Enhanced transfer of genetic instructions to effector immune cells

Abstract

A method for preparing immune effector cells for transduction, comprising a step for increasing expression of low density lipoprotein receptor (LDLR) and, optionally, a step for inhibiting intracellular antiviral defense mechanisms of the immune effector cells.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 391,672, filed July 22, 2022, U.S. Provisional Patent Application No. 63 / 391,676, filed July 22, 2022, and U.S. Provisional Patent Application No. 63 / 391,677, filed July 22, 2022, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION In some embodiments, provided herein are methods for generating bioengineered natural killer (NK) cells or T cells, such as NK cells or T cells that express chimeric antigen receptor-natural killer (CAR-NK cells or chimeric antigen receptor-T, CAR-T cells), and uses thereof. Also provided herein, in some embodiments, are compositions comprising CAR-NK cells or CAR-T cells, and uses thereof for treating diseases or disorders. Summary of the Invention

[0003] In one aspect, provided herein is a method for preparing immune effector cells for transduction, the method comprising increasing expression of low-density lipoprotein receptor (LDLR).

[0004] In some embodiments, the immune effector cells are autologous or allogeneic.

[0005] In some embodiments, the immune effector cell is a cytotoxic cell, optionally, the cytotoxic cell is a natural killer cell.

[0006] In some embodiments, the immune effector cells are gamma-delta T cells.

[0007] In some embodiments, the immune effector cells are alpha-beta T cells.

[0008] In some embodiments, the step of increasing expression of low density lipoprotein receptor (LDLR) comprises contacting the immune effector cells with a statin.

[0009] In some embodiments, the statin is rosuvastatin.

[0010] In some embodiments, the statin is atorvastatin.

[0011] In some embodiments, the method further comprises a step for inhibiting an intracellular antiviral defense mechanism of an immune effector cell.

[0012] In some embodiments, the step of inhibiting an intracellular antiviral defense mechanism of an immune effector cell comprises contacting the immune effector cell with an inhibitor of 3-phosphoinositide-dependent kinase 1 (PDK1).

[0013] In some embodiments, the inhibitor of PDK1 is BX795.

[0014] In some embodiments, the method further comprises providing one or more transduction enhancers.

[0015] In some embodiments, the transduction enhancer is Vectofusin.

[0016] In some embodiments, the transduction enhancer is retronectin.

[0017] In some embodiments, the method further comprises introducing the nucleic acid into an immune effector cell.

[0018] In some embodiments, the nucleic acid comprises a promoter.

[0019] In some embodiments, the promoter is selected from the group consisting of CAG, PGK, EF1a, and EFS promoters.

[0020] In some embodiments, the nucleic acid is delivered via a lentiviral particle.

[0021] In some embodiments, the lentiviral particles are pseudotyped with vesicular stomatitis virus G (VSV-G).

[0022] In some embodiments, the nucleic acid encodes an exogenous functional receptor.

[0023] In some embodiments, the exogenous functional receptor is a chimeric antigen receptor (CAR) or a TCR.

[0024] In one aspect, provided herein is a method for preparing immune effector cells for transduction, the method comprising contacting the immune effector cells with an agent that increases expression of low-density lipoprotein receptor (LDLR).

[0025] In some embodiments, the immune effector cells are autologous or allogeneic.

[0026] In some embodiments, the immune effector cell is a cytotoxic cell, optionally, the cytotoxic cell is a natural killer cell.

[0027] In some embodiments, the immune effector cells are gamma delta T cells.

[0028] In some embodiments, the immune effector cells are alpha beta T cells.

[0029] In some embodiments, the step of increasing expression of low density lipoprotein receptor (LDLR) comprises contacting the immune effector cells with a statin.

[0030] In some embodiments, the statin is rosuvastatin.

[0031] In some embodiments, the statin is atorvastatin.

[0032] In some embodiments, the method further comprises a step for inhibiting an intracellular antiviral defense mechanism of an immune effector cell.

[0033] In some embodiments, the step of inhibiting an intracellular antiviral defense mechanism of an immune effector cell comprises contacting the immune effector cell with an inhibitor of 3-phosphoinositide-dependent kinase 1 (PDK1).

[0034] In some embodiments, the inhibitor of PDK1 is BX795.

[0035] In some embodiments, the method further comprises providing one or more transduction enhancers.

[0036] In some embodiments, the transduction enhancer is a vectofusin.

[0037] In some embodiments, the transduction enhancer is retronectin.

[0038] In some embodiments, the method further comprises introducing the nucleic acid into an immune effector cell.

[0039] In some embodiments, the nucleic acid comprises a promoter.

[0040] In some embodiments, the promoter is selected from the group consisting of CAG, PGK, EF1a, and EFS promoters.

[0041] In some embodiments, the nucleic acid is delivered via a lentiviral particle.

[0042] In some embodiments, the lentiviral particles are pseudotyped with vesicular stomatitis virus G (VSV-G).

[0043] In some embodiments, the nucleic acid encodes an exogenous functional receptor.

[0044] In some embodiments, the exogenous functional receptor is a chimeric antigen receptor (CAR) or a TCR.

[0045] In one aspect, there is provided a method for preparing immune effector cells for transduction, comprising: i. a first step to increase the expression of low density lipoprotein receptor (LDLR); ii. a second step of inhibiting the intracellular antiviral defense mechanisms of immune effector cells; Methods are provided herein in which the first step can be performed before, simultaneously with, or after the second step.

[0046] In some embodiments, the immune effector cells are autologous or allogeneic.

[0047] In some embodiments, the immune effector cell is a cytotoxic cell, optionally, the cytotoxic cell is a natural killer cell.

[0048] In some embodiments, the method further comprises providing one or more transduction enhancers.

[0049] In some embodiments, the transduction enhancer is vectofusin. In some embodiments, the transduction enhancer is retronectin.

[0050] In some embodiments, the method further comprises introducing the nucleic acid into an immune effector cell. In some embodiments, the nucleic acid comprises a promoter. In some embodiments, the promoter is selected from the group consisting of CAG, PGK, EF1a, and EFS promoters.

[0051] In some embodiments, the nucleic acid is delivered via a lentiviral particle. In some embodiments, the lentiviral particle is pseudotyped with vesicular stomatitis virus G (VSV-G). In some embodiments, the nucleic acid encodes an exogenous functional receptor.

[0052] In some embodiments, the exogenous functional receptor is a chimeric antigen receptor (CAR) or a TCR.

[0053] In some embodiments, the first step comprises contacting the immune effector cells with a statin. In some embodiments, the statin is rosuvastatin.

[0054] In some embodiments, the second step comprises contacting the immune effector cells with an inhibitor of 3-phosphoinositide-dependent kinase 1 (PDK1). In some embodiments, the inhibitor of PDK1 is BX795.

[0055] In another aspect, there is provided a method for preparing immune effector cells for transduction, comprising: i. contacting immune effector cells with a first agent that increases expression of low density lipoprotein receptor (LDLR); ii. contacting the immune effector cells with a second agent that inhibits the immune effector cells' intracellular antiviral defense mechanisms; iii. contacting the immune effector cells with a third agent that enhances transduction.

[0056] In yet another aspect, a method comprising: i. contacting immune effector cells with a first agent that increases expression of low density lipoprotein receptor (LDLR); ii. contacting the immune effector cells with a second agent that inhibits the immune effector cells' intracellular antiviral defense mechanisms; iii. contacting the immune effector cells with a third agent that enhances transduction; iv. introducing the nucleic acid into an immune effector cell. [Brief explanation of the drawings]

[0057] [Figure 1A] Figure 1A shows that CAR expression was downregulated over time in both NK-like cell lines and primary NK cells. Figure 1A shows downregulation in the NK-like cell line NK-92. [Figure 1B] Figure 1B shows that CAR expression was downregulated over time in both NK-like cell lines and primary NK cells. Figure 1B shows downregulation in the NK-like cell line NK-L. [Figure 1C] Figure 1C shows that CAR expression was downregulated over time in both NK-like cell lines and primary NK cells. Figure 1C shows downregulation in primary NK cells. [Figure 2A] Figure 2A shows that the PDK1 inhibitor BX795 stabilized surface CAR expression in primary NK cells in a dose-dependent manner. [Figure 2B] Figure 2B and C show that the PDK1 inhibitor BX795 stabilized surface CAR expression in primary NK cells in a dose-dependent manner. [Figure 2C]Figure 2B and C show that the PDK1 inhibitor BX795 stabilized surface CAR expression in primary NK cells in a dose-dependent manner. [Figure 3A] Figure 3A shows that increased low-density lipoprotein receptor (LDLR) expression on primary NK cells was associated with increased antigen-2 CAR expression. Figure 3A shows increased LDLR expression on primary NK cells after exposure of the cells to the statin rosuvastatin. [Figure 3B] Figure 3B shows that increased low-density lipoprotein receptor (LDLR) expression on primary NK cells was associated with increased antigen-2 CAR expression. Figure 3B shows that increased antigen-2 CAR expression is associated with increased LDLR expression. [Figure 4] We show that the presence of the transduction enhancers retronectin and vectofusin during lentiviral transduction of primary NK cells was associated with increased antigen-3 CAR expression. [Figure 5A] We show that lentiviral transduction of primary NK cells in the presence of statin, BX795, retronectin, and vectofusin increased CAR stability on primary NK cells. Experiments were performed in triplicate (Figures 5A-C). [Figure 5B] We show that lentiviral transduction of primary NK cells in the presence of statin, BX795, retronectin, and vectofusin increased CAR stability on primary NK cells. Experiments were performed in triplicate (Figures 5A-C). [Figure 5C] We show that lentiviral transduction of primary NK cells in the presence of statin, BX795, retronectin, and vectofusin increased CAR stability on primary NK cells. Experiments were performed in triplicate (Figures 5A-C). [Figure 5D] Figure 5D shows that lentiviral transduction of primary NK cells in the presence of statin, BX795, retronectin, and vectofusin increased CAR stability on primary NK cells. Figure 5D specifically shows the expression of the antigen-3 CAR on primary NK cells 14 days after lentiviral transduction of primary NK cells under different conditions. [Figure 6] 1 shows that lentiviral transduction of primary αβ T cells in the presence of statin, BX795, retronectin, and vectofusin increases CAR expression. [Figure 7] Enhancer: % surviving primary γδ T cells after lentiviral transduction in the presence of statin, BX795, and vectofusin. LV: lentiviral vector, UTD: non-transduced. [Figure 8] Shown is the % of primary γδ T cells that were antigen-4 CAR positive after lentiviral transduction in the presence of statin, BX795, and vectofusin. [Figure 9A] Shown are the % of primary γδ T cells that were CAR positive after lentiviral transduction in the presence of atorvastatin (FIGS. 9A-9C). [Figure 9B] Shown are the % of primary γδ T cells that were CAR positive after lentiviral transduction in the presence of atorvastatin (FIGS. 9A-9C). [Figure 9C] Shown are the % of primary γδ T cells that were CAR positive after lentiviral transduction in the presence of atorvastatin (FIGS. 9A-9C). [Figure 9D] Shown are the % of primary γδ T cells that were CAR positive after lentiviral transduction in the presence of rosuvastatin (FIGS. 9D-9F). [Figure 9E] Shown are the % of primary γδ T cells that were CAR positive after lentiviral transduction in the presence of rosuvastatin (FIGS. 9D-9F). [Figure 9F] Shown are the % of primary γδ T cells that were CAR positive after lentiviral transduction in the presence of rosuvastatin (FIGS. 9D-9F). [Figure 10A] Shown is LDLR expression, presented as geometric mean, after lentiviral transduction in the presence of atorvastatin (FIG. 10A) at the concentrations (μM) indicated on the X-axis. [Figure 10B] LDLR expression, shown as geometric mean, is shown after lentiviral transduction in the presence of rosuvastatin (FIG. 10B) at the concentrations (μM) indicated on the X-axis. [Figure 11A]Shows the frequency of the Vγ9Vδ2 subset of primary γδ T cells after lentiviral transduction in the presence of the statin atorvastatin (FIG. 11A) at the concentrations (μM) indicated on the X-axis. [Figure 11B] Shown is the frequency of the Vγ9Vδ2 subset of primary γδ T cells after lentiviral transduction in the presence of the statin rosuvastatin (FIG. 11B) at the concentrations (μM) indicated on the X-axis. Detailed Description of the Invention

[0058] Adoptive transfer of immune cells genetically modified to recognize malignant tumor-associated antigens is a promising new approach to treating cancer (see, e.g., Brenner et al., Current Opinion in Immunology, 22(2):251-257 (2010); Rosenberg et al., Nature Reviews Cancer, 8(4):299-308 (2008)).

[0059] Genetically engineered T cells expressing chimeric antigen receptors (CARs) have emerged as one of the most powerful cancer treatments. While the therapeutic outcomes of CAR-T therapy are unprecedented, this therapy is significantly limited by its autologous characteristics. For example, T cells cannot be harvested from every patient, and the quality of T cells may not meet manufacturing standards. In addition, conventional CAR-T therapies have inherent manufacturing issues, such as production failures, time delays, insufficient cell expansion, or heterogeneous product yields, which can be harmful to recipient patients. Such highly personalized treatments also inevitably come at a high cost.

[0060] Natural killer (NK) cells are part of the innate immune system and act as the first line of defense against cancer and viral infections. NK cells have attracted increasing attention as a promising alternative effector cell type for CAR engineering due to their rapid cytotoxicity against tumors, their safety profile (low cytokine storm, minimal graft-versus-host disease, and low immune effector cell-associated neurotoxicity syndrome), and their potential use as off-the-shelf cell therapy. However, because NK cells have been evolutionarily selected for resistance to viral infections, optimizing highly efficient and clinically applicable gene transfer protocols for NK cells has been challenging. Therefore, there is a need in the art for improved gene transfer protocols for NK cell-based immunotherapy.

[0061] The present disclosure is based in part on novel methods or processes for preparing immune effector cells (e.g., NK cells) for transduction and their use to perform cell therapy to treat diseases or disorders.

[0062] 5.1.Definition The techniques and procedures described or referenced herein include those generally well understood and / or commonly employed by those skilled in the art using conventional techniques, such as the widely used methods described in Sambrook et al: A Laboratory Manual (3rd ed. 2001), Current Protocols in Molecular Biology (Ausubel et al. eds., 2003), Therapeutic Monoclonal Antibodies: From Bench To Clinic (An ed. 2009), Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010), and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds., 2nd ed. 2010).

[0063] Unless otherwise defined herein, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. For purposes of interpreting this specification, the following explanations of terms shall apply, and where appropriate, terms used in the singular shall also include the plural and vice versa. In the event that any explanation of a term provided conflicts with any document incorporated herein by reference, the explanation of the term provided below shall prevail.

[0064] As used herein, the term "immune effector cell" refers to any of various types of cells that actively respond to stimuli and effect some change to defend the body in an immune response.

[0065] As used herein, the term "cytotoxic cell" or "cytotoxic lymphocyte" refers to a specific subtype of immune effector cell that kills their cognate infected or transformed targets via the cytotoxic granule exocytosis pathway. In some embodiments, the cytotoxic cell is a natural killer cell (NK cell). In some embodiments, the cytotoxic cell is a cytotoxic T lymphocyte (CTL). In some embodiments, the cytotoxic cell is an alpha-beta (αβ) T cell. In some embodiments, the cytotoxic cell is a gamma-delta (γδ) T cell.

[0066] As used herein, the term "natural killer cell" or "NK cell" refers to a type of cytotoxic cell that is important to the innate immune system. NK cells have the ability to recognize and kill stressed cells in the absence of antibodies and MHC, allowing for a much faster immune response.

[0067] As used herein, the term "alpha-beta (αβ) T cells" refers to a subset of T lymphocytes in peripheral blood that express the αβ T cell receptor (TCR).

[0068] As used herein, the term "gamma-delta (γδ) T cells" refers to a subset of T lymphocytes in peripheral blood that express the γδ T cell receptor (TCR). In some embodiments, the γδ T cells comprise the Vγ9Vδ2 subset of γδ T cells.

[0069] As used herein, the term "cytotoxic T lymphocyte" or "CTL" refers to a type of cytotoxic cell that resides in the cytosol or adjacent nuclear compartment and destroys virus-infected cells, tumor cells, and tissue transplants. CTLs are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface and associate with MHC class I molecules.

[0070] The term "low-density lipoprotein receptor" or "LDLR" refers to a class of cell surface proteins involved in receptor-mediated endocytosis of specific ligands. LDLR binds to particles called low-density lipoproteins (LDL), which are the major carriers of cholesterol in the blood.

[0071] The term "pseudotyping" refers to producing a virus or viral vector by combining it with a foreign viral envelope protein. The result is a pseudotyped virus that contains envelope proteins from a different virus.

[0072] The term "vesicular stomatitis virus G" or "VSV-G" refers to the viral fusion protein of vesicular stomatitis virus (VSV). Viral fusion proteins are essential for enveloped virus infection. VSV-G mediates the fusion of the viral envelope with the host cell membrane, allowing the viral genome to be released into the host cell.

