PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(51) International Patent Classification 7 :
`
`(11) International Publication Number:
`
`WO 00/42072
`
`C07K 16/00, A61K 39/395, C12N 15/13,
`15/85, 5/10, G01N 33/68, 33/577 // C07K
`16/28
`
`A2
`
`(43) International Publication Date:
`
`20 July 2000 (20.07.00)
`
`(21) International Application Number:
`
`PCT/USOO/00973
`
`(22) International Filing Date:
`
`14 January 2000 (14.01.00)
`
`(30) Priority Data:
`60/116,023
`
`15 January 1999 (15.01.99)
`
`US
`
`(71) Applicant: GENENTECH, INC. [US/US]; 1 DNA Way, South
`San Francisco, CA 94080—4990 (US).
`
`(81) Designated States: AE, AL, AM, AT, AU, AZ, BA, BB, BG,
`BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE,
`ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP,
`KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA,
`MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU,
`SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG,
`UZ, VN, YU, ZA, ZW, ARIPO patent (GH, GM, KE, LS,
`MW, SD, SL, SZ, TZ, UG, ZW), Eurasian patent (AM, AZ,
`BY, KG, KZ, MD, RU, TJ, TM), European patent (AT, BE,
`CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC,
`NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA,
`GN, GW, ML, MR, NE, SN, TD, TG).
`
`(72) Inventor: PRESTA, Leonard, G.; 1900 Gough Street, #206,
`San Francisco, CA 94109 (US).
`
`Published
`
`(74) Agents: LEE, Wendy, M. et al.; Genentech, Inc.,
`South San Francisco, CA 94080—4990 (US).
`
`1 DNA Way,
`
`Without international search report and to be republished
`
`upon receipt of that report.
`
`
`(54) Title: POLYPEPTIDE VARIANTS WITH ALTERED EFFECTOR FUNCTION
`
`(57) Abstract
`
`The present invention concerns polypeptides comprising a variant Fc region. More particularly, the present invention concerns Fc
`region—containing polypeptides that have altered effector function as a consequence of one or more amino acid modifications in the Fc
`region thereof.
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Zimbabwe
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`Albania
`ES
`LS
`Lesotho
`51
`Slovenia
`Spain
`FI
`Armenia
`Finland
`LT
`Lithuania
`SK
`Slovakia
`Austria
`FR
`France
`LU
`SN
`Luxembourg
`Senegal
`Australia
`GA
`Gabon
`LV
`Latvia
`SZ
`Swaziland
`GB
`MC
`TD
`Monaco
`Chad
`Azerbaijan
`United Kingdom
`GE
`MD
`TG
`Bosnia and Herzegovina
`Georgia
`Republic of Moldova
`Togo
`Barbados
`GH
`Ghana
`MG
`TJ
`Madagascar
`Tajikistan
`GN
`Guinea
`MK
`Belgium
`TM
`Turkmenistan
`The former Yugoslav
`Burkina Faso
`GR
`Greece
`TR
`Republic of Macedonia
`Turkey
`HU
`TT
`Mali
`Bulgaria
`Hungary
`Trinidad and Tobago
`Benin
`IE
`Ireland
`UA
`Ukraine
`Mongolia
`Brazil
`IL
`Israel
`Mauritania
`UG
`Uganda
`Belarus
`IS
`Iceland
`Malawi
`US
`United States of America
`Canada
`IT
`Mexico
`UZ
`Uzbekistan
`Italy
`JP
`VN
`Viet Nam
`Central African Republic
`Japan
`Niger
`KE
`Netherlands
`Y U
`Congo
`Kenya
`Yugoslavia
`Switzerland
`KG
`ZW
`Kyrgyzstan
`Norway
`KP
`COIe d’Ivoire
`New Zealand
`Democratic People‘s
`Cameroon
`Poland
`Republic of Korea
`China
`Republic of Korea
`Portugal
`Cuba
`Kazakstan
`Romania
`Saint Lucia
`Russian Federation
`Czech Republic
`Liechtenstein
`Sudan
`Germany
`Denmark
`Sri Lanka
`Sweden
`Estonia
`Liberia
`Singapore
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`KR
`KZ
`LC
`LI
`LK
`LR
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`

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`WO 00/42072
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`PCT/USOO/00973
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`POLYPEPTIDE VARIANTS WITH ALTERED EFFECTOR FUNCTION
`
`BACKGROUND OF THE INVENTION
`
`Field of the Invention
`
`The present invention concerns polypeptides comprising a variant Fc region. More
`
`particularly, the present invention concerns Fc region-containing polypeptides that have altered
`
`effector function as a consequence of one or more amino acid modifications in the Fc region
`
`thereof.
