(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`1 November 2007 (01.11.2007)
`
`(51) International Patent Classification:
`A613 17/122 (2006.01)
`
`(21) International Application Number:
`PCT/US2007/007983
`
`(22) International Filing Date: 30 March 2007 (30.03.2007)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/787,972
`60/787,987
`
`30 March 2006 (30.03.2006)
`31 March 2006 (31.03.2006)
`
`US
`US
`
`(71) Applicant (for all designated States except US): UNIVER-
`SITY OF CALIFORNIA [US/US]; Office Of Technology
`Transfer, 2150 Shaltuck Avenue, Suite 510, Berkeley, CA
`94720— 1620 (US).
`
`(71)
`(72)
`
`Applicants and
`Inventors (for all designated States except US): ALLI-
`SON, James [US/US]; 1275 York Avenue, NY (US).
`CURRAN, Michael [US/7]; 1275 York Avenue, NY
`(US).
`
` (10) International Publication Number
`
`W0 2007/1237 37 A2
`
`(81) Designated States (unless otherwise indicated, for ever
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BH, 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, GT, HN, HR, HU, ID, IL, IN,
`IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR,
`LS, LT, LU, LY, MA, MD, MG, MK, MN, MW, MX, MY,
`MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RS,
`RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84) Designated States (unless otherwise indicated, for ever
`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, 1E, 18, 1T, LT, LU, LV, MC, MT, 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:
`
`without international search report and to be republished
`upon receipt of that report
`
`(74) Agent: LORENZ, Todd, A.; Dorsey & Whitney LLP,
`555 California Street, 3rd Floor, San Francisco, CA 94104—
`1513 (US).
`
`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: METHODS AND COMPOSITIONS FOR LOCALIZED SECRETION OF ANTI—CTLA —4 ANTIBODIES
`
`BamHl
`Ascl
`——> MH’l/MHZ
`
`Bglll
`
`pCMV
`
`9D9 Light
`
`909 Heavy
`
`Kc<—~— mlgGZB
`
`(57) Abstract: The present invention provides compositions and methods for effectuating the localized expression of anti—CTLA—4
`antibody proximal to a target tissue in a patient.
`
`Xhol
`
`
`
`W02007/123737A2|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`

`

`WO 2007/123737
`
`PCT/US2007/007983
`
`METHODS AND COMPOSITIONS FOR LOCALIZED SECRETION OF ANTI-CTLA-4
`
`ANTIBODIES
`
`'[001] The disclosure claims benefit under 35 U.S.C. § 119(e) of U.S. "Provisional
`
`Application Serial No. 60/787,972 filed March 30, 2006, and U.S. Provisional Application
`
`Serial No. 60/787,987 filed March 31, 2006, each of which is incorporated herein by
`
`reference in its entirety.
`
`FIELD OF THE INVENTION
`
`.[002] The disclosure relates to methods and compositions for the localized secretion of
`
`anti—CTLA—4 antibodies and their use in immunotherapy.
`
`BACKGROUND
`
`[003] Cytotoxic T lymphocyte antigen-4 (CTLA—4) is a member of the CD28-B?
`
`.immunoglobulin superfamily of immune regulatory molecules. Greenwald et al., Ann. Rev.'
`
`Immunol. 23:515—548 (2005). Although initially mischaracterized as a positive regulator
`
`based on homology to its co-stimulatory counterpart CD28, CTLA-4 has now been
`
`recognized as one of the key negative regulators of adaptive immune responses in general,
`
`and T cell proliferation and effector functions in particular. Peggs et al., Curr. Opin.
`Immunol. 18:206-213 (2006). Unlike constitutively-expressed CDZB, CTLA-4 expression is
`
`‘tightly regulated and short-lived on activated T cells, and exhibits significantly higher
`
`affinities for the B7 ligands it shares with 0028.
`
`[004] A number of clinical efforts are underway to therapeutically exploit the important
`
`biological function of this molecule. On the one hand, CTLA-4lg fusion molecules have been
`
`created and employed as immune suppressants in vivo based on its higher affinity for BT
`
`and the consequent inhibition of CD28—B7 mediated costimulation. Bluestone et at,
`Immunity 242233—38 (2006). The hCTLA-lg fusion protein OrenciaTM (abatacept) recently
`
`received FDA approval as a first-in-class antagonist of CD28 costimulation in rheumatoid
`arthritis.
`
`[005] On the other hand, CTLA-4 blockade is being explored as a promising approach to
`
`.cancer immunotherapy, empioying monoclonal antibodies directed against CTLA-4 to
`
`. prevent its negative regulation and thereby enhance the cellular immune response. Peggs,
`
`