[0073] The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and are used in the broadest sense, specifically encompassing, for example, polyclonal or monoclonal antibodies (including agonist, antagonist, neutralizing, and full-length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, monovalent antibodies, polyvalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single-chain antibodies, and fragments thereof, as described below. Antibodies may be human, humanized, chimeric, and / or affinity matured, as well as antibodies from other species, e.g., mouse, rabbit, llama, etc. The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides that are capable of binding to a specific molecular antigen and are composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids, and the carboxy-terminal portion of each chain containing a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2nd ed. 1995) and Kuby, Immunology (3rd ed. 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies such as those derived from Camelidae species (e.g., llamas and alpacas) or humanized variants thereof, intracellular antibodies, anti-idiotypic (anti-Id) antibodies, and functional fragments of any of the above (e.g., antigen-binding fragments), and refer to portions of antibody heavy or light chain polypeptides that retain some or all of the binding activity of the antibody from which the fragment is derived.Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFv) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen-binding domains or molecules containing an antigen-binding site that binds to an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989), Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995), Huston et al., 1993, Cell Biophysics 22:189-224, Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515, and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules. The antibody can be an agonist antibody or an antagonist antibody. An antibody may be neither an agonist nor an antagonist.

[0074] An "antigen" is a structure to which an antibody can selectively bind. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen is associated with a cell, e.g., present on or within a cell.

[0075] An "intact" antibody is one that contains an antigen-binding site as well as a CL and at least the heavy chain constant regions CH1, CH2, and CH3. The constant region may include a human constant region or a variant thereof. In certain embodiments, an intact antibody has one or more effector functions.

[0076] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising a VH antibody domain and a VL antibody domain connected in a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a description of sFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0077] The term "binding" or "binding" refers to interactions between molecules, including, for example, forming a complex. The interaction can be a non-covalent interaction, including, for example, hydrogen bonding, ionic bonding, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the overall non-covalent interactions between a single antigen-binding site of an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope. The ratio of the dissociation rate (koff) to the association rate (kon) of a binding molecule (e.g., an antibody) for a monovalent antigen (koff / kon) is the dissociation constant KD, which is inversely related to affinity. The lower the KD value, the higher the affinity of the antibody. The value of KD varies for different antibody-antigen complexes and depends on both kon and koff. The dissociation constant KD of the antibodies provided herein can be determined using any of the methods provided herein or any other method known to those of skill in the art. Affinity at one binding site does not necessarily reflect the true strength of the interaction between an antibody and an antigen. When a complex antigen containing multiple repeating antigenic determinants, such as a multivalent antigen, comes into contact with an antibody containing multiple binding sites, the interaction of the antibody with the antigen at one site will increase the probability of reaction at a second site. The strength of multiple interactions between such a multivalent antibody and an antigen is called avidity.

[0078] In the context of the binding molecules described herein, terms such as "binds to," "specifically binds to," and similar terms are also used interchangeably herein to refer to binding molecules of an antigen-binding domain that specifically binds to an antigen, such as a polypeptide. Binding molecules or antigen-binding domains that bind to or specifically bind to an antigen can be identified, for example, by immunoassays, Octet®, Biacore®, or other techniques known to those of skill in the art. In some embodiments, a binding molecule or antigen-binding domain binds to or specifically binds to an antigen if it binds to the antigen with higher affinity than any cross-reactive antigens as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective response is at least two times the background signal or noise, and may exceed ten times the background signal or noise. For a discussion of binding specificity, see, e.g., Fundamental Immunology 332-36 (Paul, ed., 2d ed. 1989). In certain embodiments, the extent of binding of a binding molecule or antigen-binding domain to a "non-target" protein is less than about 10% of the binding of the binding molecule or antigen-binding domain to its particular target antigen, as determined, for example, by FACS analysis or RIA. Binding molecules or antigen-binding domains that bind to an antigen include those that can bind the antigen with sufficient affinity so that the binding molecule is useful, for example, as an antigen-targeted therapeutic and / or diagnostic agent. In certain embodiments, a binding molecule or antigen-binding domain that binds to an antigen has a dissociation constant (KD) of 1 μM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM or less. In certain embodiments, the binding molecule or antigen-binding domain binds to an epitope of an antigen that is conserved among antigens of different species.

[0079] In certain embodiments, binding molecules or antigen-binding domains may comprise "chimeric" sequences in which a portion of the heavy and / or light chain is identical to or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567 and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55). Chimeric sequences may include humanized sequences.

[0080] In certain embodiments, a binding molecule or antigen-binding domain may comprise a portion of a "humanized" form of a non-human (e.g., camelid, murine, non-human primate) antibody containing sequences from a human immunoglobulin (e.g., recipient antibody) in which native CDR residues are replaced by residues from a corresponding CDR of a non-human species (e.g., donor antibody) such as camel, mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some cases, one or more FR region residues of the human immunoglobulin sequence are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. The heavy or light chain of a humanized antibody can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, a humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-25 (1986); Riechmann et al., Nature 332:323-29 (1988); Presta, Curr. Op. Struct. Biol., 2:593-96 (1992); Carter, et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); U.S. Patent Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213, and 6,054,297.

[0081] In certain embodiments, a binding molecule or antigen-binding domain can comprise a portion of a "fully human antibody" or "human antibody," which terms are used interchangeably herein and refer to antibodies comprising a human variable region and, for example, a human constant region. A binding molecule can comprise a single-domain antibody sequence. In specific embodiments, these terms refer to antibodies comprising variable and constant regions of human origin. A "fully human" antibody can also encompass, in certain embodiments, antibodies that bind to a polypeptide and are encoded by nucleic acid sequences that are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequences. The term "fully human antibody" includes antibodies having variable and constant regions that correspond to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and / or has been produced using any of the techniques for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)) and yeast display libraries (Chao, et al., Nature Protocols, 1:755-68 (2006)).Furthermore, human monoclonal antibodies can be prepared using the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985), Boerner et al., J. Immunol. 147(1):86-95 (1991), and van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., mice, that have been modified to produce such antibodies in response to antigen challenge, but whose endogenous gene loci have been disabled (see, e.g., Jakobovits, Curr. Opin. Biotechnol. 6(5):561-66 (1995); Bruggemann and Taussing, Curr. Opin. Biotechnol. 8(4):455-58 (1997); and U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, e.g., Li, et al., Proc. Natl. Acad. Sci. USA 103:3557-62 (2006) (regarding human antibodies generated by human B-cell hybridoma technology).

[0082] In certain embodiments, a binding molecule or antigen-binding domain may comprise a portion of a "recombinant human antibody," a phrase that includes human antibodies prepared, expressed, created, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from animals (e.g., mice or cows) that are transgenic and / or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, LD, et al., Nucl. Acids Res. 20:6287-6295 (1992)), or antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally exist within the human antibody germline repertoire in vivo.

[0083] In certain embodiments, a binding molecule or antigen-binding domain can comprise a portion of a "monoclonal antibody," a term used herein to refer to an antibody obtained from a population of substantially homogeneous antibodies, where the individual antibodies comprising the population are identical except for minor naturally occurring mutations and well-known post-translational modifications, such as amino acid isomerization and deamidation, oxidation of methionine, and deamidation of asparagine and glutamine, and each monoclonal antibody typically recognizes a single epitope on an antigen. In specific embodiments, a "monoclonal antibody," as used herein, is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to a particular method for producing the antibody. For example, monoclonal antibodies useful in the present disclosure can be prepared by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or can be made using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222:581-97 (1991). Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art. See, for example, Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002).

[0084] A typical four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). In the case of IgG, the four-chain unit generally has a size of approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus followed by three constant domains (CH) for the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus followed by a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. Particular amino acid residues are thought to form an interface between the light and heavy chain variable domains. The pairing of VH and VL together forms a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994) and Immunobiology (Janeway et al. eds., 5th ed. 2001).

[0085] The term "Fab" or "Fab region" refers to the region of an antibody that binds to an antigen. Conventional IgGs typically contain two Fab regions, each located in one of the two arms of the Y-shaped IgG structure. Each Fab region typically consists of one variable region and one constant region from each of the heavy and light chains. More specifically, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are the VL and CL regions. The VH, CH1, VL, and CL regions in the Fab region can be arranged in various ways to confer antigen-binding capability according to the present disclosure. For example, the VH and CH1 regions can be on one polypeptide, while the VL and CL regions can be on separate polypeptides, as in the Fab region of a conventional IgG. Alternatively, the VH, CH1, VL, and CL regions can all be present on the same polypeptide and oriented in a different order, as described in more detail in the following sections.

[0086] The terms "variable region," "variable domain," "V region," or "V domain" refer to a portion of an antibody's light or heavy chain, located generally at the amino terminus of the light or heavy chain, approximately 120-130 amino acids in length for heavy chains and approximately 100-110 amino acids in length for light chains, that is used to determine the binding and specificity of each particular antibody to its particular antigen. The variable region of a heavy chain may be referred to as "VH." The variable region of a light chain may be referred to as "VL." The term "variable" refers to the fact that certain segments of the variable region vary significantly in sequence among antibodies. The V region mediates antigen binding and determines the specificity of a particular antibody for a particular antigen. However, the variability is not uniform across the 110-amino acid span of the variable region. Instead, the V region consists of less variable (e.g., relatively invariant) stretches of approximately 15-30 amino acids called framework regions (FRs), separated by shorter regions of greater variability (e.g., extreme variability) called "hypervariable regions," each approximately 9-12 amino acids in length. The heavy and light chain variable regions each contain four FRs, mostly in a β-sheet structure, connected by three hypervariable regions, which form loops connecting the β-sheet structure and, in some cases, form part of the β-sheet structure. The hypervariable regions within each chain are held in close proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant regions are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The sequences of the variable regions vary significantly among different antibodies. In a specific embodiment, the variable regions are human variable regions.

[0087] The terms "Kabat variable region residue numbering" or "amino acid position numbering as in Kabat," and variations thereof, refer to the numbering system used in the heavy or light chain variable regions of the antibody compilations of Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening of, or insertion into, the FRs or CDRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by aligning the sequence of that antibody with the "standard" Kabat numbering sequence at the region of homology. The Kabat numbering system is generally used when referring to residues of the variable domain (e.g., residues 1-107 for the light chain and residues 1-113 for the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is generally used when referring to residues within the heavy chain constant region of an immunoglobulin (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0088] The term "heavy chain" when used with respect to antibodies refers to a polypeptide chain of approximately 50 to 70 kDa, the amino-terminal portion of which contains a variable region of approximately 120 to 130 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The constant region can be one of five distinct types (e.g., isotypes), designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains vary in size, with α, δ, and γ containing approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with light chains, these distinct types of heavy chains give rise to five well-known classes (e.g., isotypes) of antibodies: IgA, IgD, IgE, IgG, and IgM (including the four subclasses of IgG, i.e., IgG1, IgG2, IgG3, and IgG4).

[0089] The term "light chain" when used with respect to antibodies refers to a polypeptide chain of approximately 25 kDa, the amino-terminal portion of which contains a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two different types, called kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain.

[0090] As used herein, the terms "hypervariable region," "HVR," "complementarity-determining region," and "CDR" are used interchangeably. "CDR" refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of an immunoglobulin (Ig or antibody) VH β-sheet framework, or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of an antibody VL β-sheet framework. Thus, CDRs are variable region sequences interspersed within framework region sequences.

[0091] CDR regions are well known to those skilled in the art and are defined by well-known numbering systems. For example, Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra). "Chothia" instead refers to the position of the structural loop (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies from H32 to H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34). AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Dubel, eds., 2nd ed. 2010)). The "contact" hypervariable regions are based on an analysis of available complex crystal structures. Another universal numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System® (Lafranc, et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is an integrated information system dedicated to immunoglobulins (IGs), T cell receptors (TCRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. CDRs are referred to herein in terms of both amino acid sequence and location within a light or heavy chain.Because the "location" of CDRs within the structure of immunoglobulin variable domains is conserved among species and resides in structures called loops, CDR and framework residues are readily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to graft and replace CDR residues from one species of immunoglobulin into an acceptor framework, typically from a human antibody. An additional numbering system (AHon) was developed by Honegger and Pluckthun, J. Mol. Biol. 309:657-70 (2001). The correspondence between numbering systems, including, for example, Kabat numbering and the IMGT-specific numbering system, is well known to those skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). Residues from each of these hypervariable regions or CDRs are illustrated in Table 1 below.

[0092] [Table 1]

[0093] The boundaries of a given CDR may vary depending on the scheme used for identification. Thus, unless otherwise specified, the terms "CDR" and "complementarity-determining region" of a given antibody or region thereof, such as a variable region, and individual CDRs of an antibody or region thereof (e.g., CDR-H1, CDR-H2) should be understood to encompass the complementarity-determining regions defined by any of the known schemes described hereinabove. In some cases, a scheme for identifying a particular CDR or CDRs is specified, such as CDRs defined by the IMGT, Kabat, Chothia, or Contact methods. In other cases, specific amino acid sequences of the CDRs are included. Note that CDR regions can also be defined by a combination of various numbering systems, for example, a combination of the Kabat numbering system and the Chothia numbering system, or a combination of the Kabat numbering system and the IMGT numbering system. Thus, terms such as "CDRs represented in a particular VH" include any CDR1 defined by a system, including, but not limited to, the exemplary CDR numbering systems described above. Given a variable region (eg, VH or VL), one of skill in the art will understand that the CDRs within the region may be defined by different numbering systems or combinations thereof.

[0094] The hypervariable regions may include "extended hypervariable regions" such as: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH.

[0095] The term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains that are not directly involved in binding the antibody to an antigen, but which exhibit various effector functions, such as interaction with Fc receptors. This term refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portion of the immunoglobulin, the variable region, which contains the antigen-binding site. The constant region can include the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0096] The term "framework" or "FR" refers to variable domain residues that flank the CDRs. FR residues are present, for example, in chimeric, humanized, and human antibodies, domain antibodies (e.g., single-domain antibodies), diabodies, linear antibodies, and bispecific antibodies. FR residues are variable domain residues other than hypervariable region or CDR residues.

[0097] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native-sequence Fc regions, recombinant Fc regions, and variant Fc regions. While the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is often defined to stretch from the amino acid residue at position Cys226, or from the amino acid residue at position Pro230, to the carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, an intact antibody composition can include an antibody population in which all K447 residues have been removed, an antibody population in which the K447 residue has not been removed, and an antibody population having a mixture of antibodies with and without the K447 residue. A "functional Fc region" possesses the "effector functions" of a native-sequence Fc region. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require that the Fc region be combined with a binding region or domain (e.g., an antibody variable region or domain) and can be assessed using a variety of assays known to those of skill in the art. A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, the variant Fc region comprises at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., from about 1 to about 10 amino acid substitutions, or from about 1 to about 5 amino acid substitutions in the native-sequence Fc region or the Fc region of a parent polypeptide. The variant Fc region herein may have at least about 80% homology to a native sequence Fc region and / or the Fc region of a parent polypeptide, or at least about 90% homology thereto, for example, at least about 95% homology thereto.

[0098] As used herein, "epitope" is a term of art that refers to a localized region of an antigen to which a binding molecule (e.g., an antibody comprising a single domain antibody sequence) can specifically bind. An epitope can be a linear or conformational epitope, a non-linear epitope, or a discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be consecutive amino acids of a polypeptide (a "linear" epitope), or an epitope can include amino acids from two or more non-contiguous regions of a polypeptide (a "conformational," "non-linear," or "discontinuous" epitope). In general, it will be understood by those skilled in the art that a linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a group of amino acids regardless of whether the amino acids are folded into the native three-dimensional protein structure. In other embodiments, the binding molecule requires that the amino acid residues that make up the epitope exhibit a particular conformation (eg, a bend, twist, turn, or fold) in order to recognize and bind to the epitope.

[0099] "Percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a particular peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0100] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to graft one or more antigen specificities onto immune effector cells, such as T cells or NK cells. In some embodiments, a CAR comprises an extracellular antigen-binding domain specific for one or more antigens (such as tumor antigens), a transmembrane domain, and an intracellular signaling domain of an NK cell and / or other receptor. "CAR-NK cell" refers to an NK cell that expresses a CAR.

[0101] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The term also refers to amino acid polymers that are naturally modified or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. The definition also includes polypeptides containing one or more analogs of an amino acid, including, but not limited to, unnatural amino acids, as well as other modifications known in the art. Because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in certain embodiments, a "polypeptide" can occur as a single chain or as two or more related chains.

[0102] "Polynucleotide" or "nucleic acid," as used interchangeably herein, refer to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. As used herein, "oligonucleotide" refers to a short, generally single-stranded, synthetic polynucleotide, generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally fully applicable to oligonucleotides. Cells producing the binding molecules of the present disclosure can include parent hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acid encoding the antibody has been introduced. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end. The left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction. The region of the sequence on the DNA strand that has the same sequence as the RNA transcript at the 5' to 5' end of the RNA transcript is referred to as the "upstream sequence," and the region of the sequence on the DNA strand that has the same sequence as the RNA transcript at the 3' to 3' end of the RNA transcript is referred to as the "downstream sequence."

[0103] An "isolated nucleic acid" is a nucleic acid, e.g., RNA, DNA, or mixed nucleic acid, that is substantially separated from other genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, that are naturally associated with the native sequence. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. In specific embodiments, one or more nucleic acid molecules encoding a single domain antibody or an antibody described herein are isolated or purified. This term encompasses a nucleic acid sequence that has been removed from its naturally occurring environment, including recombinant or cloned DNA isolates and chemically synthesized analogs, or biologically synthesized analogs produced by heterologous systems. A substantially pure molecule can include isolated forms of the molecule. Specifically, an "isolated" nucleic acid molecule encoding a CAR described herein is a nucleic acid molecule that has been identified and isolated from at least one contaminant nucleic acid molecule with which it is normally associated in the production environment.

