(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`
`International Bureau
`
`(43) International Publication Date
`1 March 2001 (01.03.2001)
`
`PCT
`
`(10) International Publication Number
`wo 01/14424 A2
`
`(51) International Patent Classification7:
`
`C07K 16/00
`
`(21) International Application Number:
`
`PCT/USOO/23356
`
`(22) International Filing Date: 24 August 2000 (24.08.2000)
`
`(25) Filing Language:
`
`(26) Publication Language:
`.
`.
`(30) Priority Dam"
`60/150,452
`
`English
`
`English
`
`24 August 1999 (24.08.1999)
`
`US
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): KORMAN, Alan,
`J. [US/US]; 301 E1 CerI-ito Avenue, Piedmont, CA 94611
`(US). HALK, Edward, L. [US/US]; 1004 Edmonds Court,
`Sunnyvale, CA 94087 (US). LONBERG, Nils [US/US];
`780 West California Way, Woodside, CA 94062 (US).
`
`(74) Agents: SERAFINI, Andrew, T. et a1.; Townsend and
`Townsend and Crew LLP, Two Embarcadero Center, 8th
`floor, San Francisco, CA 94111—3834 (US).
`
`(63) Related by continuation (CON) or continuation-in-part
`(am to earlier application;
`US
`Filed on
`
`60/150,452 ((311))
`24 August 1999 (24.08.1999)
`
`except US):
`all designated States
`(for
`(71) Applicant
`MEDAREX,
`INC.
`[US/US];
`1545 Route 22 East,
`Annandale, NJ 08801 (US).
`
`(81) Designated States (national): AE, AG, AL: AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ,
`DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR,
`HU, ID, IL, IN, IS, JR KE, KG, KR KR, KZ, LC, LK, LR,
`LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ,
`NO, NZ, PL, PI, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM,
`TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW.
`
`[Continued on next page]
`
`(54) Title: HUMAN CTLA—4 ANTIBODIES AND THEIR USES
`
`Comm. Lymphomas
`
`(57) Abstract: The present invention provides novel human
`sequence antibodies against human CTLA74 and methods of
`treating human diseases, infections and other conditions us—
`ing these antibodies.
`
`PHA activated
`lymphocytes
`
`3
`
`Without Ab
`
`30
`
`20
`
`Counts
`
`Hulth-FITC
`
`l47—FITC
`
`10D1-FlTC
`
`10
`10D1-FITC
`
`Without Ab
`
`HulgG 1-FlTC
`
`147-FITC
`
`WO01/14424A2
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`WO 01/14424 A2
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`||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`Published:
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian 7 Without international search report and to be republished
`patent (Alvl, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
`upon receipt of that report,
`patent (AT, BE, CH, CY, DE, DK, ES, FT, FR, GB, GR, IE,
`IT, LU, MC, NL, PT, SE), OAPI patent (BF, B J, CF, CG,
`CI, CM, GA, GN, GW, NIL, MR, NE, SN, TD, TG).
`“
`
`For two-letter codes and other abbreviations, refer to the ”Guid-
`ance Notes on Codes andAbbreviations ” appearing at the begin-
`ning ofeach regular issue of the PCT Gazette.
`
`

`

`WO 01/14424
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`PCT/US00/23356
`
`HUMAN CTLA-4 ANTIBODIES AND THEIR USES
`
`REFERENCE TO RELATED APPLICATIONS
`
`This application claims the benefit of US. provisional patent application
`
`Serial No. 60/150,452, the disclosure of which is incorporated herein in its entirety.
`
`FIELD OF THE INVENTION
`
`The present invention relates generally to molecular immunology and the
`
`treatment of human diseases. In particular, it relates to novel human sequence antibodies
`
`against human CTLA-4 and methods of treating human diseases and infections using
`
`these antibodies.
`
`10
`
`BACKGROUND OF THE INVENTION
`
`The vertebrate immune system requires multiple signals to achieve optimal
`
`immune activation; see, e.g., Janeway, Cold Spring Harbor Symp. Quant. Biol. 5421-14
`
`(1989); Paul William E., ed. Raven Press, N.Y., Fundamental Immunology, 4th edition
`
`(1998), particularly chapters 12 and 13, pages 411~to 478. Interactions between T
`
`15
`
`lymphocytes (T cells) and antigen presenting cells (APC) are essential to the immune ‘
`
`response. Levels of many cohesive molecules found on T cells and APC’s increase during
`
`an immune response (Springer et al.., A. Rev. Immunol. 5:223-252 (1987); Shaw and
`
`Shimuzu, Current Opinion in Immunology, Eds. Kindt and Long, 1:92—97 (1988)); and
`
`Hemler, Immunology Today 91109-1 13 (1988)). Increased levels of these molecules may
`
`help explain why activated APC’s are more effective at stimulating antigen-specific T cell
`
`proliferation than are resting APC’s (Kaiuchi et al.., J. Immunol. 131 :109—1 14 (1983);
`
`Kreiger et al.., J. Immunol. 135:2937-2945 (1985); McKenzie, J. Immunol. 141:2907-
`
`2911 (1988); and Hawrylowicz and Unanue, J. Immunol. 141:4083-4088 (1988)).
