(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`‘
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`Organization
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`International Bureau
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`é,
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`~/
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`(43) International Publication Date
`7 January 2016 (07.01.2016)
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`WIPOIPCT
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`(10) International Publication Number
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`WO 2016/004383 A1
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`(51)
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`International Patent Classification:
`C07K16/28 (2006.01)
`C07H 21/04 (2006.01)
`C07K 16/00 (2006.01)
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`(21)
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`International Application Number:
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`PCT/US2015/039103
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`(22)
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`International Filing Date:
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`(25)
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`(26)
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`(30)
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`(71)
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`(72)
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`(74)
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`(81)
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`Filing Language:
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`Publication Language:
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`2 July 2015 (02.07.2015)
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`English
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`English
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`Priority Data:
`62/020,806
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`3 July 2014 (03.07.2014)
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`US
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`Applicants: CITY OF HOPE [US/US]; 1500 East Duarte
`Road, Duarte, CA 91010-3000 (US). THOMAS JEFFER-
`SON UNIVERSITY [US/US]; 1020 Locust Street, Jeffer-
`son Alumni Hall, Suite M34, Philadelphia, PA 19107
`(US).
`
`Inventors: WILLIAMS, John, C.; 462 Stedman Place,
`Monrovia, CA 91016 (US). RODECK, Ulrich; 758 N
`22nd Street, Philadelphia, PA 19130 (US).
`
`Agents: HETZER-EGGER, Claudia H. et a1; MintZ
`Levin Cohn Ferris Glovsky And Popco PC, 3580 Carmel
`Mountain Road, Suite 300, San Diego, CA 92130 (US).
`
`Designated States (unless otherwise indicated, for every
`kind ofnational protection available): AE, AG, AL, AM,
`
`A0, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG,
`MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM,
`PA, PE, PG, PH, PL, PT, QA, Ro, RS, RU, RW, SA, SC,
`SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84)
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`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ,
`TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,
`TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE,
`DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU,
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK,
`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, KM, ML, MR, NE, SN, TD, TG).
`Published:
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`with international search report (Art. 21(3))
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`before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments (Rule 482(k))
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`with sequence listing part ofdescription (Rule 5.2(a))
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`(54) Title: TUMOR-SELECTIVE CTLA-4 ANTAGONISTS
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`(57) Abstract: Provided herein are recombinant masking proteins and recombinant ligand proteins useful in treating cancer, neuro —
`degenerative disease, and cardiovascular disease. The recombinant masking proteins provided herein may, inter alia, be used as non-
`covalent masks of antagonists of, for example, cellular growth factors (e.g., TNF) or cell surface proteins (e. g., CTLA—4).
`
`
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`TUMOR-SELECTIVE CTLA—4 ANTAGONISTS
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`CROSS-REFERENCES TO RELATED APPLICATIONS
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`[0001[ This application claims the benefit of US. Provisional Application No. 62/020,806, filed
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`July 3, 2014, the content of which is incorporated herein by reference in its entirety and for all
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`purposes.
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`REFERENCE TO A "SEQUENCE LI STING," A TABLE, OR A COMPUTER
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`PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII FILE
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`[0002[
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`The Sequence Listing written in file 48440—530001WO_ST25.TXT, created on July 1,
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`2015, 50,482 bytes, machine format IBM-PC, MS Windows operating system, is hereby
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`incorporated by reference.
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`STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER
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`FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
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`[0003[ This invention was made with government support under Grant Nos. R21 CAl35216 and
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`P30 CA033572 awarded by the National Cancer Institute. The Government has certain rights in the
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`invention.
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`BACKGROUND OF THE INVENTION
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`[0004[ Most protein—based targeted therapies currently in use target molecular mechanisms but not
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`disease sites such as monoclonal antibodies which bind to diseased cells or to T cells (as in the case
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`of ipilimumab and the related CTLA-4 antagonist tremelimumab). However, engagement of these
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`targets in normal tissues gives rise to adverse events with various degrees of severity depending on
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`the molecular target. If the therapeutic agent induces autoimmune phenomena the toxicity not only
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`leads to significant morbidity, but also often necessitates the administration of immunosuppressants
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`(corticosteroids, TNF-a inhibitors) which interfere with therapeutic intent.