`
`Description of Related Art
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`Antibodies are proteins which exhibit binding specificity to a specific antigen. Native
`
`antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two
`identical light (L)_chains and two identical heavy (H) chains. Each light chain is linked to a heavy
`chain by one covalent disulfide bond, while the number of disulfide linkages varies between the
`heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularty
`spaced intrachairi-disulfide bridges. Each heavy chain has at one end a variable domain (VH)
`followed by a nurnber of constant domains. Each light chain has a variable domain at one end
`(VL) and a constant domain at its other end; the constant domain of the light chain is aligned with
`the first constant domain of the heavy chain, and the light chain variable domain is aligned with
`the variable domain'of the heavy chain. Particular amino acid residues are believed to form an
`interface between the light and heavy chain variable domains.
`
`The term "variable'l refers to the fact that certain portions of the variable domains differ
`
`extensively in sequence among antibodies and are responsible forthe binding specificity of each
`
`particular antibody for its particular antigen. However, the variability is not evenly distributed
`
`through the variable domains of antibodies.
`
`It
`
`is concentrated in three segments called
`
`complementarity determining regions (CDRs) both in the light chain and the heavy chain variable
`
`domains. The more highly conserved portions of the variable domains are called the framework
`
`regions (FRs). The variable domains of native heavy and light chains each comprise four FRs,
`
`largely adopting a B-sheet configuration, connected by three CDRs, which form loops connecting,
`
`and in some cases forming part of, the B-sheet structure. The CDRs in each chain are held
`
`together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the
`
`formation of the antigen binding site of antibodies (see Kabat et aI., Sequences of Proteins of
`
`Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda,
`
`MD. (1991)).
`
`The constant domainsare not involved directly in binding an antibody to an antigen, but
`
`exhibit various effector functions. Depending on the amino acid sequence of the constant region
`
`of their heavy chains, antibodies or immunoglobulins can be assigned to different classes. There
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`are five (major classes of immunoglobulins: IgA, lgD, lgE, IgG and IgM, and several of these may
`
`be further divided into subclasses (isotypes), e.g. igG1, lgGZ, lgG3, and lgG4; lgA1 and lgA2.
`
`The heavy chain constant regions that correspond to the different classes of immunoglobulins
`
`are called a, 8, s, y, and )1, respectively. Of the various human immunoglobulin classes, only
`
`human lgG1, lgGZ, lgG3 and lgM are known to activate complement; and human IgG1 and lgG3
`
`mediate ADCC more effectively than lgG2 and lgG4.
`
`A schematic representation of the native lgG1 structure is shown in Fig. 1, where the
`
`various portions of the native antibody molecule are indicated. Papain digestion of antibodies
`
`produces two identical antigen binding fragments, called Fab fragments, each with a single
`
`antigen binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize
`
`readily. The crystal structure of the human lgG Fc region has been determined (Deisenhofer,
`
`Biochemistry 20:2361-2370 (1981)). In human lgG molecules, the Fc region is generated by
`
`papain cleavage N—terminal to Cys 226. The Fc region is central to the effector functions of
`antibodies.
`
`The effector functions mediated by the antibody Fc region can be divided into two
`
`categories: (1) effector functions that operate after the binding of antibody to an antigen (these
`
`functions involve the participation of the complement cascade or Fc receptor (FcR)—bearing cells);
`
`and (2) effector functions that operate independently of antigen binding (these functions confer
`
`persistence in the circulation and the ability to be transferred across cellular barriers by
`
`transcytosis). Ward and Ghetie, Therapeutic Immunology 2:77-94 (1995).
