(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`
`(19) World Intellectual Property Organization (APO
`International Bureau
`
` (10) International Publication Number
`
`(43) International Publication Date
`11 May 2006 (11.05.2006)
`
`WO 2006/048749 Al
`
`(51) International Patent Classification:
`CO07K 16/28 (2006.01)
`A61K 39/395 (2006.01)
`
`(21) International Application Number:
`PCT/IB2005/003307
`
`(22) International Filing Date: 24 October 2005 (24.10.2005)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/624,856
`
`4 November2004 (04.11.2004)
`
`US
`
`(71)
`
`(72)
`(75)
`
`Applicant(for all designated States except US): PFIZER
`PRODUCTSINC. [US/US]; Eastern Point Road, Groton,
`CT 06340 (US).
`
`Inventor; and
`GOMEZ-
`only):
`US
`(for
`Inventor/Applicant
`[ES/US]; Pfizer Global Research
`NAVARRO, Jesus
`and Development, 50 Pequot Avenue, New London, CT
`06320 (US).
`
`(74)
`
`Agents: FULLER,Grover, F., Jr. et al.; Pfizer Inc., 201
`Tabor Road, Morris Plains, NJ 07950 (US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`
`AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
`GB, GD, GE, GH, GM, HR, HU,ID,IL, IN,IS, JP, KE,
`KG, KM,KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, LY,
`MA, MD,MG,MK,MN, MW, MX, MZ, NA, NG, NI, NO,
`NZ, OM,PG, PH, PL, PT, RO, RU, SC, SD, SE, SG, SK,
`SL, SM, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ,
`VC, VN, YU, ZA, ZM, ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM,KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU,TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK,EE,ES, FI,
`FR, GB, GR, HU,IE,IS, IT, LT, LU, LV, MC, NL,PL,PT,
`RO,SE,SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA,
`GN, GQ, 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 receipt of
`amendments
`
`For two-letter codes and other abbreviations, refer to the "Guid-
`ance Notes on Codes and Abbreviations" appearing at the begin-
`ning of each regular issue of the PCT Gazette.
`
`(54) Title: CTLA-4 ANTIBODY AND AROMATASEINHIBITOR OR COMBINATION TREATMENTFOR BREAST CAN-
`CER
`
`4.1.1 Heavy Chain cDNA (SEQ ID NO:1)
`atggagtttg ggctgagetg ggttttccte gttgetcttt taagaggtgt ccagtgtcag
`gtgcagctgg togagtctagg ggqgaggegtg gtccagectg ggaggtccct gagactctcc
`tgtgtagegt ctggattcac cttcagtage catggcatge actgggtceg ccaggcetcca
`ggeaagggge taggagtgggt ggceagttata tggtatgatg gaagaaataa atactatgca
`gactecgtga agggccgatt caccatctce agagacaatt ccaagaacac getgtttctg
`Caaatgaaca gcectgagagc cgaggacacg gctgtgtatt actgtgegag aggaggtcac
`tteggtcctt Ltgactactg gggccagqgqga accctggtca ecgtctectc agectccacc
`aagggcccat cggtcttcee ectggegcca tgctccagga gcacctccga gagcacagcg
`geectggget gectggtcaa ggactacttc cccgaacegg tgacggtgtc gtggaactca
`ggegetctga ccageggegt gcacaccttc ccagctgtce tacagtcctce aggactctac
`teectcagca gegtggtgac cgtgecctce agcaacttcg gcacccagac ctacacctge
`aacgtagatc acaagcccag caacaccaag gtggacaaga cagttgagcg caaatgttgt
`gtcgagtgce caccgtgece agcaccacct gtggcaggac cgtcagtctt cetcttccce
`eccaaaaccca aggacaccct catgatctce cggaccactg aggtcacgtg egtggtggtg
`gacgtgagee acgaagacce cgaggtccag ttcaactggt acgtggacgg cgtggaggtg
`cataatgcca agacaaagee acgggaggag cagttcaaca gceacgttecg tgtggtcage
`gtectcaccg ttgtgcacca ggactggctg aacggcaagg agtacaagtg caaggtctcc
`aacaaaggce tcccagecce catcgagaaa accatctcca aaaccaaagg gcagccccga
`Qaaccacagg tgtacaccct gecccccatce coggaggaga tgaccaagaa ccaqgtcage
`ctgacctgce tqgtcaaagg cttctaccce agcgacatcg ccgtggagtg ggagagcaat
`gggeagecgg agaacaacta caagaccaca cctcccatge tggactcega cggctccttc
`ttectctaca gcaagctcac cgtggacaag agcaggtgge agcaggggaa cgtcttctca
`tgctcegtga tgcatgagge tctgcacaac cactacacge agaagagect ctecectgtct
`
`60
`120
`180
`240
`300
`360
`420
`480
`540
`600
`660
`720
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`840
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`
`
`006/048749A.IMMATARIININIANTTINIANIYGTTANMTRRO
`
`
`
`
`
`N (57) Abstract: The inventionrelates to administration of an anti-CTLA4antibody,particularly human antibodies to human CTLA4,
`© such as those having amino acid sequences of antibodies 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1,
`12.3.1.1, 12.9.1.1, and 10DI (MDX-010), in combination with an aromatase inhibitor, for treatment of breast cancer. More particu-
`WwW
`larly, the invention relates to administration of an anti-CTLA4 antibody and exemestane for treatment of breast cancer.
