PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`Intematlonal Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`
`C07K 16/24, C12N 15/13, 15/63, 5/10,
`C07K 16/00, A61K 39/395, G01N
`33/577, C12P 21/08,. A61P 43/00
`
`(11) International Publication Number:
`
`WO 00/56772
`
`
`(51) International Patent Classification 7 :
`
`
`
`
`
`
`
`(43) International Publication Date:
`
`28 September 2000 (28.09.00)
`
`-
`-
`-
`b
`N
`t
`IA 1
`t
`I t
`1
`pp lca um um er
`(2 ) n erna Iona
`(22) International Filing Date:
`
`.
`.
`
`PCT/USOO/07946
`
`24 March 2000 (24.03.00)
`
`.
`-
`-
`(30) Priority Data.
`60/126,603
`
`.
`25 M h 1999 25.03.99
`ale
`(
`
`)
`
`US
`
`-
`-
`-
`-
`-
`t
`t
`C N
`t
`t
`-
`-P t
`(63) Rel?é‘}‘i,;’fo%$l'igraggfi(cagog or C0“ "ma 10" m M
`US
`'
`60/126 603 (CIP)
`Filed on
`25 March 1999,950399)
`
`(71) Applicants (for all designated States except US): BASF AK—
`TIENGESELLSCHAFT [DE/DE]; Carl—Bosch Strasse 38,
`D—67056 Ludwigshafen (DE). GENETICS INSTITUTE
`INC. [US/US]; 87 CambridgePark Drive, Cambridge, MA
`02140 (US).
`
`(72) Inventors; and
`Jochen,
`SALFELD,
`(75) Inventors/Applicants (for US only):
`G.
`[DE/US]; 177 Old Westborogh Road, North Grafton,
`MA 01536 (US). ROGUSKA, Michael
`[US/US];
`16
`Hilldale Road, Ashland, MA 01721 (US). PASKIND,
`Michael
`[US/US]; 253 Redemption Rock Trail, Sterling,
`
`
`
`25
`[IN/US];
`MA 01564 (US). BANERJEE, Subhashis
`.
`Hapgood Way, Shrewsbury, MA 01545 (US). TRACEY,
`Damelr E~ [US/US]; 149 Shaker Roadr Harvard, MA 01451
`(US). WHITE, Michael
`[US/US];
`30 Angelica Drive,
`Framingham, MA 01701 (US). KAYMAKCALAN, Zehra
`TR/US]; 4 Picadilly Way, Westborough, MA 01581 (US).
`,
`[
`LABKOVSKY, Borls
`[US/US];
`107A—5 Broadmeadow
`Road, Marlborough, MA 01752 (US). SAKORAFAS,
`Paul [US/US]; 47 Court Street, Newton, MA 02160 (US).
`FRIEDRICH, Stuart [CA/US]; 20 Albion Street, Melrose,
`MA 02176 (US). MYLES, Angela [US/US]; Apartment #7,
`1 1 Crescent Drive, Andover, MA 01810 (US). VELDMAN,
`Geertruida, M.
`[NL/US]; 60 Woodmere Drive, Sudbury,
`MA 01776 (US). VENTURINI, Amy [US/US]; 207 Wobum
`Street, Lexington, MA 02420 (US). WARNE, Nicholas, W.
`[US/US]; 27 Farrwood Drlve, Andover, MA 01810 (US)-
`WIDOMY Angela [US/US]:
`19 Cherokee Road, Acton.
`MA 01720 (Us) ELVIN, John, G- [GB/GB]; 36 Perowne
`Street, Cambridge CB1 ZAY (GB?' DUNCAN’ Alexander,
`R. [GB/GB]; 3 Hauthon Road, thtle Shelford, Cambridge
`CB2 5H] (GB). DERBYSHIRE, Elaine, J.
`[GB/GB];
`15
`The Brambles, Studlands Rise, Royston, Hertfordshire SG8
`9NQ (,GB) CARMEN’ Sara [GB/GB]; 136 Sturton Street,
`Cambridge C31 ZQF (GB)- SMITH»,StCPh?“ [GB/GEL
`33 Church Road, Wicken, Ely, Cambrldgeshlre. CB7 SXT
`(GB). HOLTETr Thor: Las [BK/GB];
`‘3 “former Road;
`Royston, Hertfordshxre (GB). DU FOU, Saral, L. [GB/GB],
`76 Ickleford Road, I-litchen, Hertfordshire SG5 1TL (GB).
