(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`3 December 2015 (03.12.2015)
`
`WIPOIPCT
`
`\Zo
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`(10) International Publication Number
`WO 2015/184009 Al
`
`6)
`
`International Patent Classification:
`
`C07K 16/28 (2006.01)
`
`(21)
`
`International Application Number:
`
`PCT/US2015/032745
`
`(22)
`
`International Filing Date:
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`(25)
`
`Filing Language:
`
`(26)
`
`Publication Language:
`
`27 May 2015 (27.05.2015)
`
`English
`
`English
`
`US
`US
`US
`US
`US
`
`(30)
`
`(71)
`
`(72)
`(71)
`
`Priority Data:
`62/003,136
`62/003,104
`62/003,908
`62/020,199
`62/110,338
`
`27 May 2014 (27.05.2014)
`27 May 2014 (27.05.2014)
`28 May 2014 (28.05.2014)
`2 July 2014 (02.07.2014)
`30 January 2015 (30.01.2015)
`
`Applicant: ACADEMIA SINICA; 128 Academia Road,
`Scction 2, Nankang, Taipei, 11529 (CN).
`
`Inventor; and
`Applicant : WONG,Chi-Huey [US/US]; P.O. Box 8154,
`Rancho Santa Fe, California 92067 (US).
`
`(72)
`
`(74)
`
`(81)
`
`Inventors: WU, Chung-Yi; No. 78-1, Dongshi Street, Xi-
`Zhi District, New Taipei City, 221 (CN). MA, Che; c/o
`Academia Sinica,
`128 Academia Road, Section 2,
`Nankang, Taipei, 11529 (CN).
`
`Agent: NORTON, Vicki; Duane Morris LLP, Suite 2900,
`750 B Street, San Diego, California 92101 (US).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CII, CL, CN, CO, CR, CU, CZ, DE, Dk, DM,
`DO, DZ, EC, EE, EG, ES, FL, GB, GD, GE, GH, GM, GT,
`HN, HR, HU,ID,IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG,
`MK,MN, MW,MX, MY, MZ, NA, NG, NI, NO, NZ, OM,
`PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC,
`SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ,
`TZ, UG, ZM, ZW), Furasian (AM, AZ, BY, KG, KZ, RU,
`
`[Continued on next page]
`
`(54) Titles COMPOSITIONS AND METHODS RELATING TO UNIVERSAL GLYCOFORMS FOR ENHANCED ANTIBODY
`EFFICACY
`
`
`
`Endoglycosidase
`and
`
`Heterogeneous Antibodies
`Move than 50 glycans on Fe Asn297
`
`Ae HS
`Eee
`28-NSCT
`SOME OCS
`
`
`
`Fig. 1b
`
`
`
`.
`
`orfucosidase ¢
`
`Slycosynthase, es Ry, aH
`ermal niteta
`B
`Homogencous antibody with
`optimized glyenn at the Fe region
`Therapeutic activity improvement
`
`
`
`
`(57) Abstract: The present disclosure relates to glycopro-
`teins, particularly monoclonal antibodies, comprising a gly-
`coengincered Fe region, wherein said Fe region comprises an
`optimized N-glycan having the
`structure of Sia,(o2-
`6)Gal,GlceNAc;Man3GlcNAc2. The glycoengineered Fe re-
`gion binds FceyRILA or FcyRIA with a greateraffinity, relat-
`ive to comparable monoclonal antibodies comprising the
`wild-type Fc region. The monoclonal antibodies of the inven-
`tion are particularly useful in preventing, treating, or amelior-
`ating one or more symptoms associated with a disease, dis-
`order, or infection where an enhanced efficacy of effectorcell
`function (e.g., ADCC) mediated by FcyR is desired, e.g., can-
`cer, autoimmune, infectious disease, and in enhancing the
`therapeutic efficacy of therapeutic antibodies the effect of
`whichis mediated by ADCC.