`
`Page 1 of 78
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`TRANSGENE/BIOINVENT
`EXHIBIT 1070
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`TRANSGENE/BIOINVENT
`EXHIBIT 1070
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`WO 2006/048749
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`PCT/IB2005/003307
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`
`CTLA4 ANTIBODY AND AROMATASE INHIBITOR
`
`COMBINATION TREATMENT FOR BREAST CANCER
`
`Background of the Invention
`
`Each year, more than 180,000 women are diagnosed with breast cancer in the United States.
`
`If
`
`current breast cancer rates remain constant, each woman born today has a one-in-ten chance of
`
`developing breast cancer at a median onset age of about 60 to 65.
`
`Approximately two-thirds of post-menopausal breast cancer patients have estrogen-dependent
`
`disease thereby rendering them amenable to antiestrogen therapy. Estrogens promote growth and
`
`proliferation of specific target cells, such as breast epithelium and estrogen-dependentbreast carcinoma
`
`10
`
`cells (Brueggemeier Amer J Therapeutics 8:333-344 (2001)). Thus, systemic adjuvant treatment therapy,
`
`administered in addition to primary treatment of localized breast cancer (@.g., breast-conserving surgery
`
`and radiation or mastectomy) nas focused on agents that block the estrogen receptor on tumorcells.
`
`Such antihormonal agents include tamoxifen (NOVALDEX D, SOLTAMOX, TAMOFEN), which inhibits
`
`binding of estrogen with the estrogen receptor, fulvestrant (FASLODEX), which degradesthe receptor, as
`
`15
`
`well as aromatase inhibitors (e.g., anastrozole (ARIMIDEX),
`
`letrozole (FEMARA), and exemestane
`
`(AROMASIN)), which block aromatase enzyme catalysis of androgens to estrogens thereby decreasing
`
`the amount of hormoneligand available to bind with the receptor.
`
`Despite advances in hormonal or other breast cancer therapies,
`
`it
`
`is estimated that 40,921
`
`women will die of recurrent or advanced breast cancer in 2004 (American Cancer Society, Cancer
`
`20
`
`Statistics (2004)). Currently, there is no curative treatment available for patients with extensive disease
`
`and therapy is essentially palliative. Thus, novel therapies, such as combining antihormonal agent with
`
`immunotherapy, are important
`
`in the treatment of breast cancer. More specifically, one cancer
`
`immunotherapy approach targets cytotoxic T lymphocyte-associated antigen 4 (CTLA4; CD152), which is
`
`a cell surface receptor expressed on activated T cells. Binding of CTLA4 to its natural ligands, B7.1
`
`25
`
`(CD80) and B7.2 (CD86), delivers a negative regulatory signal to T cells, and blocking this negative signal
`
`results in enhanced T cell
`
`immune function and antitumor activity in animal models (Thompson and
`
`Allison Immunity 7:445-450 (1997); McCoy and LeGros /mmunol.& Cel! Biol. 77:1-10 (1999)). Several
`studies have demonstrated that CTLA4 blockade using antibodies markedly enhancesT cell-mediated
`killing of tumors and can induce antitumor immunity (Leach et al., Science 271:1734-1736 (1996); Kwon
`et al. Proc. Natl. Acad. Sci. USA 94:8099-8103 (1997); Kwon et al., Natl. Acad. Sci. USA 96:15074-15079
`(1999)).