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`[0005[ There is a substantial, unmet need in the clinic to develop targeted therapies with reduced
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`side effects and enhanced efficacy. Often referred to as “magic bullets”, monoclonal antibodies
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`(mAbs) preferentially target diseased tissue and are generally better tolerated than traditional
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`chemotherapy. In most cases, therapeutic mAbs bind to an antigen that is ‘self’ but overexpressed in
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`the tumor, such as the Erbb family members (e.g., EGFR, Her2). However, systemic administration
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`of these mAbs, at therapeutic doses, leads the mAb to engage antigen expressed on normal tissues,
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`and thus, can lead to serious adverse side effects. As an example, the mAbs cetuximab (ErbituXTM)
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`and trastuzumab (HereeptinTM) that are currently used to treat neck and colon cancers and breast
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`cancer, also give rise to acneiform skin eruptions, gastrointestinal irritation and cardiotoxicity.
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`Serious side effects due to off-target effects have been also been observed with other mAbs, such as
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`efalizumab (RaptivaTM). Indeed, the adverse effects of efalizumab in treatment of psoriasis, recently
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`resulted in efalizumab being withdrawn from the market due to the development of progressive
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`multifocal leukoencephalopathy. Adverse side effects of mAbs in clinical use reduce the efficacy of
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`these agents, cause additional, substantial costs related to monitoring these side effects, and
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`ultimately reduce patients’ quality of life. In fact, the psychological aspect of the severe skin rashes
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`alone has led patients to discontinue cetuXimab, an effective treatment approved for various
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`epithelial malignancies. Thus, there is a need in the art for treatment options which avoid these and
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`other adverse effects. Provided herein are solutions to these and other problems in the art.
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`BRIEF SUMMARY OF THE INVENTION
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`[0006[ Accordingly, herein are provided, inter alia, compositions, kits, and methods for treating
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`diseases using recombinant masking proteins and recombinant ligand proteins.
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`[0007 [
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`Provided herein are protein compositions. In one aspect these are recombinant masking
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`proteins including two identical masking protein domains. Each of the masking protein domains
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`includes (1) a masking dimerizing domain; (2) a ligand—masking binding domain; and (3) a
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`cleavable masking linker connecting the ligand-masking binding domain to the masking dimerizing
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`domain. The masking protein domains are bound together.
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`[0008[ Also provided herein are pharmaceutical compositions. In one aspect, the pharmaceutical
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`compositions include a pharmaceutically acceptable exeipient, a recombinant masking protein as
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`described herein, including embodiments thereof, and a recombinant ligand protein as described
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`herein, including embodiments thereof.
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`[0009[ The recombinant protein compositions and pharmaceutical compositions may also be
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`included in kits described herein. In one aspect this is a kit that includes a recombinant masking
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`protein as described herein, including embodiments thereof, and a recombinant ligand protein as
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`described herein, including embodiments thereof.
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`[0010[
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`Provided herein are methods of treating a disease in a subject in need thereof. In one
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`aspect, the method includes administering to a subject a therapeutically effective amount of a
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`recombinant masking protein and a recombinant ligand protein as described herein including
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`embodiments thereof
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`[0011[
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`FIG. 1: Predicted mode of action of the masked CTLA-4 antagonist modified lipocalin2
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`(m—Lcn2): A) In normal tissues M-Lcn2 is masked by the recombinant CTLA-4 fragment tethered to
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`it by N-terminal linkage and hence does not bind to T cells but in prostate cancer tissue, tumor-
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`associated MMP9 cleaves the CTLA—4 mask leading to native CTLA—4 engagement and activation
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`of tumor—infiltrating lymphocytes (TILs) (the design principle can be applied to monovalent or
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`bivalent m-Lcn2 to exploit the avidity gain of such ligands); B) Predicted atomic structure of the
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`bivalent CTLA-4 mask containing an Fe fragment tethered to MMP9-cleavable extensions that
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`consist of CTLA-4-derived peptides that fit the m-Lcn2 binding pocket.