`
`(74) Agents: KARA, Catherine, J. et al.; Lahive & Cockfield, LLP,
`28 State Street, Boston, MA 02109 (US).
`
`(81) Designated States: AE, AG, AL, AM, AT, AU, AZ, BA, BB,
`BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM,
`DZ, 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, TI‘, TZ,
`UA, UG, US, 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, Fl, 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).
`
`Published
`
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`(54) Title: HUMAN ANTIBODIES THAT BIND HUMAN lL—12 AND METHODS FOR PRODUCING
`
`(57) Abstract
`
`Human antibodies, preferably recombinant human antibodies, that specifically bind to human interleukin—12 (hIL—12) are disclosed.
`Preferred antibodies have high affinity for hIL—12 and neutralize hIL—12 activity in vitro and in viva. An antibody of the invention can be
`a full~length antibody or an antigen—binding portion thereof. The antibodies, or antibody portions, of the inveniton are useful for detecting
`hIL—12 and for inhibiting hIL—12 activity, e.g.,
`in a human subject suffering from a disorder in which hIL—12 activity is detrimental.
`Nucleic acids, vectors and host cells for expressing the recombinant human antibodies of the invention, and methods of synthesizing the
`recombinant human antibodies, are also encompassed by the invention.
`
`AMGEN INC.
`
`Exhibit 1039
`
`
`
`
`
`Ex. 1039 - Page 1 of 377
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`Ex. 1039 - Page 1 of 377
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`AMGEN INC.
`Exhibit 1039
`
`

`

`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Céte d’Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Gemrany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People’s
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`SZ
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`US
`UZ
`VN
`YU
`ZW
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`Zimbabwe
`
`
`
`
`
`
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`Ex. 1039 - Page 2 of 377
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`WO 00/56772
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`PCT/USOO/07946
`
`-1-
`
`HUMAN ANTIBODIES THAT BIND HUMAN IL-12
`
`AND METHODS FOR PRODUCING
`
`Related Applications
`
`This application is a non-provisional application claiming priority to US.
`
`provisional application Serial No. 60/126,603, filed March 25, 1999, the contents of
`
`which are hereby incorporated by reference.
`
`Background of the Invention
`
`Human interleukin 12 (IL-12) has recently been characterized as a cytokine with
`
`a unique structure and pleiotropic effects (Kobayashi, et al. (1989) J Exp Med. 1702827-
`
`845; Seder, et a1. (1993) Proc. Natl. Acad. Sci. 90:10188-10192; Ling, et a]. (1995) J.
`
`Exp Med 1542116-127; Podlaski, et a]. (1992) Arch. Biochem. Biophys. 294:230—237).
`
`IL—12 plays a critical role in the pathology associated with several diseases involving
`
`immune and inflammatory responses A review of IL—12, its biological activities, and its
`
`role in disease can be found in Gately et a]. (1998) Ann. Rev. Immunol. 16: 495—521.
`
`Structurally, IL-12 is a heterodimeric protein comprising a 35 kDa subunit (p3 5)
`
`and a 40 kDa subunit (p40) which are both linked together by a disulfide bridge (referred
`
`to as the "p70 subunit"). The heterodimeric protein is produced primarily by antigen—
`
`presenting cells such as monocytes, macrophages and dendritic cells. These cell types
`
`also secrete an excess of the p40 subunit relative to p70 subunit. The p40 and p35
`
`subunits are genetically unrelated and neither has been reported to possess biological
`
`activity, although the p40 homodimer may function as an IL-12 antagonist.