`

`WO 2007/123737
`
`PCT/U82007/007983
`
`supra. Ongoing clinical investigations utilizing systemic administration of these antibodies
`.have provided dramatic successes, but have also produced undesirable toxicities.
`In
`
`particular, adverse immune events (AIE) such as immune-mediated colitis, hypophysitis,
`
`uveitis and hepatitis have been observed, and serious AlE often correlate with antitumor
`
`responses or freedom from relapse. Attia et a/., J. Clin. Oncol. 23:6043-53 (2005).
`
`Accordingly, although the current systemic approach may be clinically acceptable in a
`
`’patient population having advanced disease and very limited treatment options, further
`
`improvement is clearly warranted to dissociate the positive anti-tumor effect from the
`
`negative adverse events.
`
`SUMMARY OF THE INVENTION
`
`[006]
`
`The present invention resolves this unmet need in the art by effectuating localized CTLA-4
`
`blockade in vivo, such that the distribution and effect of the desired CTLA-4 blockade is substantially
`
`restricted to a particular target tissue. More specifically, cells, viral vectors, compositions and
`methods are provided for the localized, in vivo secretion of anti—CTLA—4 antibodies from in vivo
`
`modified endogenous cells or ex vivo modified autologous or allogeneic cellular vaccines that are
`subsequently administered.
`
`In one aspect, the invention provides a viral vector encoding an anti-CTLA-4 antibody (Ab)
`[007]
`'that binds to the extracellular domain of CTLA—4 and inhibits CTLA—4 signaling.
`In preferred
`
`embodiments for ex vivo transformation, the viral vector can be an integrative viral vector capable of
`providing constitutive expression when transfected into a human cell such as, e.g., adeno-associated
`viral (AAV) vectors and lentiviral vectors.
`In preferred embodiments for in vivo transformation, the
`
`viral vector can be a non-integrative viral vector capable of providing transient expression when
`
`transfected into a human cell such as, e.g., an adenoviral vector or an alpha virus vector.
`
`In certain
`
`preferred embodiments, the vector is an alpha virus vector selected from the group consisting of an
`SFV and an VEE vector.
`
`[008] The vector can further include a nucleic acid encoding a cytokine.
`the cytokine is GM-CSF.
`
`In preferred embodiments,
`
`[009]
`
`In another aspect, the invention provides a modified human cell expressing an anti-
`
`‘CTLA-4 antibody that specifically binds to the extracellular domain of CTLA-4 and inhibits
`
`CTLA—4 signaling. Preferably, the modified human cell is capable of expressing the anti-
`
`CTLA-4 antibody proximal to a tumor cell in a cancer patient in need thereof.
`
`In one
`
`embodiment, the cell is an endogenous cell modified in vivo in a patient to effectuate
`
`localized expression of anti-CTLA—4 antibody in a target tissue.
`
`In an alternative
`
`