`
`30
`
`Although use of anti-CTLA4 antibodies to induce an anti-tumor response holds great promise in
`
`the treatment of cancer, and despite the promise of antihormonal therapies in the treatment of breast
`
`cancer, there is a need to develop novel therapies to treat breast cancer. The present invention meets
`
`35
`
`this need.
`
`Summary of the Invention
`
`The invention includes a method for the treatment of breast cancer in a patient in need of such
`
`40
`
`treatment. The method comprises administering to the patient a therapeutically effective amount of an
`anti-CTLA4 antibody, or antigen-binding portion thereof,
`in combination with a therapeutically effective
`amount of an aromataseinhibitor.
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`In one aspect, the aromataseinhibitor is at least one inhibitor selected from the group consisting
`of anastrozole, letrozole and exemestane.
`!n another aspect, the aromataseinhibitor is exemestane.
`In one aspect, the therapeutically effective amount of a human anti-CTLA4 antibody amount
`ranges from about 1 mg/kg to 40 mg/kg.
`In a further aspect, the therapeutically effective amount of a human anti-CTLA4 antibody amount
`ranges from about 3 mg/kg to 15 mg/kg.
`In yet another aspect, the therapeutically effective amount of exemestane ranges from about 25
`mg per day to 200 mg perday.
`In a further aspect, the therapeutically effective amount of exemestane is about 25 mg perday.
`In one aspect, the treatment is selected from the group consisting of a neoadjuvant therapy, an
`10
`adjuvanttherapy, a first line treatment, a secondline treatment, andathird line treatment.
`In another aspect, the antibody is selected from a group consisting of a non-human mammalian
`antibody, a chimeric antibody, and a human antibody.
`In one aspect, the antibody is a human anti-CTLA4 antibody.
`In another aspect, the anti-CTLA4 antibody, or antigen-binding portion thereof,
`antibody selected from the group consisting of:
`(a)
`a humanantibody having a binding affinity for CTLA4 of about 10° or greater,
`and whichinhibits binding between CTLA4 and B7-1, and binding between CTLA4 and B7-2:
`least one
`(b)
`a human antibody having an amino acid sequence comprising at
`human CDR sequence that corresponds to a CDR sequence from an antibody selected from the group
`consisting of 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1., 12.3.1.1, 12.9.1.1, and
`40D1;
`
`15
`
`20
`
`is at least one
`
`25
`
`a humanantibody having the heavy and light chain amino acid sequences of an
`(c)
`antibody selected from the group consisting of 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1,
`11.7.1., 12.3.1.1, and 12.9.1.1;
`(d)
`a human antibody having the amino acid sequences of a heavy chain variable
`region andalight chain variable region of an antibody selected from the group consisting of 4.1.1, 4.8.1,
`4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1., 12.3.1.1, 12.9.1.1, and 10D1;
`(e)
`an antibody, or antigen-binding portion thereof, that competes for binding with
`CTLA4 with at least one antibody having the heavy andlight chain amino acid sequences of an antibody
`selected from the group consisting of 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1.,
`12.3.1.1, 12.9.1.1, and 10D1; and
`(f)
`an antibody, or antigen-binding portion thereof, that cross-competes for binding
`with CTLA4 with at least one antibody having the heavy and light chain amino acid sequences of an
`antibody selected from the group consisting of 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1,
`41.7.1., 12.3.1.1, 12.9.1.1, and 10D1.
`In another aspect, the antibody is a human antibody having the heavy andlight chain amino acid
`sequencesof antibody 11.2.1.
`
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`-3-
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`In yet another aspect, the antibody comprises a heavy chain anda light chain wherein the amino
`acid sequences of the heavy chain variable region of the heavy chain andthelight chain variable region of
`the light chain are selected from the group consisting of:
`(a)
`the amino acid sequence of SEQ ID NO:3 and the amino acid sequence
`of SEQ ID NO:9;
`(b)
`the amino acid sequence of SEQ ID NO:15 and the amino acid sequence
`of SEQ ID NO:21 ;
`(c)
`the amino acid sequence of SEQ ID NO:27 and the amino acid sequence
`
`of SEQ ID NO:33;
`(d)
`the amino acid sequence encoded by the nucleic acid sequence of SEQ
`ID NO:1 and the amino acid sequence encoded by the nucleic acid sequence of SEQ ID
`
`NO:7;
`
`the amino acid sequence encoded bythe nucleic acid sequence of SEQ
`(e)
`ID NO:13 and the amino acid sequence encodedby the nucleic acid sequence of SEQ ID
`
`NO:19;
`
`the amino acid sequence encoded by the nucleic acid sequence of SEQ
`(f)
`ID NO:25 and the amino acid sequence encodedby the nucleic acid sequence of SEQ ID
`
`10
`
`15
`
`NO:31;
`(g) the amino acid sequence of a heavy chain variable region andalight
`
`chain variable region of antibody 10D7.