`
`T cell immune response is a complex process that involves cell-cell
`
`interactions (Springer et al.., A. Rev. Immunol. 5:223-252 (1987)), particularly between T
`
`and accessory cells such as APC’s, and production of soluble immune mediators
`(cytokines or lymphokines) (Dinarello (1987) New Engl. Jour. Med 317:940—945;
`
`Sallusto (1997) J. Exp. Med. 179:1109—1'118). This response is regulated by several T—cell
`
`20
`
`25
`
`surface receptors, including the T-cell receptor complex (Weiss (1986) Ann. Rev.
`
`30
`
`Immunol. 4:593-619) and other “accessory” surface molecules (Allison (1994) Curr.
`
`

`

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`Opin. Immunol. 62414-419; Springer (1987) supra). Many of these accessory molecules
`
`are naturally occurring cell surface differentiation (CD) antigens defined by the reactivity
`
`of monoclonal antibodies on the surface of cells (McMichael, Ed., Leukocyte Typing III,
`
`I
`
`Oxford Univ. Press, Oxford, NY. (1987)).
`
`Early studies suggested that B lyrnphocyte activation requires two signals
`
`(Bretscher (1970) Science 169:1042-1049) and now it is believed that all lymphocytes
`
`require two signals for their optimal activation, an antigen specific or clonal signal, as
`
`well as a second, antigen non-specific signal. (Janeway, supra). Freeman (1989) J.
`
`Immunol. 143:2714-2722) isolated and sequenced a cDNA clone encoding a B cell
`
`10
`
`activation antigen recognized by MAb B7 (Freeman (1987) J Immunol. 13823260). COS
`
`cells transfected with this cDNA have been shown to stain by both labeled MAb B7 and
`
`MAb BB-l (Clark (1986) Human Immunol. 16:100-113; Yokochi (1981) J. Immunol.
`1282823; Freeman et al.., (1989) supra; Freeman et al.. (1987), supra). In addition,
`
`expression of this antigen has been detected on cells of other lineages, such as monocytes
`
`15
`
`(Freeman et al.., supra).
`
`T helper cell (Th) antigenic response requires signals provided by APC’s.
`
`The first signal is initiated by interaction of the T cell receptor complex (Weiss, J. Clin.
`Invest. 86: 1015 (1990)) with antigen presented in the context of class II major
`r
`
`histocompatibility complex (MI-1C) molecules on the APC (Allen, Immunol. Today 8:270
`
`20
`
`(1987)). This antigen—specific signal is not sufficient to generate a full response, and in
`
`the absence of a second signal may actually lead to clonal inactivation or anergy
`
`(Schwartz, Science 248: 1349 (1990)). The'requirement for a second “costimulatory?
`
`signal provided by the MHC has been demonstrated in a number of experimental systems
`
`(Schwartz, supra; Weaver and Unanue, Immunol. Today 11:49 (1990)). The molecular
`
`25'
`
`nature of this second signal is not completely understood, although it is clear in some
`
`cases that both soluble molecules such as interleukin (IL)-1 (Weaver and Unanue, supra)
`
`and membrane receptors involved in intercellular adhesion (Springer, Nature 346:425
`
`(1990)) can provide costimulatory signals.
`
`CD28 antigen, a homodimeric glycoprotein of the immunoglobulin
`
`30
`
`superfamily (Aruffo and Seed, Proc. Natl. Acad. Sci. 84:8573-8577 (1987)), is an
`
`accessory molecule found on most mature human T cells (Damle et al.., J. Immunol.
