(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(l9) Worl~~;:::~~:! Property ~ 1111111111111111 IIIIII IIIII IIIII IIIII IIII I II Ill 111111111111111 lllll lllll 11111111111111111111111
`~ (tow)
`lntoern2atoio2na2I /Plu b8h3" caoti7on2NAumlber
`International Bureau
`(43) International Publication Date
`;;;;;..,,-""
`01 September 2022 (01.09.2022) WI PO I PCT
`
`Published:
`with international search report (Art. 21 (3))
`before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments (Rule 48.2(h))
`with sequence listing part of description (Rule 5.2(a))
`
`(51) International Patent Classification:
`C07K 14/145 (2006.01)
`C12N 15/867 (2006.01)
`
`(21) International Application Number:
`PCT/US2022/018027
`
`(22) International Filing Date:
`25 February 2022 (25.02.2022)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`63/154,639
`
`26 February 2021 (26.02.2021) US
`
`INC.
`(71) Applicant: KELONIA THERAPEUTICS,
`[US/US]; 5 Channel Center, Suite 501, Boston, MA 02210
`(US).
`
`(72) Inventors: PERKINS, Molly R.; 64 Bradlee Rd., Mil(cid:173)
`ton, Massachusetts 02186 (US). FRIEDMAN, Kevin M.;
`4 Clover Circle, Melrose, Massachusetts 02176 (US).
`
`(74) Agent: SUN, Eileen, S. et al.; Seed Intellectual Property
`Law Group LLP, Suite 5400, 701 Fifth Avenue, Seattle,
`Washington 98104-7064 (US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, EH, EN, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN,
`HR, HU, ID, IL, IN, IR, IS, IT, JM, JO, JP, KE, KG, KH,
`KN, KP, KR, KW, 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, ST, SV, SY, TH, TJ, TM,
`TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, WS, ZA, ZM,
`ZW.
`
`(84) 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).
`
`iiiiiiiiiiii
`
`-iiiiiiiiiiii
`!!!!!!!! -!!!!!!!!
`
`--
`
`-!
`
`!!!!!!! -
`
`!!!!!!!!
`iiiiiiiiiiii
`iiiiiiiiiiii
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`!!!!!!!!
`
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`t---= "">
`N M (57) Abstract: Provided herein are lentiviral vectors comprising a mutated, heterologous envelope protein, a targeting protein, and
`
`a0
`,...,i (54) Title: LYMPHOCYTE TARGETED LENTIVIRAL VECTORS
`
`Q
`at least one transgene for delivery to and expression by a cell characterized by the targeting protein. Also provided are methods and
`M materials for producing the lentiviral vectors described herein, methods for transducing target cells, and cells transduced by lentiviral
`0 vectors according to the present disclosure.
`~
`
`Page 1 of 162
`
`KELONIA EXHIBIT 1005
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`

`

`WO 2022/183072
`
`PCT/0S2022/018027
`
`LYMPHOCYTE TARGETED LENTIVIRAL VECTORS
`
`STATEMENT REGARDING SEQUENCE LISTING
`
`The Sequence Listing associated with this application is provided in text format
`
`5
`
`in lieu of a paper copy, and is hereby incorporated by reference into the
`
`specification. The name of the text file containing the Sequence Listing is
`
`930207 _ 401WO_SEQUENCE_LISTING.txt. The text file is 158 KB, was created on
`
`February 25, 2022, and is being submitted electronically via EFS-Web.
`
`10 BACKGROUND
`
`Lentiviral vectors play a critical role in gene-modified cell therapies,
`
`particularly T cell therapies. Recently approved T cell therapies rely on retroviral
`
`vectors to transduce the therapeutic molecule (e.g., chimeric antigen receptor (CAR))
`
`into T lymphocytes. An associated risk to CART cell production is the transduction of
`
`15
`
`other cell types with the transgene. The use of integrating vectors with broad cell
`
`tropism, e.g., lentiviral vectors pseudotyped with a VSV-G envelope protein, can
`
`represent a serious, though rare, safety concern.
`
`BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
`
`20
`
`FIG. 1 is a schematic representation of helper plasmids suitable for use in a
`
`third generation LVV production system.