`
`Singapore Exhibit 2011
`Singapore Exhibit 2011
`Lassen v. Singapore et al.
`Lassen v. Singaporeetal.
`PGR2019-00053
`PGR2019-00053
`
`
`
`wo2015/184009A1[IUIIMIIIN0TMOCRITNTANATIAUTAATTMAA
`
`

`

`WO 2015/184009 AIQUEIAATMATA TACTYA
`
`TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE,
`DK, EE, ES, FL FR, GB, GR, HR, HU,IE,IS, IT, LT,
`LU, LV, MC, MK, MT, NL, NO,PL, PT, RO, RS, SE,
`SL, SK, SM, TR), OAPI (BF, BJ, CF, CG, Cl, CM, GA,
`GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG).
`
`Published:
`
`with international search report (Art. 21(3))
`
`before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments (Rule 48.2(h))
`
`

`

`WO 2015/184009
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`PCT/US2015/032745
`
`COMPOSITIONS AND METHODS RELATING TO UNIVERSAL GLYCOFORMS
`
`FOR ENHANCED ANTIBODY EFFICACY
`
`RELATED APPLICATIONS
`
`[0001] This applications claims the benefit of priority to US provisional applications USSerial
`
`No. (USSN)62/003, 136, filed May 27, 2014, USSN 62/003,104, filed May 27, 2014, USSN
`
`62/003,908, filed May 28, 2014, USSN 62/020,199,filed July 2, 2014, and USSN 62/110,338,
`
`filed January 30, 2015. The contents of each of which is hereby incorporated by referenceinits
`
`entirety.
`
`BACKGROUND OF THE INVENTION
`
`[0002] Antibody-based therapies have a proven record of efficacy against many diseases
`
`including inflammatory disorders, cancers, infectious diseases, and solid organ transplant
`
`rejection. Currently more than 40 therapcutic monoclonal antibodics (mAbs) are approved for
`
`clinical use in USA, EU and several other countries. Most of them are for therapy of cancer and
`
`immune diseases. Examples of therapeutic antibodies with anti-tumoractivities include anti-
`
`CD20, anti-Her2, anti-EGFR,anti-CD40, anti-CTLA-4, and anti-PD-1 antibodies.
`
`[0003] Most of therapeutic antibodies are monoclonal and prepared by the hybridoma
`
`technology in which transgenic humanized mice were incorporated to express murine/human
`
`chimeric or humanized antibodies to avoid undesired immunological responses derived from
`
`species difference. Recently, the development of fully humanantibodies has become a major
`
`trend and its impressive progress is beneficial from the utilization of phage-displayed antibody
`
`libraries or single B cells.
`
`[0004] Like many other mammalian proteins, antibodies are heterogeneously glycosylated, and
`
`the glycosylation in the Fe region has becn an important issuc in the developmentofcfficacious
`
`and safe therapeutic monoclonal antibodies because the glycan can significantly affect the
`
`antibody’s activity through interaction with the Fc receptors. Consequently, there is a need for
`
`the development of homogeneous monoclonal antibodies with well-defined Fc-glycan to
`
`understand these interactions and to improvethe safety and efficacy in medication. Towardthis
`
`goal, it has been reported that the removal of the core fucose residue would enhance the
`
`antibody-dependentcellular cytotoxicity (ADCC)activity of IgGs due to the increased
`
`interaction between Fc-glycan and human FcyRIIIa receptor. The two FDA approved glyco-
`engineered antibodies, mogamulizumab (POTELLIGENT*) and obinutuzuman (GA101), are
`defucosylated antibodies in which POTELLIGENT®was produced by the FUT8 knockout CHO
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`WO 2015/184009
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`PCT/US2015/032745
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`cell line and GA101 was from the GnT-III overexpressing system. In addition, more FcyII]a was
`
`expressed on the monocytes of long-term RA, and the tendency of more fucosylation was also
`
`found in the IgG heavy chain of RA patients, implying the possibility of RA treatment and
`
`remission with afucosylated pharmaceutical antibodies, which not only neutralize
`
`proinflammatory cytokines but also compete with autologous autoantibodies for FeyII[a.