`
`131:2296-2300 (1983)). Current evidence suggests that this molecule functions in an
`
`alternative T cell activation pathway distinct from that initiated by the T-cell receptor
`
`complex (June et al.., Mol. Cell. Biol. 7:4472-4481 (1987)). Monoclonal antibodies
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`2
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`(MAbs) reactive with CD28 antigen can augment T cell responses initiated by various
`
`polyclonal stimuli (reviewed by June et al.,, supra). These stimulatory effects may result
`
`from MAb-induced cytokine production (Thompson et al.., Proc. Natl. Acad. Sci
`
`86:1333-1337 (1989); and Lindsten et al., Science 244:339-343 (1989)) as a consequence
`
`of increased mRNA stabilization (Lindsten et al.. (1989), supra). Anti-CD28 mAbs can
`
`also have inhibitory effects, 1'. e. , they can block autologous mixed lymphocyte reactions
`
`(Damle et al.., Proc. Natl. Acad. Sci. 78:5096—6001 (1981)) and activation of antigen—
`specific T cell clones (Lesslauer et al., Eur. J. Immunol. 16:1289-1296 (1986)).
`
`Some studieshave indicated that CD28 is a counter-receptor for the B cell
`
`10
`
`activation antigen, B7/BB-1 (Linsley et al., Proc. Natl. Acad. Sci. USA 87:5031-5035
`
`(1990)). The B7/BB-1 antigen is hereafier referred to as the “B7 antigen”. The B7
`
`ligands are also members of the immunoglobulin superfamily but have, in contrast to
`
`CD28, two lg domains in their extracellular region, an N-terminal variable (V)-1ike
`
`domain followed by a constant (C)-1ike domain.
`
`15
`
`Delivery of a non-specific costimulatory signal to the T cell requires at
`
`least two homologous B7 family members found on APC’s, B7-l (also called B7, B7.l,
`
`_ or CD80) and B7-2 (also called 372 or CD86), both of which can deliver costimulatory
`signals to T cells via CD28. Costimulation thrOugh CD28 promotes T cell activation.
`
`. Using genetic fusions of the extracellular portions of B7 antigen and CD28
`
`20
`
`receptor, and Immuno globulin (1g) C. gammal (constant region heavy chains),
`
`interactions between CD28 and B7 antigen have been characterized (Linsley et al., J.
`
`Exp. Med. 173:721-730 (1991)). Immobilized B7Ig fusion protein, as well as B7 positive
`
`CHO cells, have been shown to costimulate T cell proliferation.
`
`T cell stimulation with B7 positive CHO cells also specifically stimulates
`
`25
`
`increased levels of transcripts for IL-2. Additional studies have shown that anti-CD28
`
`MAb inhibited IL-2 production induced in certain T cell leukemia cell lines by cellular
`interactions with a B cell leukemia line (Kohno et al.., Cell. Immunol. 131-1-10 (1990)).
`
`CD28 has a single extracellular variable region (V)-1ike domain (Aruffo
`
`and Seed, supra). A homologous molecule, CTLA-4 has been identified by differential
`
`30
`
`screening of a murine cytolytic—T cell cDNA library (Brunet (1987) Nature 328:267-270).
`
`CTLA—4 is a T cell surface molecule that was originally identified by
`
`differential screening of a murine cytolytic T cell cDNA library (Brunet et al.., Nature
`
`328:267-270(1987)). CTLA—4 is also a member of the immunoglobulin (lg) superfamily;
`
`CTLA—4 comprises a single extracellular Ig domain. CTLA-4 transcripts have been
`
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`found in T cell populations having cytotoxic activity, suggesting that CTLA-4 might
`
`function in the cytolytic response (Brunet et al.. , supra ; Brunet er al.., Immunol. Rev.
`
`103-21-36 (1988)). Researchers have reported the cloning and mapping of a gene for the
`
`human counterpart of CTLA-4 (Dariavach et al.., Eur. J. Immunol. 18:1901-1905 (1988))
`
`5
`
`to the same chromosomal region (2q33—34) as CD28 (Lafage-Pochitaloff et al..,
`
`'
`
`Immunogenetics 31 : 198-201 (1990)). Sequence comparison between this human CTLA-4
`
`DNA and that encoding CD28 proteins reveals significant homology of sequence, with
`
`the greatest degree of homology in the juxtamembrane and cytoplasmic regions (Brunet et
`
`al.., 1988, supra; Dariavach et al.., 1988, supra).
`
`10
`
`Some studies have suggested that CTLA—4 has an analogous function as a
`
`secondary costimulator (Linsley et al., J Exp. Med. 176:1595-1604 (1992); Wu et al.., J
`
`Exp. Med. 185:1327-1335.(1997) Lindsley, P. et al.. US. Patent Nos. 5,977,318;
`
`5,968,510; 5,885,796; and 5,885,579). However, others have reported that CTLA-4 has
`
`an opposing role as a dampener of T cell activation (Krummel (1995) J. Exp. Med.