`
`FIGS. 2A-2B depict graphs (FIG. 2A) and FACS plots (FIG. 2B) showing on(cid:173)
`
`target and off-target entry of Jurkat T cells and Raji B cells by lentiviral vectors bearing
`
`mutated VSV-G envelope to abolish LDL receptor binding (Trop-002, Trop-051, Trop-
`
`25
`
`052, Trop-055, and Trop-061) and T cell targeting protein CD80. In FIG. 2A, on-target
`
`entry is the left bar and off-target is the right bar of each sample. Binding of the T cell
`
`targeting protein to its cognate ligand on T cells leads to entry of the lentiviral vector,
`
`and subsequent expression of reporter green fluorescent protein (GFP) is measured.
`
`I
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`FIGS. 3A-3B depicts graphs showing: (FIG. 3A) T cell targeting protein CD80
`
`expressed from the VSV-G packaging plasmid is expressed at relatively equivalent
`
`levels as the mutated VSV-G on the surface ofHEK293 producer cells; and (FIG. 3B)
`
`L VV generated with this approach can transduce targeted Jurkat T cells but do not
`
`5
`
`transduce Raji B cells.
`
`FIG. 4 depicts graphs showing: (top row) expression levels of CD80 and
`
`mutated VSV-G on the surface of HEK293 T producer cells using L VV generated by
`
`cloning CD80 targeting protein into the Rev packaging plasmid or into the mutated
`
`VSV-G packaging plasmid; and (bottom) LVV generated by cloning CD80 targeting
`
`10
`
`protein into the Rev packaging plasmid or into the mutated VSV-G packaging plasmid
`
`transduce targeted Jurkat T cells.
`
`FIG. 5 depicts (left) CD80 targeting protein expression on surface ofHEK293T
`
`producer cells using a five plasmid packaging system as a function of CD80 plasmid
`
`concentration; and (right) LVV generated with the five plasmid packaging system
`
`15
`
`transduce targeted Jurkat T cells and transduction efficiency was associated with CD80
`
`packaging plasmid concentration.
`
`FIGS. 6A-6D depict titers of lymphocyte targeting L VV produced in adherent
`
`HEK293 or suspension HEK293 producer cells. L VV harvested from HEK293
`
`adherent cell culture medium by centrifugation (FIG. 6A) or by anion exchange
`
`20
`
`chromatography followed by tangential flow filtration (FIG. 6B). LVV harvested from
`
`HEK293 suspension cell culture medium by anion exchange chromatography (FIG.
`
`6C). Concentration by AEX/TFF resulted in L VV preparations with a high level of
`
`purity and recovery (FIG. 6D).
`
`FIG. 7 depicts a schematic for testing T cell transduction in PBMCs from
`
`25
`
`healthy human donors with L VV comprising a BCMA CAR transgene or T cell
`
`targeting LVV (anti-CD3 and CD80) comprising a BCMA CAR trasngene. Graphs
`
`shown on lower right indicate that even at low MOI, the T cell targeting L VV
`
`transduced T cells at a higher level than standard L VV and that T cell targeting LVV is
`
`capable of transducing T cells without the presence of IL-2 and exogenous activating
`
`30
`
`antibodies (anti-CD3 and anti-CD28) in contrast to standard LVV.
`
`2
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`FIG. 8 depicts graphs showing T cell expansion from PBMCs obtained from
`
`three different donors and transduced using standard L VV or T cell redirected L VV
`
`(anti-CD3 and CD80) in the presence or absence of exogenous activating anti-CD3 and
`
`anti-CD28 antibodies
`
`5
`
`FIG. 9 depcits a schematic for testing T cell transduction in PBMCs from
`
`healthy human donors with LVV comprising a CD19 CAR transgene or T cell targeting
`
`LVV (anti-CD3 and CD80) comprising a CD19 CAR trasngene. Graphs shown on
`
`lower right show that the T cell targeting L VV transduced T cells at a higher level than
`
`standard L VV and that T cell targeting L VV is capable of transducing T cells without
`
`10
`
`the presence of exogenous activating antibodies (anti-CD3 and anti-CD28) in contrast
`
`to standard L VV.
`
`FIG. 10 depicts graphs showing levels of T cell transduction efficiency and T
`
`cell activation with anti-CD3 targeting proteins (12F6 in VH-VL orientation and VL(cid:173)
`
`VH orientation) used to generate T cell targeting LVV.