`
`Functionally, IL—12 plays a central role in regulating the balance between antigen
`
`specific T helper type (Th1) and type 2 (Th2) lymphocytes. The Th1 and Th2 cells
`
`govern the initiation and progression of autoimmune disorders, and IL-12 is critical in
`
`the regulation of Thl-lymphocyte differentiation and maturation. Cytokines released by
`
`the Th1 cells are inflammatory and include interferon y (IFNy), IL-2 and lymphotoxin
`
`(LT). Th2 cells secrete IL-4, lL-5, IL-6, IL-10 and IL-13 to facilitate humoral immunity,
`
`allergic reactions, and immunosuppression.
`
`10
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`15
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`20
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`25
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`30
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`Consistent with the preponderance of Th1 responses in autoimmune diseases and
`
`the proinflammatory activities oleNy, IL—12 may play a major role in the pathology
`
`associated with many autoimmune and inflammatory diseases such as rheumatoid
`
`arthritis (RA), multiple sclerosis (MS), and Crohn's disease.
`
`Human patients with MS have demonstrated an increase in lL—12 expression as
`
`documented by p40 mRNA levels in acute MS plaques. (Windhagen et (1]., (1995) J
`
`Exp. Med. 182: 1985-1996). In addition, ex vivo stimulation of antigen—presenting cells
`
`with CD40L—expressing T cells from MS patients resulted in increased IL—12 production
`
`compared with control T cells, consistent with the observation that CD40/CD40L
`
`interactions are potent inducers of IL-12.
`
`Elevated levels of IL-12 p70 have been detected in the synovia of RA patients
`
`compared with healthy controls (Morita et a1 (1998) Arthritis and Rheumatism. 41: 306—
`
`314). Cytokine messenger ribonucleic acid (mRNA) expression profile in the RA
`
`synovia identified predominantly Th1 cytokines. (Bucht et al., (1996) Clin. Exp.
`
`Immunol. 103: 347-367). 1L-12 also appears to play a critical role in the pathology
`
`associated with Crohn's disease (CD). Increased expression of INFy and 1L—12 has been
`
`observed in the intestinal mucosa of patients with this disease (Fais et a]. (1994) J.
`
`Interferon Res. 142235—238; Parronchi et al., (1997) Am. J. Path. 150:823-832;
`
`Monteleone et at, (1997) Gastroenterology 112:1169-1178, and Berrebi et al., (1998)
`
`Am. J Path 152:667-672). The cytokine secretion profile of T cells from the lamina
`
`propria of CD patients is characteristic of a predominantly Th1 response, including
`
`greatly elevated IFNy levels (Fuss, et al., (1996) J. Immunol. 157:1261—1270).
`
`Moreover, colon tissue sections from CD patients show an abundance of IL—12
`
`expressing macrophages and IFNy expressing T cells (Parronchi et a1 (1997) Am. J
`
`10
`
`15
`
`20
`
`25
`
`Path. 150:823-832).
`
`Due to the role of human 1L-12 in a variety of human disorders, therapeutic
`
`strategies have been designed to inhibit or counteract IL-12 activity. In particular,
`
`antibodies that bind to, and neutralize, 1L—12 have been sought as a means to inhibit IL-
`
`12 activity. Some of the earliest antibodies were murine monoclonal antibodies (mAbs),
`
`30
`
`secreted by hybridomas prepared from lymphocytes of mice immunized with IL-12 (see
`
`e. g, World Patent Application Publication No. WO 97/15327 by Strober et al.; Neurath
`
`et a1. (1995) J. Exp. Med. 182:1281—1290; Duchmann et a]; (1996) J. Immunol. 26:934-
`
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`938). These murine IL—12 antibodies are limited for their use in vivo due to problems
`
`associated with administration of mouse antibodies to humans. such as short serum half
`
`life, an inability to trigger certain human effector functions and elicitation of an
`
`unwanted immune response against the mouse antibody in a human (the "human anti—
`
`mouse antibody" (HAMA) reaction).
`
`In general. attempts to overcome the problems associated with use of fully-
`
`murine antibodies in humans, have involved genetically engineering the antibodies to be
`
`more "human-like." For example, chimeric antibodies, in which the variable regions of
`
`the antibody chains are murine—derived and the constant regions of the antibody chains
`
`are human-derived, have been prepared (Junghans, et a1. (1990) Cancer Res. 5021495—
`
`1502; Brown et a]. (1991) Proc. Natl. Acad. Sci. 88:2663-2667; Kettleborough et a].