`
`While binding of an antibody to the requisite antigen has a neutralizing effect that might
`
`prevent the binding of a foreign antigen to its endogenous target (e.g. receptor or ligand), binding
`
`alone may not remove the foreign antigen. To be efficient in removing and/or destructing foreign
`
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`antigens, an antibody should be endowed with both high affinity binding to its antigen, and
`efficient effector functions.
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`Fc recegtor (Fch binding
`
`The interaction of antibodies and antibody-antigen complexes with cells of the immune
`
`system effects a variety of responses, including antibody-dependent cell-mediated cytotoxicity
`
`(ADCC) and complement dependent cytotoxicity (CDC) (reviewed in Daéron, Annu. Rev.
`
`Immunol. 15:203-234 (1997); Ward and Ghetie, Therapeutic Immunol. 2:77-94 (1995); as well
`
`as Ravetch and Kinet, Annu. Rev. Immunol. 9:457—492 (1991)).
`
`Several antibody effector functions are mediated by Fc receptors (FcRs), which bind the
`
`Fc region of an antibody. FcRs are defined by their specificity for immunoglobulin isotypes; Fc
`
`receptors for lgG antibodies are referred to as FCyR, for lgE as chR, for lgA as FcorR and so on.
`
`Three subclasses of FoyR have been identified: Fcle (CD64), Fclel (CD32) and Fclell (CD16).
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`Because each FcyR subclass is encoded by two or three genes, and alternative RNA spicing
`
`leads to multiple transcripts, a broad diversity in FcyR isoforms exists. The three genes encoding
`
`the Fle subclass (FcleA, FoleB and FleC) are clustered in region 1q21.1 of the long arm
`
`of chromosome 1; the genes encoding Fclel isoforms (FoyRIIA, FelelB and FolelC) and the
`
`two genes encoding Fclell (FolellA and FolellB) are all clustered in region 1q22. These
`
`different FcR subtypes are expressed on different cell types (reviewed in Ravetch and Kinet,
`
`Annu. Rev. Immunol. 9:457-492 (1991)). For example, in humans, FclellB is found only on
`
`neutrophils, whereas FoleliA is found on macrophages, monocytes, natural killer (NK) cells, and
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`a subpopulation of T-cells. Notably, FclellA is the only FcR present on NK cells, one of the cell
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`types implicated in ADCC.
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`FoyR], Fclel and Fclell are immunoglobulin superfamily (IgSF) receptors; Fcle has
`
`three lgSF domains in its extracellular domain, while FcYRll and Fclell have only two lgSF
`domains in their extracellular domains.
`
`Another type of PC receptor is the neonatal Fc receptor (FcRn). FcRn is structurally
`
`similar to major histocompatibility complex (MHC) and consists of an cx-chain noncovalently bound
`
`to BZ-microglobulin.
`
`The binding site on human and murine antibodies for FcyR have been previously mapped
`
`to the so-called "lower hinge region" consisting of residues 233-239 (EU index numbering as in
`
`Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service,
`
`National institutes of Health, Bethesda, MD. (1991)). Woof et al. Molec. Immunol. 23:319-330
`
`(1986); Duncan et al. Nature 3322563 (1988); Canfield and Morrison, J. Exp. Med. 173:1483-
`
`1491 (1991 ); Chappel et al., Proc. Natl. Acad. Sci USA 88:9036-9040 (1991 ). Of residues 233—
`
`239, P238 and 8239 have been cited as possibly being involved in binding, but these two
`
`residues have never been evaluated by substitution or deletion.
`
`Other previously cited areas possibly involved in binding to FcyR are: G316-K338 (human
`
`lgG) for human Fcle (by sequence comparison only; no substitution mutants were evaluated)
`
`(Woof et al. Molec. Immunol. 23:319-330 (1986)); K274—R301 (human lgG1)for human Fclell
`
`(based on peptides) (Sarmay et al. Molec. Immunol. 21 :43-51 (1984)); Y407-R416 (human lgG)
`
`for human Fclell (based on peptides) (Gergely et al. Biochem. Soc. Trans. 12:739-743 (1984));
`
`as well as N297 and E318 (murine lgGZb) for murine Fclel (Lund et al., Molec. Immunol.,
`
`29:53-59 (1992)).