[0104] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0105] As used herein, the term "operably linked," and similar phrases (e.g., genetically fused), when used with reference to nucleic acids or amino acids, refers to an operable linkage in which nucleic acid or amino acid sequences, respectively, are placed in a functional relationship with each other. For example, operably linked promoters, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences result in the correct production of a nucleic acid molecule (e.g., RNA). In some embodiments, operably linked nucleic acid elements result in the transcription of an open reading frame and ultimately the production of a polypeptide (i.e., expression of the open reading frame). As another example, an operably linked peptide refers to functional regions positioned at an appropriate distance from each other to confer their intended function.

[0106] The term "vector" refers to a substance used to carry or contain a nucleic acid sequence, including, for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) described herein, to introduce the nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain operable selection sequences or markers capable of stable integration into a host cell chromosome. In addition, a vector may contain one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that may be included provide, for example, resistance to antibiotics or toxins, complement deficiencies in auxiliary auxotrophies, or supply critical nutrients absent from the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, as are well known in the art. When two or more nucleic acid molecules are coexpressed (e.g., both the heavy and light chains of an antibody, or antibody VH and VL), both nucleic acid molecules may be inserted, for example, into a single expression vector or into separate expression vectors. For expression in a single vector, the encoding nucleic acids can be operably linked to a common expression control sequence or to different expression control sequences, such as one inducible promoter and one constitutive promoter. Introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blot or amplification of mRNA by polymerase chain reaction (PCR), immunoblotting for expression of gene products, or other suitable analytical methods for testing expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that nucleic acid molecules will be expressed in amounts sufficient to produce the desired product, and will further understand that expression levels can be optimized to obtain sufficient expression using methods well known to those skilled in the art.

[0107] As used herein, the term "host" refers to an animal, such as a mammal (e.g., a human).

[0108] As used herein, the term "host cell" refers to a particular subject cell into which a nucleic acid molecule can be transfected, and to the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur during the subsequent generation or integration of the nucleic acid molecule into the host cell genome.

[0109] As used herein, the term "autologous" is meant to refer to any material derived from the same individual that is later reintroduced into the individual.

[0110] As used herein, the term "allogeneic" refers to a graft derived from a different individual of the same species.

[0111] The terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

[0112] As used herein, the term "isolation" or "isolating" refers to a process of increasing the percentage of a particular substance in a composition. For example, isolating a certain type of cell from a cell population refers to the process of creating a cell population in which the percentage of this type of cell is increased compared to the percentage of this type of cell in the original cell population. Thus, when used in the context of a certain type of cell, the term "isolated" does not mean that the isolated cell population contains 100% of this type of cell, but rather that the percentage of this type of cell in the cell population after the isolation process is increased.

[0113] As used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in animals, and more particularly for use in humans, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias.

[0114] The term "excipient" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, encapsulating material, etc. Excipients include, for example, encapsulating materials or additives such as absorption enhancers, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavorings, wetting agents, lubricants, fragrances, preservatives, propellants, release agents, bactericides, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or vehicle.

[0115] In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; proteins such as low molecular weight (e.g., less than about 10 amino acid residues) polypeptides, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990).

[0116] In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other components of the pharmaceutical formulation and suitable for use in contact with the tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit / risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005, Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and The American Pharmaceutical Association: 2009, Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007, Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, a pharmaceutically acceptable excipient is nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. In some embodiments, the pharmaceutically acceptable excipient is an aqueous pH buffered solution.

[0117] In some embodiments, the excipient may be a sterile liquid, such as water or oil, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, or sesame oil. Water is an exemplary excipient when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid excipients, particularly for injectable solutions. Excipients may also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerin, propylene, glycol, water, ethanol, or the like. If desired, the composition may further contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition may take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, or the like. Oral composition containing formulations can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0118] A composition containing a pharmaceutical compound can include, for example, a binding molecule (eg, an antibody) in isolated or purified form, together with a suitable amount of excipients.

[0119] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a single domain antibody or therapeutic molecule, including drugs and single domain antibodies or pharmaceutical compositions provided herein, that is sufficient to bring about a desired result.

[0120] The terms "subject" and "patient" can be used interchangeably herein. As used herein, in certain embodiments, a subject is a mammal, either non-primate or primate (e.g., human). In specific embodiments, a subject is a human. In one embodiment, a subject is a mammal, e.g., a human, diagnosed with a disease or disorder. In another embodiment, a subject is a mammal, e.g., a human, at risk of developing a disease or disorder.

[0121] "Administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance present outside the body to a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0122] As used herein, the terms "treatment" and "treatment / treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or condition resulting from the administration of one or more therapies. Treatment may be determined by assessing whether there has been a decrease, alleviation, and / or alleviation of one or more symptoms associated with the underlying disease, such that an improvement is observed in the patient, even though the patient may still be suffering from the underlying disease. The term "treating" includes both disease management and remission. The terms "manage," "managing," and "management" refer to the beneficial effects a subject derives from treatment, which does not necessarily result in a cure of the disease.

[0123] The terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of the occurrence (or recurrence) of a disease, disorder, condition, or associated symptom (e.g., diabetes or cancer).

[0124] The terms "about" and "approximately" refer to within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0125] As used in this disclosure and the claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0126] Whenever an embodiment is described herein with the term "comprising," it is understood that other similar embodiments otherwise described in terms of "consisting of" and / or "consisting essentially of" are also provided. Whenever an embodiment is described herein with the phrase "consisting essentially of," it is also understood that similar embodiments otherwise described in terms of "consisting of" are also provided.

[0127] The term "between" when used in phrases such as "between A and B" or "between A and B" refers to a range that includes both A and B.

[0128] The term "and / or" used herein in phrases such as "A and / or B" is intended to include both A and B, A or B, A alone, and B alone. Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A alone, B alone, and C alone.

[0129] 5.2. Methods for Preparing Immune Effector Cells for Transduction In one aspect, provided herein is a method for preparing immune effector cells for transduction.

[0130] In some embodiments, the immune effector cells are cytotoxic cells. In certain embodiments, the cytotoxic cells are natural killer cells. In certain embodiments, the cytotoxic cells are cytotoxic T lymphocytes. In certain embodiments, the immune effector cells are alpha beta T cells. In certain embodiments, the immune effector cells are gamma delta T cells.

[0131] In some embodiments, the immune effector cells are contacted with one or more agents that increase expression of the low density lipoprotein receptor (LDLR). In some embodiments, the immune effector cells are contacted with one or more first agents that increase expression of the low density lipoprotein receptor (LDLR). In some embodiments, the immune effector cells are contacted with one or more second agents that inhibit the intracellular antiviral defense mechanisms of the immune effector cells. In some embodiments, the immune effector cells are contacted with one or more third agents that enhance transduction.

[0132] In some embodiments, immune effector cells are contacted with one or more first agents that increase expression of low-density lipoprotein receptor (LDLR) and one or more second agents that inhibit the intracellular antiviral defense mechanisms of the immune effector cells. In certain embodiments, the immune effector cells are contacted with the first agent before contacting with the second agent. In certain embodiments, the immune effector cells are contacted with the first agent after contacting with the second agent. In certain embodiments, the immune effector cells are contacted with the first agent simultaneously with contacting with the second agent.

[0133] In some embodiments, immune effector cells are contacted with one or more first agents that increase expression of low-density lipoprotein receptor (LDLR) and one or more third agents that enhance transduction. In certain embodiments, immune effector cells are contacted with the first agent before contacting with the third agent. In certain embodiments, immune effector cells are contacted with the first agent after contacting with the third agent. In certain embodiments, immune effector cells are contacted with the first agent simultaneously with contacting with the third agent.

[0134] In some embodiments, the immune effector cells are contacted with one or more second agents that inhibit the immune effector cells' intracellular antiviral defense mechanisms and one or more third agents that enhance transduction. In certain embodiments, the immune effector cells are contacted with the second agent before contacting with the third agent. In certain embodiments, the immune effector cells are contacted with the second agent after contacting with the third agent. In certain embodiments, the immune effector cells are contacted with the second agent simultaneously with contacting with the third agent.

[0135] In some embodiments, immune effector cells are contacted with one or more first agents that increase expression of low-density lipoprotein receptor (LDLR), one or more second agents that inhibit the intracellular antiviral defense mechanisms of the immune effector cells, and one or more third agents that enhance transduction. In certain embodiments, immune effector cells are contacted with the first agent, then the second agent, then the third agent. In certain embodiments, immune effector cells are contacted with the first agent, then the third agent, then the second agent. In certain embodiments, immune effector cells are contacted with the second agent, then the first agent, then the third agent. In certain embodiments, immune effector cells are contacted with the second agent, then the third agent, then the first agent. In certain embodiments, immune effector cells are contacted with the third agent, then the first agent, then the second agent. In certain embodiments, immune effector cells are contacted with the third agent, then the first agent, then the second agent. In certain embodiments, immune effector cells are contacted with the third agent, then the first agent, then the second agent. In certain embodiments, immune effector cells are contacted with a first agent and then simultaneously with a second agent and a third agent. In certain embodiments, immune effector cells are contacted with a second agent and a third agent simultaneously, and then contacted with the first agent. In certain embodiments, immune effector cells are contacted with a second agent and then simultaneously with a first agent and a third agent. In certain embodiments, immune effector cells are contacted with a first agent and a third agent simultaneously, and then contacted with the second agent. In certain embodiments, immune effector cells are contacted with a third agent and then simultaneously with the first agent and the second agent. In certain embodiments, immune effector cells are contacted with a first agent and a second agent simultaneously, and then contacted with the third agent. In certain embodiments, immune effector cells are contacted with a first agent and a second agent simultaneously, and then contacted with the third agent. In certain embodiments, immune effector cells are contacted with a first agent, a second agent, and a third agent simultaneously.

[0136] In certain embodiments, the first agent is a small molecule. In certain embodiments, the first agent is a large molecule. In certain embodiments, the first agent is organic. In certain embodiments, the first agent is inorganic. In certain embodiments, the first agent is a statin. In certain embodiments, the statin is rosuvastatin. In certain embodiments, the statin is atorvastatin. In certain embodiments, the statin is fluvastatin. In certain embodiments, the statin is lovastatin. In certain embodiments, the statin is pitavastatin. In certain embodiments, the statin is pravastatin. In certain embodiments, the statin is simvastatin.

[0137] In certain embodiments, the second agent is an inhibitor of 3-phosphoinositide-dependent kinase 1 (PDK1). In certain embodiments, the PDK1 inhibitor is a small molecule. In certain embodiments, the PDK1 inhibitor is a large molecule. In certain embodiments, the PDK1 inhibitor is organic. In certain embodiments, the PDK1 inhibitor is inorganic. In certain embodiments, the PDK1 inhibitor is BX795.

[0138] In certain embodiments, the third agent is a transduction enhancer. In certain embodiments, the third agent is a small molecule. In certain embodiments, the third agent is a large molecule. In certain embodiments, the third agent is organic. In certain embodiments, the third agent is inorganic. In certain embodiments, the third agent is a vectofusin. In certain embodiments, the third agent is retronectin.

[0139] In certain embodiments, immune effector cells are contacted with a first agent that increases expression of low-density lipoprotein receptor (LDLR), a second agent that inhibits the immune effector cell's intracellular antiviral defense mechanism, and two third agents that enhance transduction. In certain embodiments, the first agent is rosuvastatin. In certain embodiments, the second agent is BX795. In certain embodiments, the third agent is vectofusin and retronectin.

[0140] In some embodiments, the method further comprises transducing the nucleic acid into immune effector cells. In certain embodiments, the nucleic acid is delivered via a viral particle. In certain embodiments, the nucleic acid is delivered via a lentiviral particle. In certain embodiments, the lentiviral particle is pseudotyped. In certain embodiments, the lentiviral particle is pseudotyped with vesicular stomatitis virus G (VSV-G).

[0141] In certain embodiments, the nucleic acid comprises a promoter. In certain embodiments, the promoter is selected from the group consisting of CAG, PGK, EF1a, and EFS promoters. In certain embodiments, the promoter is a CAG promoter. In certain embodiments, the promoter is a PGK promoter. In certain embodiments, the promoter is an EF1a promoter. In certain embodiments, the promoter is an EFS promoter.

[0142] In certain embodiments, the nucleic acid encodes an exogenous functional protein. In certain embodiments, the nucleic acid encodes an exogenous functional receptor. In certain embodiments, the exogenous functional receptor is a chimeric antigen receptor (CAR). In other embodiments, the exogenous functional receptor is a TCR.

[0143] 5.3. METHODS FOR PRODUCING IMMUNE EFFECTOR CELLS EXPRESSING EXOGENOUS FUNCTIONAL RECEPTORS In yet another aspect, provided herein is a method for producing immune effector cells expressing chimeric antigen receptors (CARs). Methods for preparing immune effector cells for transduction of CAR-encoding nucleic acids are described in detail in Section 4.2 above. In some embodiments, the immune effector cells are cytotoxic cells. In certain embodiments, the cytotoxic cells are natural killer cells. In certain embodiments, the cytotoxic cells are cytotoxic T lymphocytes.

[0144] 5.3.1. Chimeric Antigen Receptors In some embodiments, a CAR provided herein comprises a polypeptide comprising: (a) an extracellular antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0145] signal peptide In certain embodiments, the CARs provided herein may include a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide. Generally, a signal peptide is a peptide sequence that targets a polypeptide to a desired site within a cell. In some embodiments, the signal peptide targets the effector molecule to the cell's secretory pathway, allowing the effector molecule to be incorporated into and anchored to the lipid bilayer. Signal peptides, including signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences, that are suitable for use in the CARs described herein will be apparent to those of skill in the art. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain.

[0146] Extracellular antigen-binding domain The extracellular antigen-binding domain of the CAR described herein comprises one or more antigen-binding domains. In some embodiments, the extracellular antigen-binding domain of the CAR provided herein is monospecific. In other embodiments, the extracellular antigen-binding domain of the CAR provided herein is multispecific. In some embodiments, the extracellular antigen-binding domain is It comprises two or more antigen-binding domains that are fused directly to each other via a peptide bond or via a peptide linker.

[0147] In some embodiments, the extracellular antigen-binding domain comprises an antibody or a fragment thereof. For example, the binding domain can be derived from a monoclonal antibody (including agonist, antagonist, neutralizing, full-length or intact monoclonal antibodies), an antibody with polyepitopic or monoepitopic specificity, a polyclonal or univalent antibody, a multivalent antibody, a multispecific antibody formed from at least two intact antibodies (e.g., a bispecific antibody, so long as it exhibits the desired biological activity), a single-chain antibody, and fragments thereof (e.g., domain antibodies). The antibody can be human, humanized, chimeric, and / or affinity matured, and can be an antibody from another species, e.g., mouse, rabbit, llama, etc. In some embodiments, antibodies include polypeptide products of B cells within the immunoglobulin class of polypeptides that are capable of binding to a specific molecular antigen and are composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids, and the carboxy-terminal portion of each chain containing a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2nd ed. 1995) and Kuby, Immunology (3rd ed. 1997). The term "antibody" also includes, but is not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies derived from Camelidae species (e.g., llamas and alpacas) or their humanized variants, intracellular antibodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above. These fragments refer to portions of antibody heavy or light chain polypeptides that retain some or all of the binding activity of the antibody from which they are derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFvs) (including, e.g., monospecific and bispecific Fvs), Fab fragments, F(ab') fragments, F(ab') fragments, F(ab') fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies.In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen-binding domains or molecules containing an antigen-binding site (e.g., one or more CDRs of an antibody) that bind to an antigen. Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989), Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995), Huston et al., 1993, Cell Biophysics 22:189-224, Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515, and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule. The antibody can be an agonist antibody or an antagonist antibody. The antibody may be neither an agonist nor an antagonist.

[0148] In a specific embodiment, the extracellular antigen-binding domain of the CAR of the invention comprises a single-chain Fv (sFv or scFv). ScFv is an antibody fragment comprising a VH antibody domain and a VL antibody domain connected in a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. See Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0149] In another specific embodiment, the extracellular antigen-binding domain of a CAR of the invention comprises one or more single domain antibodies (sdAbs). The sdAbs may be of the same or different origins and may be of the same or different sizes. Exemplary sdAbs include heavy chain-only antibodies (e.g., VHH or VHH). NAR ), a binding molecule naturally lacking a light chain, a single domain (V) derived from a conventional four-chain antibody H or V L Such sdAbs include, but are not limited to, humanized heavy chain-only antibodies, human single domain antibodies produced by transgenic mice or rats expressing human heavy chain segments, and engineered domains and single domain scaffolds other than those derived from antibodies. Any sdAb known in the art or developed by the present disclosure, such as the single domain antibodies described above in this disclosure, can be used to construct the CARs described herein. sdAbs can be derived from any species, including, but not limited to, mouse, rat, human, camel, llama, lamprey, fish, shark, goat, rabbit, and cow. Single domain antibodies contemplated herein also include naturally occurring single domain antibody molecules from species other than camelids and sharks.