`
`15
`
`FIG. 11 depicts graphs showing that BCMA CART cells exhibited increased
`
`expression of T cell effector cytokines (TNFa - left; TNFa and IFNy - right) after
`
`culture with BCMA-positive cell lines that is not observed with BCMA-negative cell
`
`lines whether generated by standard LVV or T cell-redirected LVV (anti-CD3 and
`
`CD80).
`
`20
`
`FIGS. 12A-12B depict graphs showing in vivo delivery of transgene using T
`
`cell targeting LVV: (FIG. 12A) T cell targeting LVV (anti-CD3 and CD80) specificly
`
`transduce human T cells (CD3+) and not human B cells (CD20+) in humanized mouse
`
`model (n=5); and (FIG. 12B) T cell targeting L VV transduce both CD8+ and CD8-
`
`(CD4) T cells compared to standard LVV which did not.
`
`25
`
`FIG. 13 depicts graphs showing that CD80 targeting L VV s enhance
`
`transduction of CD4 T cells compared to standard L VV s.
`
`FIG. 14 depicts graphs showing that T cell targeting (anti-CD3 and CD80)
`
`L VV s transduce target Jurkat T cells but do not transduce off-target tumor cells (Raji,
`
`Ramos, Jeko-1, and NALM-6) compared to standard LVV.
`
`30
`
`3
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`DETAILED DESCRIPTION
`
`Engineered lentiviral vectors are described herein. The lentiviral vectors
`
`include a mutated, heterologous envelope protein, a targeting protein, and at least one
`
`transgene for delivery to and expression by a cell characterized by the targeting protein.
`
`5
`
`In some embodiments, the targeting protein is selected to target an immune cell,
`
`including, for example a lymphocyte or a T cell. In certain such embodiments, the
`
`lentiviral vectors described herein are capable of selectively targeting and efficiently
`
`transducing resting lymphocytes, e.g., T cells. In some embodiments, lentiviral vectors
`
`described herein are capable of transducing and/or activating T cells in the absence of
`
`10
`
`an exogenous T cell stimulating agent. In some embodiments, lentiviral vectors
`
`described herein enhance transduction of CD4 T cells compared to standard lentiviral
`
`vectors.
`
`In some embodiments, the lentiviral vectors incorporating a mutated env and a
`
`targeting protein as described herein are capable of producing a high titer L VV product,
`
`15
`
`as compared to standard LVV incorporating another fusogenic env protein (e.g., cocal
`
`env, paramyxovirus env, truncated VSV-G env).
`
`Also provided are methods and materials for producing the lentiviral vectors described
`
`herein, methods for transducing target cells, and cells transduced by lentiviral vectors
`
`20
`
`according to the present disclosure. In some embodiments, a lentiviral vector as
`
`described herein and/or cells transduced by such a vector may be used in treating a
`
`disease or disorder responsive to the presence of cells expressing the transgene
`
`delivered by the vector.
`
`25 Definitions
`
`Prior to setting forth this disclosure in more detail, it may be helpful to an
`
`understanding thereof to provide definitions of certain terms to be used herein.
`
`In the present description, any concentration range, percentage range, ratio
`
`range, or integer range is to be understood to include the value of any integer within the
`
`30
`
`recited range and, when appropriate, fractions thereof (such as one tenth and one
`
`hundredth of an integer), unless otherwise indicated. Also, any number range recited
`
`4
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`herein relating to any physical feature, such as polymer subunits, size or thickness, are
`
`to be understood to include any integer within the recited range, unless otherwise
`
`indicated. As used herein, the term "about" means± 20% of the indicated range, value,
`
`or structure, unless otherwise indicated. It should be understood that the terms "a" and
`
`5
`
`"an" as used herein refer to "one or more" of the enumerated components. The use of
`
`the alternative (e.g., "or") should be understood to mean either one, both, or any
`
`combination thereof of the alternatives. As used herein, the terms "include," "have" and
`
`"comprise" are used synonymously, which terms and variants thereof are intended to be
`
`construed as non-limiting.
