by GcoSciences, HuMax-Lymphoma being developed by Genmab and Amgen, HuMax-TAC ​​being developed by Genmab, IDEC-131, an anti-CD40L antibody being developed by IDEC Pharmaceuticals, IDEC-151 (clenoliximab), IDECan anti-CD4 antibody being developed by IDEC Pharmaceuticals, IDEC-114, an anti-CD80 antibody being developed by IDEC Pharmaceuticals, IDEC-152, an anti-CD23 antibody being developed by IDEC Pharmaceuticals, an anti-macrophage migration factor (MIF) antibody being developed by IDEC Pharmaceuticals, BEC2, an anti-idiotypic antibody being developed by Imclone, IMC-1C11, an anti-KDR antibody being developed by Imclone, DC101, an anti-flk-1 antibody being developed by Imclone, an anti-VE-cadherin antibody being developed by Imclone, CEA-Cide® (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibody being developed by Immunomedics, LymphoCide® (epratuzumab), an anti-CD22 antibody being developed by Immunomedics, Immu AFP-Cide being developed by Immunomedics, MyelomaCide being developed by Immunomedics, LkoCide being developed by Immunomedics, ProstaCide being developed by Immunomedics, MDX-010, an anti-CTLA4 antibody being developed by Medarex, MDX-060, an anti-CD30 antibody being developed by Medarex, MDX-070 being developed by Medarex, MDX-018 being developed by Medarex, Osidem® (IDM-1), and an anti-Her2 antibody being developed by Medarex and Immuno-Designed Molecules, HuMax®-CD4, an anti-CD4 antibody being developed by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibody being developed by Medarex and Genmab, CNTO148, an anti-T antibody being developed by Medarex and Centocor / J&J All of the references cited above in this section, including but not limited to, NFα antibodies, CNTO1275, an anti-cytokine antibody being developed by Centocor / J&J, MOR101 and MOR102, an anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibody being developed by MorphoSys, an anti-fibroblast growth factor receptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion® (visilizumab), an anti-CD3 antibody being developed by Protein Design Labs, HuZAF®, an anti-gamma interferon antibody being developed by Protein Design Labs, an anti-alpha5beta1 integrin being developed by Protein Design Labs, an anti-IL-12 antibody being developed by Protein Design Labs, ING-1, an anti-Ep-CAM antibody being developed by Xoma, and MLN01, an anti-beta2 integrin antibody being developed by Xoma, are hereby expressly incorporated by reference.

[0109] The Fc polypeptides of the invention may be incorporated into the aforementioned clinical candidates and products, or antibodies and Fc fusions that are substantially similar thereto. The Fc polypeptides of the invention may also be incorporated into humanized, affinity matured, engineered, or otherwise modified versions of the aforementioned clinical candidates and products.

[0110] In one embodiment, the Fc polypeptides of the invention are used to treat autoimmune, inflammatory, or transplant indications. Target antigens and clinical products and candidates associated with such diseases include anti-α4β7 integrin antibodies such as LDP-02, anti-β2 integrin antibodies such as LDP-01, anti-complement (C5) antibodies such as 5G1.1, anti-CD2 antibodies such as BTI-322, anti-CD3 antibodies such as MEDI-507 and OKT3, anti-CD4 antibodies such as SMART anti-CD3, IDEC-151, MDX-CD4, OKT4A, anti-CD11a antibodies, anti-CD14 antibodies such as IC14, anti-CD18 antibodies, anti-CD23 antibodies such as IDEC152, anti-CD25 antibodies such as Zenapax, anti-CD40L antibodies such as 5c8, anti-CD64 antibodies such as Antova, IDEC-131, and MDX-33, anti-CD80 antibodies such as IDEC-114, and anti-CD2 antibodies such as ABX-CBL. These antibodies include, but are not limited to, anti-CD147 antibodies, anti-E-selectin antibodies such as CDP850, anti-gpIIb / IIa antibodies such as ReoPro / Abcixima, anti-ICAM-3 antibodies such as ICM3, anti-ICE antibodies such as VX-740, anti-FcR1 antibodies such as MDX-33, anti-IgE antibodies such as rhuMab-E25, anti-IL-4 antibodies such as SB-240683, anti-IL-5 antibodies such as SB-240563, anti-IL-8 antibodies such as SCH55700 and ABX-IL8, anti-interferon gamma antibodies, anti-TNF (TNF, TNFa, TNFα, TNF-alpha) antibodies such as CDP571, and anti-VLA-4 antibodies such as CDP870, D2E7, infliximab, MAK-195F, and Antegren.

[0111] Fc variants of the invention, such as those with increased binding to FcRn, may be used in TNF inhibitor molecules to provide improved properties. Useful TNF inhibitor molecules include any molecule that inhibits the action of TNF-α in a mammal. Suitable examples include the Fc fusion Enbrel® (etanercept) and the antibodies Humira® (adalimumab) and Remicade® (infliximab). Monoclonal antibodies (such as Remicade and Humira) engineered using Fc variants of the invention to increase FcFn ligation may lead to greater efficacy through increased half-life.

[0112] In some embodiments, antibodies against infectious diseases are used. Antibodies against eukaryotic cells include antibodies targeting yeast cells, including, but not limited to, Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, as well as Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, Plasmodium falciparum, and Yarrowia lipolytica.

[0113] Antibodies against additional fungal cells are also useful, including target antigens associated with Candida strains (including Candida glabrata, Candida albicans, C. krusei, C. lusitaniae, and C. maltosa), as well as species of Aspergillus, Cryptococcus, Histoplasma, Coccidioides, Blastomyces, and Penicillium, among others. Antibodies against target antigens associated with protozoa include, but are not limited to, antibodies associated with Trypanosoma brucei, Leishmania species including Leishmania donovani, Plasmodium spp., Pneumocystis carinii, Cryptosporidium parvum, Giardia lamblia, Entamoeba histolytica, and Cyclospora canetanensis. Antibodies against prokaryotic antigens are also useful, including those against bacteria (Bacillus anthracis, Vibrio (e.g., Vibrio cholerae), Escherichia (e.g., enterotoxigenic E. coli), Shigella (e.g., S. dysenteriae), Salmonella (e.g., Salmonella typhi), Mycobacterium (e.g., Mycobacterium tuberculosis, Mycobacterium leprae), Clostridium (e.g., Clostridium botulinum, Clostridium tetani, C. difficile, Clostridium perfringens), Corynebacterium (e.g., Corynebacterium diphtheriae), Streptococcus (e.g., Streptococcus pyogenes, Streptococcus pneumoniae), Staphylococcus (e.g., Staphylococcus aureus), Haemophilus (e.g., Haemophilus influenzae), Neisseria (e.g., Neisseria meningitidis, Neisseria gonorrhoeae), Yersinia (e.g., Y. lamblia), The antibodies include antibodies against suitable bacteria, such as pathogenic and non-pathogenic prokaryotes, including, but not limited to, Yersinia pestis, Pseudomonas (e.g., Pseudomonas aeruginosa, P. putida), Chlamydia (e.g., Chlamydia trachomatis), Bordetella spp. (e.g., Bordetella pertussis), Treponema spp. (e.g., Treponema pallidum), Bacillus anthracis, Yersinia pestis, Brucella spp., Francisella tularensis, Burkholderia mallei, Burkholderia pseudomallei, Burkholderia mallei, Burkholderia pseudomallei, Clostridium botulinum, Salmonella spp., SEB cholera toxin B, Escherichia coli O157:H7, Listeria monocytogenes spp., Trichosporon beigelii, Rhodotorula spp., Hansenula anomala, Enterobacter spp., Klebsiella spp., Listeria monocytogenes spp., Mycoplasma spp., etc.

[0114] In some embodiments, the antibodies are directed against viral infections, including, but not limited to, orthomyxoviruses (e.g., influenza virus), paramyxoviruses (e.g., respiratory syncytial virus, mumps virus, measles virus), adenovirus, rhinovirus, coronavirus, reovirus, togavirus (e.g., rubella virus), parvovirus, poxvirus (e.g., smallpox virus, vaccinia virus), enterovirus (e.g., poliovirus, coxsackievirus), herpesviruses (including types A, B, and C), herpesviruses (e.g., herpes simplex virus, varicella-zoster virus, cytomegalovirus, Esptyne-Barr virus), rotavirus, Norwalk virus, hantavirus, arenaviruses, rhabdoviruses (e.g., rabies virus), retroviruses (including HIV, HTLV-I and II), papovaviruses (e.g., papillomavirus), polyomaviruses, and picornaviruses.

