(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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`(10) International Publication Number
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`WO 2016/011210 A2
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`(43) International Publication Date
`21 January 2016 (21.01.2016)
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`WIPOIPCT
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`(51)
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`International Patent Classification:
`CIZN 5/0783 (2010.01)
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`(21)
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`International Application Number:
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`PCT/US2015/040660
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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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`15 July 2015 (15.07.2015)
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`Eninsh
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`Eninsh
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`Priority Data:
`62/025,006
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`15 July 2014 (15.07.2014)
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`US
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`[US/US];
`INC.
`Applicant: JUNO THERAPEUTIC S,
`307 Westlake Ave, North, Suite 300, Seattle, WA 98109
`(US).
`
`Inventors: MOHLER, Kendall, M.; 307 Westlake Aven-
`ue North Suite 300, Seattle, WA 98109 (US). LEVITSKY,
`Hyam, I.; 307 Westlake Avenue North Suite 300, Seattle,
`WA 98109 (US).
`
`Agents: POTTER, Karen et al.; Morrison & Foerster
`LLP, 12531 High Bluff Drive, Suite 100, San Diego, CA
`92130-2040 (US).
`
`Designated States (unless otherwise indicated, for every
`kind of national 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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`without international search report and to be republished
`upon receipt oftlzat report (Rule 48.2(g))
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`with sequence listingpart ofdescription (Rule 5.2(a))
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`(54) Title: ENGINEERED CELLS FOR ADOPTIVE CELL THERAPY
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`(57) Abstract: Provided are engineered cells for adoptive therapy, including NK cells and T cells. Also provided are compositions
`for engineering and producing the cells, compositions containing the cells, and methods for their administration to subjects. In some
`aspects, features of the cells and methods provide specificity and/or efficacy. In some embodiments, the cells contain genetically en -
`gineered antigen receptors that specifically bind to antigens, such as chimeric antigen receptors (CARS) and eostimulatory receptors.
`In some embodiments, the cells include receptors targeting multiple antigens. In some embodiments, the cells include repression of
`one or more gene product, for example, by disruption of a gene encoding the gene product. In some embodiments, a gene encoding
`an antigen recognized by the engineered antigen receptor is disrupted, reducing the likelihood of targeting of the engineered cells.
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`ENGINEERED CELLS FOR ADOPTIVE CELL THERAPY
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`Cross-Reference to Related Applications
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`[0001] This application claims priority from US. provisional application No. 62/025,006, filed
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`July 15, 2014, the contents of which are incorporated by reference in their entirety.
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`Incorporation By Reference of Seguence Listing
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`[0002] The present application is being filed along with a Sequence Listing in electronic format.
`
`The Sequence Listing is provided as a file entitled 735042000540seqlist.txt, created July 15, 2015,
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`which is 43 kilobytes in size. The information in the electronic format of the Sequence Listing is
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`incorporated by reference in its entirety.
`
`
`Field
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`[0003] The present disclosure relates in some aspects to engineered cells for adoptive therapy,
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`including NK cells and T cells. In some aspects, the disclosure further relates to methods and
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`compositions for engineering and producing the cells, compositions containing the cells, and
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`methods for their administration to subjects. In some aspects, features of the cells and methods
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`provide specificity and/or efficacy.
`
`In some embodiments, the cells contain genetically engineered
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`antigen receptors that specifically bind to antigens, such as chimeric antigen receptors (CARs) and
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`costimulatory receptors. In some embodiments, the cells include receptors targeting multiple
`
`antigens. In some embodiments, the cells include repression of one or more gene product, for
`
`example, by disruption of a gene encoding the gene product. In some embodiments, a gene
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`encoding an antigen recognized by the engineered antigen receptor is disrupted, reducing the
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`likelihood of targeting of the engineered cells.
`
`Background
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`[0004] Various strategies are available for producing and administering engineered cells for
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`adoptive therapy. For example, strategies are available for engineering immune cells expressing
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`genetically engineered antigen receptors, such as CARS, and for suppression or repression of gene
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`expression in the cells. Improved strategies are needed, for example, to provide a wider range of
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`target antigens and diseases that may be treated using such cells, to improve specificity or
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`selectivity of the cells, e.g., to avoid off—target effects, and to improve efficacy of the cells, for
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`example, by avoiding suppression of effector functions and improving the activity and/or survival
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`of the cells upon administration to subjects. Provided are methods, cells, compositions, kits, and
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`systems that meet such needs.