[0150] In some embodiments, sdAbs are derived from naturally occurring single domain antigen binding molecules known as heavy chain antibodies that are devoid of light chains (also referred to herein as "heavy chain-only antibodies"). Such single domain molecules are disclosed, for example, in WO 94 / 04678 and Hamers-Casterman, C. et al., Nature 363:446-448 (1993). For clarity, variable domains derived from heavy chain molecules that naturally lack light chains are similar to the conventional V of four-chain immunoglobulins. HThese VHH molecules are known herein as VHHs to distinguish them from other VHH molecules. Such VHH molecules may be derived from antibodies raised in Camelidae species, such as camel, llama, vicuna, dromedary, alpaca, and guanaco. Other non-Camelidae species can produce heavy chain molecules that naturally lack light chains, and such VHHs are within the scope of the present disclosure. Additionally, humanized versions and other modifications and variants of VHHs are contemplated and within the scope of the present disclosure. In some embodiments, sdAbs are derived from the variable regions of immunoglobulins found in cartilaginous fish. For example, sdAbs may be derived from an immunoglobulin isotype known as the Novel Antigen Receptor (NAR), which is found in the serum of sharks. Methods for producing single domain molecules derived from the variable regions of NARs ("IgNARs") are described in WO 03 / 014161 and Streltsov, Protein Sci. 14:2901-2909 (2005).

[0151] In some embodiments, naturally occurring VHH domains against a particular antigen or target can be obtained from a library (naive or immune) of Camelid VHH sequences. Such methods may or may not involve screening such a library with the antigen or target, or at least one part, fragment, antigenic determinant, or epitope thereof, using one or more screening techniques known in the art. Such libraries and techniques are described, for example, in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020, and WO 03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from (naive or immune) VHH libraries can be used, such as VHH libraries obtained from (naive or immune) VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as described, for example, in WO 00 / 43507.

[0152] In some embodiments, sdAbs are recombinant, CDR-grafted, humanized, camelized, deimmunized, and / or generated in vitro (e.g., selected by phage display). In some embodiments, the amino acid sequence of the framework regions may be altered by "camelization" of specific amino acid residues within the framework regions. Camelization refers to the replacement or substitution of one or more amino acid residues in the amino acid sequence of a (naturally occurring) VH domain from a conventional four-chain antibody by one or more amino acid residues that occur at the corresponding positions in the VHH domain of a heavy-chain antibody. This can be done by methods known in the art, as will be apparent to those skilled in the art. Such "camelizing" substitutions are preferably inserted at amino acid positions that form and / or are present at the VH-VL interface and / or the so-called camelid hallmark residues, as defined herein (see, e.g., WO 94 / 04678; Davies and Riechmann FEBS Letters 339:285-290 (1994); Davies and Riechmann, Protein Engineering 9(6):531-537 (1996); Riechmann, J. Mol. Biol. 259:957-969 (1996); and Riechmann and Muyldermans, J. Immunol. Meth. 231:25-38 (1999)).

[0153] In some embodiments, the sdAb is a human single domain antibody produced by transgenic mice or rats expressing human heavy chain segments. See, e.g., U.S. Patent Application Publication No. 20090307787, U.S. Patent No. 8,754,287, U.S. Patent Application Publication Nos. 20150289489, 20100122358, and WO 2004049794.

[0154] In some embodiments, single domain antibodies are generated from traditional four-chain antibodies (see, e.g., EP 0 368 684, Ward et al., Nature, 341(6242):544-6 (1989), Holt et al., Trends Biotechnol., 21(11):484-490 (2003), WO 06 / 030220, and WO 06 / 003388).

[0155] In some embodiments, the extracellular antigen-binding domain comprises a humanized antibody or fragment thereof. A humanized antibody may comprise human framework regions and human constant region sequences.

[0156] Humanized antibodies can be produced by a variety of techniques, including CDR grafting (EP 239,400, WO 91 / 09967, and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering, or resurfacing (EP 592,106, EP 519,596, Padlan, 1991, Molecular Immunology 28(4 / 5):489-498, Studnicka et al., 1994, Protein Engineering 7(6):805-814, and Roguska et al., 1994, PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and, for example, U.S. Pat. Nos. 6,407,213, 5,766,886, WO 93 / 17105, Tan et al., J. Immunol. 169:1119 25 (2002), Caldas et al., Protein Eng. 13(5):353-60 (2000), Morea et al., Methods 20(3):267 79 (2000), Baca et al., J. Biol. Chem. 272(16):10678-84 (1997), Roguska et al., Protein Eng. 9(10):895 904 (1996), Couto et al., Cancer Res. 55(23 Supp):5973s-5977s (1995), Couto et al., Cancer Res. 55(8):1717-22 (1995), Sandhu JS, Gene 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol. 235(3):959-73 (1994). See also U.S. Patent Application Publication No. 2005 / 0042664(A1) (February 24, 2005), each of which is incorporated herein by reference in its entirety.

[0157] Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, and are typically taken from an "import" variable domain. Humanization can be performed, for example, by substituting hypervariable region sequences for the corresponding sequences of a human antibody according to the methods of Jones et al., 1986, Nature 321:522-25; Riechmann, et al., Nature, 1988, 332:323-27; and Verhoeyen, et al., Science, 1988, 239:1534-36).

[0158] In some cases, humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six CDRs of a parent non-human antibody (e.g., a rodent) are grafted onto a human antibody framework. For example, Padlan et al. determined that only about one-third of the CDR residues actually contact the antigen, and called these "specificity-determining residues" or SDRs (Padlan et al., 1995, FASEB J. 9:133-39). In the SDR grafting technique, only the SDR residues are grafted onto a human antibody framework (see, for example, Kashmiri et al., 2005, Methods 36:25-34).

[0159] The selection of human variable domains, both light and heavy, used to create a humanized antibody can be important to reduce antigenicity. For example, according to the so-called "best-fit" method, the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent human sequence can be selected as the human framework for the humanized antibody (Sims et al., 1993, J. Immunol. 151:2296-308, and Chothia et al., 1987, J. Mol. Biol. 196:901-17). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89, and Presta et al., 1993, J. Immunol. 151:2623-32). In some cases, the framework is derived from the consensus sequences of the most abundant human subclasses, VL6 subgroup I (VL6I) and VH subgroup III (VHIII). In other methods, human germline genes are used as the source of the framework regions.

[0160] In an alternative paradigm based on CDR comparison, called superhumanization, the homology of FRs is irrelevant. This method involves comparing non-human sequences with a functional human germline gene repertoire. Genes encoding canonical structures identical to or closely related to the mouse sequence are then selected. Next, among genes that share canonical structures with non-human antibodies, the gene with the highest homology within the CDR is selected as the FR donor. Finally, non-human CDRs are grafted onto these FRs (see, for example, Tan et al., 2002, J. Immunol. 169:1119-25).

[0161] Furthermore, it is generally desirable for antibodies to be humanized with retention of affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformations of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, 2000, Protein Eng. 13:819-24), Modeller (Sali and Blundell, 1993, J. Mol. Biol. 234:779-815), and Swiss PDB Viewer (Guex and Peitsch, 1997, Electrophoresis 18:2714-23). Inspection of these displays permits analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, for example, analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is achieved. In general, the hypervariable region residues are directly and most substantially involved in antigen binding.

[0162] Another method for antibody humanization is based on a metric for antibody humanity called Human String Content (HSC). This method compares the mouse sequence with the human germline gene repertoire and scores the differences as HSC. The target sequence is then humanized by maximizing its HSC, rather than using an overall identity measure to generate diverse humanized variants (Lazar et al., 2007, Mol. Immunol. 44:1986-98).

[0163] In addition to the methods described above, empirical methods can be used to generate and select humanized antibodies. These methods involve generating large libraries of humanized variants and selecting the best clones using enrichment or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosomal, and yeast display libraries, as well as by bacterial colony screening (see, e.g., Hoogenboom, 2005, Nat. Biotechnol. 23:1105-16; Dufner et al., 2006, Trends Biotechnol. 24:523-29; Feldhaus et al., 2003, Nat. Biotechnol. 21:163-70; and Schlapschy et al., 2004, Protein Eng. Des. Sel. 17:847-60).

[0164] In the FR library approach, multiple residue variants are introduced at specific positions in the FR, and the library is then screened to select the FR that best supports the grafted CDR. The substituted residues can include some or all of the "vernier" residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, 1992, J. Mol. Biol. 224:487-99), or those from the more limited set of target residues identified by Baca et al. (1997, J. Biol. Chem. 272:10678-84).

[0165] In FR shuffling, entire FRs are combined with non-human CDRs rather than creating a combinatorial library of selected residue variants (see, e.g., Dall'Acqua et al., Methods, 2005, 36:43-60). The library can be screened for binding in a two-step process, first humanizing the VL, followed by humanizing the VH. Alternatively, a one-step FR shuffling process can be used. Such a process has been shown to be more efficient than two-step screening, as the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., 2007, Mol. Immunol. 44:3049-60).

[0166] The "humaneering" method is based on the experimental identification of essential minimum specificity determinants (MSDs) and the sequential substitution of non-human fragments into a human FR library and evaluation of binding. Humaneering begins with the CDR3 regions of the non-human VH and VL chains, and gradually substitutes other regions of the non-human antibody, including CDR1 and CDR2 of both VH and VL, with human FRs. This approach typically identifies multiple subclasses of antibodies that retain the epitope but have distinct CDRs in the human V segments. Humaneering allows the isolation of antibodies that are 91-96% homologous to human germline antibodies (see, e.g., Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007).

[0167] "Human engineering" methods involve modifying non-human antibodies or antibody fragments, such as murine or chimeric antibodies or antibody fragments, by making specific changes to the antibody's amino acid sequence to generate modified antibodies that reduce immunogenicity in humans while retaining the desired binding characteristics of the original non-human antibody. Generally, the techniques involve classifying amino acid residues in non-human (e.g., murine) antibodies as "low risk," "moderate risk," or "high risk" residues. Classification is performed using a global risk / benefit calculation that assesses the predicted benefit (e.g., for immunogenicity in humans) of making a particular substitution against the risk that the substitution will affect the folding of the resulting antibody. Specific human amino acid residues to be substituted at a given position (e.g., low risk or moderate risk) in a non-human (e.g., murine) antibody sequence can be selected by aligning amino acid sequences from the variable regions of the non-human antibody with the corresponding regions of specific or consensus human antibody sequences. Amino acid residues at low or moderate risk positions in the non-human sequence can be substituted with the corresponding residue in the human antibody sequence depending on the alignment. Techniques for producing human engineered proteins are described in detail in Studnicka et al., 1994, Protein Engineering 7:805-14, U.S. Patent Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619, and WO 93 / 11794.

[0168] Composite human antibodies can be produced, for example, using Composite Human Antibody™ technology (Antitope Ltd., Cambridge, United Kingdom). To produce composite human antibodies, fragments of multiple human antibody variable region sequences are engineered to avoid T-cell epitopes, thereby minimizing the immunogenicity of the resulting antibody. Such antibodies may contain human constant region sequences, such as a human light chain constant region and / or a human heavy chain constant region.

[0169] A deimmunized antibody is an antibody from which T cell epitopes have been removed. Methods for producing deimmunized antibodies have been described. See, for example, Jones et al., Methods Mol Biol. 2009;525:405-23, xiv, and De Groot et al., Cell. Immunol. 244:148-153 (2006). Deimmunized antibodies contain a T cell epitope-depleted variable region and a human constant region. Briefly, the VH and VL of an antibody are cloned, and then T cell epitopes are identified by examining overlapping peptides derived from the VH and VL of the antibody in a T cell proliferation assay. T cell epitopes are identified by in silico methods to identify peptides that bind to human MHC class II. Mutations are introduced into the VH and VL to abolish binding to human MHC class II. The VH and VL are then used to generate deimmunized antibodies.

[0170] In certain embodiments, the extracellular antigen-binding domain comprises multiple binding domains. In some embodiments, the extracellular antigen-binding domain comprises a multispecific antibody or fragment thereof. In other embodiments, the extracellular antigen-binding domain comprises a multivalent antibody or fragment thereof. The term "specificity" refers to the antigen-binding protein's selective recognition of a specific epitope of an antigen. As used herein, the term "multispecific" denotes that the antigen-binding protein has two or more antigen-binding sites, at least two of which bind different antigens. As used herein, the term "valency" refers to the presence of a specific number of binding sites in the antigen-binding protein. A full-length antibody has two binding sites and is bivalent. Thus, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" refer to the presence of two, three, four, five, and six binding sites in the antigen-binding protein, respectively.

[0171] Multispecific antibodies, such as bispecific antibodies, are antibodies that have binding specificities for at least two different antigens.Methods for producing multispecific antibodies are known in the art, for example, by co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (see, for example, Milstein and Cuello, 1983, Nature 305:537-40).For further details on the production of multispecific antibodies (e.g., bispecific antibodies), see, for example, Bispecific Antibodies (Kontermann ed., 2011).

[0172] Antibodies of the present disclosure may be multivalent antibodies (e.g., tetravalent antibodies) with two or more antigen-binding sites, which can be readily produced by recombinant expression of nucleic acids encoding the antibody polypeptide chains. In certain embodiments, the multivalent antibody comprises (or consists of), for example, three to about eight antigen-binding sites. In one such embodiment, the multivalent antibody comprises (or consists of) four antigen-binding sites. A multivalent antibody comprises at least one polypeptide chain (e.g., two polypeptide chains), wherein the polypeptide chain comprises two or more variable domains. For example, the polypeptide chain may comprise VD1-(X1)n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain may comprise a VH-CH1-flexible linker-VH-CH1-Fc region chain; or a VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein may further comprise at least two (e.g., four) light chain variable domain polypeptides. The multivalent antibody herein may comprise, for example, from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated herein comprise a light chain variable domain and optionally further comprise a CL domain.

[0173] When multiple binding domains are present in the extracellular antigen-binding domain of the CAR of the present invention, the various domains may be fused to each other via peptide linkers. In some embodiments, the domains are fused directly to each other without any peptide linkers. The peptide linkers may be the same or different. Each peptide linker may have the same or different length and / or sequence depending on the structural and / or functional characteristics of the various domains. Each peptide linker may be independently selected and optimized. The length, flexibility, and / or other properties of the peptide linker used in the CAR may have some effect on properties including, but not limited to, affinity, specificity, or avidity for one or more specific antigens or epitopes. In some embodiments, the peptide linker contains flexible residues (such as glycine and serine) to allow adjacent protein domains to move freely relative to each other. For example, a glycine-serine doublet may be a suitable peptide linker.

[0174] The peptide linker may have a naturally occurring or non-naturally occurring sequence. For example, a sequence from the hinge region of a heavy chain-only antibody can be used as a linker. See, e.g., WO 1996 / 34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n , (GGGS) n , and (GGGGS) nand the like, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Other linkers known in the art, for example, as described in International Publication Nos. 2016014789, 2015158671, 2016102965, U.S. Patent Application Publication No. 20150299317, WO 2018067992, U.S. Patent No. 7741465, Colcher et al., J. Nat. Cancer Inst. 82:1191-1197 (1990), and Bird et al., Science 242:423-426 (1988), the disclosures of each of which are incorporated herein by reference, can also be included in the CARs provided herein.

[0175] In some embodiments, the extracellular antigen-binding domain provided in the CAR of the present invention recognizes an antigen that acts as a cell surface marker on target cells associated with a particular disease state. In some embodiments, the antigen is a tumor antigen. Tumors express multiple proteins that can serve as target antigens for immune responses, particularly T cell-mediated immune responses. The antigen targeted by the CAR may be an antigen on a single diseased cell, or it may be an antigen expressed on different cells, each of which contributes to the disease. The antigen targeted by the CAR may be directly or indirectly involved in the disease.

[0176] Tumor antigens are proteins produced by tumor cells that are capable of eliciting an immune response, particularly a T-cell mediated immune response. Exemplary tumor antigens include glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survivin, and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF), insulin-like ... These include, but are not limited to, IGF-I, IGF-II, IGF-I receptor, and mesothelin.

[0177] In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express numerous proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and gp100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER2 / Neu / ErbB-2. Yet another group of target antigens are oncofetal antigens, such as carcinoembryonic antigen (CEA).

[0178] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSAs are unique to tumor cells and are not present on other cells in the body. TAA-associated antigens are not unique to tumor cells, but instead are also expressed on normal cells under conditions that cannot induce a state of immune tolerance to the antigen. Expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. TAAs can be antigens expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or they can be antigens that are normally present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0179] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, and TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER2 / neu; unique tumor antigens resulting from chromosomal translocations; BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7.

[0180] Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0181] Hinge Area In some embodiments, the CAR provided herein comprises a hinge domain located between the extracellular antigen-binding domain and the transmembrane domain. A hinge domain is generally an amino acid segment found between two domains of a protein, and can allow the protein to be flexible and one or both domains to move relative to each other. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen-binding domain relative to the transmembrane domain of the effector molecule can be used.

[0182] Hinge domains of antibodies (e.g., IgG, IgA, IgM, IgE, or IgD antibodies) are also suitable for use in the pH-dependent chimeric receptor systems described herein. In some embodiments, the hinge domain is a hinge domain connecting constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge domain is an antibody hinge domain and comprises an antibody hinge domain and one or more antibody constant regions. In some embodiments, the hinge domain comprises an antibody hinge domain and an antibody CH3 constant region. In some embodiments, the hinge domain comprises an antibody hinge domain and antibody CH2 and CH3 constant regions. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises an IgG1 antibody hinge region and a CH2 and CH3 constant region. In some embodiments, the hinge region comprises an IgG1 antibody hinge region and a CH3 constant region.

[0183] Non-naturally occurring peptides can also be used as hinge domains in the chimeric receptors described herein. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain of an Fc receptor is a peptide linker, such as a (GxS)n linker, where x and n can independently be integers between 3 and 12, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

[0184] The hinge domain can contain any one of about 10 to 100 amino acids, e.g., about 15 to 75 amino acids, 20 to 50 amino acids, or 30 to 60 amino acids. In some embodiments, the hinge domain is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length.