`20
`In one aspect, the antibody, or antigen-binding portion thereof, is an antibody selected from the
`group consisting of:
`(a) an antibody having a heavy chain variable region comprising the amino acid
`sequencesset forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6,and further having a light
`chain variable region comprising the amino acid sequences set forth in SEQ ID NO:10, SEQ ID
`NO:11 and SEQ ID NO:12;
`(b)
`an antibody having a heavy chain variable region comprising the amino acid
`sequencessetforth in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, andfurther having a
`light chain variable region comprising the amino acid sequencesset forth in SEQ ID NO:22, SEQ
`ID NO:23 and SEQ ID NO:24;
`(c)
`an antibody having a heavy chain variable region comprising the amino acid
`sequencessetforth in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, and further having a
`light chain variable region comprising the amino acid sequencessetforth in SEQ ID NO:34, SEQ
`ID NO:35 and SEQ ID NO:36; and
`(d)
`an antibody having a heavy chain variable region comprising the amino acid
`sequencesof the heavy chain CDR1, CDR2, and CDR3 of antibody 10D1, further having a light
`chain variable region comprising the amino acid sequencesof the light chain CDR1, CDR2, and
`CDR3ofantibody 10D1.
`In another aspect, the method further comprises administering to the patient at least one agent
`selected from the group consisting of an alkylating agent, a folate antagonist, a pyrimidine antagonist, an
`
`40
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`-4-
`
`anthracycline antibiotic, a platinum compound, a taxane, a vinca alkaloid, a camptothecin analog, a toll-
`like receptor stimulating agent, a heat shock protein-based tumor vaccine, an antigen presenting cell-
`based therapy, a mammalian target of rapamycin inhibitor, an erbB2 inhibitor, an EGFR inhibitor, a VEGF
`inhibitor, a VEGFR inhibitor, an angiogenesis inhibitor, an antibody, an immunomodulator, a selective
`estrogen receptor modulator, a cytokine, a tumor vaccine, an antiproliferative agent, an immune
`costimulatory molecule, and a cytokine.
`the treatment of breast cancer
`The invention includes a pharmaceutical composition for
`comprising: a therapeutically effective amount of an anti-CTLA4 antibody; a therapeutically effective
`amountof an aromataseinhibitor; and a pharmaceutically acceptablecarrier.
`In one aspect, the aromataseinhibitor is at least one aromataseinhibitor selected from the group
`consisting of anastrozole, letrozole, and exemestane.
`In a further aspect, the aromataseinhibitor is exemestane.
`The invention includes a use of an amount of an anti-CTLA4 antibody in the preparation of a
`composition for the treatment of breast cancer in a patient wherein the treatment further comprises
`administering to the patient an amount of an aromataseinhibitor.
`In one aspect, the aromataseinhibitor is exemestane and further wherein the antibody is a human
`antibody having the heavy andlight chain variable region amino acid sequencesof antibody 11.2.1.
`
`Brief Description of the Drawings
`The foregoing summary, as well as the following detailed description of the invention, will be
`better understood when read in conjunction with the appended drawings. For the purposeofillustrating
`the invention there are shownin the drawings embodiment(s) which are presently preferred. It should be
`understood, however, that the invention is notlimited to the precise arrangements and instrumentalities
`
`shown:
`
`In the drawings:
`Figure 1, comprising Figures 1A-1D, shows the nucleotide and amino acid sequences for anti-
`CTLA4 antibody 4.1.1. Figure 1A shows thefull length nucleotide sequence for the 4.1.1 heavy chain
`(SEQ ID NO:1). Figure 1B shows the full length amino acid sequence for the 4.1.1 heavy chain (SEQ ID
`NO:2), and the amino acid sequencefor the 4.1.1 heavy chain variable region (SEQ ID NO:3) designated
`between brackets “[ J”. The amino acid sequence of each 4.1.1 heavy chain CDRis underlined. The
`CDR sequences are as follows: CDR1: GFTFSSHGMH (SEQ ID NO:4); CDR2: VIWYDGRNKYYADSV
`(SEQ ID NO:5); and CDR3: GGHFGPFDY(SEQ ID NO:6). Figure 1¢ shows the nucleotide sequence for
`the 4.1.1 light chain (SEQ ID NO:7). Figure 1D shows the amino acid sequenceof the full length 4.1.1
`light chain (SEQ ID NO:8), and the variable region as indicated between brackets “[ J” (SEQ ID NO:9).