`
`[0005] Thus, it is of great interest to generate therapeutic monoclonal antibodies with optimized
`
`Fe glycoforms.
`
`SUMMARYOF THE INVENTION
`
`[0006] The present disclosure is based on the discovery of glyco-optimized Fe for monoclonal
`
`antibodies, specifically a homogeneous population of monoclonal antibodies (“glycoantibodies’’).
`
`The optimized glycoform exhibits an enhanced efficacy of effector cell function (e.g., ADCC).
`
`[0007] The term “glycoantibodies” was coined by the inventor, Dr. Chi-Huey Wong,to refer to
`
`a homogencouspopulation of monoclonal antibodics (preferably, thcrapcutic monoclonal
`
`antibodies) having a single, uniform N-ghycan on Fe. The individual glycoantibodies comprising
`
`the homogeneous population are substantially identical, bind to the same epitope, and contain
`
`the same Fe glycan with a well-defined glycan structure and sequence.
`
`[0008] "Substantially identical” means the objects being compared have such close resemblance
`
`as to be essentially the same - as understood by one having ordinary skill in the art.
`
`"Substantially identical" encompasses "identical".
`
`[0009] As used herein, the term “glycoantibodies” (“GAbs”) refers to a homogeneous
`
`population of IgG molecules having the same N-glycan on Fe. The term “glycoantibody”
`
`(“GAb”) refers to an individual IgG molecule in the glycoantibodies.
`
`[0010] Accordingly, one aspect of the present disclosure relates to a composition of a
`
`homogencous population of monoclonal antibodics comprisimg a single, uniform N-glycan on Fe,
`
`wherein the structure is an optimized N-glycan structure for enhancing the efficacy of effector
`
`cell function.
`
`[0011] In preferred embodiments, the N-glycan is attached to the Asn-297 of the Fe region.
`
`[0012] In preferred embodiments, wherein the N-glycan consists of the structure of Siaa{a2-
`
`6)GalbGIcNAco>ManiGleNAc».
`
`[0013] The glycoantibodies described herein may be produced in vitro. The glycoantibodies
`
`may be generated by Fe glycoengineering. In certain embodiments, the glycoantibodies are
`
`enzymatically or chemoenzymatically engineered from the monoclonal antibodies obtained by
`
`mammalian cell culturing.
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`[0014] In some embodiments, the Fc region of the glycoantibodies described herein exhibits an
`
`increased binding affinity for FeyRIA or FeyRILA relative to a wild-type Fe region in the
`
`corresponding monoclonal antibodies.
`
`[0015] In some embodiments, the glycoantibodies described herein exhibit an enhanced
`
`antibody-dependent cell mediated cytotoxicity (ADCC)activity relative to wild-type
`
`immunoglobulins.
`
`[0016] In some embodiments, the glycoantibodies are selected from a group consisting of
`
`human IgG1, IgG2, IgG3, and IgG4.
`
`[0017] The monoclonal antibodies may be humanized, human or chimeric.
`
`[0018] The glycoantibodies described herein may bind to an antigen associated with cancers,
`
`autoimmunedisorders, inflammatory disorders or infectious diseases.
`
`[0019] In some embodiments, the glycoantibody described herein is a glycoengineered anti-
`
`CD20. In some cxamples, the glycoantibody described herein is a glycocngineered Rituximab
`
`(Rituxan®),
`
`[0020] In some embodiments, the glycoantibody described herein is a glycoengineered anti-
`
`HER2. In some examples, the glycoantibody described herein is a glycoengineered
`
`Trastuzumab (Herceptin®).
`
`[0021] In some embodiments, the glycoantibody described herein is a glycoengineered anti-
`
`TNFo. In some examples, the glycoantibody described herein is a glycoengineered
`
`Adalimumab (Humira®).