[0185] In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein. The hinge domain of any protein known in the art to contain a hinge domain is suitable for use in the chimeric receptors described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein, which confers flexibility to the chimeric receptor.

[0186] Transmembrane domain The CAR of the present disclosure comprises a transmembrane domain that can be directly or indirectly fused to an extracellular antigen-binding domain. The transmembrane domain can be derived from either natural or synthetic sources. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. A transmembrane domain suitable for use in the CAR described herein can be obtained from a naturally occurring protein. Alternatively, the transmembrane domain can be a synthetic, non-naturally occurring protein segment (e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane).

[0187] Transmembrane domains are classified based on their three-dimensional structure. For example, transmembrane domains can form an α-helix, a complex of two or more α-helices, a β-barrel, or any other stable structure that can span a cellular phospholipid bilayer. Furthermore, transmembrane domains can also or alternatively be classified based on their transmembrane domain topology (e.g., the number of times the transmembrane domain passes through the membrane and the orientation of the protein). For example, single-pass membrane proteins cross the cellular membrane once, while multi-pass membrane proteins cross the cellular membrane at least twice (e.g., 2, 3, 4, 5, 6, 7, or more times). Membrane proteins can be defined as type I, type II, or type III depending on their termini relative to the inside and outside of the cell and the topology of the transmembrane segments. Type I membrane proteins have a single transmembrane region and are oriented such that the N-terminus of the protein is present on the extracellular side of the cellular lipid bilayer and the C-terminus of the protein is present on the cytoplasmic side. Type II membrane proteins also have a single transmembrane region, but are oriented such that the C-terminus of the protein is on the extracellular side of the cell's lipid bilayer and the N-terminus of the protein is on the cytoplasmic side. Type III membrane proteins have multiple transmembrane segments and can be further subclassified based on the number of transmembrane segments and the location of the N- and C-termini.

[0188] In some embodiments, the transmembrane domain of a CAR described herein is derived from a type I single-pass membrane protein. In some embodiments, transmembrane domains from multi-pass membrane proteins may also be adapted for use in the CARs described herein. Multi-pass membrane proteins may comprise complex (at least 2, 3, 4, 5, 6, 7, or more) alpha helices or beta sheet structures. In some embodiments, the N- and C-termini of a multi-pass membrane protein are on opposite sides of a lipid bilayer (e.g., the N-terminus of the protein is on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is on the extracellular side).

[0189] The transmembrane domains used in the CARs described herein can also comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is about 15-100 amino acids. In some embodiments, the protein segment is at least approximately 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art and are described, for example, in U.S. Pat. No. 7,052,906 and WO 2000 / 032776, the relevant disclosures of which are incorporated herein by reference.

[0190] The transmembrane domains provided herein may comprise a transmembrane region and a cytoplasmic region located C-terminal to the transmembrane domain. The cytoplasmic region of the transmembrane domain may comprise three or more amino acids, and in some embodiments, helps orient the transmembrane domain within the lipid bilayer. In some embodiments, one or more cysteine residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain comprises a positively charged amino acid. In some embodiments, the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.

[0191] In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane domain of a CAR provided herein comprises an artificial hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan, and valine may be present at the C-terminus of the transmembrane domain. In some embodiments, the transmembrane region comprises mostly hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the tissue region is hydrophobic. In some embodiments, the transmembrane region comprises a poly-leucine-alanine sequence. The hydropathy, i.e., the hydrophobic or hydrophilic characteristics, of a protein or protein segment can be assessed by any method known in the art (e.g., Kyte and Doolittle hydropathy analysis).

[0192] In some embodiments, the transmembrane domain of the CAR is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDI la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL-2Rβ, IL-2Rγ, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI ld, ITGAE, CD103, ITGAL, CDI la, LFA-1, ITGAM, CDI lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT and comprising a transmembrane domain selected from AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0193] Intracellular signaling domains The intracellular signaling domain in the CAR provided herein is involved in activating at least one of the normal effector functions of immune effector cells expressing the CAR. The term "effector function" refers to a specialized function of a cell. The effector function of NK cells may be, for example, cytolytic activity, including cytokine secretion. Thus, the term "cytoplasmic signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs a cell to perform a specialized function. Typically, the entire cytoplasmic signaling domain can be used, but in many cases, it is not necessary to use the entire chain. To the extent that a cytoplasmic signaling domain can be used, such a truncated portion can be used in place of the intact chain, as long as it transmits the effector function signal. Thus, the term cytoplasmic signaling domain is meant to include any truncated portion of the cytoplasmic signaling domain that is sufficient to transmit the effector function signal.

[0194] In some embodiments, the intracellular signaling domain comprises a major intracellular signaling domain of an immune effector cell. In some embodiments, the CAR comprises an intracellular signaling domain consisting essentially of a major intracellular signaling domain of an immune effector cell. A "major intracellular signaling domain" refers to a cytoplasmic signaling sequence that acts stimulatory to induce immune effector function. In some embodiments, the major intracellular signaling domain contains a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. As used herein, "ITAM" is a conserved protein motif typically present in the tail portion of signaling molecules expressed in many immune cells. This motif may contain two repeats of the amino acid sequence YxxL / I, separated by 6 to 8 amino acids, producing the conserved motif YxxL / Ix(6-8)YxxL / I (each x independently being any amino acid). ITAMs within signaling molecules are important for signal transduction within cells, mediated at least in part by phosphorylation of tyrosine residues within the ITAM following activation of the signaling molecule. ITAMs may also serve as docking sites for other proteins involved in signaling pathways. Exemplary ITAM-containing major cytoplasmic signaling sequences include those derived from CD3z, FcRγ (FCER1G), FcRβ (FcεRib), CD3γ, CD3Δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0195] Costimulatory signaling domain In some embodiments, the CAR comprises at least one costimulatory signaling domain. As used herein, the term "costimulatory signaling domain" refers to at least a portion of a protein that mediates intracellular signal transduction to induce an immune response, such as an effector function. Many immune effector cells require costimulation in addition to stimulating antigen-specific signals to promote cell proliferation, differentiation, and survival, as well as to activate cell effector functions.

[0196] The costimulatory signaling domain of the chimeric receptors described herein can be a cytoplasmic signaling domain derived from a costimulatory protein that transmits a signal and regulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils. A "costimulatory signaling domain" can be the cytoplasmic portion of a costimulatory molecule. The term "costimulatory molecule" refers to a cognate binding partner on an immune cell (such as an NK cell) that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by the immune cell, including, but not limited to, proliferation and survival.

[0197] In some embodiments, the intracellular signaling domain comprises a single costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (such as about any of 2, 3, 4, or more) costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more identical costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains from different costimulatory proteins, such as any two or more costimulatory proteins described herein. In some embodiments, the intracellular signaling domain comprises a major intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3z) and one or more costimulatory signaling domains. In some embodiments, the one or more costimulatory signaling domains and the major intracellular signaling domain (such as the cytoplasmic signaling domain of CD3z) are fused to each other via any peptide linker. The major intracellular signaling domain and one or more costimulatory signaling domains can be arranged in any suitable order. In some embodiments, the one or more costimulatory signaling domains are located between the transmembrane domain and the major intracellular signaling domain (such as the cytoplasmic signaling domain of CD3z). Multiple costimulatory signaling domains can provide additive or synergistic stimulatory effects.

[0198] Activation of a costimulatory signaling domain in a host cell (e.g., an immune cell) can induce the cell to increase or decrease cytokine production and secretion, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. The costimulatory signaling domain of any costimulatory molecule can be adapted for use in the CARs described herein. The type of costimulatory signaling domain is selected based on factors such as the type of immune effector cell on which the effector molecule will be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effect). Examples of costimulatory signaling domains for use in CARs include members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6); members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF 8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α, and TNF RII / TNFRSF1B); members of the SLAM family (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150);and the cytoplasmic signaling domain of any other costimulatory molecule (CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA class I, HLA-DR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen-1 (LFA-1), and NKG2C). In some embodiments, the one or more costimulatory signaling domains are selected from the group consisting of a ligand that specifically binds to CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83;

[0199] In some embodiments, the intracellular signaling domain in a CAR of the present disclosure comprises a costimulatory signaling domain derived from CD137 (i.e., 4-1BB). In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of CDz and a costimulatory signaling domain of CD137. In some embodiments, the costimulatory signaling domain is a variant of any of the costimulatory signaling domains described herein, such that the costimulatory signaling domain can modulate the immune response of an immune cell. In some embodiments, the costimulatory signaling domain comprises up to 10 (e.g., 1, 2, 3, 4, 5, or 8) amino acid residue variants compared to the wild-type counterpart. Such costimulatory signaling domains comprising one or more amino acid variants may be referred to as variants. Mutation of amino acid residues in the costimulatory signaling domain may result in increased signaling and enhanced stimulation of an immune response compared to a costimulatory signaling domain that does not comprise the mutation. Mutation of amino acid residues in the costimulatory signaling domain may result in decreased signaling and reduced stimulation of an immune response compared to a costimulatory signaling domain that does not comprise the mutation.

[0200] Exemplary CAR In some embodiments, the CARs provided herein comprise the amino acid sequence of any one of the CARs exemplified below in Section 6. In some embodiments, the CARs provided herein comprise the amino acid sequence of any one of the CARs known to those of skill in the art.

[0201] In some embodiments, the CARs provided herein comprise an amino acid sequence having a certain percent identity to any one of the CARs exemplified in Section 6 below. In some embodiments, the CARs provided herein comprise or consist of an extracellular domain having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of the CARs exemplified in Section 6 below. In some embodiments, the CARs provided herein comprise an amino acid sequence having a certain percent identity to any one of the CARs known to those of skill in the art. In some embodiments, a CAR provided herein comprises or consists of an extracellular domain having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of the CARs known to one of skill in the art.

[0202] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) (modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993)). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, for example, to score=100 and word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameters set, for example, to score 50 and word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997). Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When using BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998).Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0203] In some embodiments, modifications of the amino acid sequence of the CAR described herein are contemplated. For example, it may be desirable to optimize the binding affinity and / or other biological properties of the extracellular domain (including, but not limited to, specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility). Thus, in addition to the extracellular domains described herein, it is contemplated that variants of the domains described herein can be prepared. For example, scFv variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesizing the desired antibody or polypeptide. Those skilled in the art, aware of the amino acid changes, can alter the post-translational processing of antibodies.

[0204] The variations may be substitutions, deletions, or insertions of one or more codons encoding the polypeptide, resulting in a change in the amino acid sequence compared to the native sequence antibody or polypeptide. Target sites for substitutional mutagenesis include the CDRs and FRs.

[0205] Amino acid substitutions may result from substituting one amino acid for another with similar structural and / or chemical properties, such as substituting a serine for a leucine, e.g., a conservative amino acid substitution. Standard techniques known to those of skill in the art can be used to introduce mutations into the nucleotide sequences encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions. Insertions or deletions can optionally range from about 1 to 5 amino acids. In certain embodiments, substitutions, deletions, or insertions comprise fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions compared to the original molecule. In certain embodiments, substitutions are conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. Acceptable variations can be determined by systematically inserting, deleting, or substituting amino acids in a sequence and testing the resulting variants for activity exhibited by the parent antibody.

[0206] Antibodies generated by conservative amino acid substitutions are included in the present disclosure. In conservative amino acid substitutions, an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. As discussed above, the art has defined families of amino acid residues with side chains that have similar charges. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed and the activity of the protein determined. Conservative substitutions (e.g., within a group of amino acids with similar properties and / or side chains) can be made to maintain or not significantly alter properties.

[0207] Amino acids can be grouped according to the similarity of their side chain properties (see, e.g., Lehninger, Biochemistry 73-75 (2d ed. 1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H)). Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. For example, any cysteine residue not involved in maintaining the proper conformation of the antibody can be substituted with another amino acid, such as alanine or serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Non-conservative substitutions would involve exchanging a member of one of these classes for a member of another class.

[0208] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have a modification (e.g., an improvement) in certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated (e.g., using phage-display-based affinity maturation techniques such as those described herein). Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0209] Alterations (e.g., substitutions) can be made in CDRs, for example, to improve antibody affinity. Such alterations can be made in CDR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or SDRs (a-CDRs), and the resulting mutant antibodies or fragments thereof are tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves a CDR-directed approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling.

[0210] In some embodiments, substitutions, insertions, or deletions can occur within one or more CDRs, as long as such changes do not significantly reduce the antibody's ability to bind antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not significantly reduce binding affinity can be made in a CDR. In some embodiments, each CDR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0211] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham and Wells, Science, 244:1081-1085 (1989). In this method, a target residue or group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution.

[0212] Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Mutants can be screened to determine whether they contain the desired properties.

[0213] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0214] Modifications can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, PCR mutagenesis, etc. Site-directed mutagenesis (see, e.g., Carter, Biochem J. 237:1-7 (1986); and Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315-23 (1985)), or other known techniques can be performed on cloned DNA to produce antibody variant DNA.

[0215] Furthermore, all of the above descriptions in this disclosure relating to CAR also apply to engineered T cell receptors (TCRs) in addition to CARs. Thus, in another aspect, a method for producing immune effector cells expressing TCRs is provided herein. In some embodiments, the immune effector cells are cytotoxic cells. In certain embodiments, the cytotoxic cells are natural killer cells. In certain embodiments, the cytotoxic cells are cytotoxic T lymphocytes.

[0216] The TCRs provided herein include recombinant or engineered TCRs, which, as used herein, are included as a type of functional exogenous receptor provided herein and refer to peptides expressed in immune cells. The function of a recombinant or engineered TCR can include, for example, redirecting the immune activity of immune cells against desired types of cells, such as cancer and infected cells, that have specific markers on their surface. They can replace or be co-expressed with endogenous TCRs. In some embodiments, such recombinant TCRs are single-chain TCRs containing an open reading frame in which the variable Vα and Vβ domains are paired with a protein linker. This involves molecular cloning of TCR genes known to be specific for a selected antigen. These chains are then introduced into T cells, usually by retroviral vector. The resulting expression of the cloned TCRα and TCRβ genes confers functional specificity to the transduced T cells, determined by the pairing of these new genes. A component of a recombinant or engineered TCR is any functional subunit of a TCR, such as recombinant TCR alpha and TCR beta, that is encoded by an exogenous polynucleotide sequence introduced into a cell.

[0217] Polynucleotides In certain embodiments, the present disclosure provides a polynucleotide encoding a CAR described herein. The polynucleotide of the present disclosure can be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, which can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or non-coding (antisense) strand. In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0218] The present disclosure further relates to variants of the polynucleotides described herein, where the variants encode, for example, fragments, analogs, and / or derivatives of the antibodies or CARs of the present disclosure. In certain embodiments, the present disclosure provides polynucleotides, including polynucleotides having a nucleotide sequence at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98%, or 99% identical to a polynucleotide encoding a CAR of the present disclosure. As used herein, the phrase "a polynucleotide having a nucleotide sequence at least, e.g., 95% "identical" to a reference nucleotide sequence" is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides of the reference sequence may be deleted or replaced with alternative nucleotides, or up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence. These variations in the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere between these terminal positions, and may be scattered individually among the nucleotides of the reference sequence or in one or more contiguous groups within the reference sequence.

[0219] Polynucleotide variants can contain alterations in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that result in silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, polynucleotide variants contain silent substitutions that do not result in changes to the amino acid sequence of a polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can be generated for a variety of reasons, such as to optimize codon expression for a particular host (i.e., changing codons in human mRNA to those preferred by a bacterial host, such as E. coli). In some embodiments, polynucleotide variants contain at least one silent mutation in a non-coding or coding region of the sequence.

[0220] In some embodiments, polynucleotide variants are produced to modulate or alter expression (or expression levels) of an encoded polypeptide. In some embodiments, polynucleotide variants are produced to increase expression of an encoded polypeptide. In some embodiments, polynucleotide variants are produced to decrease expression of an encoded polypeptide. In some embodiments, polynucleotide variants increase expression of an encoded polypeptide compared to the parent polynucleotide sequence. In some embodiments, polynucleotide variants decrease expression of an encoded polypeptide compared to the parent polynucleotide sequence.

[0221] Vectors Also provided are vectors comprising the polynucleotides described herein. In one embodiment, the nucleic acid molecules can be incorporated into a recombinant expression vector.

[0222] The present disclosure provides vectors for cloning and expressing any one of the CARs described herein. In some embodiments, the vector is suitable for replication and integration in eukaryotic cells, such as mammalian cells. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex viral vectors, and derivatives thereof. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.

[0223] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to genetically engineered mammalian cells in vitro or ex vivo. Many different retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Numerous adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying immunomodulatory agent (such as immune checkpoint inhibitor) coding sequences and / or self-inactivating lentiviral vectors carrying chimeric antigen receptors can be packaged using protocols known in the art. The resulting lentiviral vectors can be used to transduce mammalian cells (such as primary human NK cells) using methods known in the art. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of the transgene and its propagation in progeny cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.

[0224] In some embodiments, the vector comprises any one of the nucleic acids encoding a CAR described herein. The nucleic acid can be cloned into the vector using any molecular cloning method known in the art, such as, for example, using a restriction endonuclease site and one or more selectable markers. In some embodiments, the nucleic acid is operably linked to a promoter. A variety of promoters have been investigated for gene expression in mammalian cells, and any promoter known in the art can be used in the present disclosure. Promoters can be broadly classified as constitutive promoters or regulatable promoters (e.g., inducible promoters).