`

`WO 2007/123737
`
`PCT/U82007/007983
`
`embodiment, the human cell is an autologous or allogeneic cell modified ex vivo to
`
`effectuate localized expression of anti-CTLA-4 antibody upon administration to a target
`
`tissue.
`
`In a preferred embodiment, the target tissue is tumor tissue.
`
`[0010]
`
`In a further embodiment, the human cell is further modified to secrete at least one
`
`additional immune effector molecule such as, e.g., G—CSF, GM-CSF, lL—2 and the like.
`
`[0011]
`
`In preferred embodiments, human cells suitable for modification in accordance with
`
`the invention include tumor cells, T cells and antigen-presenting cells such as, e.g., dendritic
`
`cells. Such cells may be endogenous and/or autologous to the patient for personalized
`
`cellular therapies, or allogeneic as in the case of cell line-derived cancer vaccines.
`
`In a
`
`preferred embodiment, cells administered in accordance with the invention will be rendered
`
`short-lived by any of the various methods well known in the art, such as, e.g., irradiation.
`
`[0012]
`
`In preferred embodiments, anti-CTLA-4 antibodies will bind to the extracellular
`
`domain of CTLA-4, and may advantageously be full length antibodies as well as fragments
`
`thereof including, e.g., Fab fragments, Fd fragments, Fv fragments, F(ab‘)2 fragments,
`
`"bivalent fragments comprising two linked Fab fragments and/or single chain Fv fragments.
`
`In a particularly preferred embodiment, the anti-CTLA—4 antibody is an scFv fragment.
`
`[0013]
`
`In preferred embodiments, the anti-CTLA-4 antibodies demonstrate reduced effector
`
`function. For example, the anti-CTLA—4 antibody can have reduced binding to Fc receptors.
`In certain variations, the anti—CTLA-4 antibody lacks an Fc region.
`In other variations, the '
`
`-anti-CTLA—4 antibody has all or part of an Fc region, but is engineered to have reduced Fc
`
`binding.
`
`In still other variations, the anti—CTLA-4 antibody can be engineered to have
`
`reduced dimerization, thereby reducing the capacity for ADCC effects.
`
`[0014] Therapeutic compositions comprising human cells in the case of ex vivo
`
`manipulation, and expression vectors in the case of in vivo manipulation, are also provided.
`
`.Suitable formulations as appropriate depending on the nature of the therapeutic composition
`
`are also contemplated.
`
`[0015]
`
`In one aspect, methods for expressing an anti-CTLA-4 antibody proximal to a target
`
`tissue in a patient are provided, comprising transforming a human cell with an anti—CTLA—4
`
`expression vector to express an anti-CTLA-4 antibody proximal to a target tissue.
`
`In
`
`accordance with the subject methods, the human cell may be transformed ex vivo and
`
`subsequently administered proximal to a target tissue in a patient. Alternatively, the human
`
`

`

`WO 2007/123737
`
`PCT/U82007/007983
`
`cell may be transformed in vivo. The human cell that is transformed in vivo to express anti-
`
`CTLA-4 Ab is preferably a tumor cell or a tumor-associated cell, such as a support cell
`’situated proximal to a tumor cell, or an antigen—presenting cell (APC) (e.g. a dendritic cell).
`
`In each instance, anti—CTLA—4 antibody is expressed by the transformed cell and inhibits
`
`CTLA-4 mediated negative regulation in T cells proximal to a tumor cell.
`
`[0016]
`
`In one embodiment, the anti-CTLA-4 expression vector used is a viral vector.
`
`In a
`
`preferred embodiment, the viral vector is selected from the group consisting of retroviral
`
`'vectors, adeno-associated viral (AAV) vectors, adenoviral vectors, lentiviral vectors, and
`
`alpha virus vectors.
`viral vector.
`
`In another embodiment, the anti-CTLA-4 expression vector is a non-
`
`[0017]
`
`In one embodiment, an anti-CTLA—4 expression vector comprises an anti-CTLA—4 Ab
`
`encoding nucleotide sequence operably linked to an expression control region which, taken
`
`together, may be integrated into the genome of the transformed human cell.
`
`In a preferred
`
`embodiment, such vectors are viral vectors.
`
`In a preferred embodiment, such viral vectors
`
`are selected from the group consisting of retroviral vectors, Ientiviral vectors, and AAV
`
`vectors.
`
`In a preferred embodiment, such viral vectors are used for the stable
`
`transformation of human cells ex vivo.
`
`.[0018]
`
`In another embodiment, the anti—CTLA—4 expression vector comprises an anti—CTLA-
`
`4 Ab encoding nucleotide sequence that remains extrachromosomal in the transformed
`
`human cell.
`
`In a preferred embodiment, the anti—CTLA—4 expression vector is a viral vector
`
`selected from the group consisting of adenoviral vectors and alpha virus vectors, more
`
`preferably an alpha virus vector, and still more preferably an SFV or VEE vector.
`
`In a
`
`preferred embodiment, such viral vectors are used for the transient expression of anti-CTLA-
`
`'4 antibodies in vivo in a transformed human cell.
`
`[0019]
`
`In a preferred embodiment, the anti-CTLA-4 expression vector provides for high
`
`level, transient expression of anti-CTLA-4 Ab in the transformed human cell. High level
`
`transient expression is achievable. for example. with a SFV anti—CTLA—4 Ab viral vector.
`
`[0020]
`
`In one aspect, the invention provides pharmaceutical compositions for local delivery
`
`to solid tumors that are useful for inhibiting the growth of solid tumors.
`
`[0021]
`
`In one embodiment, the pharmaceutical composition comprises a non-viral anti-
`
`CTLA—4 expression vector and a gene delivery agent.
`
`