[0115] Optimized IgG variant properties

[0116] The present application also provides IgG variants that are optimized for various therapeutically relevant properties. In the present application, IgG variants that are engineered or predicted to display one or more optimized properties are referred to as " Optimized IgG variants" refers to an antibody that exhibits increased binding affinity to FcRn at low pH (e.g., pH 6.0), such as the pH associated with endosomes, while maintaining decreased affinity at high pH (e.g., 7.4), to allow for increased uptake into endosomes with a normal release rate. Similarly, these antibodies with modulated FcRn binding may optionally have other desirable properties, such as modulated FcγR binding, such as those outlined in patent applications entitled "IgG Immunoglobulin Variants with Optimized Effector Function," filed October 21, 2005, having U.S. Ser. Nos. 11 / 174,287, 11 / 124,640, 10 / 822,231, 10 / 672,280, 10 / 379,392, and 11 / 256,060. Thus, optimized properties include, but are not limited to, enhanced or decreased affinity for FcγRs. In one optional embodiment, IgG variants are optimized to have enhanced affinity for a human activating FcγR, preferably FcγRIIIa, in addition to their FcRn binding profile. In yet another optional alternative embodiment, the IgG variants are optimized to have reduced affinity for the human inhibitory receptor FcγRIIb. That is, specific embodiments include the use of antibodies that exhibit increased binding to FcRn and decreased binding to FcγRIIIa. Other embodiments utilize the use of antibodies that exhibit decreased binding to FcRn and increased binding to FcγRIIIa. These embodiments are expected to provide IgG polypeptides with enhanced human therapeutic properties, e.g., enhanced effector function and greater anti-cancer activity. In alternative embodiments, the IgG variants are optimized to have increased or decreased affinity for FcRn and increased or decreased affinity for human FcγRs (including, but not limited to, allelic variants of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb).These embodiments are expected to provide IgG polypeptides with improved therapeutic properties in humans, such as increased serum half-life and reduced effector function. In another embodiment, the IgG variants provide enhanced affinity for FcRn and one or more FcγRs, and decreased affinity for one or more other FcγRs. For example, the IgG variants may have enhanced binding to FcRn and FcγRIIIa, and decreased binding to FcγRIIb. Alternatively, the IgG variants may have decreased binding to FcRn and FcγRs. In another embodiment, the IgG variants may have decreased affinity for FcRn and FcγRIIb, and decreased affinity for one or more activating FcγRs. In yet another embodiment, the IgG variants may have increased serum half-life and reduced effector function.

[0117] While preferred embodiments include optimization of binding to human FcRn and FcγR, in alternative embodiments, the IgG variants have enhanced or decreased affinity for FcRn and FcγR from non-human organisms, including, but not limited to, rodents and non-human primates. IgG variants optimized for binding to non-human FcRn may find use in experiments. For example, mouse models are available for various diseases, allowing for testing of properties such as efficacy, toxicity, and pharmacokinetics of a given drug candidate. As is well known in the art, cancer cells can be transplanted or injected into mice to mimic human cancer, a process known as xenografting. Testing of IgG variants, including FcRn-optimized IgG variants, can provide useful information regarding the clearance characteristics of the protein, its clearance mechanism, and the like. IgG variants may also be optimized for improved functionality and / or solution properties of aglycosylated forms. Fc ligands include, but are not limited to, FcRn, FcγR, C1q, and proteins A and G, and may be derived from any source, preferably human, including, but not limited to, human, mouse, rat, rabbit, or monkey. In preferred embodiments, the IgG variants are optimized to be more stable and / or soluble than the aglycosylated form of the parent IgG variant.

[0118] IgG variants can include modifications that modulate interactions with Fc ligands other than FcRn and FcγR, including, but not limited to, complement proteins, and Fc receptor homologs (FcRH), including, but not limited to, FcRH1, FcRH2, FcRH3, FcRH4, FcRH5, and FcRH6 (Davis et al., 2002, Immunol. Reviews 190:123-136, incorporated by reference in its entirety).

[0119] Preferably, the specificity of an IgG variant's Fc ligand determines its therapeutic utility. The therapeutic utility of a given IgG variant depends on the epitope or form of the target antigen and the disease or indication being treated. For most targets and indications, enhanced FcRn binding may be preferable because it can result in an increased serum half-life. A longer serum half-life allows for less frequent or lower dosing. This is particularly preferable when the therapeutic agent is given for an indication requiring repeated administration. For some targets and indications, reduced FcRn affinity may be preferable. This may be particularly preferable when a mutant Fc with increased clearance or a reduced serum half-life is desired, for example, in Fc polypeptides used as contrast agents or radiotherapeutic agents.

[0120] IgG variants may be used, including IgG variants that provide enhanced affinity for FcRn with enhanced activating FcγRs and / or decreased affinity for inhibitory FcγRs. For some targets and indications, it may be more beneficial to use IgG variants that offer different selectivity for different activating FcγRs; for example, in some cases, increased binding to FcγRIIa and FcγRIIIa but not FcγRI may be desired, while in other cases, increased binding to only FcγRIIa may be desired. For certain targets and indications, it may be preferable to use IgG variants that alter FcRn binding and enhance both FcγR- and complement-mediated effector function, while in other cases, it may be advantageous to use IgG variants that enhance either FcRn binding or serum half-life, and FcγR- and complement-mediated effector function. For some targets or cancer indications, it may be advantageous to reduce or eliminate one or more effector functions, for example, by inactivating binding to C1q, one or more FcγRs, FcRn, or one or more other Fc ligands. For other targets and indications, it may be preferable to use IgG variants that provide increased binding to inhibitory FcγRIIb yet reduced binding to WT levels of activating FcγRs. This may be particularly useful, for example, when the goal of the IgG variant is to suppress inflammation or autoimmune disease or modulate the immune system in some way. Because autoimmune diseases are generally long-term and treatment involves repeated administration, treating these with Fc variants that have increased half-life from increased FcRn is preferred.

[0121] Modifications may be made to improve IgG stability, solubility, function, or clinical use. In a preferred embodiment, the IgG variants may include modifications to reduce immunogenicity in humans. In a most preferred embodiment, the immunogenicity of the IgG variants was reduced using the methods described in USSN 11 / 004,590, which is incorporated by reference in its entirety. In an alternative embodiment, the IgG variants are humanized (Clark, 2000, Immunol Today 21:397-402, which is incorporated by reference in its entirety).

[0122] IgG variants can include modifications that reduce immunogenicity. Modifications to reduce immunogenicity can include modifications that reduce binding of processed peptides derived from the parent sequence to MHC proteins. For example, amino acid modifications can be engineered to have high affinity and to have no or minimal immune epitopes predicted to bind to relevant MHC alleles. Several methods for identifying MHC-binding epitopes of protein sequences are well known in the art and can be used to score epitopes of IgG variants. For example, International Publication No. WO98 / 52976, WO02 / 079232, WO00 / 3317, USSN09 / 903,378, USSN10 / 039,170, USSN60 / 222,697, USSN 10 / 754,296, PCT No. WO01 / 21823, and PCT No. WO02 / 00165, Mallios, 1999, Bioinformatics 15: 432-439, Mallios,2001,Bioinformatics 17:942-948, Sturniolo et al.,1999,Nature Biotech. 17:555-561, WO98 / 59244, WO02 / 069232, WO02 / 77187, Marchall et al., 1995, J. Immunol. 154:5927-5933, and Hammer et al., 1994, J. Exp. Med. 180:2353-2358, all of which are incorporated by reference in their entireties. Sequence-based information can be used to determine the binding score of a given peptide-MHC interaction (see, e.g., Mallios, 1999, Bioinformatics 15:432-439; Mallios, 2001, Bioinformatics 17:p942-948; Sturniolo et al., 1999, Nature Biotech. 17:555-561, all of which are incorporated by reference in their entireties).

[0123] Engineering IgG mutants

[0124] The variants of the present invention may be designed by various methods. As described herein, the variants may be insertions, deletions, substitutions, other modifications, or combinations thereof and other changes. A particularly novel embodiment of the present invention is the design of insertions and deletions that increase or decrease binding of an Fc polypeptide to an Fc ligand. As disclosed herein, the insertions or deletions are made to increase or decrease the affinity of the Fc polypeptide for FcRn. The insertions and deletions may be designed by rational methods or methods that include a random component, such as the use or generation or screening of random or semi-random libraries. In alternative embodiments, substitutions that increase or decrease the affinity of an Fc polypeptide for FcRn are disclosed.