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`[0012[
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`FIG. 2: Variant m-Lcn2 and mask designs to facilitate tumor targeting: A) To create a
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`bivalent analog, m-Lcn2 is fused to the human IgG1 Fe domain; B) To create a bivalent mask,
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`CTLA-4 is fused to the same Fe domain, but the linker contains a MMP9 site; C) To enhance tumor
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`targeting, an HA scFv may be fused to the C-terminus of the m-Lcn2-Fc construct (MMP9 cleavage
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`site may added to enhance engagement TILs at tumor sites) and a non—cleavable linker may also be
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`generated; D) The same targeting principle may be applied to the CTLA4—Fc mask (please refer to
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`Fig. 13).
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`[0013[
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`F1G. 3: Characterization of mask prototype complex with m-Lcn2-Fc and CTLA-4-Fc: A)
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`SEC indicates the formation ofa complex (orange trace) at l 1.4 mL; B) SPR traces indicate m-
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`Lcn2—Fc binds with high affinity to CTLA-4-MMP9-Fc (highest concentration is 300 nM). C) SDS-
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`PAGE of CTLA-4-MMP9-Fc before and after 8hrs of MMP9 treatment (control is a mutated the
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`MMP9 site that was not cleaved by MMP9 (right panel)).
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`[0014[
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`FIG. 4: Proposed mutations in the CTLA-4 mask to facilitate unmasking and block CTLA-
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`4 receptor binding on cells.
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`[0015[
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`FIG. 5: Representation of non-covalent CTLA4-Fc mask system. CTLA4 antagonist
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`prodrug is systemically administered as a 1:1 mLCNZ—Fc:CTLA4—Fc complex. A Non—covalent
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`CTLA4—Fc MMP mask inhibits mLCNZ—Fc from binding endogenous CTLA4 in normal, healthy
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`tissue. B Once the prodrug cntcrs the tumor microcnvironmcnt, local protcasc ovcrcxprcssion
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`(shown here as MMP) activates the prodrug by cleaving one or both MMP sites on the ligand mask.
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`Avidity and, therefore, affinity are lost between the mask and mLCNZ-Fc, permitting dissociation of
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`mask and enabling mLCNZ-Fc to bind to CTLA4 within the tumor.
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`[0016[
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`FIG. 6: Binding stoichiometry of mLCNZ-Fc and CTLA4-Fc MMP9 is dependent on initial
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`protein concentrations. A High initial protein concentrations (“Fast " mixture) resulted in multiple
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`high—order binding mixtures, as demonstrated by SEC. Most protein was bound l:l when lower
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`initial concentrations were used (“Slow ” mixture). Peaks 1, 2, and 3 of initial B “Fast” mixture and
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`C “Slow ” mixture were isolated and reanalyzed by SEC.
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`[0017[
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`FIG. 7: CTLA4-Fc MMP9 inhibits the engagement of mLCNZ-Fc and murinc T-cclls.
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`Murine splenocytes were isolated and cultured in the absence or presence of tumor supernatant
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`(naive or primed, respectively). After 24 h, 20 jig CTLA4-Fc MMPQ was added in the competition
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`condition. Subsequently 20 jig 647'mLCN2-Fc was added to all treatment wells. Cells were
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`analyzed by flow cytometry for CD4+ (top row), CD8+ (bottom), and 647'mLCN2-Fc staining.