`
`(1991) Protein Engineering. 4:773—783). However, because these chimeric and
`
`humanized antibodies still retain some murine sequences, they still may elicit an
`
`unwanted immune reaction, the human anti—chimeric antibody (HACA) reaction,
`
`especially when administered for prolonged periods.
`
`A preferred IL—12 inhibitory agent to murine antibodies or derivatives thereof
`
`(e. g. , chimeric or humanized antibodies) would be an entirely human anti—IL—12
`
`antibody, since such an agent should not elicit the HAMA reaction, even if used for
`
`prolonged periods. However, such antibodies have not been described in the art and,
`
`10
`
`15
`
`20
`
`therefore are still needed.
`
`Summary of the Invention
`
`The present invention provides human antibodies that bind human IL—12. The
`
`invention also relates to the treatment or prevention of acute or chronic diseases or
`
`25
`
`conditions whose pathology involves IL-12, using the human anti-IL—12 antibodies of
`
`the invention.
`
`In one aspect, the invention provides an isolated human antibody, or an antigen-
`
`binding portion thereof, that binds to human IL-12.
`
`In one embodiment, the invention provides a selectively mutated human IL-12
`
`30
`
`antibody, comprising:
`
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`-4-
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`a human antibody or antigen—binding portion thereof, selectively mutated at a
`
`preferred selective mutagenesis position, contact or hypermutation position with an
`
`activity enhancing amino acid residue such that it binds to human IL-l2.
`
`In a preferred embodiment. the invention provides a selectively mutated human
`
`IL—12 antibody, comprising:
`
`a human antibody or antigen-binding portion thereof, selectively mutated at a
`
`preferred selective mutagenesis position with an activity enhancing amino acid residue
`
`such that it binds to human IL—l2.
`
`In another preferred embodiment, the selectively mutated human IL—12 antibody
`
`or antigen-binding portion thereof is selectively mutated at more than one preferred
`
`selective mutagenesis position, contact or hypermutation positions with an activity
`
`enhancing amino acid residue. In another preferred embodiment, the selectively mutated
`
`human IL—l2 antibody or antigen—binding portion thereof is selectively mutated at no
`
`more than three preferred selective mutagenesis positions, contact or hypermutation
`
`positions. In another preferred embodiment, the selectively mutated human IL-12
`
`antibody or antigen—binding portion thereof is selectively mutated at no more than two
`
`preferred selective mutagenesis position, contact or hypermutation positions. In yet
`
`another preferred embodiment, the selectively mutated human IL-l2 antibody or
`
`antigen-binding portion thereof, is selectively mutated such that a target specificity
`
`affinity level is attained, the target level being improved over that attainable when
`
`selecting for an antibody against the same antigen using phage display technology. In
`
`another preferred embodiment, the selectively mutated human IL—12 antibody further
`
`retains at least one desirable property or characteristic, e. g, preservation of non-cross
`
`reactivity with other proteins or human tissues, preservation of epitope recognition,
`
`production of an antibody with a close to a germline immunoglobulin sequence.
`
`In another embodiment, the invention provides an isolated human antibody, or
`
`antigen-binding portion thereof, that binds to human IL-12 and dissociates from human
`
`IL-12 with a Koff rate constant of 0.1 5‘1 or less, as determined by surface plasmon
`
`resonance, or which inhibits phytohemagglutinin blast proliferation in an in vitro
`
`phytohemagglutinin blast proliferation assay (PHA assay) with an IC50 of 1 x 10'6 M or
`
`less. More preferably, the isolated human antibody or an antigen—binding portion
`
`thereof, dissociates from human IL-12 with a Koff rate constant of 1 x 10'2 5'1 or less, or
`
`10
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`15
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`20
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`25
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`30
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`-5-
`
`inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC50 of
`
`1 x 10'7 M or less. More preferably, the isolated human antibody. or an antigen-binding
`
`portion thereof. dissociates from human IL—12 with a Koff rate constant of l x 10‘3 3'1 or
`
`less, or inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an
`
`IC50 of l x 10'8 M or less. More preferably, the isolated human antibody, or an antigen—
`
`binding portion thereof, dissociates from human IL—12 with a Koff rate constant of l x
`
`10'4 S'1 or less, or inhibits phytohemagglutinin blast proliferation in an in vitro PHA
`
`assay with an IC50 of l x 10'9 M or less. More preferably, the isolated human antibody,
`
`or an antigen-binding portion thereof, dissociates from human IL—l2 with a Koff rate
`
`constant of l x 10‘5 5'1 or less, or inhibits phytohemagglutinin blast proliferation in an in
`
`vitro PHA assay with an IC50 of l X 10"0 M or less. Even more preferably, the isolated
`
`human antibody, or an antigen—binding portion thereof, dissociates from human IL-l2
`
`with a Koff rate constant of l x 10'5 5'1 or less, or inhibits phytohemagglutinin blast
`
`proliferation in an in vitro PHA assay with an ICso of l x 10'1 1 M or less.