`

`WO 2007/123737
`
`PCT/U82007/007983
`
`In a preferred embodiment, the pharmaceutical composition comprises a viral anti-
`[0022]
`.CTLA-4 expression vector.
`
`[0023]
`
`In one embodiment, the pharmaceutical compositions comprise a modified human
`
`cell capable of expressing an anti-CTLA—4 antibody.
`
`In one aspect, the invention provides pharmaceutical compositions for local delivery
`[0024]
`.to solid tumors that are useful for increasing the anti-tumor T cell response directed against
`the solid tumor.
`
`[0025]
`
`In one embodiment, the pharmaceutical composition comprises a non-viral anti-
`
`CTLA-4 Ab expression vector and a gene delivery agent.
`
`In a preferred embodiment, the pharmaceutical composition comprises a viral anti-
`[0026]
`'CTLA—4 Ab expression vector.
`
`[0027]
`
`In one embodiment, the pharmaceutical compositions comprise a modified human
`
`cell capable of expressing an anti-CTLA-4 antibody.
`
`In one aspect, the invention provides pharmaceutical compositions for local delivery
`[0028]
`.to solid tumors that are useful for the treatment of patients having solid tumors.
`
`[0029]
`
`In one embodiment, the pharmaceutical composition comprises a non-viral anti—
`
`CTLA-4 Ab expression vector and a gene delivery agent.
`
`[0030]
`
`In a preferred embodiment, the pharmaceutical composition comprises a viral anti—
`
`CTLA—4 Ab expression vector.
`
`[0031]
`
`In one embodiment, the pharmaceutical compositions comprise a modified human
`
`cell capable of expressing an anti—CTLA—4 antibody.
`
`[0032]
`
`In a further aspect, the modified human cell is administered to a patient.
`
`In certain
`
`embodiments, administration of the cell to the patient can be systemic, intramuscular or
`
`.subcutaneous, or at the site of a tumor cell vaccination.
`
`In other embodiments, the cell can
`
`be administered at or proximal to the tumor site.
`
`In still other embodiments, the cell can be
`
`administered at or proximal to one or more lymph nodes of the patient.
`
`In an additional aspect, the invention provides methods for producing a medicament
`[0033]
`useful for the treatment of a patient having a tumor, such as a solid tumor.
`
`

`

`WO 2007/123737
`
`PCT/U82007/007983
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`'[0034] Figure 1 depicts the cloning strategy for producing the 9D9 antibody.
`
`[0035] Figure 2 depicts the sequences of the light and heavy chain primers used in
`
`constructing the 9D9 antibody.
`
`[0036] Figure 3 depicts VL and VH constructs used to produce the 909 antibody.
`
`-[0037] Figure 4 depicts the 9D9 scFv sequence.
`
`[0038] Figure 5 depicts an expression vector used for production of the myc—HIS tailed 9D9
`scFv molecule.
`
`[0039] Figure 6 depicts an expression vector used for production of the lgG1 tailed version
`of the 909 scFv molecule.
`
`[0040] Figure 7 depicts a CD spectrum of scFv antibodies against DT230 cells expressing
`
`surface CTLA—4 protein.
`
`[0041] Figure 8 depicts various CD spectra of scFv antibodies against DT230 cells
`
`expressing surface CTLA—4 protein.
`
`[0042] Figure 9 depicts DT230/LMtK comparative histograms of the 909 antibody and
`various forms of the scFv molecules.
`
`[0043] Figure 10 depicts CD spectra of B16-GM-transduced with 9D93cFv.
`
`[0044] Figure 11 depicts the tumor/matrigel size in mice 18 days after treatment with the
`
`shown anti-CTLA-4 monoclonal antibodies and scFv molecules.
`
`[0045] Figure 12 depicts the lntra-tumor T—cell profile in mice 18 days after treatment with
`
`the shown anti-CTLA-4 monoclonal antibodies and scFv molecules.
`
`[0046] Figure 13 depicts DT230 staining of B16-GM-909scFv lg cell lines with DT230 raw
`supernatant and anti-mouse PE.
`
`[0047] Figure 14 depicts the delivery of the scFv anti-CTLA antibody at the site of tumor cell
`vaccine.
`
`