`
`Pr0331 in lgG3 was changed to Ser, and the affinity of this variant to target cells analyzed.
`
`The affinity was found to be six—fold lower than that of unmutated lgG3, indicating the involvement
`
`of Pro331 in Fle binding. Morrison 91‘ al., Immunologist, 2:1 19—124 (1994); and Canfield and
`
`Morrison, J. Exp. Med. 173:1483-91 (1991).
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`C1q binding
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`C1q and two serine proteases, C1r and C15, form the complex C1, the first component
`
`of the complement dependent cytotoxicity (CDC) pathway. C1q is a hexavalent molecule with a
`
`molecular weight of approximately 460,000 and a structure likened to a bouquet of tulips in which
`
`six collagenous “stalks” are connected to six globular head regions. Burton and Woof, Advances
`
`in Immunol. 51 :1—84 (1992). To activate the complement cascade, it is necessary for C1q to bind
`
`to at least two molecules of lgG1, lgG2, or lgG3 (the consensus is that lgG4 does not activate
`
`complement), but only one molecule of lgM, attached to the antigenic target. Ward and Ghetie,
`
`Therapeutic Immunology 2:77-94 (1995) at page 80.
`
`Based upon the results of chemical modifications and crystallographic studies, Burton et
`
`al.
`
`(Nature, 288:338-344 (1980)) proposed that
`
`the binding site for
`
`the complement
`
`subcomponent C1q on lgG involves the last two (C—terminal) B-strands of the CH2 domain. Burton
`
`later suggested (Molec. Immunol., 22(3):161-206 (1985)) that the region comprising amino acid
`
`residues 318 to 337 might be involved in complement fixation.
`
`Duncan and Winter (Nature 332:738—40 (1988)), using site directed mutagenesis, reported
`
`that Glu318, Lys320 and Ly3322 form the binding site to C1q. The data of Duncan and Winter
`
`were generated by testing the binding of a mouse lgG2b isotype to guinea pig C1q. The role of
`
`Glu318, Lys320 and Lys322 residues in the binding of C1q was confirmed by the ability of a short
`
`synthetic peptide containing these residues to inhibit complement mediated lysis. Similar results
`
`are disclosed in U.S. Patent No. 5,648,260 issued on July 15, 1997, and U.S. Patent No.
`
`5,624,821 issued on April 29, 1997.
`
`The residue Pr0331 has been implicated in C1q binding by analysis of the ability of human
`
`lgG subclasses to carry out complement mediated cell lysis. Mutation of Ser331 to Pro331 in
`
`lgG4 conferred the ability to activate complement. (Tao et al., J. Exp. Med., 178:661-667 (1993);
`
`Brekke et al., Eur. J. Immunol., 24:2542-47 (1994)).
`
`From the comparison of the data of the Winter group, and the Tao et al. and Brekke et al.
`
`papers, Ward and Ghetie concluded in their review article that there are at least two different
`
`regions involved in the binding of C1q: one on the B-strand of the CH2 domain bearing the
`
`Glu318, Lys320 and Lys322 residues, and the other on a turn located in close proximity to the
`
`same B-strand, and containing a key amino acid residue at position 331.
`
`Other reports suggested that human lgG1 residues Leu235, and Gly237, located in the
`
`lower hinge region, play a critical role in complement fixation and activation. Xu et al., Immunol.
`
`150:152A (Abstract) (1993). W094/29351 published December 22, 1994 reports that amino acid
`
`residues necessary for C1q and FcR binding of human lgG1 are located in the N-terminal region
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`of the CH2 domain, i.e. residues 231 to 238.
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`lt has further been proposed that the ability of lgG to bind C1q and activate the
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`complement cascade also depends on the presence, absence, or modification of the
`
`carbohydrate moiety positioned between the two CH2 domains (which is normally anchored at
`
`Asn297). Ward and Ghetie, Therapeutic Immunology 2:77-94 (1995) at page 81.