`
`In another embodiment, the invention provides an isolated human antibody, or an
`
`antigen-binding portion thereof, which has the following characteristics:
`
`a)
`
`inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay
`
`with an IC50 ofl x 10'6 M or less;
`
`b)
`
`has a heavy chain CDR3 comprising the amino acid sequence of SEQ ID
`
`10
`
`15
`
`20
`
`NO: 1; and
`
`c)
`
`has a light chain CDR3 comprising the amino acid sequence of SEQ ID
`
`NO: 2.
`
`In a preferred embodiment, the isolated human antibody, or an antigen-binding
`
`25
`
`30
`
`portion thereof, has a heavy chain CDR2 comprising the amino acid sequence of SEQ
`
`ID NO: 3; and has a light chain CDR2 comprising the amino acid sequence of SEQ ID
`
`NO: 4. In a preferred embodiment, the isolated human antibody, or an antigen-binding
`
`portion thereof, has a heavy chain CDRI comprising the amino acid sequence of SEQ
`
`ID NO: 5; and has a light chain CDRl comprising the amino acid sequence of SEQ ID
`
`NO: 6. In a preferred embodiment, the isolated human antibody, or antigen binding
`
`portion thereof, has a heavy chain variable region comprising the amino acid sequence
`
`of SEQ ID NO: 7; and has a light chain variable region comprising the amino acid
`
`sequence of SEQ ID NO: 8.
`
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`-6—
`
`In another embodiment. the invention provides an isolated human antibody, or an
`
`antigen-binding portion thereof. which has the following characteristics:
`
`a)
`
`inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay
`
`with an IC50 of 1 x 10'9 M or less;
`
`b)
`
`has a heavy chain CDR3 comprising the amino acid sequence of SEQ ID
`
`NO: 9; and
`
`c)
`
`has a light chain CDR3 comprising the amino acid sequence of SEQ ID
`
`NO: 10.
`
`In a preferred embodiment. the isolated human antibody, or an antigen-binding
`
`portion thereof, has a heavy chain CDR2 comprising the amino acid sequence of SEQ
`
`ID NO: 1 1; and has a light chain CDR2 comprising the amino acid sequence of SEQ ID
`
`NO: 12.
`
`In a preferred embodiment. the isolated human antibody, or an antigen-binding
`
`portion thereof, has a heavy chain CDRl comprising the amino acid sequence of SEQ
`
`ID NO: 13; and has a light chain CDRl comprising the amino acid sequence of SEQ ID
`
`NO: 14. In a preferred embodiment, the isolated human antibody has a heavy chain
`
`variable region comprising the amino acid sequence of SEQ ID NO: 15; and has a light
`
`chain variable region comprising the amino acid sequence of SEQ ID NO: 16.
`
`In another embodiment, the invention provides an isolated human antibody, or an
`
`antigen—binding portion thereof, which
`
`a)
`
`inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay
`
`10
`
`15
`
`20
`
`with an IC50 of] x 10'9 M or less;
`
`b)
`
`has a heavy chain CDR3 comprising the amino acid sequence of SEQ ID
`
`NO: 17; and
`
`c)
`
`has a light chain CDR3 comprising the amino acid sequence of SEQ ID
`
`25
`
`NO: 18.