[0225] In some embodiments, the nucleic acid encoding the CAR is operably linked to a constitutive promoter. A constitutive promoter allows a heterologous gene (also referred to as a transgene) to be constitutively expressed in a host cell. Exemplary constitutive promoters contemplated herein include, but are not limited to, the cytomegalovirus (CMV) promoter, human elongation factor-1 alpha (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerokinase promoter (PGK), simian virus 40 early promoter (SV40), and chicken beta-actin promoter (CAGG) combined with the CMV early enhancer. The efficiency of such constitutive promoters in promoting transgene expression has been extensively compared in numerous studies.

[0226] In some embodiments, the nucleic acid encoding the CAR is operably linked to an inducible promoter. Inducible promoters belong to the category of regulatable promoters. Inducible promoters can be induced by one or more conditions (such as physical conditions), the microenvironment of the engineered immune effector cells, or the physiological state of the engineered immune effector cells, an inducer (i.e., an inducer), or a combination thereof.

[0227] In some embodiments, the inducing conditions do not induce expression of an endogenous gene in the genetically engineered mammalian cell and / or in a subject receiving the pharmaceutical composition, hi some embodiments, the inducing conditions are selected from the group consisting of an inducer, irradiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox conditions, the tumor environment, and an activation state of the genetically engineered mammalian cell.

[0228] In some embodiments, the vector also contains a selectable marker gene or reporter gene to select cells expressing the CAR from a population of host cells transfected via the lentiviral vector. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. For example, the vector can contain transcription and translation terminators, initiation sequences, and promoters useful for controlling the expression of the nucleic acid sequence.

[0229] 5.4. Immune effector cells expressing chimeric antigen receptors In yet another aspect, provided herein are immune effector cells expressing a CAR produced according to the methods provided herein (e.g., as described in Sections 4.2 and 4.3, above).

[0230] In some embodiments, the immune effector cells are cytotoxic cells. In certain embodiments, the cytotoxic cells are natural killer cells. In certain embodiments, the cytotoxic cells are cytotoxic T lymphocytes.

[0231] In some embodiments, a CAR expressed in an immune effector cell of the invention comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR further comprises one or more additional regions / domains, such as a signal peptide, a hinge region, a costimulatory signaling domain, a linker, etc., each of which may be as described above in Section 4.3.1.

[0232] Specifically, in certain embodiments, the CARs provided herein may further comprise a signal peptide at the N-terminus of the polypeptide. In some embodiments, the signal peptide targets the effector molecule to the secretory pathway of the cell and enables incorporation and anchoring of the effector molecule into the lipid bilayer. Signal peptides, including signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences, suitable for use in the CARs described herein will be apparent to those of skill in the art. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain.

[0233] The extracellular antigen-binding domain of a CAR described herein comprises one or more antigen-binding domains. In some embodiments, the extracellular antigen-binding domain comprises an antibody or a fragment thereof. In specific embodiments, the extracellular antigen-binding domain of a CAR of the invention comprises a single-chain Fv (sFv or scFv). In some embodiments, the extracellular antigen-binding domain comprises a humanized antibody or a fragment thereof.

[0234] In certain embodiments, the extracellular antigen-binding domain comprises multiple binding domains. In some embodiments, the extracellular antigen-binding domain comprises a multispecific antibody or fragment thereof. In other embodiments, the extracellular antigen-binding domain comprises a multivalent antibody or fragment thereof. When multiple binding domains are present in the extracellular antigen-binding domain of the CAR of the invention, the various domains may be fused to each other via peptide linkers. In some embodiments, the domains are fused directly to each other without any peptide linker. The peptide linkers may be the same or different. Each peptide linker may have the same or different length and / or sequence depending on the structural and / or functional characteristics of the various domains. Each peptide linker may be independently selected and optimized.

[0235] In some embodiments, the extracellular antigen-binding domain provided in the CAR of the present invention recognizes an antigen that acts as a cell surface marker on target cells associated with a particular disease state. In some embodiments, the antigen is a tumor antigen. Tumors express multiple proteins that can serve as target antigens for the immune response. The antigen targeted by the CAR may be an antigen on a single diseased cell, or it may be an antigen expressed on different cells, each of which contributes to the disease. The antigen targeted by the CAR may be directly or indirectly involved in the disease.

[0236] Tumor antigens are proteins produced by tumor cells that can induce immune responses, especially T cell-mediated immune responses. Exemplary tumor antigens include, but are not limited to, glioma-associated antigens, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mutated hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0237] In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express numerous proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and gp100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER2 / Neu / ErbB-2. Yet another group of target antigens are oncofetal antigens, such as carcinoembryonic antigen (CEA).

[0238] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSAs are unique to tumor cells and are not present on other cells in the body. TAA-associated antigens are not unique to tumor cells, but are instead expressed on normal cells under conditions that cannot induce a state of immune tolerance to the antigen. Expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. TAAs can be antigens that are expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or they can be antigens that are normally present at very low levels on normal cells but are expressed at much higher levels on tumor cells.

[0239] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, and TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER2 / neu; unique tumor antigens resulting from chromosomal translocations; BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens, such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7.

[0240] Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0241] In some embodiments, the CAR provided herein comprises a hinge domain located between the extracellular antigen binding domain and the transmembrane domain.In some embodiments, the hinge domain is the hinge domain of a naturally occurring protein.The hinge domain of any protein known in the art to contain a hinge domain is suitable for use in the chimeric receptor described herein.In some embodiments, the hinge domain is at least a part of the hinge domain of a naturally occurring protein, which provides flexibility to the chimeric receptor.

[0242] The CARs of the present disclosure comprise a transmembrane domain that can be fused directly or indirectly to an extracellular antigen-binding domain. The transmembrane domain can be derived from either natural or synthetic sources. Transmembrane domains suitable for use in the CARs described herein can be obtained from naturally occurring proteins. Alternatively, the transmembrane domain can be a synthetic, non-naturally occurring protein segment (e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane). In some embodiments, the transmembrane domain is derived from a type I, type II, or type III membrane protein. In some embodiments, the transmembrane domain of a CAR described herein is derived from a type I single-pass membrane protein. In some embodiments, transmembrane domains from multi-pass membrane proteins can also be suitable for use in the CARs described herein. The transmembrane domain used in the CARs described herein can also comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the protein segment is about 15-100 amino acids. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is at least approximately 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids.

[0243] The transmembrane domains provided herein may comprise a transmembrane region and a cytoplasmic region located C-terminal to the transmembrane domain. In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues.

[0244] In some embodiments, the transmembrane domain of the CAR is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDI la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL-2Rβ, IL-2Rγ, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI ld, ITGAE, CD103, ITGAL, CDI la, LFA-1, ITGAM, CDI lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT and comprising a transmembrane domain selected from AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0245] The intracellular signaling domain within the CARs provided herein is responsible for activating at least one of the normal effector functions of immune effector cells expressing the CAR. In some embodiments, the intracellular signaling domain comprises a major intracellular signaling domain of an immune effector cell. In some embodiments, the CAR comprises an intracellular signaling domain consisting essentially of a major intracellular signaling domain of an immune effector cell. In some embodiments, the major intracellular signaling domain contains a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Exemplary ITAM-containing major cytoplasmic signaling sequences include those derived from CD3z, FcRγ (FCER1G), FcRβ (FcεRib), CD3γ, CD3Δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0246] In some embodiments, the CAR comprises at least one costimulatory signaling domain. The costimulatory signaling domain of the chimeric receptors described herein can be a cytoplasmic signaling domain derived from a costimulatory protein that transmits a signal and modulates a response mediated by an immune cell. In some embodiments, the intracellular signaling domain comprises a single costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (such as about any of two, three, four, or more) costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more identical costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains derived from different costimulatory proteins, such as any two or more costimulatory proteins described herein. In some embodiments, the intracellular signaling domain comprises a major intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3z) and one or more costimulatory signaling domains. In some embodiments, the one or more costimulatory signaling domains and the major intracellular signaling domain (such as the cytoplasmic signaling domain of CD3z) are fused to each other via an optional peptide linker. The primary intracellular signaling domain and one or more costimulatory signaling domains can be arranged in any suitable order. In some embodiments, one or more costimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3z). Multiple costimulatory signaling domains can provide additive or synergistic stimulatory effects.

[0247] The costimulatory signaling domain of any costimulatory molecule may be adapted for use in the CARs described herein. Examples of costimulatory signaling domains for use in CARs include members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6); members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF 8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α, and TNF RII / TNFRSF1B); members of the SLAM family (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150);and the cytoplasmic signaling domain of any other costimulatory protein, including, but not limited to, CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA class I, HLA-DR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C. In some embodiments, the one or more costimulatory signaling domains are selected from the group consisting of a ligand that specifically binds to CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83;

[0248] In some embodiments, the CARs provided herein comprise the amino acid sequence of any one of the CARs exemplified below in Section 6. In some embodiments, the CARs provided herein comprise the amino acid sequence of any one of the CARs known to those of skill in the art.

[0249] In some embodiments, the CARs provided herein comprise an amino acid sequence having a certain percent identity to any one of the CARs exemplified in Section 6 below. In some embodiments, the CARs provided herein comprise or consist of an extracellular domain having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of the CARs exemplified in Section 6 below. In some embodiments, the CARs provided herein comprise an amino acid sequence having a certain percent identity to any one of the CARs known to those of skill in the art. In some embodiments, a CAR provided herein comprises or consists of an extracellular domain having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of the CARs known to one of skill in the art.

[0250] Pharmaceutical Compositions In one aspect, the present disclosure further provides a pharmaceutical composition comprising an engineered immune effector cell (e.g., an NK cell, an alpha-beta T cell, or a gamma-delta T cell) that expresses a CAR, such as those described in Section 4.4 above. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an engineered immune effector cell (e.g., an NK cell, an alpha-beta T cell, or a gamma-delta T cell) of the present disclosure and a pharmaceutically acceptable excipient.

[0251] In specific embodiments, the term "excipient" can refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), carrier, or vehicle. Pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerin, propylene, glycol, water, ethanol, and the like. If desired, the compositions can further contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Such compositions will contain a prophylactically or therapeutically effective amount of the active ingredients provided herein, such as in purified form, in combination with a suitable amount of excipients to provide a form for proper administration to a patient. The formulation should suit the mode of administration.

[0252] In some embodiments, the choice of excipient will be determined in part by the particular cells and / or by the method of administration, and thus a variety of suitable formulations exist.

[0253] Typically, acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants (such as ascorbic acid, methionine, vitamin E, sodium metabisulfite, and the like); preservatives, tonicity agents, stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents (such as EDTA and / or nonionic surfactants).

[0254] Buffers can be used to control the pH within a range that optimizes therapeutic efficacy, especially when stability is pH-dependent. Buffers suitable for use in the present disclosure include both organic and inorganic acids and their salts, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, and acetate. Additionally, buffers can include histidine and trimethylamine salts (such as Tris).

[0255] Preservatives may be added to retard microbial growth. Suitable preservatives for use in the present disclosure include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chlorides, bromides, iodides), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens (such as methyl or propyl paraben); catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0256] Tonicity adjusting agents, sometimes known as "stabilizers," may be present to adjust or maintain the tonicity of the liquid in the composition. When used with large charged biomolecules (such as proteins and antibodies), tonicity adjusting agents are often called "stabilizers" because they can interact with the charged groups of amino acid side chains, thereby reducing the possibility of inter- and intra-molecular interactions. Exemplary tonicity adjusting agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0257] Further exemplary excipients include (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to container walls. Such excipients include polyhydric sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myo-inisitose, myo-inositol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polysaccharides, and the like. Examples of suitable reducing agents include ethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides (e.g., raffinose); and polysaccharides such as dextrin or dextran.

[0258] A non-ionic surfactant or detergent (also known as a "wetting agent") may be present to aid in solubilizing the therapeutic agent and to protect the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stresses without causing denaturation of the active therapeutic protein or antibody. Suitable non-ionic surfactants include, for example, polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0259] For pharmaceutical compositions to be used for in vivo administration, pharmaceutical compositions are preferably sterile.Pharmaceutical compositions can be sterilized by filtering through a sterile filtration membrane.The pharmaceutical compositions herein can generally be placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0260] The route of administration will be in accordance with known and accepted methods, such as, for example, injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation, or by sustained or extended release means, as single or multiple boluses, or infusion over an extended period of time, in an appropriate manner.

[0261] In another embodiment, the pharmaceutical composition can be provided as a controlled-release or sustained-release system. In one embodiment, a pump can be used to achieve controlled or sustained release (see, e.g., Sefton, Crit. Ref. Biomed. Eng. 14:201-40 (1987); Buchwald et al., Surgery 88:507-16 (1980); and Saudek et al., N. Engl. J. Med. 321:569-74 (1989)). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of prophylactic or therapeutic agents (e.g., fusion proteins described herein) or compositions provided herein (see, e.g., Medical Applications of Controlled Release (Langer and Wise, eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126 (1983); Levy et al., Science 228:190-92 (1985); During et al., Ann. Neurol. 25:351-56 (1989); Howard et al. al., J. Neurosurg. 71:105-12 (1989); U.S. Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, and 5,128,326; WO 99 / 15154 and WO 99 / 20253).Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (poly(lactide-co-glycolide, PLGA), and polyorthoesters. In one embodiment, the polymers used in sustained release formulations are inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In yet another embodiment, controlled or sustained release systems can be placed in proximity to specific target tissues, such as the nasal cavity or lungs, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984). Controlled-release systems are discussed, for example, by Langer, Science 249:1527-33 (1990). Any technique known to those skilled in the art can be used to prepare sustained-release formulations containing one or more agents described herein (see, e.g., U.S. Pat. No. 4,526,938; WO 91 / 05548 and WO 96 / 20698; Ning et al., Radiotherapy & Oncology, 39:179-89 (1996); Song et al., PDA J. of Pharma. Sci. & Tech. 50:372-97 (1995); Cleek et al., Proc. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-54 (1997); and Lam et al., Proc. Int'l. Symp. Control). See Rel. Bioact. Mater. 24:759-60 (1997).

[0262] The pharmaceutical compositions described herein may also contain more than one active compound or agent, as needed for the particular condition being treated. Alternatively, or in addition, the composition may include a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressant, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts effective for the intended purpose.

[0263] The active ingredient can also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, prepared, for example, by coacervation techniques or by interfacial polymerization, respectively, in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition.

[0264] A variety of compositions and delivery systems are known and can be used with the therapeutic agents provided herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, construction of a nucleic acid as part of a recombinant cell, retrovirus or other vector capable of expressing a single domain antibody or therapeutic molecule provided herein, etc.

[0265] In some embodiments, the pharmaceutical compositions provided herein contain binding molecules and / or cells in an amount effective for treating or preventing a disease or disorder, e.g., a therapeutically or prophylactically effective amount. In some embodiments, the therapeutic or prophylactic effect is monitored by periodic evaluation of the treated subject. In the case of repeated administration over several days or longer, depending on the condition, treatment is repeated until the desired suppression of disease symptoms occurs. However, other drug administration methods may be useful and can be determined.

[0266] 5.6. Methods and Uses In another aspect, provided herein are methods for and uses of engineered immune effector cells (e.g., NK cells) that express a CAR, such as those described in Section 4.4 above.

[0267] In some embodiments, engineered immune effector cells (e.g., NK cells) expressing a CAR provided herein are useful as allogeneic CAR-NK cell therapy. In some embodiments, the CAR-NK cell therapy of the present invention has safety features not present in conventional autologous CAR-NK therapy, such as no or low cytokine storm, no stimulation of regulatory T cells, reduced autologous tissue damage, reduced autoimmune induction, reduced graft-versus-host disease, etc.

[0268] Such methods and uses include, for example, therapeutic methods and uses that involve administering the cells or compositions containing the cells to a subject having a disease or disorder. In some embodiments, the cells are administered in an amount effective to effect treatment of the disease or disorder. Uses include use of the cells in such methods and treatments, and in the preparation of medicaments for carrying out such treatments. In some embodiments, the methods are carried out by administering the cells or compositions comprising the cells to a subject having or suspected of having a disease or condition. In some embodiments, the methods thereby treat the disease or disorder in the subject.

[0269] In some embodiments, the treatments provided herein result in a complete or partial improvement or reduction of a disease or disorder, or its associated symptoms, adverse effects or outcomes, or phenotype. Desirable effects of treatment include, but are not limited to, suppression of disease occurrence or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, suppression of metastasis, reduction of the rate of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis. These terms include, but do not imply, complete cure of the disease or complete elimination of any or all symptoms or effects on outcomes.

[0270] As used herein, in some embodiments, the treatments provided herein delay the onset of a disease or disorder, e.g., postpone, prevent, slow, retard, stabilize, inhibit, and / or postpone the onset of a disease (such as cancer). This delay can be of varying duration, depending on the disease history and / or the individual receiving treatment. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease or disorder. For example, late-stage cancer, such as the development of metastases, can be delayed. In other embodiments, the methods or uses provided herein prevent a disease or disorder.

[0271] In some embodiments, the CAR-NK cell therapy of the present invention is used to treat solid tumor cancers. In other embodiments, the CAR-NK cell therapy of the present invention is used to treat hematological cancers. In other embodiments, the disease or disorder is an autoimmune disease or an inflammatory disease.

