Enhanced transfer of genetic instructions to effector immune cells

Abstract

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

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 391,672, filed on July 22, 2022, U.S. Provisional Patent Application No. 63 / 391,676, filed on July 22, 2022, and U.S. Provisional Patent Application No. 63 / 391,677, filed on July 22, 2022. The entire contents of the above applications are incorporated herein by reference in their entirety. Technical Field

[0003] In some embodiments, provided herein are methods and uses thereof for producing bioengineered natural killer (NK) cells or T cells, for example, NK cells or T cells expressing chimeric antigen receptors (CAR-NK cells or CAR-T cells). In some embodiments, provided herein are compositions comprising CAR-NK cells or CAR-T cells and uses of the compositions for treating diseases or disorders. Summary of the invention

[0004] In one aspect, provided herein is a method for preparing immune effector cells for transduction, the method comprising a step for increasing the expression of low-density lipoprotein receptor (LDLR).

[0005] In some embodiments, the immune effector cells are autologous or allogeneic.

[0006] In some embodiments, the immune effector cell is a cytotoxic cell, and wherein optionally the cytotoxic cell is a natural killer cell.

[0007] In some embodiments, the immune effector cells are γ-δ T cells.

[0008] In some embodiments, the immune effector cells are α-β T cells.

[0009] In some embodiments, the step of increasing the expression of low density lipoprotein receptor (LDLR) comprises contacting the immune effector cells with a statin.

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

[0011] In some embodiments, the statin is atorvastatin.

[0012] In some embodiments, the method further comprises a step for suppressing the intracellular antiviral defense mechanisms of immune effector cells.

[0013] In some embodiments, the step of 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.

[0014] In some embodiments, the PDK1 inhibitor is BX795.

[0015] In some embodiments, the method further comprises providing one or more transduction enhancers.

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

[0017] In some embodiments, the transduction enhancer is Retronectin.

[0018] In some embodiments, the method further comprises introducing the nucleic acid into an immune effector cell.

[0019] In some embodiments, the nucleic acid comprises a promoter.

[0020] In some embodiments, the promoter is selected from the group consisting of a CAG promoter, a PGK promoter, an EF1a promoter, and an EFS promoter.

[0021] In some embodiments, the nucleic acid is delivered via lentiviral particles.

[0022] In some embodiments, the lentiviral particles are pseudotyped with vesicular stomatitis virus G (VSV-G).

[0023] In some embodiments, the nucleic acid encodes an exogenous functional receptor.

[0024] In some embodiments, the exogenous functional receptor is a chimeric antigen receptor (CAR) or TCR.

[0025] In one aspect, provided herein is a method for preparing an immune effector cell for transduction, the method comprising contacting the immune effector cell with an agent that increases expression of a low-density lipoprotein receptor (LDLR).

[0026] In some embodiments, the immune effector cells are autologous or allogeneic.

[0027] In some embodiments, the immune effector cell is a cytotoxic cell, and wherein optionally the cytotoxic cell is a natural killer cell.

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

[0029] In some embodiments, the immune effector cells are αβ T cells.

[0030] In some embodiments, the step of increasing the expression of low density lipoprotein receptor (LDLR) comprises contacting the immune effector cells with a statin.

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

[0032] In some embodiments, the statin is atorvastatin.

[0033] In some embodiments, the method further comprises a step for suppressing the intracellular antiviral defense mechanisms of immune effector cells.

[0034] In some embodiments, the step of 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.

[0035] In some embodiments, the PDK1 inhibitor is BX795.

[0036] In some embodiments, the method further comprises providing one or more transduction enhancers.

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

[0038] In some embodiments, the transduction enhancer is Retronectin.

[0039] In some embodiments, the method further comprises introducing the nucleic acid into an immune effector cell.

[0040] In some embodiments, the nucleic acid comprises a promoter.

[0041] In some embodiments, the promoter is selected from the group consisting of a CAG promoter, a PGK promoter, an EF1a promoter, and an EFS promoter.

[0042] In some embodiments, the nucleic acid is delivered via lentiviral particles.

[0043] In some embodiments, the lentiviral particles are pseudotyped with vesicular stomatitis virus G (VSV-G).

[0044] In some embodiments, the nucleic acid encodes an exogenous functional receptor.

[0045] In some embodiments, the exogenous functional receptor is a chimeric antigen receptor (CAR) or TCR.