`
`[0022] In some embodiments, the glycoantibody described herein is a glycoengineered F16
`
`antibodies.
`
`[0023] Another aspect of the present disclosure features a pharmaceutical composition
`
`comprising a composition of glycoantibodics described herein and a pharmaccutically
`
`acceptable carrier. The pharmaceutical composition may be used in therapeutics such as
`
`oncology, autoimmunedisorders, inflammatory disorders and infectious diseases.
`
`[0024] In some embodiments, the pharmaceutical composition is used for preventing, treating,
`
`or ameliorating one or more symptomsassociated with a disease, disorder, or infection where an
`
`enhancedefficacy of effector cell function (e.g., ADCC) mediated by FcyR is desired, e.g.,
`
`cancer, autoimmune, infectious disease, and in enhancingthe therapeutic efficacy of therapeutic
`
`antibodies the effect of which is mediated by ADCC.
`
`[0025] Disclosed herein also include methods for enhancing antibody-dependent cell mediated
`
`cytotoxicity (ADCC)activity, the method comprising administering to a subject an amount of
`
`glycoantibodies described herein.
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`[0026] Further, disclosed herein include methodsfor preventing, treating, or ameliorating one or
`
`more symptomsassociated with a discasc, disorder, or infection, the method comprising
`
`administering to a subject in need thereof a therapeutically effective amount of the
`
`pharmaceutical composition described herein. The disease, disorder, or infection may be
`
`selected from a group consisting of cancers, autoimmune disorders, inflammatory disorders and
`
`infectious infections.
`
`[0027] Another aspect of the present disclosure features a method for treating a viral disease in a
`
`human subject in need thereof, comprising (a) administering to the subject a first compoundthat
`
`blocks an inhibitory receptor of an NK cell, and (b) administering to the subject a therapeutically
`
`effective amount of the pharmaceutical composition described herein.
`
`[0028] In these treatment methods described herein, the pharmaceutical composition of
`
`glycoantibodies can be administered alone or in conjunction with a second therapeutic agent
`
`such as a second antibody, or a chcmothcrapcutic agent or an immunosuppressive agent.
`
`[0029] This application refers to various issued patent, published patent applications, journal
`
`articles, and other publications, all of which are incorporated herein by reference.
`
`[0030] The details of one or more embodiments of the invention are set forth in the description
`
`below. Other features or advantages of the present invention will be apparent from the following
`
`drawings and detailed description of several embodiments, and also from the appending claims.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0031] Figure 1. shows(a) general strategy for the preparation of homogeneousantibody
`
`through remodeling of the glycan structures on the Fe region of IgG1 (b).
`
`[0032] Figure 2. showsthat antibody dependent B-cell depletion activity of various
`
`glycoengineered Rituximab. The depletion of human B cells was conducted using freshly
`
`prepared human PBMCcells and analyzed on FACS, based on the CD19+ CD2- B cells. (A)
`
`Compared to a series of different glycoengineered Riruximabs, the 2,6-NSCT Rituximab
`
`showed higher depletion ability. (B) In the whole blood B-cell depletion activity of 10 donors,
`
`the 2,6-sialylated Rituximab wassignificantly more active than the non-treated Rituximab with a
`
`p value of 0.0016, whereas the mono-GlcNAc Rituximab showed the lowest activity. (C) The
`
`prepared Rituximab-resistant cells of Ramos and Raji express lower level of CD20 oncell
`
`surface. (D, E) The 2,6-NSCT Rituximab showed a remarkable ADCCefficacy towards both
`
`normal andresistant cells, whereas non-treated antibody dramatically lostits activity towards
`
`resistant strains.