`
`10
`
`Terms understood by those in the art of antibody technology are each given the
`
`meaning acquired in the art, unless expressly defined differently herein. The term
`
`"antibody" is used in the broadest sense and includes polyclonal and monoclonal
`
`antibodies. An "antibody" may refer to an intact antibody comprising at least two
`heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as
`
`15
`
`an antigen-binding portion ( or antigen-binding domain) of an intact antibody that has or
`
`retains the capacity to bind a target molecule. An antibody may be naturally occurring,
`
`recombinantly produced, genetically engineered, or modified forms of
`
`immunoglobulins, for example intrabodies, peptibodies, nanobodies, single domain
`
`antibodies, SMIPs, multispecific antibodies (e.g., bispecific antibodies, diabodies,
`
`20
`
`triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv, ADAPTIR). A monoclonal
`
`antibody or antigen-binding portion thereof may be non-human, chimeric, humanized,
`
`or human, preferably humanized or human. Immunoglobulin structure and function are
`
`reviewed, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual,
`
`Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). "Antigen-
`
`25
`
`binding portion" or "antigen-binding domain" of an intact antibody is meant to
`
`encompass an "antibody fragment," which indicates a portion of an intact antibody and
`
`refers to the antigenic determining variable regions or complementary determining
`
`regions of an intact antibody. Examples of antibody fragments include, but are not
`
`limited to, Fab, Fab', F(ab')2, and Fv fragments, Fab' -SH, F(ab')2, diabodies, linear
`
`30
`
`antibodies, scFv antibodies, VH, and multispecific antibodies formed from antibody
`
`fragments. A "Fab" (fragment antigen binding) is a portion of an antibody that binds to
`
`antigens and includes the variable region and CHI of the heavy chain linked to the light
`
`5
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`chain via an inter-chain disulfide bond. An antibody may be of any class or subclass,
`
`including IgG and subclasses thereof (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.
`
`The term "variable region" or "variable domain" in the context of an antibody
`
`refers to the domain of an antibody heavy or light chain that is involved in binding of
`
`5
`
`the antibody to antigen. The variable domains ( or regions) of the heavy chain and light
`
`chain (VH and VL, respectively) of a native antibody generally have similar structures,
`
`with each domain comprising four conserved framework regions (FRs) and three
`
`complementary determining regions (CDRs). (See, e.g., Kindt et al. Kuby Immunology,
`
`6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be
`
`10
`
`sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a
`
`particular antigen may be isolated using a VH or VL domain from an antibody that
`
`binds the antigen to screen a library of complementary VL or VH domains,
`
`respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et
`
`al., Nature 352:624-628 (1991).
`
`15
`
`The terms "complementarity determining region" and "CDR," which are
`
`synonymous with "hypervariable region" or "HVR," are known in the art to refer to
`
`non-contiguous sequences of amino acids within antibody variable regions, which
`
`confer antigen specificity and/or binding affinity. In general, there are three CDRs in
`
`each heavy chain variable region (HCDRl, HCDR2, HCDR3) and three CDRs in each
`
`20
`
`light chain variable region (LCDRl, LCDR2, LCDR3).
`
`As used herein, the terms "binding domain", "binding region", and "binding
`
`moiety" refer to a molecule, such as a peptide, oligopeptide, polypeptide, or protein that
`
`possesses the ability to specifically and non-covalently bind, associate, unite, recognize,
`
`or combine with a target molecule (e.g., tumor antigen). A binding domain includes
`
`25
`
`any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding
`
`partner for a biological molecule or other target of interest. In some embodiments, the
`
`binding domain is an antigen-binding domain, such as an antibody or functional binding
`
`domain or antigen-binding portion thereof. Exemplary binding domains include single
`
`chain antibody variable regions (e.g., domain antibodies, sFv, scFv, Fab), receptor
`
`30
`
`ectodomains (e.g., TNF-a), ligands (e.g., cytokines, chemokines), or synthetic
`
`polypeptides selected for the specific ability to bind to a biological molecule.
`
`"Major histocompatibility complex molecule" (MHC molecule) refers to a
`
`glycoprotein that delivers a peptide antigen to a cell surface. MHC class I molecules
`
`6
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`are heterodimers composed of a membrane spanning a chain (with three a domains)
`
`and a non-covalently associated ~2 microglobulin. MHC class II molecules are
`
`composed of two transmembrane glycoproteins, a and~' both of which span the
`
`membrane. Each chain has two domains. MHC class I molecules deliver peptides
`
`5
`
`originating in the cytosol to the cell surface, where peptide:MHC complex is recognized
`
`by CD8+ T cells. MHC class II molecules deliver peptides originating in the vesicular
`
`system to the cell surface, where they are recognized by CD4+ T cells. An MHC
`
`molecule may be from various animal species, including human, mouse, rat, or other
`
`mammals.