[0046] In one aspect, provided herein is a method for preparing immune effector cells for transduction, the method comprising:

[0047] i. a first step for increasing the expression of low-density lipoprotein receptor (LDLR); and

[0048] ii. The second step of the intracellular antiviral defense mechanism for suppressing immune effector cells;

[0049] The first step may be performed before, simultaneously with or after the second step.

[0050] In some embodiments, the immune effector cells are autologous or allogeneic.

[0051] In some embodiments, the immune effector cell is a cytotoxic cell, and wherein optionally the cytotoxic cell is a natural killer cell.

[0052] In some embodiments, the method further comprises providing one or more transduction enhancers.

[0053] In some embodiments, the transduction enhancer is Vectofusin. In some embodiments, the transduction enhancer is Retronectin.

[0054] In some embodiments, the method further comprises introducing the nucleic acid into the immune effector cell. In some embodiments, the nucleic acid comprises a promoter. In some embodiments, the promoter is selected from the group consisting of a CAG promoter, a PGK promoter, an EF1a promoter, and an EFS promoter.

[0055] 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.

[0056] In some embodiments, the exogenous functional receptor is a chimeric antigen receptor (CAR) or TCR.

[0057] In some embodiments, the first step comprises contacting the immune effector cells with a statin. In some embodiments, the statin is rosuvastatin.

[0058] In some embodiments, the second step comprises contacting the immune effector cells with a 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor. In some embodiments, the PDK1 inhibitor is BX795.

[0059] In another aspect, the present invention provides a method for preparing immune effector cells for transduction, the method comprising:

[0060] i. contacting the immune effector cells with a first agent that increases expression of the low-density lipoprotein receptor (LDLR);

[0061] ii. contacting the immune effector cells with a second agent that inhibits the intracellular antiviral defense mechanisms of the immune effector cells; and

[0062] iii. contacting the immune effector cells with a third agent that enhances transduction.

[0063] In another aspect, there is provided a method comprising:

[0064] i. contacting the immune effector cells with a first agent that increases expression of the low-density lipoprotein receptor (LDLR);

[0065] ii. contacting the immune effector cells with a second agent that inhibits the intracellular antiviral defense mechanisms of the immune effector cells;

[0066] iii. contacting the immune effector cells with a third agent that enhances transduction; and

[0067] iv. Introduction of nucleic acids into immune effector cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figures 1A to 1C It was shown that CAR expression was downregulated over time in both NK-like cell lines and primary NK cells. Figure 1A Downregulation of the NK-like cell line NK-92 is shown. Figure 1B Downregulation of the NK-like cell line NK-L is shown. Figure 1C Downregulation of primary NK cells is shown.

[0069] FIG. 2A to FIG. 2C It was shown that the PDK1 inhibitor BX795 stabilized surface CAR expression in primary NK cells in a dose-dependent manner. Figure 2A A schematic schedule of the experiment is shown. The experiment was run in duplicate ( Figure 2B and Figure 2C ).

[0070] Figure 3A and Figure 3B It was shown that elevated expression of low-density lipoprotein receptor (LDLR) on primary NK cells correlated with increased antigen-2 CAR expression. Figure 3A Shown is the increased expression of LDLR on primary NK cells following exposure of the cells to the statin, rosuvastatin. Figure 3B Increased Antigen-2 CAR expression associated with elevated expression of LDLR is shown.

[0071] Figure 4 It was shown that the presence of the transduction enhancers Retronectin and Vectofusin correlated with increased Antigen-3 CAR expression during lentiviral transduction of primary NK cells.

[0072] FIG. 5A to FIG. 5D It is shown that lentiviral transduction of primary NK cells in the presence of statins, BX795, Retronectin and Vectofusin increased CAR stability on primary NK cells. Experiments were run in triplicate ( FIG. 5A to FIG. 5C ). Figure 5D Specifically shown is the expression of antigen-3CAR on primary NK cells 14 days after lentiviral transduction of primary NK cells under different conditions.

[0073] Figure 6 It was shown that lentiviral transduction of primary αβ T cells in the presence of statins, BX795, Retronectin, and Vectofusin increased CAR expression.

[0074] Figure 7 Shown are the percentages of live primary γδ T cells after lentiviral transduction in the presence of statins, BX795 and the Vectofusin enhancer. LV: lentiviral vector; UTD: untransduced.

[0075] Figure 8 Shown are the percentages of primary γδ T cells positive for Antigen-4 CAR following lentiviral transduction in the presence of statin, BX795, and Vectofusin.