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`Summary
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`[0005] Provided are cells, including engineered cells, such as engineered immune or
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`immunostimulatory cells, as well as methods for producing and using the cells, such as in adoptive
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`therapy, and compositions, such as pharmaceutical compositions, containing the cells. Among the
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`cells are those having one or more features, such as dual—antigen targeting features and/or gene
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`disruptions, which on their own or collectively provide improved safety, specificity, selectivity,
`
`and/or efficacy, and/or allow for the targeting of a broader range of antigens or diseases by adoptive
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`cell therapy.
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`[0006] In some embodiments, the cells are engineered immune cells including: a genetically
`
`engineered antigen receptor that specifically binds to a target antigen; and a disruption in a gene
`
`encoding the target antigen, said disruption resulting in reduced expression of the target antigen in
`
`the engineered immune cell. In some aspects, the target antigen is an antigen expressed on the
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`surface of resting T cells, activated T cells, or both. In some aspects, the target antigen is expressed
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`on the cell surface in a cancer, such as a hematologic cancer, an immune cancer, a leukemia, a
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`lymphoma, and/or a myeloma, such as multiple myeloma.
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`[0007] In some aspects, such as where the antigen receptor induces an activating signal or one
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`that causes an immune response directed at cells expressing the target antigen, the target antigen is
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`one that is expressed on a disease or condition to be treated, such as cancer but that also is
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`ordinarily expressed on the cell being engineered or sued for adoptive cell therapy. In some
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`aspects, that target antigen is a universal tumor antigen, in some aspects one that is expressed
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`naturally on or in the engineered cells and/or expressed on or in or the expression of which is
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`upregulated on or in, activated T cells. In some aspects, the universal tumor antigen is a human
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`telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2),
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`cytochrome P450 1B1 (CYPlB), HER2/neu, Wilms’ tumor gene 1 (WTl), livin, alphafetoprotein
`
`(AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUCl, prostate— specific membrane
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`antigen (PSMA), p53 or cyclin (D1). For example, the target antigen is hTERT or survivin. In
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`some aspects, the target antigen is CD38. In other aspects, the target antigen is CD33 or TllVI—3. In
`
`other aspects, it is CD26, CD30, CD53, CD92, CD100, CD148, CD150, CD200, CD261, CD262, or
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`CD362.
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`[0008] In some aspects, such as where the receptor induces a suppressive or inhibitory, e. g.,
`
`immunosuppressive, signal, the antigen is one that is not expressed in the disease or condition. In
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`some aspects, wherein the genetically engineered antigen receptor is capable of inducing an
`
`inhibitory or immunosuppressive or repressive signal to the cell upon recognition of the target
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`antigen.
`
`In some aspects, the antigen is an antigen that is not expressed on the surface of a cancer
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`cell or infected cell or the expression of which is downregulated on a cancer cell or an infected cell.
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`Exemplary of such antigens are MHC—class I molecules.
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`[0009] In some embodiments, the antigen is a gene product that is naturally expressed in the cell
`
`type of the engineered cell. In some embodiments, expression of the target antigen in the engineered
`
`immune cell is reduced by at least 50, 60, 70, 80, 90, or 95 % as compared to the expression in the
`
`immune cell in the absence of said gene disruption. In some embodiments, the disruption comprises
`
`a deletion of at least a portion of at least one exon of the gene; comprises a deletion, mutation,
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`and/or insertion in the gene resulting in the presence of a premature stop codon in the gene; and/or
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`comprises a deletion, mutation, and/or insertion within a first or second exon of the gene.
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`[0010] Among the cell types are T cells, NK cells, CD4+ T cells, CD8+ T cells, and stem cells,
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`such as an induced pluripotent stem cell (iPS cell).