`"Chimeric antigen receptor" (CAR) refers to a chimeric fusion protein
`
`10
`
`comprising two or more distinct domains linked together in a way that does not occur
`
`naturally in a host cell and can function as a receptor when expressed on the surface of
`
`a cell. CARs are generally composed of an extracellular domain comprising a binding
`
`domain that binds a target antigen, an optional extracellular spacer domain, a
`
`15
`
`transmembrane domain, and an intracellular signaling domain (e.g., comprising an
`
`immunoreceptor tyrosine-based activation motif (ITAM)), and optionally an
`
`intracellular costimulatory domain). In certain embodiments, an intracellular signaling
`
`domain of a CAR has an ITAM (e.g., CD3~) containing intracellular signaling domain
`
`and an intracellular costimulatory domain (e.g., 4-lBB). In certain embodiments, a
`
`20
`
`CAR is synthesized as a single polypeptide chain or is encoded by a nucleic acid
`
`molecule as a single chain polypeptide.
`
`A variety of assays are known for identifying binding domains of the present
`
`disclosure that specifically bind a particular target, as well as determining binding
`
`domain affinities, such as Western blot, ELISA, analytical ultracentrifugation,
`
`25
`
`spectroscopy, surface plasmon resonance (BIACORE®) analysis, and MHC tetramer
`
`analysis (see also, e.g., Scatchard et al., Ann. NY Acad Sci. 51:660, 1949; Wilson,
`
`Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; Altman et al.,
`
`Science 274:94-96, 1996; and U.S. Patent Nos. 5,283,173, 5,468,614, or the
`
`equivalent). As used herein, "specifically binds" refers to an association or union of a
`
`30
`
`binding domain, or a fusion protein thereof, to a target molecule with an affinity or Ka
`
`(i.e., an equilibrium association constant of a particular binding interaction with units of
`
`1/M) equal to or greater than 105 M·1, while not significantly associating or uniting with
`
`any other molecules or components in a sample.
`
`7
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`The terms "antigen" and "Ag" refer to a molecule that is capable of inducing an
`
`immune response. The immune response that is induced may involve antibody
`
`production, the activation of specific immunologically-competent cells, or both.
`
`Macromolecules, including proteins, glycoproteins, and glycolipids, can serve as an
`
`5
`
`antigen. Antigens can be derived from recombinant or genomic DNA As
`
`contemplated herein, an antigen need not be encoded (i) solely by a full-length
`
`nucleotide sequence of a gene or (ii) by a "gene" at all. An antigen can be generated or
`
`synthesized, or an antigen can be derived from a biological sample. Such a biological
`
`sample can include, but is not limited, to a tissue sample, a tumor sample, a cell, or a
`
`10
`
`biological fluid.
`
`The term "epitope" or "antigenic epitope" includes any molecule, structure,
`
`amino acid sequence or protein determinant within an antigen that is specifically bound
`
`by a cognate immune binding molecule, such as an antibody or fragment thereof (e.g.,
`
`scFv), T cell receptor (TCR), CAR, or other binding molecule, domain or protein.
`
`15
`
`Epitopic determinants generally contain chemically active surface groupings of
`
`molecules, such as amino acids or sugar side chains, and can have specific three(cid:173)
`
`dimensional structural characteristics, as well as specific charge characteristics. An
`
`epitope may be a linear epitope or a conformational epitope.
`
`As used herein, an "effector domain" is an intracellular portion of a fusion
`
`20
`
`protein or chimeric receptor that can directly or indirectly promote a biological or
`
`physiological response in a cell expressing the effector domain when receiving the
`
`appropriate signal. In certain embodiments, an effector domain is part of a protein or
`
`protein complex that receives a signal when bound. In other embodiments, the effector
`
`domain is part of a protein or protein complex that binds directly to a target molecule,
`
`25 which triggers a signal from the effector domain. For example, in response to binding
`
`of a CAR to a target molecule, the effector domain may transduce a signal to the
`
`interior of the host cell, eliciting an effector function. An effector domain may directly
`
`promote a cellular response when it contains one or more signaling domains or motifs.