[0125] Backbone modifications: insertions and deletions

[0126] A variant Fc polypeptide may be formed by substituting a variant amino acid for a parent amino acid at a position in an Fc polypeptide. By substituting one or more variant amino acids in the Fc polypeptide, the side chains at those positions are altered. Most useful substitutions modify Fc properties by altering the Fc side chains. The substituted side chains may directly or indirectly interact with Fc binding partners associated with Fc function or properties. At least one substitution alters the covalent structure of one or more side chains of the parent Fc polypeptide.

[0127] Alternatively, mutant Fc polypeptides may be formed by altering the covalent structure of the backbone of a parent Fc polypeptide. The backbone atoms in a protein are peptide nitrogen, alpha carbon, carbonyl or peptide carbon, and carbonyl oxygen. Altering the covalent structure of the backbone provides an additional method for modifying the properties of an Fc polypeptide. The covalent structure of the Fc backbone may be altered by adding atoms to the backbone (e.g., inserting one or more amino acids) or removing atoms from the backbone (e.g., deleting one or more amino acids). The covalent bonds of the backbone may also be altered by changing individual atoms of the backbone to other atoms (Deechongkit et al., J Am Chem Soc. 2004. 126(51):16762-71, incorporated by reference in its entirety). As is well known in the art and as demonstrated herein, insertion or deletion of amino acids in an Fc polypeptide may be achieved by inserting or deleting the corresponding nucleotides in DNA encoding the Fc polypeptide. Alternatively, amino acid insertions or deletions may be made during synthesis of the Fc polypeptide, as is well known in the art.

[0128] The design of amino acid insertions or deletions that alter the interaction of an Fc polypeptide with one or more binding partners (e.g., FcγR, FcRn, C1q) may be made taking into account the structure of the complex of the Fc polypeptide and its binding partners. In less preferred embodiments, the design may be made taking into account information about the structure of the Fc polypeptide and the Fc region associated with binding of the binding partners. This information may be obtained by mutagenesis experiments, homology comparisons, computer modeling, or other means.

[0129] Preferred positions within the amino acid sequence for insertions or deletions that affect Fc binding interactions but do not affect the overall structure, stability, expression, or use of the Fc polypeptide are within the loops responsible for Fc / Fc-binding partner interactions. To alter FcRn binding to an Fc polypeptide, positions 244-257, 279-284, 307-317, 383-390, and 428-435 are preferred loop positions for insertions or deletions (numbering from the EU index of Kabat et al., 1994, Nature, 372:379-383; Martin et al., 2001, Mol Cell 7:867-877, all of which are incorporated by reference in their entireties). To alter Fcγ receptor binding to an Fc polypeptide, positions 229-239, 266-273, 294-299, and 324-331 are preferred loop positions for insertion or deletion (numbering from the EU index of Kabat et al., PDB code 1E4K.pdb; Sondermann et al., Nature. 2000 406:267, all of which are incorporated by reference in their entireties). Loops are regions of a polypeptide that are not involved in alpha helix or beta sheet structure. Loop positions are positions that are not within either alpha helix or beta sheet structure (van Holde, Johnson and Ho. Principles of Physical Biochemistry. Prentice Hall, New Jersey 1998, Chapter 1 pp2-67, incorporated by reference in their entirety). Loop positions are preferred because their backbone atoms are typically more flexible and less likely to participate in hydrogen bonds compared to the backbone atoms of alpha helices and beta sheets. Thus, lengthening or shortening the loop by inserting or deleting one or more amino acids is less likely to lead to large, disruptive changes to the Fc polypeptide, including stability or other problems.

[0130] Insertions and deletions may be used to alter the length of a polypeptide. For example, in loop regions, altering the length of the loop results in changes in the flexibility and conformational entropy of the loop. Generally, insertions into loops increase the conformational entropy of the loop, which may be defined as the Boltzmann constant multiplied by the natural logarithm of the number of possible conformations (van Holde, Johnson and Ho. Principles of Physical Biochemistry. Prentice Hall, New Jersey 1998, pp. 78, incorporated by reference in its entirety). By inserting at least one amino acid into a polypeptide, the total number of possible conformations of the polypeptide increases. These additional conformations may be beneficial for forming a favorable Fc / Fc binding partner interaction, since the polypeptide may use one of the additional conformations to bind an Fc-binding protein. In this case, the insertion may result in a stronger Fc / Fc binding partner interaction. If the additional conformations are not used at the binding interface, the insertions may result in weaker Fc / Fc binding partner interactions because the additional conformations may compete with competent conformations for binding. Similarly, deletions of polypeptide fragments may result in either stronger or weaker Fc / Fc binding partner interactions. If deletions of the fragments, which reduce the number of possible backbone conformations, remove competent conformations for binding, the deletions may result in weaker Fc / Fc binding partner interactions. If deletions do not remove competent conformations for binding, the deletions may result in stronger Fc / Fc binding partner interactions because the deletions may remove conformations that compete with competent conformations for binding.

[0131] Insertions and deletions may be used to alter the position and orientation of amino acids within an Fc polypeptide. Because insertions and deletions result in changes to the covalent structure of the backbone, they necessarily result in changes to the positions of backbone atoms. Figure 7 compares the backbone positions of several loop fragments, marked L1-L4, in three different backbones. The reference backbone structure contains the loop fragment, whereas the deletion backbone lacks fragment L1, and the insertion fragment contains an additional fragment before, or at the N-terminus of, fragment L1. Deletions and insertions result in the greatest change in the backbone structure near the site of insertion or deletion. By removing a fragment near the N-terminus of a loop, e.g., fragment L1, the loop is shortened, and the remaining fragment is moved to a position closer to the end of the N-terminus of the loop. This has the effect of moving the L2 fragment toward the original position of the L1 fragment and toward the end of the N-terminus of the loop. This change in the position of the L2 fragment relative to the L1 fragment enhances binding of the Fc / Fc binding partner complex and is preferred when there is prior information suggesting that an amino acid or amino acids located in L2 favorably interact with the Fc binding partner. For example, if L2 contains alanine and tyrosine and substitution of two L1 amino acids with alanine and tyrosine previously resulted in an Fc variant with increased binding, deletion of L1 can result in an Fc variant with increased affinity for the Fc binding partner.

[0132] Similarly, insertion of a polypeptide fragment into an Fc polypeptide N-terminal to the loop moves the position of the loop fragment toward the C-terminus of the loop. In Figure 7, insertion of one or more amino acids before, or N-terminal to, fragment L1 alters the backbone conformation, shifting the L1 fragment toward the C-terminus of the loop. This type of insertion is preferred when an amino acid located in fragment L1 is said to have a favorable interaction when located in position L2, because the insertion can result in a stronger Fc / Fc binding partner interaction. If a weaker Fc / Fc binding partner interaction is desired, an insertion can be used to move an unfavorable amino acid to a new position. The inserted fragment, deleted fragment, and reference fragment (L1-L4 in Figure 7) can be one or more amino acids within the Fc polypeptide.

[0133] Alternatively, insertions or deletions may be used at the C-terminal end of the loop in a manner similar to insertions or deletions at the N-terminal end of the loop. Insertions at the C-terminal end of the loop may result in the N-terminal position of the insertion being shifted toward the N-terminal end of the loop. Deletions at the C-terminal end of the loop may result in the N-terminal position of the deletion being shifted toward the C-terminal end of the loop. The choice of using insertions or deletions at the N- or C-terminal end of the loop depends on the amino acids located within the loop, the increased or decreased affinity of the Fc / Fc binding partner, and the desired position shift.

[0134] Insertions or deletions may be used in any region of an Fc polypeptide, including loops, alpha helices, and beta sheet regions. Preferred locations for insertions and deletions include loop regions that are not alpha helices or beta sheet regions. Loops are generally preferred because they are more responsive to backbone changes than alpha helices or beta sheets. Particularly preferred locations for insertions or deletions that result in stronger protein / protein interactions are the N- or C-terminal ends of the loop. When loop side chains are responsible for Fc / Fc binding partner interactions, insertions or deletions at the ends are less likely to result in significant adverse changes in binding interactions. Deletions within the exact center of a loop are more likely to remove important residues at the Fc / Fc binding partner interface, while insertions within the exact center of a loop are more likely to cause unfavorable interactions at the Fc / Fc binding partner interface. The number of deleted or inserted residues is determined by the size of the backbone change desired, with insertions or deletions of 15 or fewer residues being preferred, insertions or deletions of 10 or fewer residues being more preferred, and insertions or deletions of 5 or fewer residues being most preferred.