`

`WO 2007/123737
`
`PCT/U82007/007983
`
`. DETAILED DESCRIPTION
`
`[0048] The viral vectors and transformed human cells of the present invention express anti-
`
`CTLA-4 antibodies that inhibit CTLA-4 mediated negative signaling.
`
`In a preferred
`
`embodiment, the viral vector or human cells expressing the anti-CTLA—4 antibody are
`
`capable of expressing the antibody proximal to a tumor.
`
`'[0049] Human cells that can be used include tumor cells, antigen-presenting cells (e.g.
`
`dendritic cells). B cells and T cells. The presently disclosed cells provide for localized
`
`expression of anti-CTLA-4 antibodies by cells proximal to a tumor. The cells can be
`
`modified in vivo, or alternatively cells modified ex vivo can be administered to a patient by a
`
`variety of methods, such as by injection. By creating and/or introducing the transformed
`
`cells proximal to a tumor, the CTLA-4 blockade can be localized to the tumor.
`
`[0050]
`
`in one embodiment, the cell is a tumor cell. For ex vivo transformation, such tumor
`
`cells can be irradiated to eliminate the ability of the cell to replicate. as known in the art.
`
`while maintaining the transient expression of anti-CTLA—4 antibodies after administration.
`
`For in vivo transformation, non-integrative expression vectors may be preferred.
`
`-[0051]
`
`In certain preferred embodiments, the tumor cell is autologous or endogenous.
`
`In the
`
`former instance, the tumor cell is taken from a patient, transfected or transduced with a
`
`construct encoding an anti-CTLA—4 antibody, and re—introduced to the patient, for example
`
`after irradiation.
`
`In the latter instance, the tumor cell is transformed in vivo by local
`
`administration of an appropriate construct as described herein.
`
`.[0052]
`
`In an alternative embodiment, the modified tumor cell is allogeneic. The aliogeneic
`
`tumor cell thus can be maintained in a cell line.
`
`In this instance, the tumor cell can be
`
`selected from the cell line, irradiated, and introduced to the patent.
`
`[0053]
`
`In another alternative embodiment, the modified human cells are antigen—presenting
`
`cells such as dendritic cells, or monocytes.
`
`.[0054] Without being bound to a particular theory or mode of action, dendritic cells can
`
`serve as hubs where T-cells become primed to attack tumor cells bearing a given antigen.
`
`Dendritic cells are laden with surface B7 molecules, which could switch off potentially
`
`responsive T—cells that express CTLA-4 prematurely (e.g. while still in the vicinity of the APC,
`
`