`
`[0011] In some embodiments, the genetically engineered antigen receptor is capable of inducing
`
`an activating signal to the cell. In some aspects, the genetically engineered antigen receptor
`
`comprises an intracellular domain with an ITAM—containing motif. In some aspects, the genetically
`
`engineered antigen receptor is a T cell receptor (TCR) or a functional non—TCR antigen recognition
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`receptor. In some aspects, it is a chimeric antigen receptor (CAR), such as an activating or
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`stimulatory CAR, an inhibitory CAR and/or a co stimulatory CAR. Among the CARS are those with
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`U.)
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`an extracellular antigen—recognition domain that specifically binds to the target antigen and an
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`intracellular signaling domain comprising an ITAM, such as an intracellular domain of a CD3—zeta
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`(CD3Q) Chain those that further comprise a costimulatory signaling region, such as a signaling
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`domain of CD28 or 41BB. In some aspects, the CAR comprises an extracellular antigen—
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`recognition domain that specifically binds to the target antigen and an intracellular signaling domain
`
`that comprises a signaling portion of an immune checkpoint molecule, such as PD—l or CTLA4.
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`[0012] In some aspects, the cell comprises another genetically engineered antigen receptor, such
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`as a costimulatory receptor, such as a chimeric costimulatory receptor, that specifically binds to
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`another antigen and is capable of inducing a costimulatory signal to the cell. In some aspects, such
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`another target antigen and the first target antigen recognized by the first receptor are distinct. In
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`some embodiments, at least one of such antigens is selected from among a human telomerase
`
`reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome
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`P450 1B1 (CYPlB), HER2/neu, Wilms’ tumor gene 1 (WTl), livin, alphafetoprotein (AFP),
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`carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1 , prostate-specific membrane antigen
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`(PSMA), p53 or cyclin (D1), and the other is another antigen expressed on a tumor or cancer, and in
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`some cases is expressed on a particular type of tumor or cancer, and not on one or more certain
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`other types of cancers. In some aspects, such other antigen is a multiple myeloma—associated or
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`multiple myeloma—specific antigen such as CD38 or CD138 or BCMA or CS—l; in some aspects,
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`such other antigen is expressed on one or more blood cancers or one or more solid tumor types. In
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`some aspects, the target antigen and said another antigen are distinct and are selected, individually,
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`from the group consisting of CD38 and CD138.
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`In some such aspects, the cell further comprises an
`
`additional genetically engineered antigen receptor which recognizes an antigen expressed on a
`
`disease or condition to be treated and induces a stimulatory or activating signal, which is dampened
`
`by the first genetically engineered antigen receptor.
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`[0013] Also provided are methods for producing the cells and cells produced by such methods.
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`In some embodiments, the methods are carried out by (a) introducing into an immune cell a
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`genetically engineered antigen receptor that specifically binds to a target antigen; and (b) effecting
`
`repression of expression of the target antigen in the immune cell, thereby producing a genetically
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`engineered immune cell in which expression of the target antigen is repressed. In some aspects,
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`steps (a) and (b) are carried out simultaneously or sequentially in any order.
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`[0014] In some embodiments, the effecting in (b) comprises disrupting a gene encoding the
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`target antigen, the disruption comprises disrupting the gene at the DNA level and/or the disruption
`
`is not reversible; and/or the disruption is not transient. In some aspects, the disruption comprises
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`introducing into the immune cell a DNA binding protein or DNA—binding nucleic acid that
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`specifically binds to or hybridizes to the gene.
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`[0015] In some embodiments, the disruption comprises introducing: (a) a fusion protein
`
`comprising a DNA—targeting protein and a nuclease or (b) an RNA—guided nuclease. For example,
`
`in some embodiments, the DNA—targeting protein or RNA— guided nuclease comprises a zinc finger
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`protein (ZFP), a TAL protein, or a Clustered regularly interspaced short palindromic nucleic acid
`
`(CRISPR) specific for the gene.
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`In some embodiments, the disruption comprises introducing a zinc
`
`finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or and a CRISPR-Cas9 combination that
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`specifically binds to, recognizes, or hybridizes to the gene. In some embodiments, the introducing
`
`is carried out by introducing into the cell a nucleic acid comprising a sequence encoding the DNA—
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`binding protein, DNA—binding nucleotide, and/or complex comprising the DNA—binding protein or
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`DNA—binding nucleotide. In some embodiments, the nucleic acid is a viral vector.