[0135] Once the location and size of the Fc deletion variant is designed, the entire polypeptide sequence is completely determined and the polypeptide may be constructed by methods well known in the art.

[0136] However, Fc insertion mutants have the additional step of designing the sequence of at least one inserted amino acid. Insertion of polar residues, including Ser, Thr, Asn, Gln, Ala, Gly, and His, is preferred at positions expected to be exposed within the Fc polypeptide. Smaller amino acids, including Ser, Thr, and Ala, are particularly preferred because their small size makes them less likely to sterically interfere with Fc / Fc binding partner interactions. Ser and Thr also have the ability to hydrogen bond with atoms on the Fc binding partner.

[0137] Insertions also provide added flexibility, allowing the inserted polypeptide to be designed to favorably interact with the Fc binding partner, as may be desired when stronger Fc / Fc binding partner binding is desired. The length of the backbone insertion may be determined by modeling a variant backbone with a simple generic sequence to be inserted. For example, polyserine, polyglycine, or polyalamin insertions of different lengths may be constructed and modeled. Modeling may be performed by a variety of methods, including homology modeling based on the known three-dimensional structure of the homolog containing the insertion, and by computer modeling, including MODELLER (MAMarti-Renom et al., Annu. Rev. Biophys. Biomol. Struct. 29, 291-325, 2000) and ROSETTA (Kuhlman et al. (2003). Science 302, 1364-8), both of which are incorporated by reference in their entireties. Typically, various backbone conformations are generated first, and the final backbone structure may be determined after the side chain identities are established. Side chains may be designed by PDA® algorithms (see U.S. Pat. Nos. 6,188,965, 6,269,312, 6,403,312, 6,801,861, 6,804,611, 6,792,356, 6,950,754, and U.S. Ser. Nos. 09 / 782,004, 09 / 927,790, 10 / 101,499, 10 / 666,307, 10 / 666311, 10 / 218,102, all of which are incorporated by reference in their entireties).

[0138] Insertions and deletions may be used to alter the binding of Fc polypeptides to FcγR in a manner similar to that described for altering FcRn binding properties. The Fc domain binds to FcγR at the position shown in Figure 1. Structures of Fc / FcγR complexes, including PDB codes 1T89 and 1IIS (Radaev S et al., J Biol Chem. v276, pp. 16469-16477, incorporated by reference in their entireties), show the interacting residues and loops between the two structures. Mutagenesis results, such as those found in US 11 / 124620 and US 6737056 (both incorporated by reference in their entireties), all have utility in determining appropriate shifts in scaffold positioning.

[0139] Insertions and deletions may be engineered into any polypeptide adjacent to the Fc polypeptide using the methods described herein. For example, insertions or deletions within the TNF superfamily member, APRIL, may be engineered using its three-dimensional structure (PDB code 1XU1.pdb, Hymowitz, et al., (2005) J. Biol. Chem. 280:7218, incorporated by reference in its entirety). Insertions or deletions may be designed to increase binding of APRIL to its receptor, TACI. Preferred loop residues for insertion or deletion are Ser118-Val124, Asp164-Phe167, Pro192-Ala198, and Pro221-Lys226. These loops interact with TACI in the APRIL / TACI complex and mediate binding.

[0140] Variants incorporating polypeptides

[0141] IgG variants can be based on human IgG sequences, which are thus used as "base" sequences to which other sequences, including but not limited to sequences from other organisms (e.g., rodent and primate sequences), are compared. IgG variants may also include sequences from other immunoglobulin classes, such as IgA, IgE, IgD, and IgM. While IgG variants are engineered in the context of one parent IgG, it is contemplated that the variants can be engineered or "transferred" to another, second parent IgG. This is typically done by determining "equivalent" or "corresponding" residues and substitutions between the first and second IgGs based on sequence or structural homology between the IgG sequences. To establish homology, the amino acid sequence of the first IgG outlined in this application is directly compared to the sequence of the second IgG. After aligning the sequences, one or more homology alignment programs known in the art are used to define residues equivalent to specific amino acids in the primary sequence of the first IgG variant (e.g., using conserved residues between species), while allowing for insertions and deletions necessary to maintain alignment (i.e., avoiding the removal of conserved residues through any deletions or insertions). Preferably, alignment of conserved residues should preserve 100% of such residues. However, alignment of 75% or more, or even as little as 50%, of conserved residues is sufficient to define equivalent residues. Equivalent residues may also be defined by determining structural homology between the first and second IgGs at the level of the tertiary structure of the IgG, whose structure has been determined. In this case, equivalent residues are defined as those in which the atomic coordinates (N vs. N, CA vs. CA, C vs. C, and O vs. O) of two or more main-chain atoms of a specific amino acid residue in the parent or precursor are within 0.13 nm, preferably 0.1 nm, after alignment. Alignment is achieved after the best model is oriented and positioned to give the greatest overlap of atomic coordinates of the protein's non-hydrogen protein atoms. Regardless of how equivalent or corresponding residues are determined, and regardless of the identity of the parent IgG from which the IgG is formed, the message is that the discovered IgG variants can be engineered into any second parent IgG that has significant sequence or structural homology with the IgG variant.Thus, for example, if a mutant antibody is generated where the parent antibody is a human IgG1 using the methods described above or other methods for determining equivalent residues, the mutant antibody may be engineered into another IgG1 parent antibody that binds a different antigen, a human IgG2 parent antibody, a human IgA parent antibody, a mouse IgG2a or IgG2b parent antibody, etc. Again, as noted above, the relatedness of the parent IgG variant does not affect the ability of the IgG variant to be transported to other parent IgGs.

[0142] Methods for engineering, producing, and screening IgG variants are provided. The described methods are not intended to be limited to any particular application or theory of operation. Rather, the provided methods are intended to generally illustrate that one or more IgG variants may be engineered, produced, and screened to obtain IgG variants with optimized effector function. Various methods for designing, producing, and testing antibody and protein variants are described in USSN 10 / 754,296 and USSN 10 / 672,280, both of which are incorporated by reference in their entireties.

[0143] Various protein engineering methods may be used to design IgG variants with optimized effector function. In one embodiment, structure-based engineering methods may be used, in which available structural information is used to guide substitutions, insertions, or deletions. In a preferred embodiment, computational screening methods may be used, in which substitutions are designed based on their energy compatibility in a computational calculation. See, e.g., USSN 10 / 754,296 and USSN 10 / 672,280, and the references cited therein, all of which are incorporated by reference in their entireties.

[0144] Sequence alignment may be used to guide substitutions at identified positions. Those skilled in the art will appreciate that sequence information may be used to prevent the introduction of substitutions that are potentially deleterious to protein structure. The source of the sequences can vary widely and includes the Kabat database (Northwestern University), Johnson & Wu, 2001, Nucleic Acids Res. 29:205-206; Johnson & Wu, 2000, Nucleic Acids Res. 28:214-218), the IMGT database (IMGT, the international ImMunoGeneTics information system®), Lefranc et al., 1999, Nucleic Acids Res. 27:209-212; Ruiz et al., 2000, Nucleic Acids Res. 28:219-221; Lefranc et al., 2001, Nucleic Acids Res. 29:207-209; Lefranc et al., 2003, Nucleic Acids Res. Res. 31:307-310), and VBASE, all of which are incorporated by reference in their entireties. Antibody sequence information can be obtained, compiled, and / or generated from sequence alignments of germline sequences or naturally occurring antibody sequences from any organism, including, but not limited to, mammals. Those skilled in the art will appreciate that the use of human or substantially human sequences may have the additional advantage of being less immunogenic when administered to humans. Other databases that are more general nucleic acid or protein databases, i.e., not specifically antibody-specific, include, but are not limited to, SwissProt, GenBank Entrez, and the EMBL Nucleotide Sequence Database. Aligned sequences can include VH, VL, CH, and / or CL sequences. There are many sequence-based alignment programs and methods well known in the art, all of which are useful for generating sequence alignments.