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`[0018[
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`FIG. 8: CTLA4-Fc MMP9 inhibits mLCNZ-Fc-mediated stimulation of IFN-y production by
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`T-cells in vitro. Cytokine IFN—y production in murine splenocytes co-cultured with mLCNZ-Fc or
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`mLCN2—Fc:CTLA4—Fc (complex) was analyzed by ELISpot. Splenocytes from OT—I mice were
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`activated by A phytohaemagglutinin (PHA), B TAC—expressing B 16 melanoma cells in vitro, or C in
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`vivo vaccination with adenovirus encoding TAC antigen. Black squares represent the number of IFN-y
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`producing cells per well. Black bisecting bar is the average per condition. Statistics provided from
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`one-tail t-test. * p<0.05; ** p<0.0l; *** p<0.001.
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`[0019[
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`FIG. 9: CTLA4-Fc MMP9 binds CD80 and CD86 expressed on live cells. Human
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`lymphoblast Daudi cells, which naturally express CD80 and CD86, were incubated with 647"CTLA4-
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`Fc MMPQ. CTLA4-Fc binding was assessed by flow cytometry.
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`[0020[
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`FIG. 10: SEC analysis of CD86-Fc interactions CTLA4-Fc mask variants. Equimolar
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`solutions of recombinant human CD86—Fc and CTLA4—Fc variants were analyzed by analytical
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`SEC. Absorbance at 230 nm is reported.
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`[0021[
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`FIG.
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`1 1: SEC analysis of mLCN2-Fc interactions CTLA4-Fc mask variants. Equimolar
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`solutions of mLCNZ—Fc and CTLA4—Fc variants were analyzed by analytical SEC. Absorbance at
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`230 nm is reported.
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`DETAILED DESCRIPTION OF THE INVENTION
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`[0022[ The abbreviations used herein have their conventional meaning within the chemical and
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`biological arts. The chemical structures and formulae set forth herein are constructed according to
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`the standard rules of chemical valency known in the chemical arts.
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`[0023[ A “cell” as used herein, refers to a cell carrying out metabolic or other functions sufficient
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`to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art
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`including, for example, presence of an intact membrane, staining by a particular dye, ability to
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`produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a
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`viable offspring. Cells may include prokaryotic and eukaroytic cells. Prokaryotic cells include but
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`are not limited to bacteria. Eukaryotic cclls include but are not limited to yeast cells and cells
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`derived from plants and animals, for example mammalian, insect (e. g, spodoptera) and human cells.
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`Cells may be useful when they are naturally nonadherent or have been treated not to adhere to
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`surfaces, for example by trypsinization.
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`[0024[ The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer
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`to a polymer of amino acid residues, wherein the polymer may optionally be conjugated to a moiety
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`that does not consist of amino acids. The terms apply to amino acid polymers in which one or more
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`amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino
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`acid, as well as to naturally occurring amino acid polymers and non—naturally occurring amino acid
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`polymer. A “fusion protein” refers to a chimeric protein encoding two or more separate protein
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`sequences that is recombinantly expressed as a single moiety.
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`[0025[ The term “peptidyl” and “peptidyl moiety” means a monovalent peptide.
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`[0026[ The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as
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`amino acid analogs and amino acid mimetics that function in a manner similar to the naturally
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`occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as
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`well as those amino acids that are later modified, e. g., hydroxyproline, y-carboxyglutamate, and O-
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`phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure
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`as a naturally occurring amino acid, i.e., an (1 carbon that is bound to a hydrogen, a carboxyl group,
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`an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine
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`methyl sulfonium. Such analogs have modified R groups (e. g., norleucine) or modified peptide
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`backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino
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`acid mimctics refers to chemical compounds that have a structure that is different from the general
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`chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring
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`amino acid.
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`[0027[ Amino acids may be referred to herein by either their commonly known three letter
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`symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature
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`Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter
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`codes.
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`[0028[
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`“Conservatively modified variants” applies to both amino acid and nucleic acid sequences.