`
`SUMMARY OF THE INVENTION
`
`The present invention provides a variant of a parent polypeptide comprising an Fc region,
`
`which variant mediates antibody-dependent cell-mediated cytotoxicity (ADCC) in the presence
`
`of human effector cells more effectively, or binds an Fc gamma receptor (FcyR) with better affinity,
`
`than the parent polypeptide and comprises at least one amino acid modification in the Fc region.
`
`The polypeptide variant may, for example, comprise an antibody or an immunoadhesin. The Fc
`
`region of the parent polypeptide preferably comprises a human Fc region; e.g., a human lgG1,
`
`lgG2, lgG3 or lgG4 Fc region. The polypeptide variant preferably comprises an amino acid
`
`modification (e.g. a substitution) at any one or more of amino acid positions 256, 290, 298, 312,
`
`326, 330, 333, 334, 360, 378 or 430 of the Fc region, wherein the numbering of the residues in
`
`the Fc region is that of the EU index as in Kabat.
`
`In addition, the invention provides a polypeptide comprising a variant Fc region with
`
`altered Fc gamma receptor (FcyR) binding affinity, which polypeptide comprises an amino acid
`
`modificatibn at any one or more of amino acid positions 238, 239, 248, 249, 252, 254, 255, 256,
`
`258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295,
`
`296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334,
`
`335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437,
`
`438 or 439 of the Fc region, wherein the numbering of the residues in the Fc region is that of the
`
`EU index as in Kabat. The variant Fc region preferably comprises a variant human lgG Fc region,
`
`e.g., a variant human lgG1, tgG2, lgG3 or lgG4 Fc region.
`
`In this respect, it is noted that, in the
`
`work in the above-cited art where the parent polypeptide had a non—human murine Fc region,
`
`different residues from those identified herein were thought to impact FcR binding. For example,
`
`in the murine lgG2b/murine chRll system. lgG E318 was found to be important for binding (Lund
`
`et al. Molec. Immunol. 27(1):53-59 (1992)), whereas E318A had no effect in the human
`
`lgG/human Felel system (Table 6 below).
`
`In one embodiment, the polypeptide variant with altered FcyR binding activity displays
`
`reduced binding to an FcyR and comprises an amino acid modification at any one or more of
`
`amino acid positions 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293,
`
`294, 295, 296, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389,
`
`414, 416, 419, 434, 435, 437, 438 or 439 of the Fc region, wherein the numbering of the residues
`
`in the Fc region is that of the EU index as in Kabat.
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`For example, the polypeptide variant may display reduced binding to an Fcle and
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`comprise an amino acid modification at any one or more of amino acid positions 238, 265, 269,
`
`270, 327 or 329 of the Fc region, wherein the numbering of the residues in the Fc region is that
`
`of the EU index as in Kabat.
`
`The polypeptide variant may display reduced binding to an FcleI and comprise an amino
`
`acid modification at any one or more of amino acid positions 238, 265, 269, 270, 292, 294, 295,
`
`298, 303, 324, 327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 of the Fc region,
`
`wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat.
`
`The polypeptide variant of interest may display reduced binding to an Fclell and
`
`comprise an amino acid modification at one or more of amino acid positions 238, 239, 248, 249,
`
`252, 254, 265, 268, 269, 270, 272, 278, 289, 293, 294, 295, 296, 301, 303, 322, 327, 329, 338,
`
`340, 373, 376, 382, 388, 389, 416, 434, 435 or 437 of the Fc region, wherein the numbering of
`
`the residues in the Fc region is that of the EU index as in Kabat.
`
`In another embodiment, the polypeptide variant with altered FcyR binding affinity displays
`
`improved binding to the FcyR and comprises an amino acid modification at any one or more of
`
`amino acid positions 255, 256, 258, 267, 268, 272, 276, 280, 283, 285, 286, 290, 298, 301, 305,
`
`307, 309, 312, 315, 320, 322, 326, 330, 331, 333, 334, 337. 340, 360, 378, 398 or 430 of the Fc .
`
`region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat.
`
`For example, the polypeptide variant may display increased binding to an FclelI and,
`
`optionally, may further display decreased binding to an FcyRil. An exemplary such variant
`
`comprises amino acid modification(s) at position(s) 298 and/or 333 of the Fc region, wherein the
`
`numbering of the residues in the Fc region is that of the EU index as in Kabat.