`
`1 5
`
`182:459-465); Krummel et al.., Int ’1 Immunol. 8:519-523(1996); Chambers et al. .,
`
`Immunity. 7:885-895(1997)). It has been reported that CTLA—4 deficient mice suffer from
`
`massive lymphoproliferation (Chambers et al.., supra). It has been reported that CTLA—4
`
`blockade augments T cell responses in vitro (Walunas et al.., Immunity. 1:405-413
`
`(1994)) and in vivo (Kearney (1995).]. Immunol. 155:1032-1036), exacerbates antitumor
`
`20
`
`immunity (Leach (1996) Science. 271:1734-1736), and enhances an induced autoimmune
`
`disease (Luhdel' (1998) J Exp. Med. 187 :427—432). It has also been reported that CTLA—4
`
`has an alternative or additional impact on the initial character of the T cell immune ,
`
`response (Chambers (1997) Curr. Opin. Immunol. 92396-404; Bluestone (1997) J.
`
`Immunol. 158:1989—1993; Thompson (1997) Immunity 7:445—450). This is consistent
`
`25 . with the observation that some autoimmune patients have autoantibodies to CTLA-4. It is
`
`possible that CTLA-4 blocking antibodies have a pathogenic role in these patients
`
`(Matsui (1999) J. Immunol. 162:4328-4335).
`
`Non-human CTLA-4 antibodies have be used in the various studies
`
`discussed above. However, one of the major impediments facing the development of in
`
`30
`
`vivo therapeutic and diagnostic applications for antibodies in humans is the intrinsic
`
`immunogenicity of non—human immunoglobulins. For example, when immunocompetent
`
`human patients are administered therapeutic doses of rodent monoclonal antibodies, the
`
`’patients produce antibodies against the rodent immunoglobulin sequences; these human
`
`anti-mouse antibodies (HAMA) neutralize the therapeutic antibodies and can cause acute
`
`4
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`toxicity. These and other deficiencies in the previous antibodies are overcome by the
`
`provision of human antibodies to CTLA-4 by the present invention.
`
`SUMMARY OF THE INVENTION
`
`The present invention provides a human sequence antibody that
`
`specifically binds to human CTLA—4 and a human sequence antibody that specifically
`
`binds to human CTLA-4 which is substantially free of non-immunoglobulin associated
`
`human proteins.
`
`In a related aspect, the invention also provides a therapeutically—effective
`
`human sequence antibody that specifically binds to human CTLA—4. In some
`
`10
`
`embodiments, the therapeutically-effective human sequence antibody binds to CTLA-4
`
`on the cell surface of normal human T cells. In other embodiments, the T cell
`
`subpopulations marked by CD antigens CD4, CD8, CD25, and CD69 remain stable
`
`during and subsequent to the administration of the therapeutically-effective human
`
`15
`
`sequence antibody. In other embodiments, the therapeutically-effective human sequence
`antibody binds CTLA-4 on the cell surface of normal human T cells. In other
`embodiments, the human sequence antibody well-tolerated in a patient. In a related
`
`embodiment,
`
`Also provided is a composition of polyclonal antibodies comprising a
`
`plurality of human sequence antibodies that specifically bind to human CTLA—4. The
`
`20
`
`composition of polyclonal antibodies can comprise at least about 2, 5, 10, 50, 100, 500 or
`
`1000 different human sequence antibodies that specifically bind to human CTLA—4.
`
`The invention also provides human sequence antibodies that specifically
`bind to human CTLA—4 and which block binding of human CTLA-4 to human B7 or do
`
`not block binding of human CTLA-4 to human B7.
`
`25
`
`The invention also provides human sequence antibodies that bind to
`
`human CTLA-4 with an equilibrium association constant (Ka) of at least 108 M". Also
`
`provided are human sequence antibodies that bind to human CTLA—4 with an equilibrium
`
`association constant (Ka) of at least 109 M'l.
`
`The invention also provides human sequence antibodies that specifically
`
`30
`
`bind to human CTLA-4 that block binding of human CTLA-4 to human B7 by at least
`
`about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
`
`The invention also provides human sequence antibodies that specifically
`
`bind to human CTLA-4 having an antibody heavy chain of either IgG or IgM. The IgG
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`' antibody heavy chain can be IgGl , IgG2, IgG3 or IgG4. The invention also provides
`
`human sequence antibodies wherein the antibody light chain is a kappa light chain. The
`
`human sequence antibody can be encoded by human IgG heavy chain and human kappa
`
`light chain nucleic acids that comprise nucleotide sequences in their variable regions as
`set forth in SEQ ID N022 through SEQ ID NO:23, respectively.