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`[0016] In some embodiments, the specific binding to the gene is within an exon of the gene
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`and/or is within a portion of the gene encoding an N—terminus of the target antigen. In some
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`embodiments, the introduction thereby effects a frameshift mutation in the gene and/or an insertion
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`of an early stop codon within the coding region of the gene.
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`[0017] In some embodiments, the methods further include (c)
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`introducing another genetically
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`engineered antigen receptor, which is a chimeric costimulatory receptor that specifically binds to
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`another antigen and is capable of inducing a costimulatory signal to the cell, wherein steps (a), (b)
`
`and (c) are carried out simultaneously or sequentially in any order. Also provided are cells
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`produced by the methods.
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`[0018] In some embodiments, the engineered cells comprise: (a) a first genetically engineered
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`antigen receptor, which specifically binds to a first antigen and is capable of inducing an activating
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`signal to the cell; and (b) a second genetically engineered antigen receptor, which is a costimulatory
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`receptor such as a chimeric costimulatory receptor that specifically binds to a second antigen and is
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`capable of inducing a costimulatory signal to the cell (such as one that is necessary for full
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`activation of the cell or a particular effector function thereof following binding of the first receptor
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`to its antigen). In some such embodiments, the first and second antigens are distinct and, at least one
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`is selected from the group consisting of human telomerase reverse transcriptase (hTERT), survivin,
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`mouse double minute 2 homolog (MDMZ), cytochrome P450 1B1 (CYPlB), HERZ/neu, Wilms’
`
`tumor gene 1 (WTl), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin l6
`
`(MUC16), MUCl, prostate—specific membrane antigen (PSMA), p53 and cyclin (D l). The other of
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`the first second antigen can be a different antigen from any of hTERT, survivin, MDM2, CYPlB,
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`HERZ/neu, WTl, livin, AFP, CEA, MUC l6, MUCl, PSMA, p53 or cyclin (D l), or can be another
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`tumor antigen.
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`[0019] In some embodiments, the engineered cells comprise: (a) a first genetically engineered
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`antigen receptor, which specifically binds to a first antigen and is capable of inducing an activating
`
`signal to the cell; and (b)a second genetically engineered antigen receptor, which is a costimulatory
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`receptor such as a chimeric costimulatory receptor that specifically binds to a second antigen and is
`
`capable of inducing a costimulatory signal to the cell (such as one that is necessary for full
`
`activation of the cell or a particular effector function thereof following binding of the first receptor
`
`to its antigen). In some such embodiments, the first and second antigens are distinct and,
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`individually, are selected from the group consisting of CD38, CS— 1, and CDl38.
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`[0020] In some embodiments, the engineered cell comprises (a)a first genetically engineered
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`antigen receptor, which specifically binds to a first antigen and is capable of inducing an activating
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`signal to the cell; and (b)a second genetically engineered antigen receptor, which is a chimeric
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`costimulatory receptor that specifically binds to a second antigen and is capable of inducing a
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`costimulatory signal to the cell, wherein the first and second antigens are distinct and, individually,
`
`are selected from the group consisting of CD38 and CD 138.
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`[0021] In some embodiments, the engineered immune cell comprises (a)a first genetically
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`engineered antigen receptor that specifically binds to a first antigen and is capable of inducing an
`
`activating signal to the cell; and (b)
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`a second genetically engineered antigen receptor which is a
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`chimeric costimulatory receptor that specifically binds to a second antigen and is capable of
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`inducing a costimulatory signal to the cell, wherein the first and second antigens are distinct and the
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`first or the second antigen is CS— 1.
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`[0022] In some embodiments, the second antigen is an antigen expressed in multiple myeloma.
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`[0023] In some embodiments, the first genetically engineered antigen receptor comprises an
`
`ITAM—containing sequence, the first genetically engineered antigen receptor comprises an
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`intracellular signaling domain of a CD3—zeta (CDSC) chain, and/or the first genetically engineered
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`antigen receptor does not comprise a signaling domain from a T cell costimulatory molecule, such
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`as one having an intracellular signaling domain of a T cell costimulatory molecule, such one or
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`more molecules selected from the group consisting of CD28 and 41BB.