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`The polypeptide variant may display increased binding to an Fclel and comprise an
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`amino acid modification at any one or more of amino acid positions 255, 256, 258, 267, 268, 272,
`276, 280, 283,285, 286,290, 301, 305, 307, 309, 312, 315, 320, 322, 326, 330, 331, 337, 340,
`
`378, 398 or 430 of the Fc region, wherein the numbering of the residues in the Fc region is that
`
`of the EU index as in Kabat. Such polypeptide variants with increased binding to an Fclel may
`
`optionally further display decreased binding to an FclelI and may, for example, comprise an
`
`amino acid modification at any one or more of amino acid positions 268, 272, 298, 301, 322 or
`
`340 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU
`
`index as in Kabat.
`
`The invention further provides a polypeptide comprising a variant Fc region with altered
`
`neonatal Fc receptor (FcRn) binding affinity, which polypeptide comprises an amino acid
`
`modification at any one or more of amino acid positions 238, 252, 253, 254, 255, 256, 265, 272,
`
`286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380,382, 386. 388,
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`400, 413, 415, 424, 433, 434, 435, 436, 439 or 447 of the Fc region, wherein the numbering of
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`the residues in the Fc region is that of the EU index as in Kabat. Such polypeptide variants with
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`reduced binding to an FcRn may comprise an amino acid modification at any one or more of
`
`amino acid positions 252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 435, 436, 439 or 447
`
`of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index
`as in Kabat. The above-mentioned polypeptide variants may, alternatively, display increased
`
`binding to FcRn and comprise an amino acid modification at any one or more of amino acid
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`positions 238,256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378,
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`380, 382, 413, 424 or 434 of the Fc region, wherein the numbering of the residues in the Fc region
`is that of the EU index as in Kabat.
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`The invention also provides a composition comprising the polypeptide variant and a
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`physiologically or pharmaceutically acceptable carrier or diluent. This composition for potential
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`therapeutic use is sterile and may be lyophilized.
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`Diagnostic and therapeutic uses for the polypeptide variants disclosed herein are
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`contemplated.
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`In one diagnostic application, the invention provides a method for determining the
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`presence of an antigen of interest comprising exposing a sample suspected of containing the
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`antigen to the polypeptide variant and determining binding of the polypeptide variant to the
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`sample. In one therapeutic application, the invention provides a method of treating a mammal
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`suffering from or predisposed to a disease or disorder, comprising administering to the mammal
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`a therapeutically effective amount of a polypeptide variant as disclosed herein, or of a composition
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`comprising the polypeptide variant and a pharmaceutically acceptable carrier.
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`The invention further provides: isolated nucleic acid encoding the polypeptide variant; 3
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`vector comprising the nucleic acid, optionally, operably linked to control sequences recognized
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`by a host cell transformed with the vector; a host cell containing the vector; a method for
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`producing the polypeptide variant comprising culturing this host cell so that the nucleic acid is
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`expressed and, optionally, recovering the polypeptide variant from the host cell culture (e.g. from
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`the host cell culture medium).
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`The invention further provides a method for making a variant Fc region with altered Fc
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`receptor (FcR) binding affinity, or altered antibody—dependent cell-mediated cytotoxicity (ADCC)
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`activity, comprising:
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`(a)
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`introducing one or more amino acid modifications into an Fc region of a parent polypeptide
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`in order to generate a variant Fc region;
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`(b)
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`determining binding of the variant Fc region to an FcR, or determining ADCC activity of
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`the variant Fc region.
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`Step (b) of the method may comprise determining binding of the variant Fc region to one
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`or more FcRs in vitro. Moreover, the method may result in the identification of a variant Fc region
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`with improved FcR binding affinity, or with improved ADCC activity, in step (b) thereof. Where
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`step (b) comprises determining binding of the Fc region to an FcR, the FcR may, for example, be
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`human Fc gamma receptor ill (Folell). Where step (b) comprises determining binding of the
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`variant Fc region to at least two different FcRs, the FcRs tested preferably include human Fc
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`gamma receptor ll (chRll) and human Fc gamma receptor Ill (Fclell).