`
`The invention also provides a human sequence antibody wherein the
`
`human sequence antibody is encoded by human IgG heavy chain and human kappa light
`
`chain nucleic acids that comprise nucleotide sequences in their vaxiable regions as set
`
`forth in SEQ ID NO:16 and SEQ ID NO:6, respectively.
`
`10
`
`The invention also provides a human sequence antibody wherein the
`
`human sequence antibody is encoded by human IgG heavy chain and human kappa light
`
`chain nucleic acids that comprise nucleotide sequences in their variable regions as set
`
`forth in SEQ ID NO: 18 and SEQ ID N028, respectively.
`
`The invention also provides a human sequence antibody wherein the
`
`15
`
`human sequence antibody is encoded by human IgG heavy chain and human kappa light
`chain nucleic acids that comprise nucleotide sequences in their variable regions as set
`
`forth in SEQ ID NO:22 and SEQ ID NO:12, respectively.
`
`The invention also provides a human sequence antibody wherein the
`
`human sequence antibody is encoded by heavy chain and light chain variable region
`
`20
`
`amino acid sequences as set for the in SEQ ID NO:17 and SEQ ID NO:7, respectively.
`
`The invention provides a human sequence antibody wherein the human
`
`sequence antibody is encoded by heavy chain and light chain variable region amino acid
`
`sequences as set for the in SEQ ID NO:19 and SEQ ID N029, respectively.
`
`The invention also provides a human sequence antibody wherein the
`
`25
`
`. human sequence antibody is encoded by heavy chain and light chain variable region
`
`amino acid sequences as set for the in SEQ ID NO:23 and SEQ ID NO:13, respectively.
`
`The invention provides a human sequence antibody wherein the human
`
`sequence antibody is encoded by human IgG heavy chain and human kappa light chain
`
`nucleic acids comprising variable heavy and light chain sequences from V gene segments
`
`30
`
`VH 3-303 and VK A-27, respectively.
`
`The invention also provides a human sequence antibody wherein the
`
`human sequence antibody is encoded by human IgG heavy chain and human kappa light
`
`chain nucleic acids comprising variable heavy and light chain sequences from V gene
`
`segments VH 3-33 and VK L-15, respectively.
`
`6
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`Some human sequence antibodies of the invention comprise heavy chain
`
`CDRl, CDR2, and CDR3 sequences, SYTMH (SEQ ID N027),
`
`FISYDGNNKYYADSVKG (SEQ ID N0:32) and TGWLGPFDY (SEQ 1D N0:37),
`
`respectively, and light chain CDRl, CDR2, and CDR3 sequences, RASQSVGSSYLA
`
`(SEQ ID N024), GAFSRAT (SEQ ID N029), and QQYGSSPWT (SEQ ID N0:35),
`
`respectively.
`
`Some human sequence antibodies of the invention comprise heavy chain
`
`CDRl, CDR2, and CDR3 sequences, SYTMI-I (SEQ ID N027),
`
`FISYDGSNKHYADSVKG (SEQ ID N033) and TGWLGPFDY (SEQ ID N0:38),
`respectively, and light chain CDRI, CDR2, and CDR3 sequences, RASQSVSSSFLA
`
`10
`
`(SEQ ID N025), GASSRAT (SEQ ID N030), and QQYGSSPWT (SEQ ID N0:35),
`
`15
`
`20
`
`25
`
`30
`
`respectively.
`
`'
`
`Other human sequence antibodies of the invention comprise heavy chain
`
`CDRI, CDR2, and CDR3 sequences, SYGMI-I (SEQ ID N028),
`
`VIWYDGSNKYYADSVKG (SEQ ID N0:34) and APNYIGAFDV (SEQ ID N0:39),
`
`respectively, and light chain CDRI, CDR2, and CDR3 sequences, RASQGISSWLA
`
`(SEQ ID N026), AASSLQS (SEQ ID N03 1), and QQYNSYPPT (SEQ ID N0:36),
`respectively.
`-
`
`The invention also provides human sequence antibodies that specifically
`
`bind to human CTLA-4, wherein said human sequence antibody is produced by a
`
`transgenic non-human animal. The transgenic non-human animal can be a mouse.
`The invention also provides a human sequence antibody that .specifically
`
`bind to human CTLA—4 that is a Fab fragment.