`
`In a preferred embodiment, the isolated human antibody, or an antigen—binding
`
`portion thereof, has a heavy chain CDR2 comprising the amino acid sequence of SEQ
`
`ID NO: 19; and a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:
`
`20. In a preferred embodiment, the isolated human antibody, or an antigen-binding
`
`portion thereof, has a heavy chain CDRl comprising the amino acid sequence of SEQ
`
`ID NO: 21; and a light chain CDRI comprising the amino acid sequence of SEQ ID NO:
`
`22. In a preferred embodiment, the isolated human antibody, or an antigen-binding
`
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`portion thereof, has the heavy chain variable region comprising the amino acid sequence
`
`of SEQ ID NO: 23. and a light chain variable region comprising the amino acid
`
`sequence of SEQ ID NO: 24.
`
`In a preferred embodiment, the isolated human antibody
`
`comprises a heavy chain constant region selected from the group consisting of IgG1 ,
`
`IgG2, IgG3, IgG4, IgM, IgA and IgE constant regions or any allelic variation thereof as
`
`discussed in Kabat et al. (Kabat, E.A., el al. (1991) Sequences ofProteins of
`
`Immunological Interest, Fifth Edition, US. Department of Health and Human Services,
`
`NIH Publication No. 91-3242), included herein by reference.
`
`In a more preferred
`
`embodiment, the antibody heavy chain constant region is IgGl. In another preferred
`
`embodiment, the isolated human antibody is a Fab fragment, or a F(ab')2 fragment or a
`
`single chain FV fragment.
`
`In another embodiment, the invention provides an isolated human antibody, or an
`
`antigen-binding portion thereof, which
`
`a)
`
`inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay
`
`with an IC50 ofl x 10'9 M or less;
`
`b)
`
`has a heavy chain CDR3 comprising the amino acid sequence selected
`
`from the group consisting of SEQ ID NO: 404—SEQ ID NO: 469; and
`
`c)
`
`has a light chain CDR3 comprising the amino acid sequence selected
`
`from the group consisting of SEQ ID NO: 534—SEQ ID NO: 579.
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`In a preferred embodiment, the isolated human antibody, or an antigen-binding
`
`portion thereof, has a heavy chain CDR2 comprising the amino acid sequence selected
`
`from the group consisting of SEQ ID NO:335-SEQ ID NO: 403; and a light chain CDR2
`
`comprising the amino acid sequence selected from the group consisting of SEQ ID NO:
`
`506-SEQ ID NO: 533. In a preferred embodiment, the isolated human antibody, or an
`
`antigen-binding portion thereof, has a heavy chain CDRl comprising the amino acid
`
`sequence selected from the group consisting of SEQ ID NO: 288—SEQ ID NO: 334; and
`
`a light chain CDRl comprising the amino acid sequence selected from the group
`
`consisting of SEQ ID NO: 470-SEQ ID NO: 505. In a preferred embodiment, the
`
`isolated human antibody, or an antigen-binding portion thereof, comprising a the heavy
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`chain variable region comprising the amino acid sequence of SEQ ID NO: 23, and a
`
`light chain variable region comprising the amino acid sequence of SEQ ID NO: 24. In a
`
`preferred embodiment, the isolated human antibody comprises a heavy chain constant
`
`10
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`15
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`20
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`25
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`region. or an Fab fragment or a F(ab')2 fragment or a single chain Fv fragment as
`
`described above.
`
`In another embodiment, the invention provides an isolated human antibody. or an
`
`antigen-binding portion thereof, which
`
`a)
`
`inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay
`
`with an IC5O of I X 10'9 M or less;
`b)
`has a heavy chain CDR3 comprising the amino acid sequence of SEQ ID
`
`NO: 25; and
`
`c)
`
`has a light chain CDR3 comprising the amino acid sequence of SEQ ID
`
`10
`
`NO: 26.