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`[0024] In some aspects, (a) the first antigen is CD38 and the second antigen is CD138;
`
`(b)) the
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`first antigen is CD38 and the second antigen is CS—l; (c) the first antigen is CD138 and the second
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`antigen is CD38; (d) the first antigen is CD 138 and the second antigen is CS— 1;
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`(e) the first antigen
`
`is CS-l and the second antigen is CD38; or (f) the first antigen is CS-l and the second antigen is
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`CD138. In some cases, the cell further comprises a third genetically engineered antigen receptor
`
`recognizing a third antigen.
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`[0025] In some aspects, the first genetically engineered antigen receptor contains an
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`extracellular antigen recognition domain which specifically binds to the first target antigen at a
`
`dissociation constant (KD) of at least 10'8 M, at least 10'7 M, at least 10'6 M, at least 10'5 M, 10'5 M,
`
`or 10'4 M. In some aspects, ligation of the first genetically engineered antigen receptor and ligation
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`of the second genetically engineered antigen receptor induces a response in the cell, Which response
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`is not induced by ligation of either of the genetically engineered antigen receptors alone.
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`[0026] In some embodiments, the response is selected from the group consisting of
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`proliferation, secretion of a cytokine, and cytotoxic activity.
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`In some embodiments, the cell further
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`comprising a disruption in a gene encoding the first antigen, and/or in a gene encoding the second
`
`antigen, said disruption resulting in reduced expression of the first and/or second antigen in the
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`engineered immune cell, such as by disruption as described herein, for example, Where the disrupted
`
`gene encodes CD38.
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`[0027] Also provided are compositions, including pharmaceutical compositions comprising the
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`cells, and in some aspects, a carrier, such as a pharmaceutically acceptable carrier. The disease
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`sand conditions include cancers and infectious diseases, such as hematological cancers, leukemias,
`
`lymphomas, and multiple myeloma.
`
`Detailed Description
`
`I.
`
`Compositions and methods providing specificity and/or efficacy in adoptive cell
`therapy
`
`[0028] Provided are cells for adoptive cell therapy, e. g. , adoptive immunotherapy. The cells
`
`include immune cells such as T cells and NK cells, and generally express genetically engineered
`
`antigen receptors such as engineered TCRs and/or chimeric antigen receptors (CARS). Also
`
`provided are methods and uses of the cells, such as in adoptive therapy in the treatment of cancers
`
`including multiple myeloma (MM). Also provided are methods for engineering, preparing, and
`
`producing the cells, compositions containing the cells, and kits and devices containing and for
`
`using, producing and administering the cells. In some embodiments, the embodiments provide
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`improved selectivity, specificity, and/or efficacy of antigen-specific adoptive cell therapy, and/or
`
`expand the scope of diseases, conditions, and/or target antigens which may be targeted via adoptive
`
`cell therapy.
`
`[0029] The cells generally are engineered by introducing one or more genetically engineered
`
`nucleic acid or product thereof. Among such products are genetically engineered antigen receptors,
`
`including engineered T cell receptors (TCRs) and functional non—TCR antigen receptors, such as
`
`chimeric antigen receptors (CARS), including activating, stimulatory, and costimulatory CARs, and
`
`combinations thereof.
`
`[0030] Also among the cells are those in which certain genes have been disrupted, including
`
`genes encoding the antigen recognized by a chimeric antigen receptor (CAR) or other engineered
`
`antigen receptors expressed by the cells. In some embodiments, the gene repressed is a gene
`
`encoding an antigen targeted by the genetically engineered antigen receptor, such as an activating,
`
`inhibitory, or costimulatory CAR. Thus, also provided are nucleic acids effecting repression of
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`expression and/or disruption of endogenous genes, such as by gene editing. Also provided are cells
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`with reduced expression of a gene or genes and methods for effecting the repression.