`
`The invention provides a polyvalent complex comprising at least two
`
`human sequence antibodies each of which specifically binds to human CTLA-4. The two
`
`different antibodies can be linked to each other covalently or non-covalently.
`
`The invention provides a nucleic acid encoding a heavy chain of a human
`
`sequence antibody. The nucleic acid can comprise a nucleotide sequence as set forth in
`
`SEQ ID NO:1.
`
`The invention provides a transgenic non-human animal having a genome
`
`comprising a human sequence heavy chain transgene and a human sequence light chain
`
`transgene, which animal has been immunized with a human CTLA-4, or a fragment or an
`
`analog thereof, whereby the animal expresses human sequence antibodies to the human
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`CTLA~4. The transgenic non-human animal can be a transgenic mouse. The transgenic
`
`mouse can comprise HCo7 or HC012.
`
`The invention provides a hybridoma cell line comprising a B cell obtained
`
`fiom a transgenic non-human animal having a genome comprising a human sequence
`
`heavy chain transgene and a human sequence light chain transgene, wherein the
`
`hybridoma produces a human sequence antibody that specifically binds to human CTLA-
`
`4. In a related embodiment,the hybridoma secretes a human sequence antibody that
`
`specifically binds human CTLA-4 or binding fragment thereof, wherein the antibody is
`
`selected from the group consisting of: a human sequence antibody comprising heavy
`
`10
`
`chain heavy chain CDRl, CDR2, and CDR3 sequences, SYTMH (SEQ ID NO:27),
`
`FISYDGNNKYYADSVKG (SEQ ID NO:32) and TGWLGPFDY (SEQ ID NO:37),
`
`respectively, and light chain CDRl , CDR2, and CDR3 sequences, RASQSVGSSYLA
`
`(SEQ ID N0:24), GAFSRAT (SEQ ID N029), and QQYGSSPWT (SEQ ID NO:35),
`
`respectively, and heavy chain and light chain variable region amino acid sequences as set
`
`15
`
`forth in SEQ ID NO: 17 and SEQ ID N027, respectively; a human sequence antibody
`
`comprising heavy chain CDRl, CDR2, and CDR3 sequences, SYTMH (SEQ ID N0:27),
`
`FISYDGSNKHYADSVKG (SEQ ID NO:33) and TGWLGPFDY (SEQ ID NO:3 8),
`respectively, and light chain CDRl, CDR2, and CDR3 sequences, RASQSVSSSFLAV
`(SEQ ID NO:25), GASSRAT (SEQ ID N030), and QQYGSSPWT (SEQ ID NO:35),
`
`20
`
`respectively, and heavy chain and light chain variable region amino acid sequences as set
`
`forth in SEQ ID N0219 and SEQ ID NO:9, respectively; or a human sequence antibody of
`
`claim 1, comprising heavy chain CDRl, CDR2, and CDR3 sequences, SYGMH (SEQ ID
`
`N028), VIWYDGSNKYYADSVKG (SEQ ID NO:34) and APNYIGAFDV (SEQ ID
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`NO:39), respectively, and light chain CDRl, CDR2, and CDR3 sequences,
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`25
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`, RASQGISSWLA (SEQ ID N0126), AASSLQS (SEQ ID NO:31), and QQYNSYPPT
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`(SEQ II) NO:36), respectively, and heavy chain and light chain variable region amino
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`acid sequences as set forth in SEQ ID N0223 and SEQ ID N0:13, respectively.
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`The invention provides a pharmaceutical composition comprising a human
`
`sequence antibody that specifically binds to human CTLA—4 and a pharmaceutically
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`acceptable carrier. The pharmaceutical composition can further comprise an agent
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`effective to induce an immune response against a target antigen. Also provided are
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`chemotherapeutic agents. In addition, antibodies to immunosuppressive molecules are
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`also provided.
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`The invention provides a method for inducing, augmenting or prolonging
`an immune response to an antigen in a patient, comprising administering to the patient an
`
`effective dosage of a human sequence antibody that specifically binds to human CTLA-4,
`
`wherein the antibody blocks binding of human CTLA-4 to human B7. The antigen can be
`
`a tumor antigen, or the antigen can be from a pathogen. The tumor antigen can also be
`
`telomerase. The pathogen can be a virus, a bacterium, a fungus or a parasite. The
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`pathogen can also be an HIV. This method can further comprise administering the
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`antigen, or a fragment or an analog thereof, to the patient, whereby the antigen in
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`combination with the human sequence antibody induces, augments or prolongs the
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`immune response. The antigen can be a tumor antigen or a component of an amyloid
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`formation in the patient, such as a patient suffering from Alzheimer's disease and the
`
`antigen is AB peptide. This method can fiirther comprise administering a cytokine to the
`
`patient.