`The amino acid sequenceof each CDRisindicated as follows: CDR1: RASQSISSSFLA (SEQ ID NO:10);
`CDR2: GASSRAT(SEQ ID NO:11); and CDR3: CQQYGTSPWT(SEQ ID NO:12).
`Figure 2, comprising Figures 2A-2D, shows the nucleotide and amino acid sequences for anti-
`CTLA4 antibody 4.13.1. Figure 2A shows the full length nucleotide sequence for the 4.13.1 heavy chain
`(SEQ ID NO:13). Figure 2B showsthe full length amino acid sequence for the 4.13.1 heavy chain (SEQ
`ID NO:14), and the amino acid sequence for the 4.13.1 heavy chain variable region (SEQ ID NO:15)
`
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`-5-
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`designated between brackets “[ ]. The amino acid sequence of each 4.13.1 heavy chain CDR is
`underlined.
`The CDR sequences are as follows: CDR1: GFTFSSHGIH (SEQ ID NO:16); CDR2:
`VIWYDGRNKDYADSV (SEQ ID NO:12); and CDR3: VAPLGPLDY (SEQ ID NO:18). Figure 2C shows
`the nucleotide sequence for the 4.13.1 light chain (SEQ ID NO:19). Figure 2D shows the amino acid
`sequence of the full
`length 4.13.1 light chain (SEQ ID NO:20), and the variable region as indicated
`between brackets “[ ]” (SEQ ID NO:21). The amino acid sequence of each CDRis indicated as follows:
`CDR1: RASQSVSSYLA (SEQ ID NO:22); CDR2: GASSRAT (SEQ !D NO:23);
`and CDR3:
`CQQYGRSPFT(SEQ ID NO:24).
`Figure 3, comprising Figures 3A-3D, shows the nucleotide and amino acid sequences for anti-
`CTLA4 antibody 11.2.1. Figure 3A showsthefull length nucleotide sequencefor the 11.2.1 heavy chain
`(SEQ ID NO:25). Figure 3B showsthefull length amino acid sequencefor the 11.2.1 heavy chain (SEQ
`ID NO:26), and the amino acid sequence for the 11.2.1 heavy chain variable region (SEQ ID NO:27)
`designated between brackets “[ J’. The amino acid sequence of each 11.2.1 heavy chain CDR is
`underlined. The CDR sequences are as follows: CDR1: GFTFSSYGMH (SEQ ID NO:28); CDR2:
`VIWYDGSNKYYADSV (SEQ ID NO:29); and CDR3: DPRGATLYYYYYGMDV(SEQ ID NO:30). Figure
`3C shows the nucleotide sequence for the 11.2.1 light chain (SEQ ID NO:31). Figure 3D shows the
`amino acid sequence of the full length 11.2.1 light chain (SEQ ID NO:32), and the variable region as
`indicated between brackets “[ ]” (SEQ ID NO:33). The amino acid sequence of each CDRis indicated as
`follows: CDR1: RASQSINSYLD (SEQ ID NO:34); CDR2: AASSLQS (SEQ ID NO:35); and CDR3:
`QOQYYSTPFT(SEQ ID NO:36).
`
`Detailed Description Of The Invention
`The invention relates to anti-CTLA4 antibodies used in combination with at least one aromatase
`inhibitor, e.g., anastrozole, letrozole, and exemestane, to treat breast cancerin a patient in need of such
`treatment. The invention further relates to treatment of breast cancer by combination of the antibody-
`
`aromataseinhibitor combination with another agentor agents.