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`[0031]
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`In some embodiments, for example, in the context of an activating or costimulatory
`
`antigen receptor, e.g., activating CAR or costimulatory CAR, the repression of a gene that encodes
`
`an antigen expressed in the engineered cells avoids or reduces the likelihood of the engineered cells
`
`themselves being targeted for killing or inhibition, thereby improving efficacy of, and/or persistence
`
`or expansion of cells in, the adoptive cell therapy. Thus, in some aspects, such disruption avoids or
`
`reduces the likelihood of targeting, by the engineered cells, of the engineered cells themselves.
`
`[0032] For example, antigen—specific cells, such as antigen—specific T cells, targeting antigens
`
`also expressed by such cells, can induce fratricide killing of the cells. In some cases, fratricide
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`killing of T cells is observed in cultures of antigen—specific T cells against survivin, hTERT, p53
`
`and others, which are protein antigens expressed in activated T cells (Turksma et al. (2013) Journal
`
`of Translational Medicine, 11:152; Leisegang et al. (2010) J Clin. Invest, 120:3869—3877; Chen et
`
`al. (2007) Cancer Biol Then, 6: 1991—1996; Theoret er al. (2008) Hum Gene Ther., 19:1219-1232).
`
`Methods of detecting fratricide, such as self-killing, of the immune cells can include, for example,
`
`methods in which T cells genetically engineered with an antigen receptor (e. g. TCR or CAR or
`
`other antigen receptor) are cultured in vitro, e. g., for up to 2 weeks, and monitored over time using
`
`any of a variety of assays for cell proliferation, viability and/or cytotoxicity. In some cases, the
`
`cultured cells can be stained with 7—AAD or other staining or other reagent used to discriminate
`
`viability and cell death or apoptosis or activation of apoptotic pathways.
`
`In some examples,
`
`cytotoxicity induced by fratricide killing of antigen—specific immune cells can be assessed using a
`
`cytotoxicity assay, such as an assay to assess chromium release. In some cases, the self—killing of
`
`the immune cells themselves expressing the genetically engineered antigen receptor can limit
`
`methods of using certain antigen—specific immune cells in adoptive cell therapy, since such cells can
`
`be eliminated by the fratricide killing ex vivo prior to administration and/or are upon administration
`
`in vivo. Self—killing of engineered cells within a population may be by killing of a single cell within
`
`such population of engineered cells by the same cell or by cells within the population killing one
`
`another.
`
`[0033] Provided are cells and methods that overcome such problems. For example, in some
`
`embodiments, the expression of a gene encoding an antigen specifically bound by one or more of
`
`the genetically engineered antigen receptors is repressed in the cell. By repressing the specific
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`antigen in the immune cell, fratricide killing of the immune cells themselves is prevented or reduced.
`
`In some embodiments, the fratricide killing (6. g. as assessed by a proliferation, viability and/or
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`cytotoxicity assay) is reduced by at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, such as as
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`compared to a cell or cell population in which the cells express the engineered antigen receptor but
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`in which the gene or antigen targeted thereby or expression thereof has not been disrupted. Such
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`features in some aspects confer improved specificity and/or efficacy in the context of adoptive cell
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`therapy.
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`[0034] Among such antigens are those expressed in cancers of the immune system, such as
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`leukemias, lymphomas, and/or myelomas, such as multiple myeloma, and/or expressed on T cells
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`and/or NK cells, including activated T cells. Exemplary antigens are those expressed on activated
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`or stimulated T cells or NK cells, and subsets thereof, particularly activated cells produced by
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`stimulatory conditions used to promote the introduction of the nucleic acid encoding the CAR or
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`other engineered receptor. For example, among such antigens are those not generally expressed on
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`resting T cells but expression of which is induced upon T cell activation and/or that are expressed
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`on activated T cells. In some embodiments, the gene encodes a universal tumor antigen, such as
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`hTERT, survivin, MDM2, CYPlB, HER2/neu, WTl, livin, AFP, CEA, MUCl6, MUCl, PSMA,
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`p53 or cyclin (D1). For example, the gene encodes hTERT or survivin.
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`In some embodiments the
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`gene encodes the antigen CD38. In some embodiments, the gene encodes the antigen CD33 or TIM—
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`3; in some embodiments, it encodes the antigen CD26, CD30, CD53, CD92, CD100, CD148,
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`CD150, CD200, CD261, CD262, or CD362.