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`With respect to particular nucleic acid sequences, conservatively modified variants refers to those
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`nucleic acids which encode identical or csscntialIy idcntical amino acid scqucnccs, or where the
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`nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of
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`the degeneracy of the genetic code, a large number of functionally identical nucleic acids sequences
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`encode any given amino acid residue. For instance, the codons GCA, GCC, GCG and GCU all
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`encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the
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`codon can be altered to any of the corresponding codons described without altering the encoded
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`polypeptide. Such nucleic acid variations are “silent variations,” which are one species of
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`conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide
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`also describes every possible silent variation of the nucleic acid. One of skill will recognize that
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`each codon in a nucleic acid (cxccpt AUG, which is ordinarily the only codon for mcthioninc, and
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`TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally
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`identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide
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`is implicit in each described sequence with respect to the expression product, but not with respect to
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`actual probe sequences.
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`[0029[ As to amino acid sequences, one of skill will recognize that individual substitutions,
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`deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds
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`or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a
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`“conservatively modified variant” where the alteration results in the substitution of an amino acid
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`with a chemically similar amino acid. Conservative substitution tables providing functionally similar
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`amino acids are well known in the art. Such conservatively modified variants are in addition to and
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`do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
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`[0030[ The following eight groups each contain amino acids that are conservative substitutions for
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`one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine
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`(N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M),
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`Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W), 7) Serine (S), Threonine (T); and
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`8) Cysteine (C), Methionine (M) (see, e. g., Creighton, Proteins (1984)).
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`[0031[
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`"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in
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`either single- or double-stranded form, and complements thereof The term "polynucleotide" refers
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`to a linear sequence of nucleotides. The term "nucleotide" typically refers to a single unit of a
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`polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or
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`modified versions thcrcof. Examples of polynuclcotidcs contemplated hcrcin include single and
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`double stranded DNA, single and double stranded RNA (including siRNA), and hybrid molecules
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`having mixtures of single and double stranded DNA and RNA. Nucleic acid as used herein also
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`refers nucleic acids that have the same basic chemical structure as a naturally occurring nucleic
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`acids. Such analogues have modified sugars and/or modified ring substituents, but retain the same
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`basic chemical structure as the naturally occurring nucleic acid. A nucleic acid mimetic refers to
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`chemical compounds that have a structure that is different the general chemical structure of a nucleic
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`acid, but that functions in a manner similar to a naturally occurring nucleic acid. Examples of such
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`analogues include, without limitation, phosphorothiolates, phosphoramidates, methyl phosphonates,
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`chiral-methyl phosphonates, 2-O-mcthyl ribonucleotides, and peptide-nucleic acids (PNAs).
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`[0032[
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`“Synthetic mRNA” as used herein refers to any mRNA derived through non-natural
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`means such as standard oligonucleotide synthesis techniques or cloning techniques. Such mRNA
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`may also include non-proteinogenic derivatives of naturally occurring nucleotides. Additionally,
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`“synthetic mRNA” herein also includes mRNA that has been expressed through recombinant
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`techniques or exogenously, using any expression vehicle, including but not limited to prokaryotic
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`cells, eukaryotic cell lines, and viral methods. “Synthetic mRNA” includes such mRNA that has
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`been purified or otherwise obtained from an expression vehicle or system.
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`[0033[ The term “modulation”, “modulate”, or “modulator” are used in accordance with their
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`plain ordinary meaning and refer to the act of changing or varying one or more properties.
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`“Modulator” refers to a composition that increases or decreases the level of a target molecule or the
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`function of a target molecule or the physical state of the target of the molecule. “Modulation” refers
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`to the process of changing or varying one or more properties. For example, as applied to the effects
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`of a modulator on a biological target, to modulate means to change by increasing or decreasing a
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`property or function of the biological target or the amount of the biological target.
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`[0034[ As defined herein, the term “inhibition”, “inhibit”, “inhibiting” and the like in reference to
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`a protein-inhibitor (e.g. antagonist) interaction means negatively affecting (e.g. decreasing) the
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`activity or function of the protein relative to the activity or function of the protein in the absence of
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`the inhibitor. In embodiments inhibition refers to reduction of a disease or symptoms of disease.