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`In a preferred embodiment, the isolated human antibody, or an antigen-binding
`
`portion thereof. has a heavy chain CDR2 comprising the amino acid sequence of SEQ
`
`ID NO: 27; and a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:
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`28. In a preferred embodiment, the isolated human antibody, or an antigen—binding
`
`portion thereof, has a heavy chain CDRI comprising the amino acid sequence of SEQ
`
`ID NO: 29; and a light chain CDRI comprising the amino acid sequence of SEQ ID NO:
`
`30. In a preferred embodiment, the isolated human antibody, or an antigen—binding
`
`portion thereof, which has a heavy chain variable region comprising the amino acid
`
`sequence of SEQ ID NO: 31, and a light chain variable region comprising the amino
`
`acid sequence of SEQ ID NO: 32.
`
`In a preferred embodiment, the isolated human
`
`antibody comprises a heavy chain constant region, or an Fab fragment, or a F(ab')2
`
`fragment or a single chain Fv fragment as described above.
`
`In another embodiment, the invention provides an isolated human antibody, or an
`
`antigen-binding portion thereof, which
`
`a)
`
`inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay
`
`with an IC50 ofl x 10'6 M or less;
`
`b)
`
`comprises a heavy chain CDR3 comprising the amino acid sequence of
`
`SEQ ID NO: I, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID
`
`NO: 3 and a heavy chain CDRI comprising the amino acid sequence of SEQ ID NO: 5,
`
`or a mutant thereof having one or more amino acid substitutions at a contact position or
`
`a hypermutation position, wherein said mutant has a koff rate no more than 10-fold
`
`higher than the antibody comprising a heavy chain CDR3 comprising the amino acid
`
`15
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`20
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`25
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`30
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`sequence of SEQ ID NO: I, a heavy chain CDR2 comprising the amino acid sequence of
`
`SEQ ID NO: 3. and a heavy chain CDRl comprising the amino acid sequence of SEQ
`
`ID NO: 5; and
`
`c)
`
`comprises a light chain CDR3 comprising the amino acid sequence of
`
`SEQ ID NO: 2, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:
`
`4, and a light chain CDRl comprising the amino acid sequence of SEQ ID NO: 6, or a
`
`mutant thereof having one or more amino acid substitutions at a contact position or a
`
`hypermutation position, wherein said mutant has a koff rate no more than lO—fold higher
`
`than the antibody comprising a light chain CDR3 comprising the amino acid sequence of
`
`SEQ ID NO: 2, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:
`
`4, and a light chain CDRl comprising the amino acid sequence of SEQ ID NO: 6.
`
`In another embodiment, the invention provides an isolated human antibody, or an
`
`antigen—binding portion thereof, which
`
`a)
`
`inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay
`
`with an IC50 ofl x 10‘9 M or less;
`
`b)
`
`comprises a heavy chain CDR3 comprising the amino acid sequence of
`
`SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID
`
`NO: 1 l and a heavy chain CDRl comprising the amino acid sequence of SEQ ID NO:
`
`13, or a mutant thereof having one or more amino acid substitutions at a contact position
`
`or a hypermutation position, wherein said mutant has a koff rate no more than lO-fold
`
`higher than the antibody comprising a heavy chain CDR3 comprising the amino acid
`
`sequence of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of
`
`SEQ ID NO: 11, and a heavy chain CDRI comprising the amino acid sequence of SEQ
`
`ID NO: 13; and
`
`c)
`
`comprises alight chain CDR3 comprising the amino acid sequence of
`
`SEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence of SEQ ID
`
`NO: 12, and alight chain CDRl comprising the amino acid sequence of SEQ ID NO:
`
`14, or a mutant thereof having one or more amino acid substitutions at a preferred
`
`selective mutagenesis position, contact position or a hypermutation position, wherein
`
`said mutant has a koff rate no more than 10—fold higher than the antibody comprising a
`
`light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10, a light chain
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`10
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`15
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`20
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`25
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`30
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`CDR2 comprising the amino acid sequence of SEQ ID NO: 12. and a light chain CDRl
`
`comprising the amino acid sequence of SEQ ID NO: 14.