[0076] 9A to 9F It was shown that in atorvastatin ( 9A to 9C ) or rosuvastatin ( FIG. 9D to FIG. 9F ) after lentiviral transduction with the presence of CAR.

[0077] FIG. 10A to FIG. 10B It was shown that in atorvastatin ( Fig. 10A ) or rosuvastatin ( Fig. 10B LDLR expression shown as geometric mean after lentiviral transduction in the presence of ) at the concentrations indicated on the X-axis (μM).

[0078] FIG. 11A to FIG. 11B It is shown that in statin, namely atorvastatin ( Fig.11A ) or rosuvastatin ( Fig. 11B ) at the concentrations indicated on the X-axis (μM). DETAILED DESCRIPTION

[0079] The adoptive transfer of immune cells genetically modified to recognize malignancy-associated antigens shows promise as a 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)).

[0080] Genetically engineered T cells expressing chimeric antigen receptors (CARs) have emerged as one of the most effective cancer treatments. Although the therapeutic results of CAR-T therapy are unprecedented, they are significantly limited by their own characteristics. For example, T cells cannot be harvested from all patients, and the quality of T cells may not meet manufacturing standards. In addition, conventional CAR-T therapies have inherent manufacturing problems, such as manufacturing failure, time delays, insufficient cell expansion or heterogeneous products, which can be harmful to recipient patients. For such highly personalized programs, high costs are also inevitable.

[0081] Natural killer (NK) cells are part of the innate immune system and serve as the first line of defense for cancer and viral infection. Due to the rapid cytotoxicity of NK cells to tumors, safety characteristics (low cytokine storm, minimal graft-versus-host disease and low immune effector cell-associated neurotoxicity syndrome) and potential use as ready-made cell therapy, it has been increasingly concerned as a promising alternative effector cell type for CAR engineering. However, it is challenging to optimize the efficient and clinically applicable gene transfer scheme for NK cells, because NK cells are selected to have resistance to viral infection in evolution. Therefore, the art needs improved gene transfer schemes for immunotherapy based on NK cells.

[0082] The present disclosure is based, in part, on novel methods or processes for preparing immune effector cells (eg, NK cells) for transduction, and their use in preparing cell therapies for treating diseases or disorders.

[0083] 5.1. Definitions

[0084] The techniques and procedures described or referenced herein include those generally known to those skilled in the art and / or commonly employed using routine methods, such as, for example, the widely used methods described in Sambrook et al., Molecular Cloning: 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, Vol. 1 and Vol. 2 (Kontermann and Dübel, eds., 2nd ed., 2010).

[0085] Unless otherwise defined herein, the technical and scientific terms used in this specification have the meanings commonly understood by those of ordinary skill in the art. For the purpose of interpreting this specification, the following term descriptions will apply, and whenever appropriate, terms used in the singular will also include the plural, and vice versa. In the event of any conflict between any description of the term set forth and any document incorporated herein by reference, the description of the term set forth below will prevail.

[0086] As used herein, the term "immune effector cell" refers to any of various types of cells that protect the body in an immune response by actively responding to a stimulus and effecting some change.

[0087] As used herein, the term "cytotoxic cell" or "cytotoxic lymphocyte" refers to certain subtypes of immune effector cells that kill their homologous infection or transformed targets through the exocytosis pathway of cytotoxic granules. 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 α-β (αβ) T cell. In some embodiments, the cytotoxic cell is a γ-δ (γδ) T cell.

[0088] As used herein, the term "natural killer cell" or "NK cell" refers to a class of cytotoxic cells that are critical 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.

[0089] As used herein, the term "alpha-beta (αβ) T cells" refers to a subpopulation of T lymphocytes in peripheral blood that express the αβ T cell receptor (TCR).

[0090] As used herein, the term "γ-δ (γδ) T cells" refers to a T lymphocyte subpopulation expressing a γδ T cell receptor (TCR) in peripheral blood. In some embodiments, the γδ T cells comprise a Vγ9Vδ2 subpopulation of γδ T cells.

[0091] As used herein, the term "cytotoxic T lymphocyte" or "CTL" refers to a type of cytotoxic cell that destroys virus-infected cells, tumor cells, and tissue transplants present in the cytosol or continuous nuclear compartments. CTL is also called a CD8+ T cell because it expresses CD8 glycoprotein on its surface and associates with MHC class I molecules.

[0092] The term "low-density lipoprotein receptor" or "LDLR" refers to a class of cell surface proteins that participate 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.