`
`In other embodiments, an effector domain will indirectly promote a cellular response by
`
`30
`
`associating with one or more other proteins that directly promote a cellular response.
`
`"Junction amino acids" or "junction amino acid residues" refer to one or more
`
`(e.g., about 2-20) amino acid residues between two adjacent motifs, regions or domains
`
`of a polypeptide. Junction amino acids may result from the construct design of a
`
`8
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`chimeric protein (e.g., amino acid residues resulting from the use of a restriction
`
`enzyme site during the construction of a nucleic acid molecule encoding a fusion
`
`protein).
`
`A "disease" is a state of health of a subject wherein the subject cannot maintain
`
`5
`
`homeostasis, and wherein, if the disease is not ameliorated, then the subject's health
`
`continues to deteriorate. In contrast, a "disorder" or "undesirable condition" in a
`
`subject is a state of health in which the subject is able to maintain homeostasis, but in
`
`which the subject's state of health is less favorable than it would be in the absence of
`
`the disorder or undesirable condition. Left untreated, a disorder or undesirable
`
`10
`
`condition does not necessarily result in a further decrease in the subject's state of
`
`health.
`
`"Nucleic acid molecule" and "polynucleotide" can be in the form of RNA or
`
`DNA, which includes cDNA, genomic DNA, and synthetic DNA. A nucleic acid
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`molecule may be composed of naturally occurring nucleotides (such as
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`deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides
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`(e.g., a-enantiomeric forms of naturally occurring nucleotides), or a combination of
`
`both. Modified nucleotides can have modifications in or replacement of sugar moieties,
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`or pyrimidine or purine base moieties. Nucleic acid monomers can be linked by
`
`phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages
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`include phosphorothioate, phosphorodithioate, phosphoroselenoate,
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`phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and
`
`the like. A nucleic acid molecule may be double stranded or single stranded, and if
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`single stranded, may be the coding strand or non-coding (anti-sense strand). A coding
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`molecule may have a coding sequence identical to a coding sequence known in the art
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`or may have a different coding sequence, which, as the result of the redundancy or
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`degeneracy of the genetic code, or by splicing, can encode the same polypeptide.
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`"Encoding" refers to the inherent property of specific polynucleotide sequences,
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`such as DNA, cDNA, and mRNA sequences, to serve as templates for synthesis of
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`other polymers and macromolecules in biological processes having either a defined
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`sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino
`
`acids and the biological properties resulting therefrom. Thus, a polynucleotide encodes
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`a protein if transcription and translation of mRNA corresponding to that polynucleotide
`
`produces the protein in a cell or other biological system. Both a coding strand and a
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`non-coding strand can be referred to as encoding a protein or other product of the
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`polynucleotide. Unless otherwise specified, a "nucleotide sequence encoding an amino
`
`acid sequence" includes all nucleotide sequences that are degenerate versions of each
`
`other and that encode the same amino acid sequence.
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`As used herein, the term "endogenous" or "native" refers to a gene, protein,
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`compound, molecule or activity that is normally present in a host or host cell, including
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`naturally occurring variants of the gene, protein, compound, molecule, or activity.
`
`As used herein, "homologous" or "homolog" refers to a molecule or activity
`
`from a host cell that is related by ancestry to a second gene or activity, e.g., from the
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`same host cell, from a different host cell, from a different organism, from a different
`
`strain, from a different species. For example, a heterologous molecule or heterologous
`
`gene encoding the molecule may be homologous to a native host cell molecule or gene
`
`that encodes the molecule, respectively, and may optionally have an altered structure,
`
`sequence, expression level or any combination thereof.
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`As used herein, "heterologous" nucleic acid molecule, construct or sequence
`
`refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native
`
`to a host cell, but can be homologous to a nucleic acid molecule or portion of a nucleic
`
`acid molecule from the host cell. The source of the heterologous nucleic acid molecule,
`
`construct or sequence can be from a different genus or species. In some embodiments,
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`the heterologous nucleic acid molecules are not naturally occurring. In certain
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`embodiments, a heterologous nucleic acid molecule is added (i.e., not endogenous or
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`native) into a host cell or host genome by, for example, conjugation, transformation,
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`transfection, transduction, electroporation, or the like, wherein the added molecule can
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`integrate into the host cell genome or exist as extra-chromosomal genetic material (e.g.,
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`as a plasmid or other form of self-replicating vector), and can be present in multiple
`
`copies. In addition, "heterologous" refers to a non-native enzyme, protein or other
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`activity encoded by a non-endogenous nucleic acid molecule introduced into the host
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`cell, even if the host cell encodes a homologous protein or activity.