[0145] Alternatively, random or semi-random mutagenesis methods can be used to make amino acid modifications at desired positions. In these cases, positions are selected randomly, or amino acid changes are made using simple rules. For example, all residues can be mutated to alanine, which is called alanine scanning. Such methods can be coupled with more sophisticated engineering methods that use selection methods to screen for higher levels of sequence diversity. As is well known in the art, there are a variety of selection techniques that can be used in such methods, including, for example, display techniques such as phage display, ribosome display, and cell surface display, as described below.

[0146] Methods for producing and screening IgG variants are well known in the art. General methods for antibody molecular biology, expression, purification, and screening are described in "Antibody Engineering," edited by Duebel & Kontermann, Springer-Verlag, Heidelberg, 2001, and Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76. Reference is also made to the methods described in USSN 10 / 754,296, USSN 10 / 672,280, and USSN 10 / 822,231 and 11 / 124,620, all of which are incorporated by reference in their entireties. Preferred variants of the present invention include those shown in Figure 8. Alternatively, preferred variants of the present invention include those shown in Figure 9. Alternatively, preferred variants of the present invention include those shown in Figure 10. These variants exhibited increased binding to the Fc receptor, FcRn, as shown in the Examples.

[0147] Generation of IgG mutants

[0148] IgG variants can be generated by any method known in the art. In one embodiment, an IgG variant sequence is used to generate nucleic acids that encode member sequences, which can then be cloned into host cells, expressed, and analyzed as desired. These practices are carried out using well-known techniques, and a variety of methods that may find use are described in Molecular Cloning—A Laboratory Manual, 3 rd Ed. (Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001), and Current Protocols in Molecular Biology (John Wiley & Sons), both of which are incorporated by reference in their entireties. Nucleic acids encoding the nucleic acid IgG variants can be incorporated into expression vectors to express the protein. Expression vectors typically contain the protein operably linked, i.e., placed in functional relationship with, regulatory or control sequences, a selectable marker, any fusion partners, and / or additional elements. IgG variants can be produced by culturing host cells transfected with a nucleic acid, preferably an expression vector, containing a nucleic acid encoding the Fc variant under conditions appropriate to induce or cause expression of the protein. A wide variety of suitable host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. For example, various cell lines that may find use are described in the ATCC Cell Line Catalog available from the American Type Culture Collection, which is incorporated by reference in its entirety. Methods of introducing foreign nucleic acid into host cells are well known in the art and vary with the host cell used.

[0149] In a preferred embodiment, the IgG variants are purified or isolated after expression. Antibodies can be isolated or purified in a variety of ways well known to those skilled in the art. Standard purification methods include chromatographic techniques, electrophoresis, immunology, precipitation, dialysis, filtration, concentration, and chromatofocusing. As is well known in the art, various natural proteins bind to antibodies, for example, bacterial proteins A, G, and L, and these proteins can find use in purification. Purification can often be enabled by specific fusion partners. For example, proteins can be bound to glutathione resins when GST fusions are used, or Ni-tags when His-tags are used. +2 Purification can be achieved by affinity chromatography or, if a flag-tag is used, by using immobilized anti-flag antibodies. For a general guide to suitable purification techniques, see Antibody Purification: Principles and Practice, 3 rd Ed., Scopes, Springer-Verlag, NY, 1994, which is incorporated by reference in its entirety.

[0150] Screening for IgG variants

[0151] Fc variants can be screened using a variety of methods, including, but not limited to, in vitro assays, in vivo and cell-based assays, and those using selection techniques. Screening procedures can utilize automation and high-throughput screening techniques. Screening can use fusion partners or small molecule labels, such as, for example, immunolabels, isotopic labels, or fluorescent or colorimetric dyes.

[0152] In a preferred embodiment, the functional and / or biophysical properties of the Fc variants are screened in in vitro assays, hi a preferred embodiment, the protein is screened for functionality, e.g., its ability to catalyze a reaction or its binding affinity to its target.

[0153] As is well known in the art, a subset of screening methods are those that select for preferred members of a library. In this application, the methods are referred to as "selection methods," and these methods are useful in the present invention for screening Fc variants. When a protein library is screened using a selection method, only those members of the library that are preferred, i.e., that meet the selection criteria, are expanded, isolated, and / or observed. A variety of selection methods that may find use in the present invention for screening protein libraries are well known in the art. Other selection methods that may find use in the present invention include methods that do not rely on display, such as in vivo methods. A subset of selection methods, known as "directed evolution" methods, involve joining or breeding preferred sequences during selection, often incorporating new and differentiated mutations.

[0154] In preferred embodiments, Fc variants are screened using one or more cell-based or in vivo assays. Such assays typically involve the exogenous addition of purified or unpurified protein such that cells are exposed to individual variants or pools of variants from a library. These assays are typically, but not always, based on the function of the Fc polypeptide, i.e., its ability to bind to its target and mediate some biochemical event, e.g., effector function, ligand / receptor binding inhibition, apoptosis, etc. Such assays often involve monitoring the cellular response to IgG, e.g., cell survival, cell death, changes in cell morphology, transcriptional activation, e.g., expression of a cellular native or reporter gene. For example, such assays may measure the ability of Fc variants to induce ADCC, ADCP, or CDC. Some assays may require the addition of additional cells or components in addition to the target cells, e.g., serum complement or effector cells, such as peripheral blood monocytes (PBMCs), NK cells, macrophages, etc. Such additional cells may be derived from any organism, preferably from humans, mice, rats, rabbits, and monkeys. Antibodies may induce apoptosis of certain cell lines expressing the antibody's target, or they may mediate attack of target cells by immune cells added to the assay. Methods for monitoring cell death or viability are well known in the art and include the use of dyes, immunochemistry, cytochemistry, and radioactive reagents. Transcriptional activation may also serve as a method for assaying function in cell-based assays. Alternatively, cell-based screening is performed using cells transformed or transfected with nucleic acids encoding the Fc variants. That is, the Fc variants are not exogenously added to the cells.

[0155] The biological properties of IgG variants can be characterized through cell, tissue, and whole-organism experiments. As is well known in the art, drugs are often tested in animals, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and monkeys, to measure the drug's efficacy in treating a disease or disease model, or to measure the drug's pharmacokinetics, toxicity, and other properties. Such animals are sometimes referred to as disease models. Therapeutic drugs are often tested in mice, including, but not limited to, nude mice, SCID mice, xenograft mice, and genetically modified mice (including knock-ins and knock-outs). Such experiments can provide meaningful data for determining the potential of a protein for use as a therapeutic drug. Any organism, preferably a mammal, can be used for testing. For example, due to its genetic similarity to humans, monkeys may be a suitable therapeutic model and can therefore be used to test the efficacy, toxicity, pharmacokinetics, or other properties of IgG. Drug approval ultimately requires human testing, and therefore these experiments are naturally contemplated. Thus, the IgGs can be tested in humans to determine their therapeutic efficacy, toxicity, immunogenicity, pharmacokinetics, and / or other clinical properties.

[0156] How to Use IgG Mutants

[0157] The IgG variants may find use in a wide range of products. In one embodiment, the IgG variants are therapeutic, diagnostic, or research reagents, preferably therapeutic. The IgG variants may find use in antibody compositions, which may be monoclonal or polyclonal. In a preferred embodiment, the IgG variants are used to kill target cells bearing the target antigen, e.g., cancer cells. In an alternative embodiment, the IgG variants are used to inhibit, antagonize, or stimulate the target antigen, e.g., antagonize a cytokine or cytokine receptor. In an alternative preferred embodiment, the IgG variants are used to inhibit, antagonize, or stimulate the target antigen and kill target cells bearing the target antigen.

[0158] The IgG variants can be used for a variety of therapeutic purposes. In a preferred embodiment, antibodies, including IgG antibodies, are administered to patients to treat antibody-related diseases. patient " includes humans and other animals, preferably mammals, most preferably humans. antibody-related disorders ",or" Antibody-responsive disorders ",or" situation ",or" disease " refers to a disease that may be ameliorated by administration of a pharmaceutical composition comprising an IgG variant. Antibody-related diseases include, but are not limited to, autoimmune diseases, immune diseases, infectious diseases, inflammatory diseases, neurological diseases, and oncological and neoplastic diseases, including cancer. In this application, " cancer " and " cancerous " refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine tumors, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.