[0093] The term "pseudotype" refers to the production of a virus or viral vector that is conjugated to an exogenous viral envelope protein. The result is a pseudotyped virus that contains envelope proteins derived from a different virus.

[0094] The term "vesicular stomatitis virus G" or "VSV-G" refers to the viral fusion protein of the vesicular stomatitis virus (VSV). The viral fusion protein is essential for infection by enveloped viruses. VSV-G mediates fusion between the viral envelope and the host cell membrane, allowing the viral genome to be released into the host cell.

[0095] The terms "antibody", "immunoglobulin" or "Ig" are used interchangeably herein and in the broadest sense, and specifically encompass, for example, polyclonal or monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions with poly-epitope or mono-epitope specificity, monovalent antibodies, multivalent antibodies, or multispecific antibodies (e.g., bispecific antibodies, as long as they exhibit the desired biological activity) formed by at least two intact antibodies, single-chain antibodies, and fragments thereof (e.g., domain antibodies), as described below. Antibodies can be human, humanized, chimeric and / or affinity matured, as well as antibodies from other species such as mice, rabbits, llamas, etc. The term "antibody" is intended to include polypeptide products of B cells within the class of immunoglobulin polypeptides, which are capable of binding to a specific molecular antigen and are composed of two pairs of identical polypeptide chains, wherein each pair has one heavy chain (about 50 kDa-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain including a variable region of about 100 to about 130 or more amino acids, and each carboxyl-terminal portion of each chain including a constant region. See, e.g., Antibody Engineering (Borrebaeck, ed., 2nd ed., 1995); and Kuby, Immunology (3rd edition, 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies or humanized variants thereof, including those from camelid species (e.g., llamas or alpacas), intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen binding fragments) of any of the above, which refers 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 Fv (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. Specifically, the antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., antigen binding domains or molecules containing antigen binding sites that bind to antigens (e.g., one or more CDRs of antibodies). Such antibody fragments may 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, Vol. 22: pp. 189-224; Plückthun and Skerra, 1989, Meth. Enzymol. Vol. 178: pp. 497-515; and Day, Advanced Immunochemistry (2nd edition, 1990). The antibodies provided herein can be 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 antibodies can be agonistic antibodies or antagonistic antibodies. The antibodies may be neither agonistic nor antagonistic.

[0096] 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, an antigen is associated with a cell, e.g., is present on or in a cell.

[0097] A "complete" antibody is an antibody that comprises an antigen binding site as well as CL and at least heavy chain constant regions CH1, CH2 and CH3. The constant region may comprise a human constant region or an amino acid sequence variant thereof. In certain embodiments, the complete antibody has one or more effector functions.

[0098] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising VH and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain, which enables the sFv to form the structure required for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315, 1994.

[0099] The term "binding" refers to the interaction between molecules, including, for example, the formation of a complex. The interaction can be, for example, a non-covalent interaction, including hydrogen bonds, ionic bonds, hydrophobic interactions and / or van der Waals interactions. The complex can also include the combination of two or more molecules held together by covalent or non-covalent bonds, interactions or forces. The intensity of the total non-covalent interaction between a single antigen binding site on an antibody and a single epitope of a target molecule such as an antigen is the affinity of the antibody or functional fragment to the epitope. The ratio (koff / kon) of the dissociation rate (koff) and the association rate (kon) of a binding molecule (e.g., an antibody) to a monovalent antigen is the dissociation constant KD, which is inversely proportional to the affinity. The lower the KD value, the higher the affinity of the antibody. The KD value varies with different complexes of the antibody and the antigen, and depends on both kon and koff. The dissociation constant KD of the antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. The affinity at a binding site does not always reflect the true intensity of the interaction between the antibody and the antigen. When a complex antigen containing multiple repeating antigenic determinants (such as a multivalent antigen) contacts an antibody containing multiple binding sites, the interaction of the antibody with the antigen at one site will increase the probability of reacting at the second site. The strength of this multiple interaction between the multivalent antibody and the antigen is called avidity.

