(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)
`
`WIF'OI PCT
`
`|||||||||||||| |||||||| |||||||||| ||||| ||||||| ||| ||||| |||||||||| III" "III |||| ||||||| |||| ||||||||
`
`(10) International Publication Number
`
`WO 2015/184009 A1
`
`27 May 2015 (27.05.2015)
`.
`English
`English
`
`(51)
`
`(21)
`
`(22)
`
`International Patent Classification:
`C07K 16/28 (2006.01)
`International Application Number:
`,
`_
`_
`International Filing Date:
`
`PCTms2015/032745
`
`.
`.
`.
`(25) Flhng Language.
`(26) Publication Language:
`_
`_
`(30) Pmmy Data:
`US
`Z7 May3014(37~05~3014)
`63/001136
`US
`2.7 May 2014 (A7..05-014)
`62/003,104
`US
`28 May 2014 (28.05.2014)
`62/003,908
`US
`2 July 2014 (02.07.2014)
`62/020,199
`US
`30 January 2015 (30.012015)
`62/110,338
`(71) Applicant: ACADEMIA SINICA; 128 Academia, Road,
`Section 2’ Nankang, Twp“) 11529 (CN)
`Inventor; and
`(72)
`(71) Applicant : WONG, Chi-Huey [US/US]; PO. Box 8154,
`Rancho Santa Fe, California 92067 (US)
`
`(72)
`
`Inventors: WU, Chung-Yi; No. 78-1, Dongshi Street, Xi-
`Zhi District, New Taipei City, 221 (CN). MA, Che; c/o
`.
`.
`.
`7
`.
`.
`sittinarrjnllicafi5998(CNfadem1a Road, Section 2’
`g’
`p ’
`‘
`‘
`(74) Agent: NORTON, Vicki; Duane Morris LLP, Suite 2900,
`50 B t
`t
`D'
`l'f
`.
`2101
`7.
`S ree , San
`1ego, Ca, 1 ornia 9
`(US).
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`A01 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, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, 1N, IR, is, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, IVIE, 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» 1A» 1M: 1W-
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BVV, GH,
`GM KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ,
`TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,
`
`[Continued on nextpage]
`
`(54) Title: COMPOSITIONS AND METHODS RELATING TO [
`EFFICACY
`
`\IIVERSAL GLYCOFORMS FOR ENHANCED ANTIBODY
`
`s§
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`
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`Fig. 1b
`
`(57) Abstract: The present disclosure relates to glycopro—
`teins, particularly monoclonal antibodies, comprising a gly-
`cocnginccred Fc region, wherein said Fc region comprises an
`optimized N-glycan having the
`structure of Sia2((L-
`6)Gal;GlcNAc;Man3GlcNAcz. The glycoengineered Fc re-
`gion binds FcyRILA or FcyRIILA With a greater affinity, 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 effector cell
`function (e.g., ADCC) mediated by FcyR is desired, e.g, can-
`cer, autoimmune, infectious disease, and 111 enhancing the
`therapeutic efficacy of therapeutic antibodies the effect of
`which is mediated by ADCC.
`
`Singapore Exhibit 2011
`Singapore Exhibit 2011
`Lassen v. Singapore et al.
`Lassen V. Singapore et al.
`PGR2019-00053
`PGR2019-00053
`
`
`
`wo2015/184009A1|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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`

`

`WO 2015/184009 A1 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, Published:
`DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT,
`LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE,
`SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, — before the expiration of the time limit for amending the
`GN, GQ, GVV, KM, ML, MR, NE, SN, TD, TG),
`claims and to be republished in the event of receipt of
`amendments (Rule 48.2(h))
`
`— with international search report (Art. 21 (3))
`
`

`

`WO 2015/184009
`
`PCT/US2015/032745
`
`COMPOSITIONS AND METHODS RELATING TO UNIVERSAL GLYCOFORMS
`
`FOR ENl-LANCED ANTIBODY EFFICACY
`
`RELATED APPLICATIONS
`
`[0001] This applications claims the benefit of priority to US provisional applications US Serial
`
`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 reference in its
`
`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 therapeutic monoclonal antibodies (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-tumor activities include anti—
`
`CD20, anti-Her2, anti—EGFR, anti-CD40, anti-CTLA—4, and anti-PD—l 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 human antibodies 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 Fc region has been an important issue in the development of efficacious
`
`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
`
`undelstand these interactions and to improve the safety and efficacy in medication. Toward this
`
`goal, it has been reported that the removal of the core fucose residue would enhance the
`
`antibody—dependent cellular cytotoxieity (ADCC) activity of IgGs due to the increased
`
`interaction between Fe—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
`
`