`
`As used herein, the term "engineered," "recombinant," "mutant," "modified" or
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`"non-natural" refers to an organism, microorganism, cell, nucleic acid molecule, or
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`vector that has been modified by introduction of a heterologous nucleic acid molecule,
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`or refers to a cell or microorganism that has been genetically engineered by human
`
`intervention-that is, modified by introduction of a heterologous nucleic acid
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`molecule, or refers to a cell or microorganism that has been altered such that expression
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`of an endogenous nucleic acid molecule or gene is controlled, deregulated or
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`constitutive, where such alterations or modifications can be introduced by genetic
`
`engineering. Human-generated genetic alterations can include, for example,
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`5 modifications introducing nucleic acid molecules (which may include an expression
`
`control element, such as a promoter) encoding one or more proteins, chimeric receptors,
`
`or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other
`
`functional disruption of or addition to a cell's genetic material. Exemplary
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`modifications include those in coding regions or functional fragments thereof
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`heterologous or homologous polypeptides from a reference or parent molecule.
`
`Additional exemplary modifications include, for example, modifications in non-coding
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`regulatory regions in which the modifications alter expression of a gene or operon.
`
`As used here, the term "transgene" refers to a gene or polynucleotide encoding a
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`protein of interest (e.g., a CAR) whose expression is desired in a host cell and that has
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`been transferred by genetic engineering techniques into a cell. A transgene may encode
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`proteins of therapeutic interest as well as proteins that are reporters, tags, markers,
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`suicide proteins, etc. A transgene may be from a natural source, modification of a
`
`natural gene, or a recombinant or synthetic molecule. In certain embodiments, a
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`transgene is a component of a vector.
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`The term "overexpressed" or "overexpression" of an antigen refers to an
`
`abnormally high level of antigen expression in a cell. Overexpressed antigen or
`
`overexpression of antigen is often associated with a disease state, such as in
`
`hematological malignancies and cells forming a solid tumor within a specific tissue or
`
`organ of a subject. Solid tumors or hernatological rnalignancies characterized by
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`overexpression of a tumor antigen can be determined by standard assays knmvn in the
`
`art.
`
`As used herein, the terms ''peptide," "polypeptide," and "protein" are used
`
`interchangeably, and refer to a compound comprised of amino acid residues covalently
`
`linked by peptide bonds. A protein or peptide nmst contain at least two amino acids,
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`and no limitation is placed on the maximum number of amino acids that can comprise a
`
`protein's or peptide's sequence. Polypeptides include any peptide or protein comprising
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`two or more amino acids joined to each other by peptide bonds. As used herein, the
`
`term refors to both short chains, which also commonly are referred to in the art as
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`peptides, oligopeptides and oligomers, for example, and to longer chains, which
`
`generally are referred to in the art as proteins, of which there are many types.
`
`"Polypeptides" include, for example, biologically active fragments, substantially
`
`hornologous polypeptides, oligopeptides, homodimers, heterodirners, variants of
`
`5
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`polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among
`
`others. The polypeptides include natural peptides, recombinant peptides, synthetic
`
`peptides, or a combination thereof
`
`As used herein, the term "mature polypeptide" or "mature protein" refers to a
`
`protein or polypeptide that is secreted or localized in the cell membrane or inside
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`certain cell organelles (e,g., the endoplasmic reticulum, golgi, or endosome) and
`
`includes a partially cleaved N-terminal signal sequence ( e.g., one or more amino acids
`
`of the signal sequence remaining but less than the whole signal sequence) or does not
`
`include an N-terminal signal sequence (i.e., the N-terminal signal sequence has been
`
`entirely removed, such as by an endogenous cleavage process, from the protein or
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`polypeptide).
`
`A "signal sequence'', also referred to as ''signal peptide", "leader sequence",
`
`"leader peptide", "localization sign