`
`[0033] Figure 3. shows that ECSO of glycoengineered Herceptin in V158 FcyRII]a mediated
`
`ADCCreporter bioassay. Experiments were performed underE/T ratio of 6 tol with SKBR3 as
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`target cells and V158 FcyRIIa engineered Jurkat as effector cells. All data shown in the same
`
`graph were cxpcriments done in the same microplate and the same batch of cffector cells; bars
`
`of 95% confidence interval were plotted. (A) afucosylated Herceptin G& and commercial
`
`Herceptin showed a similar ADCCeffect, illustrating that the defucosylation advantage of anti-
`
`FeyRIIa is lost in the afucosylated Herceptin G8. (B) Bisected and its non-bisected analogue
`
`Herceptin, G9 and G4 showed similar ECS0 values, indicating that no better bisected glycan
`
`mediated ADCC function was observed in this assay. (C) Compared to glycoengineered
`
`Herceptin G1 with two galactose terminals, no significant ECS0 change in the 2,6-sialylated
`
`antibody was observed, whereas the apparent ECS0 increase was shownin the 2,3-sialylated
`
`Herceptin. The results indicated that the 2,3-sialylation on Fe would lowerthe effector cell
`
`activation but the 2,6-linked one would not. Curves of fold induction were results of induced
`
`luminescence divided by induction of no antibody control. (D) Samples with lowest EC50 in
`
`graph (A) to (C) were chosen and compared to commercial Herceptin. All samples demonstrated
`
`better activity in this ADCC reporter bioassay.
`
`[0034] Figure 4. showsthat anti-influenza antibody FI6 with a modified homogeneous SCT
`
`glycan attached to its Fe Asn297 (FI6m) significantly showed an enhancementof its ADCC
`
`activity and prophylactically protects mice from a lethal dose of HIN1 virus challenge. (a)
`
`Cytotoxicity is represented as the percentage of lysed HEK293T cells (target cells) expressed
`
`with influenza H| hemagglutinin (HA) (A/California/07/09) when incubated with PBMCs
`
`(effector cells) and various concentrations of antibodies. (b) ADCC activity was shownas fold
`
`increases of bioluminescence from a luciferase reporter assay that gave signals when ADCC
`
`signaling nuclear factor of activated T-cell pathway was activated. HA expressed HEK293T
`
`cells (target cells) were incubated with NK cells with the said luciferase reporter (effector cells)
`
`and various amounts of anti-influcnza antibody F16 and Fl6m. Curvefitting was done with
`
`software GraphPad Prism in 4PL nonlinear regression. (c) Survival of mice was monitored upon
`
`lethal dose (10 MLDS0O)infection of influenza virus A/California/07/09 (H1N1). Two hours
`
`before infection, each group of mice (N=9) was intraperitoneally given either 2.5 mg/kg of FI6,
`
`Fl6m or PBS,respectively. The FI6 and Fl6m groups had significant survival difference
`
`(p<0.01).
`
`Definitions
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0035]
`
`The practice of the present invention will employ, unless otherwise indicated,
`
`conventional techniques of molecular biology, microbiology, recombinant DNA,and
`
`immunology, which are within the skill of the art. Such techniques are explained fully in the
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`WO 2015/184009
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`literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by
`
`Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning,
`
`VolumesI and I (D. N. Glover ed., 1985); Culture Of Animal Cells (R. I. Freshney, Alan R.
`
`Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical
`
`Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press,
`
`Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,
`
`1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al.
`
`eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds.,
`
`Academic Press, London, 1987); Antibodies: A Laboratory Manual, by Harlowand Lane s
`
`(Cold Spring Harbor Laboratory Press, 1988); and Handbook Of Experimental Immunology,
`
`Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986).
`
`[0036] The term “glycoantibodies” was coined by the inventor, Dr. Chi-Huey Wong,to refer to
`
`a homogencouspopulation of monoclonal antibodics (preferably, therapcutic monoclonal
`
`antibodies) having a single, uniformed glycoform boundto the Fc region. The individual
`
`glycoantibodies comprising the essentially homogeneous population are identical, bind to the
`
`same epitope, and contain the same Fe glycan with a well-defined glycan structure and sequence.