`

`WO 2007/123737
`
`PCT/U82007/007983
`
`usually within a lymph node). Preferably. APCs expressing anti-CTLA-4 Abs can increase
`
`'the intensity and duration of the anti-tumor T-cell response primed by the APC.
`
`[0055]
`
`In another alternative embodiment, the modified human cells are T cells.
`
`In one
`
`embodiment, T cells specific for particular tumor antigens can be transformed and expanded
`
`ex vivo and re-infused into patients. Again, without being bound by a particular theory or
`
`mode of action, by expressing anti—CTLA-4 Abs the T cells may protect themselves and
`
`other endogenous and/or adoptively transferred tumor-specific T—cells from being silenced
`by lack of costimulation or suppressive APCs in the tumor environment.
`
`[0056] Modified human cells capable of producing the anti-CTLA—4 antibodies can be made
`
`by transfecting or transducing the cells with an expression vector encoding an anti—CTLA—4
`
`antibody. The anti-CTLA—4 expression vector can be made by methods well known in the art.
`
`In certain preferred embodiments, the anti-CTLA-4 antibody is an scFv molecule.
`'[0057]
`scFv molecules may be produced for example, as described by Smith et al. Gene Ther.
`
`2003 Aug; 10(15):1248-57. Likewise, scFv antibodies may be produced as described by
`
`Wang et al., J Immunol Methods, 2000 233(1—2):167-77, which is incorporated herein by
`
`reference in its entirety.
`
`In various embodiments, the anti-CTLA-4 antibody can be administered to a patient
`.[0058]
`in the form of a nucleic acid construct.
`
`[00591 In one embodiment, the construct comprises a retroviral vector. Retroviral vectors
`
`are capable of permanently integrating DNA encoding the anti-CTLA-4 antibody into the cell
`
`genome. Thus, in the case of ex vivo manipulation of autologous or allogeneic cells, stable
`
`"cell lines that constitutively produce the anti-CTLA—4 antibody can be prepared.
`
`In a
`
`preferred embodiment, the cells are irradiated prior to administration to a patient. The
`
`irradiated cells produce the anti-CTLA-4 antibody for a limited period of time
`
`[0060]
`
`In one embodiment, the anti—CTLA—4 antibody construct comprises an SFV vector,
`
`which demonstrates high levels of transient expression in mammalian cells. The SFV vector
`
`-is described, for example, in Lundstrom, Expert Opin. Biol. Ther. 3:771-777 (2003),
`
`incorporated herein by reference in its entirety. Thus, in the case of in vivo manipulation of
`
`endogenous cells in a patient, transient expression of high levels of the anti-CTLA—4
`
`antibody can be accomplished. This is to prevent constitutive expression, and permanent
`
`blockade, of the CTLA-4 signaling pathway in viva.
`
`

`

`WO 2007/123737
`
`PCT/U82007/007983
`
`[0061] Systems capable of expressing antibodies in vivo are known in the art. By way of
`example and not limitation, the system can use the 2A mediated antibody expression system
`.disclosed in Fang et al., Nature Biotech. 23(5) 2005 and US. Patent Publication
`
`2005/0003506, the disclosures of which are expressly incorporated by reference herein in
`their entirety. Other systems known in the art are contemplated, and can also be adapted to
`produce anti—CTLA-4 antibodies in vivo as described herein.
`
`[0062] Administration of the anti-CTLA-4 producing cells disclosed herein can be combined
`
`‘with administration of cytokines that stimulate antigen-presenting cells such as granulocyte-
`macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M—
`CSF), granulocyte colony stimulating factor (G—CSF), interleukin 3 (IL-3), interleukin 12 (IL-
`12), etc., or cellular vaccines capable of expressing such cytokinesln preferred
`embodiments, the anti-CTLA-4 producing cells are further modified to express such
`
`cytokines. Additional proteins and/or cytokines known to enhance T cell proliferation and
`.secretion, such as lL-1, lL-2, B7, anti-CD3 and anti-0028 can be employed simultaneously
`or sequentially with the blocking agents to augment the immune response. The present
`therapy can also be combined with any of the molecules, or conducted as described in, us.
`
`Patent No. 6,051,227, incorporated herein by reference in its entirety.
`
`Anti-C TLA-4 Vectors and Methods of Transformation
`
`'[0063] Anti-CTLA-4 expression vectors of the invention may be viral or non-viral. Viral
`
`vectors are preferred for use in vivo. Anti-CTLA-4 expression vectors of the invention
`
`comprise an anti—CTLA—4 antibody encoding nucleic acid, or a complement thereof, operably
`linked to an expression control region, or complement thereof, that is functional in a
`
`mammalian cell. The expression control region is capable of driving expression of the
`
`operably linked anti-CTLA-4 antibody encoding nucleic acid such that anti-CTLA-4 antibody
`‘is produced in a human cell transformed with the anti-CTLA-‘4 expression vector.
`
`[0064] Expression control regions are regulatory polynucleotides (sometimes referred to
`
`herein as elements), such as promoters and enhancers, that influence expression of an
`operably linked nucleic acid.
`
`-[0065] An expression control region of an anti-CTLA-4 expression vector of the invention is
`
`capable of expressing operably linked anti—CTLA-4 antibody encoding nucleic acid in a
`human cell.
`In one embodiment, the cell is a tumor cell.
`in one embodiment, the cell is a
`non-tumor cell.
`
`