`

`WO 2015/184009
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`PCT/US2015/032745
`
`cell line and GA101 was from the GnT-III overexpressing system. In addition, more FcyIIIa 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 FcyIIIa.
`
`[0005] Thus, it is of great interest to generate therapeutic monoclonal antibodies with optimized
`
`Fc glycoforms.
`
`SUMMARY OF 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 homogeneous population of monoclonal antibodies (preferably, therapeutic monoclonal
`
`antibodies) having a single. uniform Nvglycan on Fe The individual glycoantibodies comprising
`
`the homogeneous population are substantially identical, bind to the same epitope, and contain
`
`the same Fc 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 havin g the same N-giyean 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
`
`homogeneous population of monoclonal antibodies comprising a single, uniform N—glyean 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 Fc region.
`
`[0012] In preferred embodiments, wherein the N—glycan consists of the structure of Siag(d,2—
`
`6)GalgGlcNA03MangGlCNACg.
`
`[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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`WO 2015/184009
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`PCT/US2015/032745
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`[0014] In some embodiments, the Fc region of the glycoantibodies described herein exhibits an
`
`increased binding affinity for F clelA or FclellA relative to a wild-type Fc 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, IgGZ, 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,
`
`autoimmune disorders, inflammatory disorders or infectious diseases.
`
`[0019] In some embodiments, the glycoantibody described herein is a glycoengineered anti-
`
`CD20. In some examples, the glycoantibody described herein is a glycoengineered 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-
`
`TNFd. 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 glycoantibodies described herein and a pharmaceutically
`
`acceptable carrier. The pharmaceutical composition may be used in therapeutics such as
`
`oncology, autoimmune disorders, inflammatory disorders and infectious diseases.
`
`[0024] In some embodiments, the pharmaceutical composition is used for preventing, treating,
`
`or ameliorating one or more symptoms associated with a disease, disorder, or infection where an
`
`enhanced efficacy of effector cell function (e.g., ADCC) mediated by FcyR is desired, e.g.,
`
`cancer, autoimmune, infectious disease, and in enhancing the 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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`WO 2015/184009
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`PCT/US2015/032745
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`[0026] Further, disclosed herein include methods for preventing, treating, or ameliorating one or
`
`more symptoms associated with a disease, 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 compound that
`
`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
`
`glyeoantibodies can be administered alone or in conjunction with a second therapeutic agent
`
`such as a second antibody, or a chemotherapeutic 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 homogeneous antibody
`
`through remodeling of the glycan structures on the Fc region 0fIgG1 (b).
`
`[0032] Figure 2. shows that antibody dependent B-cell depletion activity of various
`
`glycoengineered Rituximab. The depletion of human B cells was conducted using freshly
`
`prepared human PBMC cells and analyzed on FACS, based on the CDl9+ 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 was significantly more active than the non-treated Rituximab with a
`
`p value of 0.001 6, whereas the mono-GlcNAc Rituximab showed the lowest activity. (C) The
`
`prepared Rituximab-resistant cells of Ramos and Raji express lower level of CD20 on cell
`
`surface. (D, E) The 2,6-NSCT Rituximab showed a remarkable ADCC efficacy towards both
`
`normal and resistant cells, whereas non-treated antibody dramatically lost its activity towards
`
`resistant strains.
`
`[0033] Figure 3. shows that ECSO of glycoengineered Herceptin in V158 FcyRIIIa mediated
`
`ADCC reporter bioassay. Experiments were performed under E/T ratio of 6 tol with SKBR3 as
`
`