`
`[0037] As used herein, the term “anti-CD20 glycoantibodies” (“anti-CD20 GAbs”) refers to a
`
`homogeneouspopulation of anti-CD20 IgG molecules having the same glycoform on Fe.
`
`[0038] The term “anti-CD20 glycoantibody” (“anti-CD20 GAb”) refers to an individual IgG
`
`antibody molecule in the anti-CD20 glycoantibodies. As used herein, “molecule”can also refer
`
`to antigen binding fragments.
`
`[0039] As used herein, the term “glycan” refers to a polysaccharide, oligosaccharide or
`
`monosaccharide. Glycans can be monomers or polymers of sugar residues and can be linear or
`
`branched. A glycan may include natural sugar residucs (c.g., glucose, N-acctylglucosamine, N-
`
`acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose,etc.)
`
`and/or modified sugars (e.g., 2’-fluororibose, 2’-deoxyribose, phosphomannose, 6' sulfo N-
`
`acetylglucosamine,etc). Glycan is also used herein to refer to the carbohydrate portion of a
`
`glycoconjugate, such as a glycoprotein, glycolipid, glycopeptide, glycoproteome, peptidoglycan,
`
`lipopolysaccharide or a proteoglycan. Glycans usually consist solely of O-glycosidic linkages
`
`between monosaccharides. For example, cellulose is a glycan (or more specifically a glucan)
`
`composedof 8-1,4-linked D-glucose, and chitin is a glycan composed of B-1,4-linked N-acetyl-
`
`D-glucosamine. Glycans can be homo or heteropolymers of monosaccharide residues, and can
`
`be linear or branched. Glycans can be found attached to proteins as in glycoproteins and
`
`proteoglycans. They are generally found on the exterior surface of cells. O- and N-linked
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`glycans are very common in eukaryotes but may also be found, although less commonly, in
`
`prokaryotes. N-Linked glycans are found attached to the R-group nitrogen (N) of asparagine in
`
`the sequon. The sequon is a Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acid
`
`except praline.
`
`[0040] As used herein, the terms “fucose”, “core fucose” and “ core fucose residue” are used
`
`interchangeably and refer to a fucose in a1,6-position linked to the N-acetylglucosamine.
`
`[0041] As used herein, the terms “N-glycan”, “N-linked glycan”, “N-linked glycosylation”, “Fe
`
`glycan”and “Fe glycosylation” are used interchangeably and refer to an N-linked
`
`oligosaccharide attached by an N-acetylglucosamine (GlcNAc) linked to the amide nitrogen of
`
`an asparagine residue in a Fc-containing polypeptide. The term “Fc-containing polypeptide”
`
`refers to a polypeptide, such as an antibody, which comprises an Fc region.
`
`[0042] As used herein, the term “glycosylation pattern” and “glycosylation profile“ are used
`
`interchangeably and refer to the characteristic “fingerprint” of the N-glycan species that have
`
`been released from a glycoprotein or antibody, either enzymatically or chemically, and then
`
`analyzed for their carbohydrate structure, for example, using LC-HPLC, or MALDI-TOF MS,
`
`and the like. See, for example, the review in Current Analytical Chemistry, Vol. 1, No. 1 (2005),
`
`pp. 28-57; herein incorporated by referencein its entirety.
`
`[0043] As used herein, the term “glycoengineered Fc” when used herein refers to N-glycan on
`
`the Fe region has beenaltered or engineered either enzymatically or chemically. The term “Fe
`
`glycoengineering”as used herein refers to the enzymatic or chemical process used to make the
`
`glycoengineered Fc. Exemplary methods of engineering are described in, for example, Wonget
`
`al USSN12/959,351, the contents of which is hereby incorporated by reference.
`
`[0044] The terms “homogeneous”, “uniform”, “uniformly” and “homogeneity”in the context of
`
`a glycosylation profile of Fc region are used interchangeably and are intended to meana single
`
`glycosylation pattern represented by one desired N-glycan species, with little or no trace amount
`
`of precursor N-glycan. In certain cmbodiments, the trace amount of the precursor N-glycan is
`
`less than about 2%.