[0159] In one embodiment, the IgG antibody is the only therapeutically active agent administered to the patient. Alternatively, the IgG antibody is administered in combination with one or more other therapeutic agents, including, but not limited to, cytotoxic agents, chemotherapeutic agents, cytokines, antiproliferative agents, antihormonal agents, kinase inhibitors, antiangiogenic agents, cardioprotective agents, or other therapeutic agents. The IgG variants may be administered in combination with one or more other therapeutic regimens. For example, the IgG variants may be administered to the patient together with chemotherapy, radiation therapy, or both chemotherapy and radiation therapy. In one embodiment, the IgG variants may be administered in combination with one or more antibodies, which may or may not be IgG variants. In another embodiment, the IgG variants and one or more other anti-cancer therapies are used to treat cancer cells in vivo. It is contemplated that such an internal treatment would be useful for bone marrow transplantation, particularly autologous bone marrow transplantation. It is, of course, contemplated that the IgG variants may be used in combination with additional therapeutic procedures, such as surgery.

[0160] A variety of other therapeutic agents may find use in administration with the IgG variant. In one embodiment, the IgG is administered with an anti-angiogenic agent. Anti-angiogenic agents " refers to a compound that blocks or inhibits blood vessel growth to some extent. For example, an anti-angiogenic factor may be a small molecule or protein, e.g., an antibody, Fc fusion, or cytokine that binds to a growth factor or growth factor receptor responsible for promoting angiogenesis. A preferred anti-angiogenic factor of the present application is an antibody that binds to endothelial growth factor (VEGF). In an alternative embodiment, the IgG is administered in conjunction with a therapeutic agent that induces or enhances an adaptive immune response, e.g., an antibody that targets CTLA-4. In an alternative embodiment, the IgG is administered in conjunction with a tyrosine kinase inhibitor. As used in this application, " tyrosine kinase inhibitors " means a molecule that inhibits to some extent the tyrosine kinase activity of a tyrosine kinase. In an alternative embodiment, the IgG variant is administered with a cytokine.

[0161] Pharmaceutical compositions are contemplated in which the IgG variants and one or more therapeutically active agents are formulated. IgG variant formulations are prepared for storage by combining IgG having the desired degree of purity with any pharmaceutically acceptable carriers, excipients, or stabilizers in the form of a lyophilized formulation or aqueous solution (Remington's Pharmaceutical Sciences 16th edition, Osol. A. Ed., 1980, incorporated by reference in its entirety). Formulations to be used for in vivo administration are preferably sterile. This is readily accomplished by filtration through sterile membranes or other methods. The IgG variants and other therapeutically active agents disclosed herein may also be formulated as immunoliposomes and / or encapsulated in microcapsules.

[0162] The concentration of the therapeutically active IgG variant in the formulation may vary from about 0.1 to 100% by weight. In a preferred embodiment, the concentration of IgG ranges from 0.003 to 1.0 molar. A therapeutically effective amount of the IgG variant may be administered to treat a patient. Therapeutically effective dose " refers to a dosage that produces the effect for which it is administered. The exact dosage will vary depending on the purpose of treatment and will be ascertainable by one of ordinary skill in the art using well-known techniques. Doses may range from 0.01 to 100 mg per kg or more of body weight, e.g., 0.01, 0.1, 1.0, 10, or 50 mg per kg of body weight, with 1 to 10 mg per kg being preferred. As is well known in the art, adjustments to proteolysis, systemic versus local delivery, and rate of de novo protease synthesis, as well as age, body weight, general health, sex, diet, time of administration, drug interactions, and severity of the condition may be necessary and will be ascertainable by one of ordinary skill in the art with routine experimentation.

[0163] Administration of the pharmaceutical composition comprising the IgG, preferably in the form of a sterile aqueous solution, may be accomplished in a variety of ways, including, but not limited to, oral, subcutaneous, intravenous, parenteral, intranasal, intraaural, intraocular, rectal, intravaginal, transdermal, topical (e.g., gel, ointment, lotion, cream, etc.), intraperitoneal, intramuscular, or pulmonary (e.g., AERx® inhalable technology sold by Aradigm, or Inhance® pulmonary delivery system sold by Nektar Therapeutics, etc.). The therapeutic agents described herein may be administered concurrently with other therapeutic agents, i.e., the therapeutic agents described herein may be administered concurrently with other treatments or therapeutic agents, including, for example, small molecules, other biologics, radiation therapy, surgery, etc. [Example]

[0164] The following examples are provided to illustrate the present invention. These examples are not meant to limit the present invention to any particular application or theory of operation. For all positions described herein, numbering follows the EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, incorporated by reference in its entirety). Those skilled in the art of antibodies will understand that this convention consists of non-contiguous numbering in specific regions of an immunoglobulin sequence, allowing for standardized reference to conserved positions within an immunoglobulin family. Thus, the positions of any given immunoglobulin defined by the EU index do not necessarily correspond to its contiguous sequence.

[0165] Example 1: DNA construction, expression, and purification of Fc variants

[0166] Amino acid modifications in the Fc region of the antibodies were engineered to improve their affinity for the neonatal Fc receptor, FcRn. Mutants were screened in the context of many different human IgG constant chains ( FIG. 2 ), including IgG1, IgG2, and a hybrid IgG sequence containing the CH1 and upper hinge of IgG1 and the Fc region of IgG2. Those skilled in the art will appreciate that these different parent Fc regions have different FcγR- and complement-mediated effector function properties due to the different interactions of the IgG1 and IgG2 Fc regions with FcγR and complement. Exemplary sequences of the Fc variants in the context of these parent IgG constant chains are shown in FIG. 3.

[0167] The Fc variants were engineered in the context of an antibody targeting vascular endothelial factor (VEGF). The heavy and light chain variable regions (VH and VL) are those of a humanized version of the antibody A4.6.1, also known as bevacizumab (Avastin®), which is approved for the treatment of various cancers. The amino acid sequences of the VH and VL regions of the antibody are shown in Figure 4.

[0168] Genes encoding the heavy and light chains of anti-VEGF antibodies were constructed in the mammalian expression vector pTT5. Human IgG1 and IgG2 constant chain genes were obtained from IMAGE clones and subcloned into the pTT5 vector. IgG1 / 2 genes were constructed using PCR mutagenesis. VH and VL genes encoding anti-VEGF antibodies were commercially synthesized (Blue Heron Biotechnologies, Bothell, WA) and subcloned into vectors encoding the appropriate CL, IgG1, IgG2, and IgG1 / 2 constant chains. Amino acid modifications were constructed using site-directed mutagenesis using QuikChange® site-directed mutagenesis (Stratagene, La Jolla, CA). All DNAs were sequenced to confirm sequence fidelity.

[0169] Plasmids containing the heavy chain genes (VH-Cγ1-Cγ2-Cγ3) were cotransfected with a plasmid containing the light chain genes (VL-Cκ) into 293E cells using Lipofectamine (Invitrogen, Carlsbad, CA) and grown in FreeStyle 293 medium (Invitrogen, Carlsbad, CA). After 5 days of growth, antibodies were purified from culture supernatants by protein A affinity using MabSelect resin (GE Healthcare). Antibody concentrations were determined by bicinchoninic acid (BCA) assay (Pierce).

[0170] Example 2. Fc-mutated antibodies maintain binding to antigen

[0171] The fidelity of the expressed mutant antibodies was confirmed by demonstrating that they maintained their specificity for the antigen. VEGF binding was monitored using surface plasmon resonance (SPR) (Biacore) performed using a Biacore3000 instrument. Recombinant VEGF (VEGF-165, PeproTech, Rocky Hill, NJ) was attached to a CM5 chip surface by conjugation with N-hydroxysuccinimide / N-ethyl-N'-(-3-methylaminopropyl)carbodiimide (NHS / EDC) using standard methods. WT and mutant antibodies were injected as analytes, yielding responses measured in resonance units (RU). Because the dissociation phase was too slow to measure an accurate equilibrium constant, relative binding was determined by measuring RU at the end of the association phase, which should be proportional to the protein concentration (held constant throughout the experiment) and the association rate coefficient. The data (Figure 6) show that the mutant anti-VEGF antibodies maintain binding to the antigen, in contrast to a negative control anti-Her2 antibody that does not bind VEGF.