[0272] In some embodiments, the methods involve administering cells or compositions containing the cells to a subject, tissue, or cell having, at risk of, or suspected of having a disease or disorder. In some embodiments, the cells, populations, and compositions are administered to a subject having the particular disease or disorder being treated, e.g., via adoptive cell therapy, such as adoptive NK cell therapy. In some embodiments, the cells or compositions are administered to a subject, such as a subject having or at risk for a disease or disorder. In some embodiments, the methods thereby treat, e.g., ameliorate, one or more symptoms of the disease or disorder.

[0273] Methods for administering cells for adoptive cell therapy are known, for example, as described in U.S. Patent Application Publication No. 2003 / 0170238, U.S. Patent No. 4,690,915, Rosenberg, Nat Rev Clin Oncol. 8(10):577-85 (2011), Themeli et al., Nat Biotechnol. 31(10):928-933 (2013), Tsukahara et al., Biochem Biophys Res Commun 438(1):84-9 (2013), and Davila et al., PLoS ONE 8(4):e61338 (2013). These methods can be used in connection with the methods and compositions provided herein.

[0274] In some embodiments, cell therapy (e.g., adoptive NK cell therapy) is performed via autologous transplantation, where cells are isolated and / or otherwise prepared from the subject receiving cell therapy or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from the subject in need of treatment, and the cells, after isolation and processing, are administered to the same subject. In other embodiments, cell therapy (e.g., adoptive NK cell therapy) is performed via allogeneic transplantation, where cells are isolated and / or otherwise prepared from a subject other than the subject receiving or ultimately receiving cell therapy (e.g., a first subject). In such embodiments, the cells are then administered to a different subject of the same species, e.g., a second subject. In some embodiments, the first subject and the second subject are genetically identical. In some embodiments, the first subject and the second subject are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0275] In some embodiments, the subject to which the cells, cell populations, or compositions are administered is a primate, such as a human. The subject may be male or female and of any suitable age, including infants, juveniles, adolescents, adults, and geriatric subjects. In some examples, the subject is a validated animal model for disease, adoptive cell therapy, and / or for assessing toxicity outcomes.

[0276] The compositions provided herein can be administered by any suitable means, for example, by injection, for example, intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subsubbulbar injection, subconjunctival injection, sub-Tenon injection, retrobulbar injection, periocular injection, or posterior juxtascleral delivery. In some embodiments, they are administered parenterally, intrapulmonaryly, and intranasally, and, if desired for local therapy, by intralesional administration. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0277] The amount of a prophylactic or therapeutic agent provided herein that is effective in the prevention and / or treatment of a disease or condition can be determined by standard clinical techniques. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. The appropriate dose of binding molecule or cell for disease prevention or treatment can depend on the type of disease or disorder being treated, the type of binding molecule, the severity and course of the disease or disorder, whether the agent is administered prophylactically or therapeutically, previous treatments, the patient's medical history and response to the agent, and the discretion of the attending physician. The compositions, molecules, and cells, in some embodiments, are suitably administered to the patient at one time or over a series of treatments. Multiple doses can be administered intermittently. An initial higher loading dose, followed by one or more lower doses, can be administered.

[0278] In the context of genetically engineered cells, in some embodiments, a subject may be administered a range of about one million to about 100 billion cells and / or that amount of cells per kilogram of body weight. In some embodiments, where the pharmaceutical composition comprises any one of the genetically engineered immune cells described herein, the pharmaceutical composition is administered at a dose of at least about 10, 10, 10, 10, 10, or 10 cells per kg of body weight of the individual. Doses may vary depending on the disease or disorder, and / or the patient, and / or other treatment-specific attributes.

[0279] In some embodiments, the pharmaceutical composition is administered a single time. In some embodiments, the pharmaceutical composition is administered multiple times (such as two, three, four, five, six, or more times). In some embodiments, the pharmaceutical composition is administered one or more times during a dosing cycle. The dosing cycle can be, for example, one week, two weeks, three weeks, four weeks, five weeks, or more, or one month, two months, three months, four months, five months, or more. The optimal dosage and treatment regimen for a particular patient can be determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0280] In some embodiments, the compositions provided herein are administered as part of a combination therapy, e.g., simultaneously with another therapeutic intervention, such as another antibody or engineered cell or receptor or agent (such as a cytotoxic or therapeutic agent), or sequentially in any order.

[0281] In some embodiments, the compositions provided herein are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some embodiments, the cells are co-administered with another therapy sufficiently close in time so that the cell population enhances the effect of the one or more additional therapeutic agents, or vice versa. In some embodiments, the compositions provided herein are administered before the one or more additional therapeutic agents. In some embodiments, the compositions provided herein are administered after the one or more additional therapeutic agents.

[0282] In certain embodiments, once the cells are administered to a mammal (e.g., a human), the biological activity of the genetically engineered cell population is measured by any of a number of known methods. Parameters evaluated include specific binding of genetically engineered or natural NK cells or other immune cells to an antigen in vivo (e.g., by imaging) or ex vivo (e.g., by ELISA or flow cytometry). In certain embodiments, the ability of the genetically modified cells to destroy target cells can be measured using any suitable method known in the art, such as the cytotoxicity assays described in Kochenderfer et al., J. Immunotherapy, 32(7):689-702 (2009) and Herman et al., J. Immunological Methods, 285(1):25-40 (2004). In certain embodiments, the biological activity of the cells can also be measured by assaying the expression and / or secretion of certain cytokines, such as CD107a, IFNγ, IL-2, and TNF. In some aspects, biological activity is measured by assessing a clinical outcome, such as reduction in tumor burden or tumor burden.

[0283] 5.7. Kits and Products Further provided are kits, unit doses, and articles of manufacture comprising any of the genetically engineered immune effector cells described herein. In some embodiments, kits are provided that comprise any one of the pharmaceutical compositions described herein, preferably providing instructions for its use.

[0284] The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, and flexible packaging (e.g., sealed Mylar or plastic bags). The kits may optionally provide additional components (such as buffers) and instructional information. Thus, the present application also provides articles of manufacture that include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.

[0285] The article of manufacture may include a container and a label or package insert attached to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed from a variety of materials, such as glass or plastic. Generally, the container holds a composition described herein that is effective for treating a disease or disorder (e.g., cancer) and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is used for treating a specific condition in an individual. The label or package insert further includes instructions for administering the composition to an individual. The label may indicate directions for reconstitution and / or use. The container holding the pharmaceutical composition may be a multi-dose vial, allowing for repeated administration of the reconstituted formulation (e.g., 2-6 administrations). "Package insert" refers to instructions typically included in commercial packaging for therapeutic products that contain information regarding the indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic products. Additionally, the article of manufacture may further comprise a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0286] The kit or article of manufacture may include multiple unit doses of the pharmaceutical composition and instructions for use packaged in an amount sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies.

[0287] For the sake of brevity, certain abbreviations are used herein. One example is the single letter abbreviations that represent amino acid residues. The amino acids and their corresponding three letter and one letter abbreviations are as follows:

[0288] [Table 2]

[0289] The present disclosure is generally disclosed herein using affirmative language to describe numerous embodiments. The present disclosure also specifically includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols, procedures, assays or analyses, is excluded in whole or in part. Thus, generally, aspects not expressly included in the present disclosure are disclosed herein, regardless of whether they are expressed herein as not including what the disclosure does not include.

[0290] A number of embodiments of the present disclosure have been described. However, it will be understood that various modifications can be made without departing from the spirit and scope of the present disclosure.

[0291] 6. Implementation form The present disclosure includes the following non-limiting embodiments.

[0292] In one set of embodiments (Set A): A1. A method for preparing immune effector cells for transduction, comprising: i. a first step to increase the expression of low density lipoprotein receptor (LDLR); ii. a second step of inhibiting the intracellular antiviral defense mechanisms of immune effector cells; A method in which the first step can be performed before, simultaneously with, or after the second step. A2. The method of embodiment A1, wherein the immune effector cells are autologous or allogeneic. A3. The method of embodiment A1, wherein the immune effector cells are cytotoxic cells, optionally wherein the cytotoxic cells are natural killer cells. A4. The method of any one of embodiments A1-A3, wherein the method further comprises providing one or more transduction enhancers. A5. The method of embodiment A4, wherein the transduction enhancer is vectofusin. A6. The method of embodiment A4, wherein the transduction enhancer is retronectin. A7. The method of any one of embodiments A1-A6, wherein the method further comprises introducing the nucleic acid into immune effector cells. A8. The method of embodiment A7, wherein the nucleic acid comprises a promoter. A9. The method of embodiment A8, wherein the promoter is selected from the group consisting of CAG, PGK, EF1a, and EFS promoters. A10. The method of any one of embodiments A7-A9, wherein the nucleic acid is delivered via a lentiviral particle. A11. The method of embodiment A10, wherein the lentiviral particles are pseudotyped with vesicular stomatitis virus G (VSV-G). A12. The method of any one of embodiments A7-A11, wherein the nucleic acid encodes an exogenous functional receptor. A13. The method of embodiment A12, wherein the exogenous functional receptor is a chimeric antigen receptor (CAR) or a TCR. A14. The method of any one of embodiments A1-A13, wherein the first step comprises contacting the immune effector cells with a statin. A15. The method of embodiment A14, wherein the statin is rosuvastatin. A16. The method of any one of embodiments A1-A15, wherein the second step comprises contacting the immune effector cells with an inhibitor of 3-phosphoinositide-dependent kinase 1 (PDK1). A17. The method of embodiment A16, wherein the inhibitor of PDK1 is BX795.

[0293] In another set of embodiments (Set B), B1. A method for preparing immune effector cells for transduction, comprising: i. contacting immune effector cells with a first agent that increases expression of low density lipoprotein receptor (LDLR); ii. contacting the immune effector cells with a second agent that inhibits the immune effector cells' intracellular antiviral defense mechanisms; iii. contacting the immune effector cells with a third agent that enhances transduction. B2. The method of embodiment B1, wherein the immune effector cells are autologous or allogeneic. B3. The method of embodiment B1, wherein the immune effector cells are cytotoxic cells, optionally wherein the cytotoxic cells are natural killer cells. B4. The method of any one of embodiments B1-B3, wherein the first agent is a statin. B5. The method of embodiment B4, wherein the statin is rosuvastatin. B6. The method of any one of embodiments B1-B5, wherein the second agent is an inhibitor of 3-phosphoinositide-dependent kinase 1 (PDK1). B7. The method of embodiment B6, wherein the inhibitor of PDK1 is BX795. B8. The method of any one of embodiments B1-B7, wherein the third agent is vectofusin and / or retronectin. B9. The method of any one of embodiments B1-B8, wherein the method further comprises transducing immune effector cells with a nucleic acid. B10. The method of embodiment B9, wherein the nucleic acid comprises a promoter. B11. The method of embodiment B10, wherein the promoter is selected from the group consisting of CAG, PGK, EF1a, and EFS promoters. B12. The method of any one of embodiments B9-B11, wherein the nucleic acid is delivered via a lentiviral particle. B13. The method of embodiment B12, wherein the lentiviral particles are pseudotyped with vesicular stomatitis virus G (VSV-G). B14. The method of any one of embodiments B9-B13, wherein the nucleic acid encodes an exogenous functional receptor. B15. The method of embodiment B14, wherein the exogenous functional receptor is a chimeric antigen receptor (CAR) or a TCR.

[0294] In another set of embodiments (Set C), C1. A method comprising: i. contacting immune effector cells with a first agent that increases expression of low density lipoprotein receptor (LDLR); ii. contacting the immune effector cells with a second agent that inhibits the immune effector cells' intracellular antiviral defense mechanisms; iii. contacting the immune effector cells with a third agent that enhances transduction; iv. introducing the nucleic acid into an immune effector cell. C2. The method of embodiment C1, wherein the immune effector cells are autologous or allogeneic. C3. The method of embodiment C1, wherein the immune effector cells are cytotoxic cells, optionally wherein the cytotoxic cells are natural killer cells. C4. The method of any one of embodiments C1-C3, wherein the first agent is a statin. C5. The method of embodiment C2, wherein the statin is rosuvastatin. C6. The method of any one of embodiments C1-C5, wherein the second agent is an inhibitor of 3-phosphoinositide-dependent kinase 1 (PDK1). C7. The method of embodiment C6, wherein the inhibitor of PDK1 is BX795. C8. The method of any one of embodiments C1-C7, wherein the third agent is vectofusin and / or retronectin. C9. The method of any one of embodiments C1-C8, wherein the nucleic acid comprises a promoter. C10. The method of embodiment C9, wherein the promoter is selected from the group consisting of CAG, PGK, EF1a, and EFS promoters. C11. The method of any one of embodiments C1-C10, wherein the nucleic acid is delivered via a lentiviral particle. C12. The method of embodiment C11, wherein the lentiviral particles are pseudotyped with vesicular stomatitis virus G (VSV-G). C13. The method of any one of embodiments C1-C12, wherein the nucleic acid encodes an exogenous functional receptor. C14. The method of embodiment C13, wherein the exogenous functional receptor is a chimeric antigen receptor (CAR) or a TCR.

[0295] In another set of embodiments (Set D), D1. A method for preparing immune effector cells for transduction, comprising a step for increasing expression of low density lipoprotein receptor (LDLR). D2. The method of embodiment D1, wherein the immune effector cells are autologous or allogeneic. D3. The method of embodiment D1, wherein the immune effector cells are cytotoxic cells, optionally wherein the cytotoxic cells are natural killer cells. D4. The method of embodiment D1, wherein the immune effector cells are gamma delta T cells. D5. The method of embodiment D1, wherein the immune effector cells are alpha beta T cells. D6. The method of embodiment D1, wherein the step of increasing expression of low-density lipoprotein receptor (LDLR) comprises contacting the immune effector cells with a statin. D7. The method of embodiment D6, wherein the statin is rosuvastatin. D8. The method of embodiment D6, wherein the statin is atorvastatin. D9. The method of any one of embodiments D1-D8, further comprising a step for inhibiting intracellular antiviral defense mechanisms of immune effector cells. D10. The method of embodiment D9, wherein the step of inhibiting the intracellular antiviral defense mechanism of the immune effector cell comprises contacting the immune effector cell with an inhibitor of 3-phosphoinositide-dependent kinase 1 (PDK1). D11. The method of embodiment D10, wherein the inhibitor of PDK1 is BX795. D12. The method of any one of embodiments D1-D11, wherein the method further comprises providing one or more transduction enhancers. D13. The method of embodiment D4, wherein the transduction enhancer is a vectofusin. D14. The method of embodiment D4, wherein the transduction enhancer is retronectin. D15. The method of any one of embodiments D1-D14, wherein the method further comprises introducing the nucleic acid into immune effector cells. D16. The method of embodiment D15, wherein the nucleic acid comprises a promoter. D17. The method of embodiment D16, wherein the promoter is selected from the group consisting of CAG, PGK, EF1a, and EFS promoters. D18. The method of any one of embodiments D15-D17, wherein the nucleic acid is delivered via a lentiviral particle. D19. The method of embodiment D18, wherein the lentiviral particles are pseudotyped with vesicular stomatitis virus G (VSV-G). D20. The method of any one of embodiments D15-D19, wherein the nucleic acid encodes an exogenous functional receptor. D21. The method of embodiment D20, wherein the exogenous functional receptor is a chimeric antigen receptor (CAR) or a TCR.

[0296] In another set of embodiments (Set E), E1. A method for preparing immune effector cells for transduction, comprising contacting the immune effector cells with an agent that increases expression of low density lipoprotein receptor (LDLR). E2. The method of embodiment E1, wherein the immune effector cells are autologous or allogeneic. E3. The method of embodiment E1, wherein the immune effector cells are cytotoxic cells, optionally wherein the cytotoxic cells are natural killer cells. E4. The method of embodiment E1, wherein the immune effector cells are gamma delta T cells. E5. The method of embodiment E1, wherein the immune effector cells are alpha beta T cells. E6. The method of embodiment E1, wherein the step of increasing expression of low density lipoprotein receptor (LDLR) comprises contacting the immune effector cells with a statin. E7. The method of embodiment E6, wherein the statin is rosuvastatin. E8. The method of embodiment E6, wherein the statin is atorvastatin. E9. The method of any one of embodiments E1-E8, further comprising a step for inhibiting intracellular antiviral defense mechanisms of immune effector cells. E10. The method of embodiment E9, wherein the step of inhibiting the intracellular antiviral defense mechanism of the immune effector cell comprises contacting the immune effector cell with an inhibitor of 3-phosphoinositide-dependent kinase 1 (PDK1). E11. The method of embodiment E10, wherein the inhibitor of PDK1 is BX795. E12. The method of any one of embodiments E1-E11, wherein the method further comprises providing one or more transduction enhancers. E13. The method of embodiment E12, wherein the transduction enhancer is vectofusin. E14. The method of embodiment E12, wherein the transduction enhancer is retronectin. E15. The method of any one of embodiments E1-E14, wherein the method further comprises introducing the nucleic acid into immune effector cells. E16. The method of embodiment E15, wherein the nucleic acid comprises a promoter. E17. The method of embodiment E16, wherein the promoter is selected from the group consisting of CAG, PGK, EF1a, and EFS promoters. E18. The method of any one of embodiments E15-E17, wherein the nucleic acid is delivered via a lentiviral particle. E19. The method of embodiment E18, wherein the lentiviral particles are pseudotyped with vesicular stomatitis virus G (VSV-G). E20. The method of any one of embodiments E15-17, wherein the nucleic acid encodes an exogenous functional receptor. E21. The method of embodiment E20, wherein the exogenous functional receptor is a chimeric antigen receptor (CAR) or a TCR. [Example]

[0297] The following is a description of various methods and materials used in testing, and is provided so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure. It is not intended to limit the scope of what the inventors regard as their disclosure, nor is it intended to represent that the experiments that follow are all that can be performed. It should be understood that exemplary descriptions written in the present tense are not necessarily performed, but rather descriptions are those that could be performed to generate data and the like relevant to the teachings of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental error and deviation should be accounted for.