`terms used in connection with the
`Unless otherwise defined herein, scientific and technical
`presentinvention shall have the meanings that are commonly understood by those of ordinary skill in the
`art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms
`shall include the singular. Generally, nomenclatures used in connection with, and techniquesof, cell and
`tissue culture, molecular biology,
`immunology, microbiology, genetics and protein and nucleic acid
`chemistry and hybridization described herein are those well known and commonly usedin theart.
`The methods and techniques of the present invention are generally performed according to
`methods well knownin the art and as described in various general and more specific references that are
`cited and discussed throughout the present specification unless otherwise indicated. Such references
`include, e.g., Sambrook and Russell, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor
`Press, Cold Spring Harbor, NY (2001), Ausubelet al., Current Protocols in Molecular Biology, John Wiley
`& Sons, NY (2002), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor
`Laboratory Press, Cold Spring Harbor, NY (1990), which are incorporated herein by reference. Enzymatic
`reactions and purification techniques are performed according to manufacturer's specifications, as
`
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`commonly accomplishedin the art or as described herein. The nomenclatures used in connection with,
`and the laboratory procedures and techniquesof, analytical chemistry, synthetic organic chemistry, and
`medicinal and pharmaceutical chemistry described herein are those well known and commonly usedin
`the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical
`preparation, formulation, and delivery, and treatmentof patients.
`As used herein, each of the following terms has the meaning associated withit in this section.
`The articles “a” and “an” are used herein to refer to one or to more than one(i.e., to at least one)
`of the grammatical object of the article. By way of example, “an element” means one element or more
`than one element.
`,
`As used herein, the twenty conventional amino acids and their abbreviations follow conventional
`usage. See Immunology--A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer
`Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.
`A "conservative amino acid substitution" is one in which an amino acid residue is substituted by
`another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or
`hydrophobicity).
`In general, a conservative amino acid substitution will not substantially change the
`functional properties of a protein.
`In cases where two or more amino acid sequences differ from each
`other by conservative substitutions, the percent sequenceidentity or degree of similarity may be adjusted
`upwardsto correct for the conservative nature of the substitution. Means for making this adjustment are
`well-knownto thoseofskill in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31 (1994).
`Examples of groups of amino acids that have side chains with similar chemical properties include
`1) aliphatic side chains: glycine, alanine,valine, leucine, and isoleucine; 2) aliphatic-hydroxy! side chains:
`serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains:
`phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side
`chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine.
`Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-
`tyrosine,lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
`Alternatively, a conservative replacement is any change having a positive value in the PAM250
`log-likelihood matrix disclosed in Gonnetet al., Science 256:1443-45 (1992), herein incorporated by
`reference. A "moderately conservative" replacement is any change having a nonnegative value in the
`PAM250 log-likelihood matrix.
`Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2)
`reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, and(4) confer or
`modify other physicochemical or functional properties of such analogs. Analogs comprising substitutions,
`deletions, and/or insertions can include various muteins of a sequence other than the specified peptide
`sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid
`substitutions) may be madein the specified sequence (preferably in the portion of the polypeptide outside
`the domain(s) forming intermolecular contacts, e.g., outside of the CDRs). A conservative amino acid
`substitution should not substantially change the structural characteristics of the parent sequence(e.g., a
`replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt
`other types of secondary structure that characterizes the parent sequence). Examples of art-recognized
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`polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular
`Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction fo Protein
`Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et
`al., Nature 354:105 (1991), which are each incorporated herein by reference.
`Sequence similarity for polypeptides is typically measured using sequence analysis software.
`Protein analysis software matches similar sequences using measures of similarity assigned to various
`substitutions, deletions and other modifications,
`including conservative amino acid substitutions. For
`instance, Genetics Computer Group (GCG available from Genetics Computer Group, Inc.), also referred
`to as the Wisconsin Package,
`is an integrated software package of over 130 programs for accessing,
`analyzing and manipulating nucleotide and protein sequences. GCG contains programs such as "Gap"
`and "Bestfit" which can be used with default parameters to determine sequence similarity, homology
`and/or sequence identity between closely related polypeptides, such as homologous polypeptides from
`different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG
`version 6.1, version 9.1, and version 10.0.