[0172] Example 3. Measurement of binding to human FcRn

[0173] Binding of mutant antibodies to human FcRn was measured at pH 6.0, the pH at which they naturally bind in endosomes. Vectors encoding beta2-microglobulin and His-tagged alpha chain genes of FcRn were constructed, co-transfected into 293T cells, and purified using nickel chromatography. Antibody affinity for human FcRn (hFcRn) was measured at pH 6.0 on a Biacore 3000 instrument by coupling human FcRn to a CM5 chip surface using standard NHS / EDC chemistry. Responses were measured in resonance units using wild-type and mutant antibodies in mobile phases ranging from 25 to 100 nM. Association and dissociation phases were obtained at pH 6.0, followed by injection of a pH 7.4 buffer to measure antibody release from the receptor at higher pHs. The antibody and buffer cycles provided only baseline responses, which were subtracted from each sample sensorgram.

[0174] Figure 7 shows Biacore sensorgrams for the binding of native IgG1 and select Fc mutant antibodies to human FcRn at two relevant pHs. The data show that wild-type and mutant antibodies readily bind to the FcRn chip at pH 6.0, slowly dissociate at that pH, as in endosomes, and then recycle to the endosomal membrane and rapidly release at pH 7.4, as in exposure to serum at higher pH.

[0175] The FcRn association / dissociation curves did not fit a simple Langmuir model, possibly due to the multivalency of the antibody and receptor or the heterogeneity of the chip. * The affinity of each mutant compared to its parent IgG was determined by fitting it to a conformational change model with the change in refractive index (RI) fixed at 0 RU. These values ​​for selected mutant antibodies are plotted in Figure 8. The relative affinity of each mutant compared to its parent IgG was calculated using the equation, fold = (WT Ka * / Mutant Ka * ) relative binding data for all Fc variants in IgG1 Fc regions are shown in Figure 9, and binding data for variants in antibodies with IgG2 Fc regions (constant chains IgG1 and IgG1 / 2) are shown in Figure 10. For many variants, binding experiments were repeated multiple times (n), for which fold binding was calculated by reference to the WT IgG parent within each particular binding experiment. Averaging of these data provided the means and standard deviations shown in Figures 9 and 10.

[0176] Figures 9 and 10 show that many of the engineered mutants bind with higher affinity to human FcRn at pH 6.0 compared to WT IgG1. The improvement depended heavily on the identity of the substitution at a given position. For example, using 2-fold as the measure of improved binding, many mutations at position 434 of IgG2 increased affinity (A, S, Y, F, and W), some were neutral (within 2-fold of WT IgG2) (G, H, M, and T), and many substitutions decreased affinity (<0.5-fold) (D, E, K, P, R, and V). Stronger binding in the context of IgG1 did not necessarily translate into stronger binding in IgG2 (e.g., 434T improved IgG1 but not IgG2). Furthermore, the improvements provided by single mutants were not always additive when combined. Figure 11a illustrates this graphically by plotting the experimental FcRn binding fold of selected double substitution mutants against the product of the FcRn binding fold of the individual single mutants that comprise them. The straight line represents perfect additivity, i.e., the value expected or predicted from the product of the single substitutions. Many double mutants fall on or are close to this line (259I / 319I, 259I / 428L, 319I / 428L, and 308F / 428L). Some mutants are less than additive (319I / 308F, 252Y / 428L, and 428L / 434M). For these mutants, especially the last two (252Y / 428L and 428L / 434M), the affinity improvements of the single substitutions appear to be incompatible with each other when combined. Unexpectedly, the improvement in FcRn affinity of mutants 259I / 308F and 428L / 434S was stronger than expected from the affinity of each of their single substitutions. When these particular single substitutions were combined, they had an unexpected synergistic improvement. The difference between the experimental affinity and that expected from the affinity of the single mutants is plotted in Figure 11b, with mutants grouped by their combined single mutants (259I, 308F, and 319I on the left, and combination with 482L on the right).Synergy can be quantified by calculating the fold of the experimental value over the predicted value, then normalizing to 1 and converting to a percentage (% synergy = 100 x [(experimental fold / predicted fold) - 1]). This analysis is plotted in Figure 11b, with mutants grouped by their combined single mutants. This graph again highlights the synergistic effects of some mutants, particularly 259I / 308F and 428L / 434S. Figures 11b and 11c also highlight the unpredictable nature of some of the best single substitution combinations from the screen. For example, the combination of 428L with 434S and 259I provides synergistic binding improvement, whereas 252Y or 434M have a negative impact when combined with 428L. The striking difference between the combination of 428L with 434S and 434M further emphasizes the importance of the specific amino acid identity of the substitution at a given position.

[0177] Example 4. Testing of variants in the context of other antibodies

[0178] Selected variants were constructed in the context of antibodies targeting other antigens, including TNF (TNFα), CD25 (TAC), EGFR, and IgE. Figure 4 provides the amino acid sequences of the VH and VL regions of the antibodies targeting these antigens used in the present invention. The WT and Fc variant anti-TNF antibodies contain the variable regions of adalimumab (Humira®), a fully human antibody currently approved for the treatment of rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), psoriatic arthritis (PsA), ankylosing spondylitis (AS), and Crohn's disease (CD). The WT and Fc variant anti-CD25 antibodies are humanized versions of the anti-TAC antibody, designated H1.8 / L1 anti-TAC (Junghans et al., 1990, Cancer Research 50:1495-1502). The WT and Fc variant anti-EGFR antibodies are humanized versions of the murine antibody C225, designated H4.42 / L3.32 C225. Finally, the WT and Fc variant anti-IgE antibodies contain the variable regions of omalizumab (Xolair®), a humanized antibody approved for the treatment of allergic asthma.

[0179] WT and mutant antibodies were constructed, expressed, and purified as described above. Antibodies were tested for binding to human FcRn at pH 6.0 by Biacore as described above. Relative binding data for mutant anti-TNF, -CD25, -EGFR, and -IgE antibodies to human FcRn is provided in Figure 12. As can be seen, the mutants improve FcRn affinity in the context of antibodies targeting different antigens.

[0180] Example 5. Pharmacokinetics experiment in human FcRn knock-in mice

[0181] To test the ability of selected variants to improve half-life in vivo, heterozygous knockout of mouse FcRn and heterozygous knock-in of human FcRn (mFcRn) were performed. - / - , hFcRn + ), B6 ​​mice (hFcRn or hFcRn in this application) + Pharmacokinetic studies were performed in mice (referred to as "mice") (Petkova et al., 2006, Int Immunol 18(12):1759-69, incorporated by reference in its entirety). A single intravenous tail vein injection (2 mg / kg) of anti-VEGF antibody was administered to groups of 4 to 7 female mice randomized by body weight (range 20-30 g). Blood (approximately 50 μl) was drawn from the orbital plexus at each time point, processed into serum, and stored at -80°C until analysis. Study duration was 28 or 49 days. No animals were harmed during these studies.

[0182] Antibody concentrations were determined using duplicate ELISA assays. In the first two studies (referred to as Study 1 and Study 2), a goat anti-human Fc antibody (Jackson Immuno Research) was applied to the plate, and the wells were washed with PBST (phosphate-buffered saline with 0.05% Tween) and blocked with 3% BSA in PBST. Serum or calibration standards were added, followed by a PBST wash, the addition of europium-labeled anti-human IgG (Perkin Elmer), and another PBST wash. Time-resolved fluorescent signals were collected. For Studies 3–5, serum concentrations were detected using a similar ELISA, but with recombinant VEGF (VEGF-165, PeproTech, Rocky Hill, NJ) as the capture reagent, biotinylated anti-human kappa antibody, and europium-labeled streptavidin. PK parameters were determined for individual mice using a noncompartmental model using WinNonLin (Pharsight Inc, Mountain View, CA). Nominal times and doses were used, with uniform weighing of time points. Time points (lambda Z range) used ranged from day 4 to the end of the study, except for the faster cleared mutants, P257N and P257L, which used all time points.

[0183] mFcRn - / - hFcRn + Five antibody PK studies were performed in mice. Figure 13 shows serum concentration data for WT and mutant IgG1 (Study 3) and IgG2 (Study 5) antibodies, respectively. mFcRn - / - hFcRn + The approximated PK parameters from all in vivo PK studies conducted in mice are provided in Figure 14. PK data include half-life, which represents the beta phase characterizing the clearance of the antibody from serum; C, which represents the maximum observed serum concentration; AUC, which represents the area under the concentration-time curve; and clearance, which represents the clearance of the antibody from serum. Also provided is the calculated fold improvement or reduction in half-life for each variant relative to the IgG1 or IgG2 parent antibody [half-life fold = half-life(variant) / half-life(WT)].