`
`[0045] “Essentially pure” protein means a composition comprising at least about 90% by weight
`
`of the protein, based on total weight of the composition, including, for example, at least about
`
`91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least
`
`about 96%, at Icast about 97%, at lcast about 98%, or at lcast about 99% by weight.
`
`[0046] "Essentially homogeneous” protein means a composition comprising at least about 98%
`
`by weight of protein, including for example, at least about 98.5 %, at least about 99% based on
`
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`total weight of the composition. In certain embodiments, the protein is an antibody, structural
`
`variants, and/or antigen binding fragmentthercof.
`
`[0047] As used herein, the terms “IgG”, “IgG molecule”, “monoclonal antibody”,
`
`“immunoglobulin”, and “immunoglobulin molecule” are used interchangeably. As used herein,
`
`“molecule” can also refer to antigen binding fragments.
`
`[0048] As used herein, the term “Fc receptor” or “FcR” describes a receptor that binds to the Fc
`
`region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a
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`preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of
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`the FeyRI (CD64), FeyRII (CD32), and FeyRIII (CD16) subclasses, includingallelic variants
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`and alternatively spliced forms of these receptors. FeyRII receptors include FcyRUA (an
`
`“activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid
`
`sequencesthat differ primarily in the cytoplasmic domainsthereof. Activating receptor FeyRIIA
`
`contains an immunorcceptortyrosinc-based activation motif (ITAM)in its cytoplasmic domain.
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`Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM)
`
`in its cytoplasmic domain. (see review M. in Daéron, Annu. Rev. Immunol. 15:203-234 (1997)).
`
`FeRsare reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,
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`Immunomethods 4:25-34 (1994); and de Haaset al., J. Lab. Clin. Med. 126:330-41 (1995). Other
`
`FcRs,including those to be identified in the future, are encompassed by the term “FcR”herein.
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`The term also includes the neonatal receptor, FecRn, which is responsible for the transfer of
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`matemal IgGs to the fetus (Guyeret al., J. fmmunol. 117:587 (1976) and Kim et al., 7. Jmmunol.
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`24:249 (1994)).
`
`[0049] The term “effector function” as used herein refers to a biochemical event that results
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`from the interaction of an antibody Fc region with an Fc receptor or ligand. Exemplary “effector
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`functions” include Clq binding; complement dependent cytotoxicity; Fe receptor binding;
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`antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation ofcell
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`surface receptors (e.g. B cell receptor; BCR), etc. Such effector functions can be assessed using
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`various assays knownintheart.
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`[0050] As used herein, the term “Antibody-dependent cell-mediated cytotoxicity” or “ADCC”
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`refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on
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`certain cytotoxic cells (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) enable
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`these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and
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`subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are
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`absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express
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`FeyRII only, whereas monocytes express FeyRI, FcyRIand FceyRIII. FcR expression on
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`hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.
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`Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC
`
`assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337 may be
`
`performed. Useful effector cells for such assays include peripheral blood mononuclearcells
`
`(PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the
`
`molecule of interest may be assessed in vivo, e.g., in a animal model suchas that disclosed in
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`Clyneset al. PNAS (USA) 95:652-656 (1998).
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`[0051] The term “Complement dependent cytotoxicity” or “CDC”as used herein refers to the
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`lysis of a target cell in the presence of complement. Activation of the classical complement
`
`pathway is initiated by the binding ofthe first componentof the complement system (C1q) to
`
`antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess
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`complementactivation, a CDC assay,e.g. as described in Gazzano-Santoroet al., .. /mmunol.
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`Methods 202:163 (1996), may be performed.