`

`WO 2007/123737
`
`PCT/U82007/007983
`
`In one embodiment, the expression control region confer regulatable expression to.
`'[0066]
`an operably linked nucleic acid. A signal (sometimes referred to as a stimulus) can increase
`or decrease expression of a nucleic acid operably linked to such an expression control
`
`region. Such expression control regions that increase expression in response to a signal are
`often referred to as inducible. Such expression control regions that decrease expression in
`response to a signal are often referred to as repressible. Typically, the amount of increase or
`decrease conferred by such elements is proportional to the amount of signal present; the
`greater the amount of signal, the greater the increase or decrease in expression.
`
`[0067] Especially preferred for use in the present invention are inducible promoters capable
`of effecting high level of expression transiently in response to a cue. When in the proximity
`of a tumor cell, a cell transformed with an anti-CTLA—4 expression vector comprising such an
`expression control sequence is induced to transiently produce a high level of anti-CTLA-4
`antibody by exposing the transformed cell to an appropriate cue.
`
`[0068] Preferred inducible expression control regions include those comprising an inducible
`promoter that is stimulated with a cue such as a small molecule chemical compound.
`Particular examples can be found, for example, in US. Pat. Nos. 5,989,910, 5,935,934,
`6,015,709, and 6,004,941, each of which is incorporated herein by reference in its entirety.
`
`[0069] Expression control regions include full-length promoter sequences, such as native
`
`promoter and enhancer elements, as well as subseq uences or polynucleotide variants which
`
`retain all or part of full-length or non—variant function. As used herein, the term "functional"
`
`and grammatical variants thereof, when used in reference to a nucleic acid sequence,
`subsequence or fragment, means that the sequence has one or more functions of native
`
`nucleic acid sequence (e.g., non-variant or unmodified sequence).
`
`[0070] As used herein, "operable linkage" refers to a physical juxtaposition of the
`
`components so described as to permit them to function in their intended manner.
`
`In the
`
`example of an expression control element in operable linkage with a nucleic acid, the
`
`.relationship is such that the control element modulates expression of the nucleic acid.
`
`Typically, an expression control region that modulates transcription is juxtaposed near the 5'
`end of the transcribed nucleic acid (i.e., "upstream"). Expression control regions can also be
`located at the 3' end of the transcribed sequence (i.e., "downstream") or within the transcript
`(e.g., in an intron). Expression control elements can be located at a distance away from the
`transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000, or more nucleotides from
`
`10
`
`