`

`WO 2015/184009
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`PCT/US2015/032745
`
`target cells and V158 FcyR111a engineered Jurkat as effector cells. All data shown in the same
`
`graph were experiments done in the same microplate and the same batch of effector cells; bars
`
`of 95% confidence interval were plotted. (A) afueosylated Herceptin G8 and commercial
`
`Herceptin showed a similar ADCC effect, illustrating that the defueosylation advantage of anti-
`
`Fcn/R111a is lost in the afueosylated Herceptin G8. (B) Bisected and its non-bisected analogue
`
`Herceptin, G9 and G4 showed similar ECSO 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 ECSO change in the 2,6—sialylated
`
`antibody was observed, whereas the apparent EC50 increase was shown in the 2,3—sialy1ated
`
`Herceptin. The results indicated that the 2,3 -sialylation on Fc would lower the 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. shows that anti-influenza antibody F16 with a modified homogeneous SCT
`
`glycan attached to its Fc Asn297 (F16m) significantly showed an enhancement of its ADCC
`
`activity and prophylactically protects mice from a lethal dose of HlNl virus challenge. (a)
`
`Cytotoxicity is represented as the percentage of lysed HEK293T cells (target cells) expressed
`
`with influenza Hl hemagglutinin (HA) (A/Califomia/O7/O9) when incubated with PBMCs
`
`(effector cells) and various concentrations of antibodies. (b) ADCC activity was shown as 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-influenza antibody F16 and F16m. Curve fitting was done with
`
`software GraphPad Prism in 4PL nonlinear regression. (c) Survival of mice was monitored upon
`
`lethal dose (10 MLDSO) infection of influenza virus A/California/07/09 (HlNl). Two hours
`
`before infection, each group of mice (N=9) was intraperitoneally given either 2.5 mg/kg of F16,
`
`Fl6m or PBS, respectively. The F16 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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`PCT/US2015/032745
`
`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,
`
`Volumes I and II (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, NY); 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 Harlow and 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 homogeneous population of monoclonal antibodies (preferably, therapeutic monoclonal
`
`antibodies) having a single, uniformed glycoform bound to the Fc region. The individual
`
`glycoantibodies comprising the essentially homogeneous population are identical, bind to the
`
`same epitope, and contain the same Fc glycan with a well-defined glycan structure and sequence.
`
`[0037] As used herein, the term “anti-CD20 glycoantibodies” (“anti-CD20 GAbs”) refers to a
`
`homogeneous population 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
`
`monosaecharide. Glycans can be monomers or polymers of sugar residues and can be linear or
`
`branched. A glycan may include natural sugar residues (e.g., glucose, N—acetylglucosamine, N—
`
`acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.)
`
`and’or modified sugars (e.g., 2'-flu0roribose, 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 monosaecharides. For example, cellulose is a glycan (or more specifically a glucan)
`
`composed of [5—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 monosaecharide 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. 0- and N-linked
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`WO 2015/184009
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`PCT/US2015/032745
`
`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 (11,6-position linked to the N—acetylglucosamine .
`
`[0041] As used herein, the terms “N-glycan”, “N-linked glycan”, “N-linked glycosylation”, “FC
`
`glycan” and “Fe glycosylation” are used interchangeably and refer to an N-linked
`
`oligosaccharide attached by an N-acetylglucosamine (GlcNAe) linked to the amide nitrogen of
`
`an asparagine residue in a Fc—containing polypeptide. The term “Fe—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. l (2005),
`
`pp. 28-57; herein incorporated by reference in its entirety.
`
`[0043] As used herein, the term “glycoengineered Fc” when used herein refers to N—glycan on
`
`the Fc region has been altered or engineered either enzymatically or chemically. The term “Fc
`
`glycoengineering” as used herein refers to the enzymatic or chemical process used to make the
`
`glycoengineered Fe. Exemplary methods of engineering are described in, for example, Wong et
`
`a1 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 mean a single
`
`glycosylation pattern represented by one desired N—glycan species, with little or no trace amount
`
`of precursor N—glycan. In certain embodiments, 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 least about 97%, at least about 98%, or at least 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
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`variants, and/or antigen binding fragment thereof.
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`[0047] As used herein, the terms “ IgG”, “IgG molecule”, “monoclonal antibody”,
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`“immunoglobulin”, and “immunoglobulin molecule” are used interchangeably. As used herein,
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`“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
`
`preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of
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`the Fcle (CD64), FcyRII (CD32), and FcyRIH (CD16) subclasses, including allelic variants
`
`and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (an
`
`“activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid
`
`sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRHA
`
`contains an immunoreceptor tyrosine—based activation motif (ITAM) in its cytoplasmic domain.
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`Inhibiting receptor FcyRHB contains an immunoreceptor tyrosine—based inhibition motif (ITIM)
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`in its cytoplasmic domain. (see review M. in Daéron, Annu. Rev. Immunol. 152203 -234 (1997)).
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`FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 91457—92 (1991); Capel et al.,
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`Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other
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`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, FcRn, which is responsible for the transfer of