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`Brief Description of the Drawings
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`Figure 1 is a schematic representation of a native lgG. Disulfide bonds are represented
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`by heavy lines between CH1 and CL domains and the two CH2 domains. V is variable domain;
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`C is constant domain; L stands for light chain and H stands for heavy chain.
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`Figure 2 shows C1q binding of wild type (wt) C288 antibody; 0288 antibody with a human
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`lgG2 constant region (lgG2); and variants K322A. K320A and E318A.
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`Figure 3 depicts C1q binding of variants P331A, P329A and K322A.
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`Figures 4A and 4B depict the amino acid sequences of E27 anti-lgE antibody light chain
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`(Fig. 4A; SEQ ID NO:1)and heavy chain (Fig. 4B: SEQ lD N022).
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`Figure 5 is a schematic diagram of the “immune complex" prepared for use in the FcR
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`assay described in Example 1. The hexamer comprising three anti-lgE antibody molecules (the
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`“Fc region-containing polypeptide”) and three lgE molecules (the “first target molecule") is shown.
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`lgE has two “binding sites” for the anti-lgE antibody (E27) in the Fc region thereof. Each lgE
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`molecule in the complex is further able to bind two VEGF molecules (“the second target
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`polypeptide"). VEGF has two “binding sites” for lgE.
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`Figure 6 shows C1 q binding results obtained for variants D270K and D270V compared
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`to wild type C288.
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`Figure 7 depicts complement dependent cytotoxicity (CDC) of variants 0270K and D270V,
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`compared to wild type 0288.
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`Figure 8 shows C1q binding ELISA results for 293 cell-produced wild type 0288 antibody
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`(293-Wt-C288), CHO—produced wild type C288 antibody (CHO-Wt—C288) and various variant
`antibodies.
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`Figure 9 shows C1q binding ELISA results obtained for wild type (wt) 0288 and various
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`variant antibodies as determined in Example 3.
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`Figure 10 depicts the three—dimensional structure of a human lgG Fc region, highlighting
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`residues: Asp270, Lys326, Pro329, Pro331, Ly3322 and Glu333.
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`Figure 11 shows C1q binding ELlSA results obtained for wild type C288 and various
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`variant antibodies as determined in Example 3.
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`Figure 12 shows C1q binding ELISA results obtained for wild type 02B8 and double
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`variants, K326M-E333S and K326A—E333A.
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`Figure 13 shows CDC of wild type C288 and double variants, K326M-E3338 and K326A—
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`E333A.
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`Figure 14 depicts C1q binding ELISA results obtained for 0288 with a human lgG4 (lgG4),
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`wild type 0288 (Wt-0288), C238 with a human lgG2 constant region (lgG2), and variant
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`antibodies as described in Example 3.
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`Figures 15A and 158 show binding patterns for parent antibody (E27) to chRllB and
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`FolellA. Figure 15A shows the binding pattern for the humanized anti-lgE E27 lgG1 as a
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`monomer (open circles), hexamer (closed squares), and immune complex consisting of multiple
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`hexamers (closed triangles) to a recombinant GST fusion protein of the human chRllB (CD32)
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`receptor or subunit. The hexameric complex (closed squares) was formed by the mixture of equal
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`molar concentrations of E27 (which binds to the Fc region of human lgE) and a human myeloma
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`lgE. The hexamer is a stable 1.1 kD complex consisting of 3 lgG molecules (150 kD each) and
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`3 lgE molecules (200 kD each). The immune complex (closed triangles) was formed sequentially
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`by first mixing equal molar concentrations of E27 and recombinant anti—VEGF lgE (human lgE
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`with Fab variable domains that bind human VEGF) to form the hexamer. Hexamers were then
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`linked to form an immune complex by the addition of 2x molar concentration of human VEGF. a
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`44 kD homodimer which has two binding sites for the anti-VEGF lgE per mole of VEGF. Figure
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`158 shows the binding pattern to a recombinant GST fusion protein of the human chRlllA
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`(CD16) receptor or subunit.