`

`WO 2007/123737
`
`PCT/U82007/007983
`
`the nucleic acid). A specific example of an expression control element is a promoter, which
`is usually located 5' of the transcribed sequence. Another example of an expression control
`element is an enhancer, which can be located 5' or 3’ of the transcribed sequence, or within
`the transcribed sequence.
`
`'[0071] Expression systems functional in human cells are well known in the art, and include
`viral systems. Generally, a promoter functional in a human cell is any DNA sequence
`capable of binding mammalian RNA polymerase and initiating the downstream (3’)
`transcription of an anti-CTLA-4 coding sequence into mRNA. A promoter will have a
`
`transcription initiating region, which is usually placed proximal to the 5’ end of the coding
`sequence. and typically a TATA box, using a located 25-30 base pairs upstream of the
`transcription initiation site. The TATA box is thought to direct RNA polymerase ii to begin
`RNA synthesis at the correct site. A promoter will also typically contain an upstream
`promoter element (enhancer element), typically located within 100 to 200 base pairs
`upstream of the TATA box. An upstream promoter element determines the rate at which
`
`transcription is initiated and can act in either orientation. Of particular use as promoters are
`.the promoters from mammalian viral genes, since the viral genes are often highly expressed
`and have a broad host range. Examples include the SV40 early promoter, mouse mammary
`tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter,
`and the CMV promoter.
`
`[0072] Typically, transcription termination and polyadenylation sequences recognized by
`mammalian cells are regulatory regions located 3’ to the translation stop codon and thus,
`together with the promoter elements, flank the coding sequence. The 3' terminus of the
`
`mature mRNA is formed by site-specific post-translational cleavage and polyadenylation.
`Examples of transcription terminator and polyadenylation signals include those derived from
`
`SV40.
`
`lntrons may also be included in expression constructs.
`
`[0073] There are a variety of techniques available for introducing nucleic acids into viable
`cells. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro
`include the use of liposomes, electroporation, microinjection, cell fusion, polymer-based
`systems, DEAE-dextran, viral transduction, the calcium phosphate precipitation method, etc.
`For in vivo gene transfer, a number of techniques and reagents may also be used, including
`liposomes; natural polymer-based delivery vehicles, such as chitosan and gelatin; viral
`.vectors are also preferred for in vivo transduction (e.g., Dzau et al., Trends in Biotechnology
`11, 205—210 [1993]).
`In some situations it is desirable to provide a targeting agent, such as _
`
`11
`
`

`

`WO 2007/123737
`
`PCT/U82007/007983
`
`an antibody or ligand specific for a tumor cell surface membrane protein. Where liposomes
`are employed, proteins which bind to a cell surface membrane protein associated with
`
`endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or
`
`fragments thereof tropic for a particular cell type, antibodies for proteins which undergo
`internalization in cycling, proteins that target intracellular localization and enhance
`
`'intracellular half-life. The technique of receptor-mediated endocytosis is described, for
`example, by Wu et al., J. Biol. Chem. 262, 4429—4432 (1987); and Wagner et al., Proc. Natl.
`
`Acad. Sci. USA 87, 3410-3414 (1 990). For review of gene therapy protocols see Anderson
`
`et al., Science 256, 808-813 (1992).
`
`[0074] Where appropriate, gene delivery agents such as, e.g. integration sequences can
`
`also be employed. Numerous integration sequences are known in the art (see for example
`Nunes-Duby et al., Nucleic Acids Res. 26:391-406. 1998; Sadwoski, J. Bacteriol., 1652341-
`
`357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999;
`
`Kootstra et al., Ann. Rev. Pharm. Toxicol., 432413-439, 2003). These include recombinases
`
`and transposases. Examples include Cre (Sternberg and Hamilton, J. Mol, Biol., 1502467—
`
`486, 1981), lambda (Nash, Nature, 247, 543-545, 1974), Flp (Broach, et al, Cell, 29:227-234,
`
`'1982) R (Matsuzaki, et al, J. Bacteriology, 172:610-618, 1990), 4:631 (see for example Groth
`
`et al., J. Mol. Biol. 335:667-678, 2004), sleeping beauty, transposases of the mariner family
`(Plasterk et al., supra), and components for integrating viruses such as AAV, retroviruses,
`
`and lentiviruses having components that provide for virus integration such as the LTR
`
`sequences of retroviruses or lentivirus and the ITR sequences of AAV (Kootstra et al., Ann.
`Rev. Pharm. Toxicol., 43:413—439, 2003).
`—
`
`Anti-CTLA-4 Viral Vectors
`
`[0075]
`
`In one aspect, the invention provides anti-CTLA-4 expression vectors that are anti-
`
`CTLA-4 Ab viral vectors. Many viral vectors useful for gene therapy are known (see, for
`
`example, Lundstrom, Trends Biotechnol., 21:117, 122, 2003.
`
`‘[0076] Preferred viral vectors include those selected from the group consisting of
`lentiviruses (LV), retroviruses (RV), adenoviruses (AV), adeno-associated viruses (AAV).
`and alpha viruses, though other viral vectors may also be-used. For in vivo uses, viral
`
`v