`
`In another embodiment. the invention provides an isolated human antibody, or an
`
`antigen-binding portion thereof, which
`
`a)
`
`inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay
`
`with an IC50 of l x 10'9 M or less;
`
`b)
`
`comprises a heavy chain CDR3 comprising the amino acid sequence of
`
`SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID
`
`NO: 19 and a heavy chain CDRl comprising the amino acid sequence of SEQ ID NO:
`
`21, or a mutant thereof having one or more amino acid substitutions at a preferred
`
`selective mutagenesis position, contact position or a hypermutation position, wherein
`
`said mutant has a koff rate no more than lO-fold higher than the antibody comprising a
`
`heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a heavy
`
`chain CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a heavy chain
`
`CDRl comprising the amino acid sequence of SEQ ID NO: 21; and
`
`c)
`
`comprises a light chain CDR3 comprising the amino acid sequence of
`
`SEQ ID NO: 18, a light chain CDR2 comprising the amino acid sequence of SEQ ID
`
`NO: 20, and a light chain CDRl comprising the amino acid sequence of SEQ ID NO:
`
`22, or a mutant thereof having one or more amino acid substitutions at preferred
`
`selective mutagenesis position, contact position or a hypermutation position, wherein
`
`said mutant has a koff rate no more than 10-fold higher than the antibody comprising a
`
`light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18, a light chain
`
`CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a light chain CDRl
`
`comprising the amino acid sequence of SEQ ID NO: 22.
`
`The invention also provides nucleic acid molecules encoding antibodies, or
`
`antigen binding portions thereof, of the invention. A preferred isolated nucleic acid
`
`encodes the heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17.
`
`The isolated nucleic acid encoding an antibody heavy chain variable region. In another
`
`embodiment, the isolated nucleic acid encodes the CDR2 of the antibody heavy chain
`
`variable region comprising the amino acid sequence of SEQ ID NO: 19. In another
`
`embodiment, the isolated nucleic acid encodes the CDRl of the antibody heavy chain
`
`variable region comprising the amino acid sequence of SEQ ID NO: 21. In another
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`10
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`15
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`20
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`25
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`3O
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`embodiment. the isolated nucleic acid encodes an antibody heavy chain variable region
`
`comprising the amino acid sequence of SEQ ID NO: 23.
`
`In another embodiment, the
`
`isolated nucleic acid encodes the light chain CDR3 comprising the amino acid sequence
`
`of SEQ ID NO: 18. The isolated nucleic acid encoding an antibody light chain variable
`
`region.
`
`In another embodiment, the isolated nucleic acid encodes the CDR2 ofthe
`
`antibody light chain variable region comprising the amino acid sequence of SEQ ID NO:
`
`20. In another embodiment, the isolated nucleic acid encodes the CDRl of the antibody
`
`light chain variable region comprising the amino acid sequence of SEQ ID NO: 22.
`
`In
`
`another embodiment, the isolated nucleic acid encodes an antibody light chain variable
`
`region comprising the amino acid sequence of SEQ ID NO: 24.
`
`In another embodiment, the invention provides an isolated human antibody, or an
`
`antigen-binding portion thereof, which
`
`a)
`
`inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay
`
`with an IC50 ofl x 10'9 M or less;
`
`b)
`
`comprises a heavy chain CDR3 comprising the amino acid sequence of
`
`SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID
`
`NO: 27 and a heavy chain CDRl comprising the amino acid sequence of SEQ ID NO:
`
`29, or a mutant thereof having one or more amino acid substitutions at a preferred
`
`selective mutagenesis position, contact position or a hypermutation position, wherein
`
`said mutant has a koff rate no more than lO-fold higher than the antibody comprising a
`
`heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25, a heavy
`
`chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a heavy chain
`
`CDRI comprising the amino acid sequence of SEQ ID NO: 29; and
`
`c)
`
`comprises a light chain CDR3 comprising the amino acid sequence of
`
`SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence of SEQ ID
`
`NO: 28, and a light chain CDRl comprising the amino acid sequence of SEQ ID NO:
`
`30, or a mutant thereof having one or more amino acid substitutions at a preferred
`
`selective mutagenesis position, contact position or a hypermutation position, wherein
`
`said mutant has a koff rate no more than lO-fold higher than the antibody comprising a
`
`light chain CDR3 comprising the amino ac