`Polypeptide sequences also can be compared using FASTA using default or recommended
`parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments
`and percent sequence identity of the regions of the best overlap between the query and search
`sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219
`(2000)). Another preferred algorithm when comparing a sequence of the invention to a database
`containing a large number of sequences from different organisms is the computer program BLAST,
`especially blastp or tblastn, using default parameters. See, e.g., Altschul et al., J. Mol. Biol. 215:403-410
`(1990); Altschul et al., Nucleic Acids Res. 25:3389-402 (1997); herein incorporated by reference.
`An “aromatase,” as the term is used herein, refers to an enzyme (e.g., 450arom) that catalyzes the
`aromatization of the A ring of an androgen substrate (mainly androstenedione)to estrone.
`“Aromatase inhibitor’ means a compound or substance that when contacted with an aromatase,
`detectably inhibits the formation of the A ring of an androgen substrate compared with the level of such
`aromatization in the absence of the compound or substance. Any assay for assessing the level of
`enzymatic activity by an aromatase can be used to assess whether there is inhibition and/or the level
`thereof. An aromatase inhibitor includes, but is not limited to, a steroidal and a non-steroidal inhibitor
`such as, inter alia, exemestane, anastrozole, letrozole, fadrozole, vorozole, and formestane, and the term
`encompasses any aromataseinhibitor whether now knownoridentified in the future.
`(L) chains inter-
`An intact "antibody" comprises at least two heavy (H) chains and two light
`connected by disulfide bonds. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed.
`RavenPress, N.Y. (1989)) (incorporated by referencein its entirety for all purposes). Each heavy chainis
`comprised of a heavy chain variable region (HCVR or Vy) and a heavy chain constant region (Cy). The
`heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Eachlight chain is
`comprised ofa light chain variable region (LCVR or V) andalight chain constant region. Thelight chain
`constant region is comprised of one domain, C,. The Vy and V, regions can be further subdivided into
`regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions
`that are more conserved, termed framework regions (FR). Each Vy and V, is composedof three CDRs
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`and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1,
`FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acids to each domain is in accordance with the
`definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health,
`Bethesda, MD (1987 and 1991)), or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987), Chothia et al.,
`Nature 342:878-883 (1989).
`The term “antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein,
`refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g.,
`CTLA4).
`It has been shown that the antigen-binding function of an antibody can be performed by
`fragments of a full-length antibody. Examples of binding fragments encompassed within the term
`"antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of
`the V,, Vu, CL and Cy1 domains;(ii) a F(ab’). fragment, a bivalent fragment comprising two Fab fragments
`linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V, and C,,1 domains;
`(iv) a Fv fragment consisting of the V, and V,, domainsof a single arm of an antibody, (v) a dAb fragment
`(Ward et al.,
`(1989) Nature 341:544-546), which consists of a Vj, domain; and (vi) an isolated
`complementarity determining region (CDR). Furthermore, although the two domainsof the Fv fragment,
`Vand Vy, are coded for by separate genes,
`they can be joined, using recombinant methods, by a
`synthetic linker that enables them to be made asa single protein chain in which the V, and Vi, regionspair
`to form monovalent molecules (known as single chain Fv (scFv)); see e.g., Bird et al. Science 242:423-
`426 (1988) and Huston et al. Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such single chain
`antibodies are also intended to beencompassedwithin the term "antigen-binding portion” of an antibody.
`Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodiesare bivalent,
`bispecific antibodies in which Vy and V° domains are expressed on a single polypeptide chain, but using a
`linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the
`domainsto pair with complementary domains of another chain and creating two antigen binding sites (see
`e.g., Holliger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al. Structure 2:1121-1123
`(1994)).
`
`Still further, an antibody or antigen-binding portion thereof may be part of larger immunoadhesion
`molecules, formed by covalent or noncovalent association of the antibody or antibody portion with one or
`more other proteins or peptides. Examples of such immunoadhesion molecules include use of the
`streptavidin core region to make a tetrameric scFv molecule (Kipriyanov et al. Human Antibodies and
`Hybridomas 6:93-101 (1995)) and use of a cysteine residue, a marker peptide and a C-terminal
`polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov et al. Mol.
`Immunol.
`31:1047-1058 (1994)). Other examples include where one or more CDRs from an antibody are
`incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin that
`specifically binds to an antigen of interest, such as CTLA4.
`In such embodiments, the CDR(s) may be
`incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain,
`or may be incorporated noncovalently. Antibody portions, such as Fab and F(ab’), fragments, can be
`prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion,
`respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion
`molecules can be obtained using s