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`Thus, in embodiments, inhibition includes, at least in part, partially or totally blocking stimulation,
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`decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating
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`signal transduction or enzymatic activity or the amount of a protein.
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`[0035[ As defined herein, the term “activation”, “activate , activating” and the like in reference
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`to a protein-activator (e.g. agonist) interaction means positively affecting (e.g. increasing) the
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`activity or function of the relative to the activity or function of the protein in the absence of the
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`activator (e. g. composition described herein). Thus, in embodiments, activation may include, at
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`least in part, partially or totally increasing stimulation, increasing or enabling activation, or
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`activating, sensitizing, or up—regulating signal transduction or enzymatic activity or the amount of a
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`protein decreased in a disease.
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`[0036[ The term “recombinan ” when used with reference, for example, to a cell, a nucleic acid, a
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`protein, or a vector, indicates that the cell, nucleic acid, protein or vector has been modified by or is
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`the result of laboratory methods. Thus, for example, recombinant proteins include proteins
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`produced by laboratory methods. Recombinant proteins can include amino acid residues not found
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`within the native (non-recombinant) form of the protein or can be include amino acid residues that
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`have been modified, e. g., labeled.
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`[0037 [ The term “heterologous” when used with reference to portions of a nucleic acid indicates
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`that the nucleic acid comprises two or more subsequences that are not found in the same relationship
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`to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having
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`two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e. g., a
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`promoter from one source and a coding region from another source. Similarly, a heterologous
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`protein indicates that the protein comprises two or more subsequences that are not found in the same
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`relationship to each other in nature (c.g., a fusion protein).
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`[0038[
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`“Antibody” refers to a polypeptide comprising a framework region from an
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`immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The
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`recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu
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`constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains
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`are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or
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`epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
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`Typically, the antigen—binding region of an antibody will be most critical in specificity and affinity
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`of binding. In some embodiments, antibodies or fragments of antibodies may be derived from
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`different organisms, including humans, mice, rats, hamsters, camels, etc. Antibodies of the invention
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`may include antibodies that have been modified or mutated at one or more amino acid positions to
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`improve or modulate a desired function of the antibody (e. g. glycosylation, expression, antigen
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`recognition, effector functions, antigen binding, specificity, etc.).
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`[0039[ An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each
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`tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light”
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`(about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a
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`variable region of about 100 to l 10 or more amino acids primarily responsible for antigen
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`recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light
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`and heavy chains respectively. The PC (i.c. fragment crystallizablc region) is the “base” or “tail” of
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`an immunoglobulin and is typically composed of two heavy chains that contribute two or three
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`constant domains depending on the class of the antibody. By binding to specific proteins the Fc
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`region ensures that each antibody generates an appropriate immune response for a given antigen.
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`The Fc region also binds to various cell receptors, such as Fc receptors, and other immune
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`molecules, such as complement proteins.
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`[0040[ Antibodies exist, for example, as intact immunoglobulins or as a number of well-
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`characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin
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`digests an antibody below the disulfide linkages in the hinge region to produce F(ab)’2, a dimer of
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`Fab which itself is a light chain joined to VH-CHl by a disulfide bond. The F(ab)’2 may be reduced
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`under mild conditions to break the disulfide linkage in the hinge region, thereby converting the
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`F(ab)’2 dimer into an Fab’ monomer. The Fab’ monomer is essentially Fab with part of the hinge
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`region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are
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`defined in terms of the digestion of an intact antibody, one of skill will appreciate that such
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`fragments may be synthesized de novo either chemically or by using recombinant DNA
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`methodology. Thus, the term antibody, as used herein, also includes antibody fragments either
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`produced by the modification of whole antibodies, or those synthesized de novo using recombinant
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`DNA methodologies (e. g., single chain Fv) or those identified using phage display libraries (see,
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`e.g., McCafferty et al., Nature 348:552—554 (1990)).