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`Figure 16A shows the binding of immune complexes using different antigen—antibody pairs
`to recombinant GST fusion protein of the chRllA receptor or subunit. Figure 16B shows the
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`binding of the same antigen-antibody pairs to the GST fusion protein of the FelellA receptor or
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`subunit. Closed circles represent binding of human lgEzanti-IgE E27 lgG1; open circles represent
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`binding of human VEGthumanized anti—VEGF lgG1.
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`Figure 17 summarizes differences in binding selectivity of some alanine variants between
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`the different chRs. Binding of alanine variants at residues in the CH2 domain of anti-lgE E27
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`IgG‘i are shown to FclelA, chRllB, and chRlllA. Type 1 abrogates binding to all three
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`receptors: D278A (265 in EU numbering). Type 2 improves binding to chRIIA and chRllB,
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`while binding to chRlllA is unaffected: 8280A (267 in EU numbering). Type 3 improves binding
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`to chRllA and FcleIB, but reduces binding to chRlllA: H281A (268 in EU numbering). Type
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`4 reduces binding to FelelA and chRllB, while improving binding to FolellA: 8317A (298 in
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`EU numbering). Type 5 improves binding to chRlllA, but does not affect binding to chRllA and
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`chRllB: E352A, K353A (333 and 334 in EU numbering).
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`Figures 18A and 188 compare the FolellA protein/protein assay and CHO GPl-chRlllA
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`cell based assay, respectively. Figure 18A illustrates binding of selected alanine variants to
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`FolellA-GST fusion protein. S317A (298 in EU numbering) and SB17A/K353A (298 and 334 in
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`EU numbering) bind better than E27 wildtype, while D278A (265 in EU numbering) almost
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`completely abrogates binding. Figure 18B illustrates that a similar pattern of binding is found on
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`CHO cells expressing a recombinant GPl—Iinked form of FolellA.
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`Figures 19A and 198 compare the FclelB protein/protein assay and CH0 GPl-F0lelB
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`cell based assay, respectively. Figure 19A illustrates binding of selected alanine variants to
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`FolelB-GST fusion protein. H281A (268 in EU numbering) binds better than E27 wildtype while
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`S317A (298 in EU numbering) shows reduced binding. Figure 193 illustrates that a similar pattern
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`of binding is found on CHO cells expressing a recombinant membrane bound form of chRllB.
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`Figure 20 shows single alanine substitutions in the CH2 domain of anti—HER2 lgG1
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`(HERCEPTIN®) that influence chRlllA binding in both the protein-protein and cell—based assays
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`alter the ability to bind to FoleilA on peripheral blood mononuclear cell (PBMC) effector cells.
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`Recombinant humanized anti-HER2 (HERCEPTIN®), which binds to HERZ-expressing SK-BR-3
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`breast
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`tumor cells, was preincubated with 51Cr—labeled SK—BR—3 cells for 30 minutes
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`(opsonization) at 100 ng/ml (filled circles) and 1.25 ng/ml (filled squares). Keeping the SK—BR-3
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`tumor target cell concentration constant, the ratio of effector cells was increased from O to 100.
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`The spontaneous cytotoxicity in the absence of antibody (hatched squares) was 20% at an
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`effectorctarget (E:T) ratio of 100:1. A single alanine mutation that did not affect FoyRIIIA binding,
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`variant G31 = R309A (292 in EU numbering), did not effect ADCC (filled triangles). A single
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`alanine mutation that only slightly increased binding to FCVRlllA, variant GBO = K307A (290 in EU
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`numbering), also showed slightly improved ADCC (i.e., a 1.1 fold improvement in ADCC activity,
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`calculated as area under the curve) at 1.25 ng/ml at all E:T ratios (filled diamonds) compared to
`wildtype antibody at 1.25-ng/ml (filled square). A single alanine mutation that decreased binding
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`to chRlllA, variant G34 = Q312A (295 in EU numbering), also showed decreased ADCC activity
`
`(filled inverted triangles).
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`Figure 21 illustrates that a single alanine mutation which had the most improved binding
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`to FolellA, variant 636 = 8317A (298 in EU numbering), in the protein-protein and cell-based
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`assays also showed the most improvement in ADCC (filled triangles) among the variants
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`co