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`maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 ( 1976) and Kim et al., J. Immunol.
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`24:249 (1994)).
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`[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 F0 receptor or ligand. Exemplary “effector
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`functions” include Clq binding; complement dependent cytotoxicity; Fc receptor binding;
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`antibody—dependent cell—mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell
`
`surface receptors (e.g. B cell receptor; BCR), etc. Such effector functions can be assessed using
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`various assays known in the art.
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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 lg 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
`
`absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express
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`ch/RHI only, whereas monocytes express FcyRI, FcyRH and FcyRIII. 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 92457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC
`
`assay, such as that described in US. Pat. No. 5,500,362 or US. Pat. No. 5,821,337 may be
`
`performed. Useful effector cells for such assays include peripheral blood mononuclear cells
`
`(PBMC) and Natural Killer (NK) cells. Altematively, or additionally, ADCC activity of the
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`molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in
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`Clynes et 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 of the first component of the complement system (C lq) to
`
`antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess
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`complement activation, a CDC assay, e. g. as described in Gazzano—Santoro et al., J. Immunol.
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`Methods 202: 163 (1996), may be performed.
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`[0052] “Chimeric” antibodies (immunoglobulins) have a portion of the heavy and/or light chain
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`identical with or homologous to corresponding sequences in antibodies derived from a particular
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`species or belonging to a particular antibody class or subclass, while the remainder of the
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`chain(s) is identical with or homologous to corresponding sequences in antibodies derived fi‘om
`
`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
`
`Morrison et 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 most part,
`
`humanized antibodies are 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
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`the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR)
`
`residues of the human immunoglobulin are replaced by corresponding non-human residues.
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`Furthermore, humanized antibodies may comprise residues which are not found in the recipient
`
`antibody or in the donor antibody. These modifications are made to further refine antibody
`
`performance such as binding affinity. Generally, the humanized antibody will comprise
`
`substantially all of at least one, and typically two, variable domains, in which all or substantially
`
`all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or
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`substantially all of the FR regions are those of a human immunoglobulin sequence although the
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`FR regions may include one or more amino acid substitutions that improve binding affinity. The
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`number of 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 humanized antibody optionally also will comprise at
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`least a portion of an immunoglobulin constant region (Fe), typically that of a human
`
`immunoglobulin. For further details, see Jones et al., Nature 321:522—525 (1986); Reichmann et
`
`al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593—596 (1992). See also
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`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).
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`[0054] As used herein, the term “antigen” is defined as any substance capable of eliciting an
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`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 proliferation.
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`[0055] As used herein, the term “immunogenicity” refers to the ability of an immunogen,
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`antigen, or vaccine to stimulate an immune response.
`
`[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 or a T cell receptor.
`
`[0057] As used herein, the term "specifically binding," refers to the interaction between binding
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`pairs (e. g., an antibody and an antigen). In various instances, specifically binding can be
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`embodied by an affinity constant of about 10-6 moles/liter, about 10-7 moles/liter, or about 10—8
`
`moles/liter, or less.
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`[0058] An “isolated” antibody is one which has been identified and separated and/0r recovered
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`from a component of its natural environment. Contaminant components of its natural
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`environment are materials which would interfere with research, diagnostic or therapeutic uses
`
`for the antibody, and may include enzymes, hormones, and other proteinaeeous or
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`nonproteinaeeous solutes.
`
`[0059] The phrase “substantially similar,” “substantially the same”, “equivalent”, or
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`“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
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`the difference between the two values to be of little or no biological and/or statistical
`
`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 values is, for example, less than
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`10
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