[0298] 7.1. Example 1 - Generation of Natural Killer CAR (CAR-NK) Cells In preparation for transduction, NK cells were first activated. On day 0, 0.5–1 × 10 cells were added per well. 6 NK cells were distributed into 24-well plates.

[0299] Thoroughly resuspend and use 1 × 10 loaded anti-biotin MACSiBead particles (Miltenyi Biotech, Cat. No. 130-094-483) for NK cell activation. 6 5 μL per 5 x 10 NK cells 5The amount of anti-biotin MACSiBead particles (100 μL of loaded anti-biotin MACSiBead particles) was transferred to an appropriate tube. 100 μL of culture medium was added to the loaded anti-biotin MACSiBead particles. The mixture was centrifuged at 300 × g for 5 minutes to wash the beads, which were then added to the plated NK cells. The supernatant was aspirated, and the loaded anti-biotin MACSiBead particles were resuspended in 5–10 μL of fresh NK MACS medium containing NK MACS supplement (Miltenyi Biotech, catalog no. 130-114-429). The prepared anti-biotin MACSiBead particles were added to the plated NK cells and gently mixed by pipetting up and down 2–3 times. The cells were then incubated at 37°C and 5% CO2.

[0300] Lentiviral transduction was performed on day 5 or 6. NK cells were resuspended to determine whether cell numbers had changed from day 0. Lentiviral particles were thawed on ice. Once thawed, the lentiviral particles were removed from the ice and allowed to equilibrate to room temperature before being added to the activated NK cells. The amount of virus required to transduce the cells was determined based on the desired multiplicity of infection (MOI). Once the NK cells were transduced with lentiviral particles, they were returned to the incubator at 37°C and 5% CO2.

[0301] Cells were monitored for surface CAR expression as early as 3-4 days post-transduction. NK cell numbers were determined. 1 x 10 5 NK cells were collected from each sample and transferred to wells of a microtiter plate. The cells in each well of the microtiter plate were washed twice with 200 μL of FACS buffer. The wells were stained with 100 μL of staining buffer containing the appropriate CAR detection antibody. The plate was incubated at 4°C for at least 30 minutes without light. The cells were then washed twice with 150 μL of FACS buffer and centrifuged at 400 × g for 4 minutes at room temperature. CAR expression on the NK cells was then measured using flow cytometry analysis.

[0302] To assess CAR transduction efficiency and surface CAR stability, NK-like cell lines (e.g., NK-L) and primary NK cells were transduced with antigen-1 CAR at an MOI of 5. Surface antigen-1 CAR expression was monitored at multiple time points post-transduction using an anti-myc tag AF647 antibody (Cell Signaling, catalog no. 2233, 1:200).

[0303] As shown in Figures 1A-1C, NK-like cell lines (NK-92 and NK-L) were efficiently transduced at early time points (day 3) compared to the low expression seen in primary NK cells. However, antigen-1 CAR expression was downregulated over time in both NK-like cell lines (NK-92 and NK-L) and primary NK cells.

[0304] 7.2. Example 2 - Optimization of Gene Delivery To improve CAR transduction efficiency and surface CAR stability, the following additional steps were performed before or simultaneously with adding lentiviral particles to the cells.

[0305] 7.2.1. Inhibitors of 3-phosphoinositide-dependent kinase 1 (PDK1) NK cells have been evolutionarily selected for their resistance to viral infections, and PDK1 inhibitors can block pattern recognition receptors that are naturally used by immune cells such as NK cells during viral infections.

[0306] To test whether PDK1 inhibitors can improve CAR transduction efficiency or surface CAR stability, primary NK cells were activated and transduced with the antigen-1 CAR in the presence or absence of BX795 (InvivoGen, catalog no. tlrl-bx7), a PDK1 inhibitor, at concentrations varying from 0.5 to 7.5 µM. BX795 was added to the cells along with the viral supernatant and incorporated into the transduction process by gentle mixing. Surface CAR expression was monitored at regular intervals using an anti-myc tag AF647 antibody (Cell Signaling, catalog no. 2233, 1:200). A schematic schedule of the experiment is shown in Figure 2A.

[0307] As shown in Figures 2B-C, primary NK cells transduced with BX795 showed improved surface CAR stability in a dose-dependent manner compared to cells transduced without BX795 (n=2).

[0308] Statins For lentivirus-pseudotyped vesicular stomatitis virus G (VSV-G), the low-density lipoprotein receptor (LDLR) functions as the primary entry receptor during internalization. Statins are commonly used to upregulate the expression of LDLR.

[0309] To test whether statins can improve CAR transduction efficiency or surface CAR stability, cells were pretreated with 5 μM rosuvastatin (Cayman Chemical, catalog number 12029) during activation prior to transduction. Activated primary NK cells were then transduced with the Antigen-2 CAR at an MOI of 5. DMSO-pretreated cells were included as a negative control.

[0310] As shown in Figure 3A, incubation of primary NK cells with rosuvastatin during activation resulted in increased LDLR expression levels on the cells (n=3). As shown in Figure 3B, increased LDLR expression levels on primary NK cells were associated with increased antigen-2 CAR expression.

[0311] 7.2.3. Transduction Enhancers Retronectin is a recombinant human fibronectin fragment that enhances lentivirus- and retrovirus-mediated gene transduction by aiding in the colocalization of target cells and viral particles. Vectofusin is a fully synthetic, nontoxic, cationic, amphipathic peptide with the ability to enhance viral transduction.

[0312] To test whether transduction enhancers can improve CAR transduction efficiency or surface CAR stability, primary NK cells were activated and transduced in the presence or absence of retronectin and vectofusin.

[0313] The day before seeding primary NK cells, Retronectin (Takara Bio Inc., catalog number T202) was diluted from a 1 μg / μL stock in sterile PBS to create a 20 μg / mL working solution. The Retronectin working solution was added to the wells and spread evenly throughout the wells. The plates were kept overnight at 4°C the day before seeding. After overnight incubation, Retronectin was removed from the wells. PBS containing 2% BSA was added to each well. The plates were incubated at room temperature for 30 minutes and washed twice with PBS. NK cells were then seeded and activated as described above in Section 6.1.

[0314] Activated primary NK cells were transduced with the antigen-2 CAR at an MOI of 5. Vectofucin (Miltenyi, catalog no. 130-111-163) with a working concentration of 10 μg / mL in the total culture volume was mixed with the viral supernatant and incubated on ice for 10–15 minutes. The mixture was equilibrated to room temperature and then added to the NK cells and mixed with the cell suspension by pipetting up and down. The cells were then incubated at 37°C and 5% CO2. CAR expression in primary NK cells was assessed on day 10.

[0315] As shown in Figure 4, the presence of the transduction enhancers retronectin and vectofusin during lentiviral transduction of primary NK cells was associated with increased antigen-3 CAR expression (n=3).

[0316] Combination To address the challenges of lentiviral transduction in primary NK cells, primary NK cells were transduced with a combination treatment of a PDK1 inhibitor, a statin, and a transduction enhancer. Activated NK cells were transduced with the Antigen-3 CAR at an MOI of 5. Cells were transduced in retronectin-coated dishes with BX795, rosuvastatin, and vectofusin. The approach for incorporating BX795, rosuvastatin, and vectofusin was the same as described above in Section 6.2. After transduction, the plates were spun down at 850 × g for 2 hours at 37°C. After spinning, the plates were returned to the incubator at 37°C and 5% CO2. At different time points after transduction, CAR expression was monitored using the anti-Antigen-3 CAR recombinant protein AF647 antibody (2 μg / mL).

[0317] As shown in Figures 5A-5D, primary NK cells transduced with the combined treatment of BX795, rosuvastatin, and vectofusin showed higher expression and stability of surface CAR compared to lentivirus alone (n=3).

[0318] To address the challenges of lentiviral transduction in primary T cells, primary αβ T cells were transduced with a combination treatment of a PDK1 inhibitor, a statin, and a transduction enhancer. Activated αβ T cells were transduced with the antigen-3 CAR (internal) at an MOI of 5. Cells were transduced in retronectin-coated dishes with BX795 (3 μM), rosuvastatin (1 μM), and vectofusin. The approach for incorporating BX795, rosuvastatin, and vectofusin was the same as described above in Section 6.2. After transduction, the plates were spun down at 850 × g for 2 hours at 37°C. After spinning, the plates were returned to the incubator at 37°C and 5% CO2. At different time points after transduction, CAR expression was monitored using the anti-antigen-3 CAR recombinant protein AF647 antibody (2 μg / mL).

[0319] As shown in Figure 6, primary αβ T cells transduced with the combined treatment of BX795, rosuvastatin, retronectin, and vectofusin showed higher CAR expression compared to lentivirus alone (n=3).

[0320] 7.3. Example 3 - Optimization of gene transfer in γδ T cells.

[0321] Lentiviral transduction of primary γδ T cells using VSV-G pseudotyped lentiviral vectors is associated with low levels of CAR expression, limiting the ability to screen for CARs. We evaluated lentiviral transduction of primary γδ T cells in the presence of compounds targeting the LDLR (statins), the antiviral pathway (BX795), and vectofusin.

[0322] Four primary γδ T cell samples from healthy donors were expanded with zoledronic acid (ZOL) for 5 days, transduced with or without enhancers (rosuvastatin, BX795, and vectofucin), and evaluated for CAR expression. Briefly, frozen PBMC cells were thawed and resuspended at 1E6 cells / ml. PBMC cells were activated with 10 μM zoledronic acid and grown in RPMI medium in the presence of IL-2 (100 IU) and IL-15 (10 ng). On day 6, cells were transduced with a lentiviral vector encoding a CAR specific for antigen-4 at an MOI of 5, involving the following steps: (1) the lentiviral vector encoding the antigen-4-specific CAR was preincubated with the statin, Vectofucin-1 (10 μg / ml) on ice for 10 minutes before being added to the cells; (2) 5 μM BX795 was added to the cells and mixed gently; (3) the cells were spun at 850 g for 2 hours at 32° C.; and (4) the cells were returned to the incubator at 37° C. Cells were analyzed for CAR expression by flow cytometry on days 3, 6, and 10.

[0323] Lentiviral transduction of primary γδ T cells in the presence of enhancers (rosuvastatin, BX795, and vectofucin) reduced cell viability compared to enhancer-free lentiviral transduction (Figure 7). Lentiviral transduction of primary γδ T cells in the presence of enhancers increased CAR levels in primary γδ T cells compared to lentiviral vector alone (Figure 8).

[0324] Conclusion: Lentiviral transduction of primary γδ T cells in the presence of enhancers (rosuvastatin, BX795, and vectofucin) increased transduction, but it was also associated with increased cell death, thus making practical implementation of this protocol difficult.

[0325] We next evaluated lentiviral transduction of primary γδ T cells using VSV-G pseudotyped lentiviral vectors in the presence of an enhancer targeting the LDLR.

[0326] Three primary γδ T cell samples from healthy donors were grown with zoledronic acid (ZOL) for 5 days and transduced with a lentiviral vector encoding an antigen-4-specific CAR in the presence or absence of different concentrations of rosuvastatin and atorvastatin, and CAR expression was assessed. Briefly, frozen PBMC cells were thawed and resuspended at 1E6 cells / ml. PBMC cells were activated with 10 μM zoledronic acid and grown in RPMI medium in the presence of IL-2 (100 IU) and IL-15 (10 ng). On day 6, the cells were transduced with a lentiviral vector encoding an antigen-4-specific CAR at an MOI of 5, involving the following steps: (1) the lentiviral vector encoding the antigen-4 CAR was preincubated with rosuvastatin on ice for 10 minutes before addition to the cells; (2) the cells were spun at 850 g for 2 hours at 32°C; and (3) the cells were returned to the incubator at 37°C. Cells were analyzed for CAR expression by flow cytometry on days 3, 6 and 10.

[0327] Lentiviral transduction of primary γδ T cells in the presence of statins was associated with higher CAR expression compared to cells transduced with lentiviral vectors alone (Figures 9A-9F). This novel protocol also achieved increased CAR expression in primary γδ T cells (Figures 9A-9F), enabling CAR screening for future experiments.

[0328] Next, we evaluated the effect of statin pretreatment on LDLR expression in lentiviral-transduced primary γδ T cells. Three primary γδ T cell samples from healthy donors were grown in ZOL for 5 days and pretreated with or without different concentrations of rosuvastatin and atorvastatin, as described above, and evaluated for LDLR expression.

[0329] Pretreatment of primary γδ T cells with either the statins atorvastatin (FIG. 10A) or rosuvastatin (FIG. 10B) increased LDLR expression compared to cells transduced with the lentiviral vector alone.

[0330] Next, we assessed the effect of statin pretreatment of lentiviral transduction of primary γδ T cells on the frequency of the Vγ9Vδ2 T cell subset within γδ T cells. Three primary γδ T cell samples from healthy donors were grown in ZOL for 5 days, pretreated with or without different concentrations of rosuvastatin or atorvastatin, as described above, and assessed for Vγ9Vδ2 expression using flow cytometry.

[0331] Pretreatment of primary γδ T cells with either the statins atorvastatin (Figure 11A) or rosuvastatin (Figure 11B) did not alter the frequency of Vγ9Vδ2 cells compared to cells transduced with lentiviral vector alone at zero statin concentration.

[0332] Those skilled in the art will appreciate that changes could be made to the embodiments described above without departing from the broad inventive concept. It is understood, therefore, that the invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined herein.

[0333] In the "Background" section and throughout this specification, various publications, articles, and patents are cited or described, and each of these references is incorporated herein by reference in its entirety. Any discussion of documents, operations, materials, devices, articles and the like which is included in the specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these items constitute part of the prior art to any invention(s) disclosed or claimed.

Claims

1. A method for preparing immunoeffector cells for transduction, A method comprising the step of increasing the expression of low-density lipoprotein receptor (LDLR).

2. The method according to claim 1, wherein the immune effector cells are autologous, allogeneic, gamma delta T cells, alpha beta T cells, cytotoxic cells, or natural killer cells.

3. The method according to claim 1, wherein the step of increasing the expression of low-density lipoprotein receptors (LDLRs) comprises contacting the immune effector cells with a statin.

4. The method according to claim 3, wherein the statin is rosuvastatin or atorvastatin.

5. (a) A step of inhibiting the intracellular antiviral defense mechanism of the immune effector cell, (b) To provide one or more transduction enhancers, (c) Introducing nucleic acids into the immune effector cells, or (d) Combinations of (a) to (c), The method according to any one of claims 1 to 4, further comprising:

6. The method according to claim 5, wherein the step for inhibiting the intracellular antiviral defense mechanism of the immune effector cells comprises contacting the immune effector cells with a 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor.

7. The method according to claim 6, wherein the inhibitor of PDK1 is BX795.

8. The method according to claim 5, wherein the transduction enhancer is bectofucin or retronectin.

9. The nucleic acid is (a) including promoters, (b) Delivered via lentiviral particles, (c) Encoding an exogenous functional receptor, or The method according to claim 5, which is a combination of (d)(a) to (c).

10. (a) The promoter is selected from the group consisting of CAG, PGK, EF1a, and EFS promoters. (b) The lentivirus particles are pseudotyped with vesicular stomatitis virus G (VSV-G), (c) The exogenous functional receptor is a chimeric antigen receptor (CAR) or a TCR, The method according to claim 9, which is a combination of (d)(a) to (c).

11. A method for preparing immunoeffector cells for transduction, comprising contacting the immunoeffector cells with a drug that increases the expression of low-density lipoprotein receptors (LDLRs).

12. The method according to claim 11, wherein the immune effector cells are autologous, allogeneic, gamma delta T cells, alpha beta T cells, cytotoxic cells, or natural killer cells.

13. The method according to claim 11, wherein the step of increasing the expression of low-density lipoprotein receptors (LDLRs) includes contacting the immune effector cells with a statin.

14. The method according to claim 13, wherein the statin is rosuvastatin or atorvastatin.

15. (a) A step of inhibiting the intracellular antiviral defense mechanism of the immune effector cell, (b) To provide one or more transduction enhancers, (c) Introducing nucleic acids into the immune effector cells, or (d) Combinations of (a) to (c), The method according to any one of claims 11 to 14, further comprising:

16. The method according to claim 15, wherein the step for inhibiting the intracellular antiviral defense mechanism of the immune effector cells comprises contacting the immune effector cells with a 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor.

17. The method according to claim 16, wherein the inhibitor of PDK1 is BX795.

18. The method according to claim 15, wherein the transduction enhancer is bectofucin or retronectin.

19. The nucleic acid is (a) including promoters, (b) Delivered via lentiviral particles, (c) Encoding an exogenous functional receptor, or The method according to claim 15, which is a combination of (d)(a) to (c).

20. (a) The promoter is selected from the group consisting of CAG, PGK, EF1a, and EFS promoters. (b) The lentivirus particles are pseudotyped with vesicular stomatitis virus G (VSV-G), (c) The exogenous functional receptor is a chimeric antigen receptor (CAR) or a TCR, The method according to claim 19, which is a combination of (d)(a) to (c).

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