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`[0041[ A single—chain variable fragment (scFv) is typically a fusion protein of the variable regions
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`of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide
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`of 10 to about 25 amino acids. The linker may usually rich in glycine for flexibility, as well as serine
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`or threonine for solubility. The linker can either connect the N-terminus of the VH with the C-
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`terminus of the VL, or vice versa.
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`[0042[
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`For preparation of suitable antibodies of the invention and for use according to the
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`invention, e.g., recombinant, monoclonal, or polyclonal antibodies, many techniques known in the
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`art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al.,
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`Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer
`
`Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow &
`
`Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles
`
`and Practice (2d ed. 1986)). The genes encoding the heavy and light chains of an antibody of interest
`
`can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a
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`hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding heavy
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`and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells.
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`Random combinations of the heavy and light chain gene products generate a large pool of antibodies
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`with different antigenic specificity (see, e.g., Kuby, Immunology (3rd ed. 1997)). Techniques for the
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`production of single chain antibodies or recombinant antibodies (U.S. Patent 4,946,778, U.S. Patent
`
`No. 4,816,567) can be adapted to produce antibodies to polypeptides of this invention. Also,
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`transgenic mice, or other organisms such as other mammals, may be used to express humanized or
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`human antibodies (see, e.g., U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;
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`5,633,425; 5,661,016, Marks et a1., Bio/Technology 10:779-783 (1992); Lonberg et a1., Nature
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`368:856—859 (1994); Morrison, Nature 368:812—13 (1994); Fishwild et al., Nature Biotechnology
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`14:845—51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern.
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`Rcv. 1mmunol. 13:65-93 (1995)). Alternatively, phage display technology can bc used to identify
`
`antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g.,
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`McCafferty et al., Nature 348:552-554 (1990); Marks et a1., Biotechnology 10:779-783 (1992)).
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`Antibodies can also be made bispecific, i.e., able to recognize two different antigens (see, e.g., WO
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`93/08829, Traunecker et a1., EMBO J. 10:3655-3 659 (1991); and Suresh et a1., Methods in
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`Enzymology 121:210 (1986)). Antibodies can also be heteroconjugates, e.g., two covalently joined
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`antibodies, or immunotoxins (see, e.g., US. Patent No. 4,676,980 , WO 91/00360; WO 92/200373;
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`and EP 03089).
`
`[0043[ Methods for humanizing or primatizing non—human antibodies are well known in the art
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`(e.g., US. Patent Nos. 4,816,567; 5,530,101; 5,859,205; 5,585,089; 5,693,761; 5,693,762;
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`5,777,085; 6,180,370; 6,210,671; and 6,329,511; WO 87/02671; EP Patent Application 0173494;
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`Jones et a1. (1986) Nature 3212522; and Verhoyen et a1. (1988) Science 239: 1534). Humanized
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`antibodies are further described in, e. g., Winter and Milstein (1991) Nature 349:293. Generally, a
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`humanized antibody has one or more amino acid residues introduced into it from a source which is
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`non-human. These non-human amino acid residues are often referred to as import residues, which
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`are typically taken from an import variable domain. Humanization can be essentially performed
`
`following the method of Winter and co—workers (see, e. g., Morrison et al., PNAS USA, 81:6851—
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`6855 (1984), Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327
`
`(1988); Morrison and Oi, Adv. 1mmunol., 44:65-92 (1988), Verhoeyen et al., Science 239:1534-
`
`1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), Padlan, Molec. 1mmun., 28:489-
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`498 (1991); Padlan, Molec. 1mmun., 31(3):169-217 (1994)), by substituting rodent CDRs or CDR
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`sequences for the corresponding sequences of a human antibody. Accordingly, such humanized
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`antibodies are chimeric antibodies (US. Patent No. 4,816,567), wherein substantially less than an
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`intact human variable domain has been substituted by the corresponding sequence from a non-
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`human species. ln practice, humanized antibodies are typically human antibodies in which some
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`CDR residues and possibly some FR residues are substituted by residues from analogous sites in
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`rodent antibodies. For example, polynucleotides comprising a first seque