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`[0035] In some embodiments, the repression is effected via disruption of the gene encoding the
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`antigen, such as by deletion, e.g., deletion of an entire gene, exon, or region, and/or replacement
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`with an exogenous sequence, and/or by mutation, e.g., frameshift or missense mutation, within the
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`gene, typically within an exon of the gene. In some embodiments, the disruption results in a
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`premature stop codon being incorporated into the gene, such that the antigen is not expressed or is
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`not expressed in a form that is recognized by the antigen receptor. The disruption is generally
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`carried out at the DNA level. The disruption generally is permanent, irreversible, or not transient.
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`In other embodiments, transient or reversible repression strategies are used, such as gene
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`knockdown using RNAi.
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`In some embodiments, by disrupting or otherwise repressing expression
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`of the antigen on the engineered cells, the methods and compositions provided here avoid or reduce
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`the likelihood of killing of the engineered cells by the engineered cells themselves, thereby
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`promoting efficacy.
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`[0036] In some embodiments, the cells include features for increased efficacy of the cells and
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`methods are provided by the disruption of gene expression in the engineered cells. In some aspects,
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`the gene disrupted encodes a checkpoint molecule, immunosuppressive molecule, such as a receptor
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`that delivers an immunosuppressive signal to the cell, and/or any molecule which could reduce the
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`robustness of the response of the engineered cell, for example, following administration in
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`connection with immunotherapy.
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`[0037] In some embodiments, for example, in the context of an antigen that is recognized by an
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`inhibitory CAR, the gene disruption prevents or reduces the likelihood of the inhibitory CAR
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`expressed by the engineered cell of itself binding to a molecule also expressed by the engineered
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`cells, thereby inducing a dampening effect on the signaling or targeted immune response by the
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`engineered cells. Exemplary of such antigens are those expressed on normal or non-targeted or off—
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`target cells (such that an inhibitory CAR molecule is included to prevent off—target effects), but is
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`also expressed on the cell type used for genetic engineering, such as the T cell or NK cell.
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`Exemplary antigens are MHC molecules, such as MHC—class I molecules, which can in some cases
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`be downregulated in the context of immune evasion, cancer, or infection, but are generally
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`expressed on nucleated cells. Other examples are any inhibitory CAR target that is also expressed
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`on a T cell, NK cell, or other cell engineered for cell therapy.
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`[0038] In other embodiments, the gene or genes disrupted is a gene other than that encoding the
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`antigen, such as a gene encoding an immunosuppressive molecule, e. g., checkpoint molecule or
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`adenosine receptor, e. g., AZAR.
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`[0039] In some aspects, the disruption is carried out by gene editing, such as using a DNA
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`binding protein or DNA—binding nucleic acid, which specifically binds to or hybridizes to the gene
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`at a region targeted for disruption. In some aspects, the protein or nucleic acid is coupled to or
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`complexed with a nuclease, such as in a chimeric or fusion protein. For example, in some
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`embodiments, the disruption is effected using a fusion comprising a DNA—targeting protein and a
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`nuclease, such as a Zinc Finger Nuclease (ZFN) or TAL—effector nuclease (TALEN), or an RNA—
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`guided nuclease such as a clustered regularly interspersed short palindromic nucleic acid (CRISPR)—
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`Cas system, such as CRISPR—Cas9 system, specific for the gene being disrupted.
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`[0040] In some embodiments, improved selectivity and specificity is achieved through
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`strategies targeting multiple antigens. Such strategies generally involve multiple antigen-binding
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`domains, which typically are present on distinct genetically engineered antigen receptors and
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`specifically bind to distinct antigens. Thus, in some embodiments, the cells are engineered with the
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`ability to bind more than one antigen. In some aspects, a plurality of genetically engineered antigen
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`receptors are introduced into the cell, which specifically bind to different antigens, each expressed
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`in or on the disease or condition to be targeted with the cells or tissues or cells thereof. Such
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`features can in some aspects address or reduce the likelihood of off—target effects. For example,
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`where a single antigen expressed in