`
`'
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`"
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`The. invention provides a method of suppressing an immune response in a
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`patient, comprising administering to the patient an effective dosage of a polyvalent
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`preparation comprising at least two human sequence antibodies to human CTLA—4 linked
`
`to each other. The invention also provides a method of suppressing an immune response
`
`in a patient, comprising administering to the patient an effective dosage of a polyclonal
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`preparation comprising at least two human sequence antibodies to human CTLA-4.
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`The present invention further provides isolated or recombinant human
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`sequence antibodies and human monoclonal antibodies which specifically bind to human
`
`CTLA-4, as well as compositions containing one or a combination of such antibodies.
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`Some of the human sequence antibodies of the invention are characterized by binding to
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`human CTLA-4 with high affinity, and/or by blocking the interaction of human CTLA-4
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`with its ligand, the human B7—l and B7-2 molecules. Accordingly, the human sequence
`
`antibodies and the human monoclonal antibodies of the invention can be used as
`
`diagnostic or therapeutic agents in vivo and in vitro.
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`The human sequence antibodies of the invention can encompass various
`
`antibody isotypes, or mixtures thereof, such as IgGl, IgG2, IgG3, IgG4, IgM, IgAl,
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`IgA2, IgAsec, IgD, and IgE. Typically, they include IgGl (e.g., Igle) and IgM
`
`isotypes. The human sequence antibodies can be full—length (e.g., an IgGl or IgG4
`antibody) or can include only an antigen-binding portion (e.g., a Fab, F(ab')2, Fv or a
`isingle chain Fv fragment). Some human sequence antibodies are recombinant human
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`sequence antibodies. Some human sequence antibodies are produced by a hybridoma
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`which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic
`
`mouse, having a genome comprising a human heavy chain transgene and a human light
`
`chain trans gene. The hybridoma can be made by, e.g., fusing the B cell to an
`
`immortalized cell. Some human sequence antibodies of the invention are produced by
`
`hybridomas referred to as 4C8, 4E10, 4E10.5, 5A8, 5C4, 5C4.1.3, 5D7, 5D7.1, SEIO,
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`5E10.12, 5G1, 5G1.4, 6A10, 6C9, 6C9.6, 6D9, 6D9.7, 6G4, 7E4, 7E4.4, 7E6, 7H8, 8E8,
`
`8E8.4, 8F8, 8F8.19, 8H1, 9810, 9A10.1, '9B9, 9C1, 9G5, 105B, 10B5.8, 10B9, 10B9.2,
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`10D1, 10D1.3,10E11,10E4,10E4.5,11B4,11D10, 11E4,11E4.1, 11E8,11F10,11F11,
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`10
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`11F9, llGl, 11G1.5, 1C7, 1H8.8, 2A7, 2A7.6, 2E2, 2E2.7, 2E7, 2E7.2, 2G1, 2G1.2,
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`3C12, 3E10, 3E10.5, 3E6, 3E6.0, 3F10, 4A1, 436 and 4B6.12. Suffixes after the decimal
`
`point indicate different clonal isolates of the same hybridoma cell lines.
`
`Some human sequence anti-CTLA-4 antibodies of the present invention
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`can be characterized by one or more of the following properties: a) specificity for human
`CTLA—4 (specifically binding to human CTLA—4); b) a binding affinity to human CTLA-
`4 with an equilibrium association constant (K,) of at least about 107 M'l, or about 109 M'1 ,
`
`or about 1010 M"1 to 1011 M'1 or higher; c) a kinetic association constant (k,) of at least
`about 103, about 104, or about 105 m'ls'l; and/or, d) a kinetic disassociation constant(kd)
`of at least about 103, about 104, or about 105 m'ls'l.
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`In another aspect, the invention provides nucleic acid molecules encoding
`
`the human sequence antibodies, or anti gen-binding portions, of the invention.
`
`Accordingly, recombinant expression vectors that include the antibody-encoding nucleic
`
`acids of the invention, and host cells transfected with such vectors, are also encompassed
`
`by the invention, as are methods of making the antibodies of the invention by culturing
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`,
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`these host cells.