`
`[0052] “Chimeric” antibodies (immunoglobulins) have a portion of the heavy and/or light chain
`
`identical with or homologousto corresponding sequences in antibodies derived from a particular
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`species or belonging to a particular antibody class or subclass, while the remainderofthe
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`chain(s) is identical with or homologous to corresponding sequences in antibodies derived from
`
`another species or belonging to another antibody class or subclass, as well as fragments of such
`
`antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and
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`Morrisonet al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Humanized antibody as used
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`herein is a subset of chimeric antibodies.
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`[0053] “Humanized”forms of non-human(e.g., murine) antibodies are chimeric antibodies
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`which contain minimal sequence derived from non-human immunoglobulin. For the mostpart,
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`humanized antibodics arc human immunoglobulins (recipient or acceptor antibody) in which
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`hypervariable region residues of the recipient are replaced by hypervariable region residues from
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`a non-human species (donor antibody) such as mouse,rat, rabbit or nonhuman primate having
`
`the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR)
`
`residues of the human immunoglobulin are replaced by corresponding non-humanresidues.
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`Furthermore, humanized antibodies may comprise residues which are not found in the recipient
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`antibody or in the donor antibody. These modifications are made to further refine antibody
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`performancesuch as binding affinity. Generally, the humanized antibody will comprise
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`substantially all of at least one, and typically two, variable domains, in whichall or substantially
`
`all of the hypervariable loops correspond to those of a non-human immunoglobulin andall or
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`substantially all of the FR regions are those of a human immunoglobulin sequencealthough the
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`FR regions may include one or more aminoacid substitutions that improve bindingaffinity. The
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`numberof these amino acid substitutions in the FR is typically no more than 6 in the H chain,
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`and in the L chain, no more than 3. The humanizedantibody optionally also will comprise at
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`least a portion of an immunoglobulin constant region (Fc), typically that of a human
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`immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmannet
`
`al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also
`
`the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy,
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`Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038
`
`(1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).
`
`[0054] As used herein, the term “antigen” is defined as any substance capableofeliciting an
`
`immune response. As used herein, the term“antigen specific” refers to a property of a cell
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`population such that supply of a particular antigen, or a fragment of the antigen, results in
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`specific cell prolifcration.
`
`[0055] As used herein, the term “immunogenicity”refers to the ability of an immunogen,
`
`antigen, or vaccine to stimulate an immuneresponse.
`
`[0056] As used herein, the term “epitope” is defined as the parts of an antigen molecule which
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`contact the antigen binding site of an antibody ora T cell receptor.
`
`[0057] As used herein, the term "specifically binding,” refers to the interaction between binding
`
`pairs (e.g., an antibody and an antigen). In various instances, specifically binding can be
`
`embodied by an affinity constant of about 10-6 moles/liter, about 10-7 moles/liter, or about 10-8
`
`moles/liter, or less.
`
`[0058] An “isolated” antibody is one which has been identified and separated and/or recovered
`
`from a componentofits natural environment. Contaminant components ofits natural
`
`environment are materials which would interfere with rescarch, diagnostic or therapcutic uscs
`
`for the antibody, and may include enzymes, hormones, and other proteinaceous or
`
`nonproteinaceoussolutes.
`
`[0059] The phrase “substantially similar,” “substantially the same”, “equivalent”, or
`
`“substantially equivalent”, as used herein, denotes a sufficiently high degree of similarity
`
`between two numeric values (for example, one associated with a molecule and the other
`
`associated with a reference/comparator molecule) such that one of skill in the art would consider
`
`the difference between the two valuesto be oflittle or no biological and/orstatistical
`
`significance within the context of the biological characteristic measured by said values(e.g., Kd
`
`values, anti-viral effects, etc.). The difference between said two valuesis, for example, less than
`
`10
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`about 50%,less than about 40%, less than about 30%, less than about 20%, and/or less than
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`about 10% as a function of the valuc for the reference/comparator molecule.
`
`[0060] The phrase “substantially reduced,” or “substantially different”, as used herein, denotes a
`
`sufficiently high degree of difference between two numeric values (generally one associated
`
`with a molecule and the other associated with a reference/comp