[0184] The data show that many engineered Fc mutant antibodies with enhanced FcRn affinity have extended in vivo half-lives at pH 6.0. Figure 15a shows a plot of in vivo half-life versus FcRn binding fold for an IgG1 antibody, with select mutants labeled. Results from replicate experiments (circled in the figure) demonstrate that the data from the in vivo model are reproducible. The best single mutants include 308F and 434S, the best double mutants include 259I / 308F, 308F / 428L, 308F / 434S, and 428L / 434S, and the best triple mutant is 259I / 308F / 428L. While there is a general correlation between affinity for FcRn and in vivo half-life, it is not completely predictive. Notably, mutants 257L and 257N improved FcRn binding by 3.4- and 3.5-fold, respectively, but reduced in vivo half-life by 0.6 and 0.3, respectively. The plot also highlights again the importance of amino acid homology for substitutions at a given position, while 308F / 434S provided a substantial half-life improvement and 308F / 434M was only marginally better than WT IgG1.

[0185] Figure 15b shows a plot of in vivo half-life versus FcRn binding fold for IgG2 mutant antibodies, with the mutants labeled. Comparing the IgG2 in vivo data with the IgG1 in vivo data (Figure 15c) yielded a surprising result: the mutants provided substantially greater improvement to in vivo half-life in the context of the IgG2 Fc region than in the IgG1 Fc region. The longest half-lives of the single and double mutants from all antibodies across all five studies were 12.2 and 16.5, respectively, provided by 434S IgG2 and 428L / 434S IgG2. Compared to IgG1, the significant improvement in half-life of the IgG2 mutants was despite the fold improvement by the mutants in IgG2 being similar to or even lower than in IgG1 (434S IgG1 fold = 3.8, 434S IgG2 fold = 4.9, 428L / 434S IgG1 fold = 17.3, 428L / 434S IgG2 fold = 14.8). Thus, unexpectedly, IgG2 antibodies may be the best application for Fc mutants to improve in vivo half-life in mammals.

[0186] Example 6. Mutant Immunoadhesins

[0187] The Fc variants of the present invention were also evaluated for their ability to improve the half-life of immunoadhesins (also referred to as Fc fusions). Select Fc variants were engineered into the anti-TNF immunoadhesin, etanercept (Enbrel®). Etanercept is a fusion of human TNF receptor 2 (TNF RII) with the Fc region of human IgG1 and is clinically approved for the treatment of rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, psoriatic arthritis, and psoriasis. An IgG2 Fc region version of this Fc fusion was also constructed, and select Fc variants were constructed in this context. The amino acid sequences of the anti-TNF immunoadhesins characterized in this invention are provided in Figure 16. Genes were constructed and subcloned into the pTT5 vector using recursive PCR, and Fc variants were constructed using QuikChange® mutagenesis. The immunoadhesin was expressed in 293E cells and purified as described above.

[0188] The binding specificity of the purified immunoadhesin was confirmed by testing its binding to recombinant TNF by Biacore. The immunoadhesin was purified using standard primary amine coupling and captured on an immobilized Protein A / G (Pierce) CM5 biosensor chip (Biacore). The immunoadhesin was immobilized on the Protein A / G surface, and recombinant TNF in serial dilutions was injected over the antibody-bound surface, followed by a dissociation phase. After each cycle, the surface was regenerated with buffer. Data were processed by zeroing the time and response before receptor injection and subtracting from the reference channel to account for changes due to injection. The kinetic data were fitted to a 1:1 binding model (Langmuir). The equilibrium association constants (Ka) obtained from these fits are provided in Figure 17. The results show that the mutant immunoadhesin retained affinity for TNF comparable to that of commercially available Enbrel.

[0189] The mutant immunoadhesins were tested for binding to human FcRn at pH 6.0 using Biacore as described above. The results (Figure 18) show that, as in the context of antibodies, the mutants improve binding to FcRn compared to their IgG1 and IgG2 parent immunoadhesin proteins.

[0190] The half-life of the mutant immunoadhesins was determined by the mFcRn - / - hFcRn +The immunoadhesins were tested in mice. Twelve mice per group were injected with 2 mg / kg of the mutant and parent IgG1 immunoadhesins. Serum concentrations were detected using an ELISA similar to that described above, except that a goat anti-human TNF RII antibody was used as the capture reagent, and detection was performed using a biotinylated anti-human kappa antibody and europium-labeled streptavidin. Figure 19 shows serum concentration data for the WT IgG1 Fc and mutant Fc immunoadhesins. The approximated PK parameters described above from the PK study are provided in Figure 20. Also provided is the calculated % increase in half-life for each mutant, calculated as 100 × the half-life of the mutant Fc fusion divided by the half-life of the WT IgG1 Fc parent. The results indicate that the mutants extend in vivo half-life in the context of the immunoadhesin.

[0191] Example 7. Pharmacokinetic studies in non-human primates

[0192] The PK properties of biologics in non-human primates are well established as predictive of their properties in humans. To evaluate the ability to improve the serum half-life of a mutant anti-VEGF antibody in non-human primates, a PK study was performed in cynomolgus monkeys (macaca fascicularis).

[0193] In preparation for PK studies in cynomolgus monkeys, we measured the binding of the mutant antibodies to cynomolgus monkey (cyno) FcRn (cFcRn) at pH 6.0. cFcRn was constructed, expressed, and purified as described above for human FcRn. Binding of the mutant anti-VEGF antibodies to cFcRn was measured using Biacore as described above. Data are provided in Figure 21. The results show that the mutants have improved affinity for cyno FcRn, as well as for human FcRn. Dissociation at higher pH (7.4) was also very rapid, similar to that observed for binding to human FcRn (data not shown). These results are not surprising given the high sequence homology between the human and cyno receptors (96% FcRn alpha chain, 91% beta-2-microglobulin).

[0194] The PK of the mutants was studied in vivo in non-human primates. Male cynomolgus monkeys (macaca fascicularis), weighing 2.3 to 5.1 kg, were randomized by weight and divided into five groups of three monkeys per group. Monkeys received a single, 1-hour peripheral intravenous infusion of 4 mg / kg antibody. Five minutes to 90 days after the end of the infusion, blood samples (1 ml) were drawn from a separate vein, processed to serum, and stored at -70°C. No animals were harmed during these studies.

[0195] Antibody concentrations were determined using the VEGF capture method described above. PK parameters were determined by fitting concentration versus time to a non-compartmental model, as was done in the mouse PK study. However, time points from 10 to 90 days were used to determine the PK parameters. The PK results are plotted in Figure 22, and the fitted parameters are provided in Figure 23. The results show that the variants improved the in vivo half-life of the antibody by up to 3.2-fold. In the best case (428L / 434S variant), the half-life increased from 9.7 days to 31.1 days. PK results obtained in cynomolgus monkeys showed that mFcRn - / - hFcRn + This is consistent with what was obtained in mice, confirming the validity of the hFcRn mouse model as a system for assessing the in vivo PK properties of mutants and supporting the conclusions from those studies.

[0196] While specific embodiments of the present invention have been described above for purposes of illustration, those skilled in the art will appreciate that many modifications of detail may be made without departing from the invention as set forth in the appended claims. All references cited in this application are incorporated herein in their entirety.

Claims

1. A polypeptide comprising an Fc variant of a human IgG Fc polypeptide, wherein the Fc variant comprises an amino acid substitution 308F / 428L and is numbered according to the EU index.

2. A polypeptide comprising an Fc variant of a human IgG Fc polypeptide, wherein the Fc variant comprises the amino acid substitutions 259I / 308F / 428L and is numbered according to the EU index.

3. A polypeptide comprising an Fc variant of a human IgG Fc polypeptide, wherein the Fc variant comprises an amino acid substitution 252Y / 428L and is numbered according to the EU index.

4. A polypeptide comprising an Fc variant of a human IgG Fc polypeptide, wherein the Fc variant comprises the amino acid substitution 307Q / 434S and is numbered according to the EU index.

5. A polypeptide comprising an Fc variant of a human IgG Fc polypeptide, wherein the Fc variant comprises the amino acid substitution 307Q / 308F / 434S and is numbered according to the EU index.

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