`
`In yet another aspect, the invention provides isolated B-cells from a
`
`transgenic non—human animal, e.g., a transgenic mouse, which are capable of expressing
`
`various isotypes (e.g., IgG, IgA and/or IgM) of human monoclonal antibodies that
`
`specifically bind to human CTLA-4. The isolated B cells can be obtained from a
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`30
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`transgenic non-human animal, e.g., a transgenic mouse, which has been immunized with
`
`a purified or enriched preparation of human CTLA—4 antigen (or antigenic fragment
`
`thereof) and/or cells expressing human CTLA-4. The transgenic non-human animal, e.g.,
`
`a transgenic mouse, can have a genome comprising a human heavy chain transgene and a
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`human light chain transgene. The isolated B—cells can be immortalized to provide a
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`source (e.g., a hybridoma) of human monoclonal antibodies to human CTLA-4.
`
`Accordingly, the present invention also provides a hybridoma capable of
`
`producing human monoclonal antibodies that specifically bind to human CTLA-4. The
`
`hybridoma can include a B cell obtained from a transgenic non-human animal, e.g. , a
`
`transgenic mouse, having a genome comprising a human heavy chain transgene and a
`
`human light chain transgene fused to an immortalized cell. The transgenic non—human
`
`animal can be immunized with'a purified or enriched preparation of human CTLA-4
`
`antigen and/or cells expressing human CTLA—4 to generate antibody—producing
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`hybridomas.
`
`In yet another aspect, the invention provides a transgenic non—human
`
`animal, such as a transgenic mouse, which express human monoclonal antibodies (also
`referred to herein as a "HuMAb-Mousem") that specifically bind to human CTLA-4. The
`
`transgenic non—human animal can be a transgenic mouse having a genome comprising a
`
`human heavy chain transgene and a human light chain transgene. The transgenic non-
`
`human animal can be immunized with a purified or enriched preparation of CTLA-4
`
`antigen (or antigenic fragment thereof) and/or cells expressing the human CTLA-4. The
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`transgenic non-human animal, 2.3., the transgenic mouse, can be capable of producing‘
`
`multiple isotypes of human monoclonal antibodies to human CTLA—4 (e.g., IgG, IgA
`
`and/or IgM) by undergoing V-D-J recombination and isotype switching. Isotype
`
`switching may occur by, e.g. , classical or non-classical isotype switching.
`
`In another aspect, the present invention provides methods for producing
`
`human sequence antibodies and human sequence monoclonal antibodies that specifically
`react with human CTLA-4. Some methods of the invention include immunizing a
`transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a
`
`I
`
`human heavy chain transgene and a human light chain transgene, with a purified or
`
`enriched preparation of human CTLA-4 antigen and/or cells expressing human CTLA—4.
`
`B cells (e.g., splenic B cells) of the animal can then be obtained and fused with myeloma
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`15
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`20
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`25
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`cells to, form immortal, hybridoma cells that secrete human monoclonal antibodies against
`human CTLA-4.
`V
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`30
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`Anti- human CTLA-4 human monoclonal antibodies of the invention, or
`
`antigen binding portions thereof (e.g., Fab), can be derivatized or linked to another
`
`functional molecule, e.g., another peptide or protein (e.g., an Fab' fragment). For
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`example, an antibody or antigen-binding portion of the invention can be functionally
`
`linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise)
`
`to one or more other molecular entities. For example, the human sequence anti-CTLA-4
`
`antibody, or antigen binding fragment thereof, can be conjugated to a therapeutic moiety,
`
`e.g., a cytotoxic drug, an enzymatically active toxin, or a fiagment thereof, a radioisotope,
`
`or a small molecule anti-cancer drug. The antibodies of the invention can also be
`
`conjugated to cytotoxic pharmaceuticals, e.g., radiolabeled with a cytotoxic agents, such
`as, e.g., 131I (e.g., Shen (1997) Cancer 80(12 Suppl):2553-2557), copper—67 (e.g.,
`I
`
`Deshpande (1988) J. Nucl. Med. 29:217-225) or, e. g., conjugation to the ribosome
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`10
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`inactivating protein gelonin (e.g., Boyle (1996) J. of Immunol. 182221-230).
`
`In another aspect, the present invention provides compositions, e.g. ,
`
`pharmaceutical and diagnostic compositions, comprising a pharmaceutically acceptable
`
`carrier and at least one human monoclonal antibody of the invention, or an antigen—
`
`binding portion thereof, which specifically binds to human CTLA—4. Some compositions
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`15
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`comprise a combination of the human sequence antibodies or antigen-binding portions
`
`thereof, preferably each of which binds to a distinct epit