[0100] Terms related to the binding molecules described herein, such as "binding", "specifically binding" and similar terms are also used interchangeably herein and refer to binding molecules that specifically bind to an antigen, such as an antigen binding domain of a polypeptide. Binding molecules or antigen binding domains that bind or specifically bind to an antigen can be detected, for example, by immunoassays, Or other techniques known to those skilled in the art to identify. In some embodiments, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), when a binding molecule or antigen-binding domain binds to an antigen with a higher affinity than any cross-reactive antigen, it binds to or specifically binds to an antigen. Typically, a specific or selective reaction will be at least twice the background signal or noise, and can be more than 10 times the background signal or noise. See, for example, Fundamental Immunology, pp. 332-336 (Paul ed., 2nd edition, 1989) for discussion of binding specificity. In certain embodiments, the degree of binding of a binding molecule or antigen-binding domain to a "non-target" protein is less than about 10% of the binding of a binding molecule or antigen-binding domain to its specific target antigen, for example, as determined by FACS analysis or RIA. A binding molecule or antigen-binding domain that binds to an antigen includes a binding molecule or antigen-binding domain that can bind to an antigen with sufficient affinity, so that the binding molecule can be used as a therapeutic agent and / or diagnostic agent for, for example, a targeted antigen. In certain embodiments, the binding molecule or antigen binding domain that binds to an antigen has a dissociation constant (KD) of less than or equal to 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. In certain embodiments, the binding molecule or antigen binding domain binds to an antigen epitope that is conserved in antigens from different species.

[0101] In certain embodiments, a binding molecule or antigen binding domain may comprise a "chimeric" sequence in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as 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, Vol. 81: pp. 6851-6855). Chimeric sequences may include humanized sequences.

[0102] In certain embodiments, binding molecules or antigen-binding domains may include a portion of a "humanized" form of a non-human (e.g., camel, murine, non-human primate) antibody, which includes a sequence from a human immunoglobulin (e.g., receptor antibody), wherein the natural CDR residues are replaced by residues from non-human species such as camels, mice, rats, rabbits or non-human primates (e.g., donor antibodies) with the desired specificity, affinity and ability of the corresponding CDR. In some cases, one or more FR region residues of the human immunoglobulin sequence are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues that are not present in the receptor antibody or donor antibody. These modifications are made to further improve antibody performance. Humanized antibody heavy or light chains may include substantially all at least one or more variable regions, wherein all or substantially all CDRs correspond to those of non-human immunoglobulins, and all or substantially all FRs are those of human immunoglobulin sequences. In certain embodiments, humanized antibodies will include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a constant region of a human immunoglobulin. For more details, see Jones et al., Nature, Vol. 321: pp. 522-525, 1986; Riechmann et al., Nature, Vol. 332: pp. 323-329, 1988; Presta, Curr. Op. Struct. Biol., Vol. 2: pp. 593-596, 1992; Carter et al., Proc. Natl. Acad. Sci. USA, Vol. 89: pp. 4285-4289, 1992; U.S. Pat. Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213, and 6,054,297.

[0103] In certain embodiments, the binding molecule or antigen binding domain may comprise a portion of a "fully human antibody" or "human antibody", wherein these terms are used interchangeably herein and refer to antibodies comprising human variable regions and, for example, human constant regions. The binding molecule may comprise a single domain antibody sequence. In specific embodiments, these terms refer to antibodies comprising variable and constant regions of human origin. In certain embodiments, a "fully human" antibody may also encompass antibodies that bind to a polypeptide and are encoded by a nucleic acid sequence that is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence. The term "fully human antibody" includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al. (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). "Human antibody" refers to an antibody having an amino acid sequence corresponding to an amino acid sequence of an antibody produced by a human and / or that has been prepared using any technique for preparing human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. Various techniques known in the art can be used to produce human antibodies, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. Vol. 227: p. 381, 1991; Marks et al., J. Mol. Biol. Vol. 222: p. 581, 1991) and yeast display libraries (Chao et al., Nature Protocols Vol. 1: p. 755-768, 2006). Methods that can also be used to prepare human monoclonal antibodies are described in Cole et al., Monoclonal Antibodies and Cancer Therapy Vol. 77, 1985; Boerner et al., J. Immunol. Vol. 147 No. 1: p. 86-95, 1991; and van Dijk and van de Winkel, Curr. Opin. Pharmacol., Vol. 5: p. 368-374, 2001.Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, such as mice (see, e.g., Jakobovits, Curr. Opin. Biotechnol., Vol. 6, No. 5: pp. 561-566, 1995; Brüggemann and Taussing, Curr. Opin. Biotechnol., Vol. 8, No. 4: pp. 455-458, 1997; and U.S. Pat. Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, Vol. 103: pp. 3557-3562, 2006 for human antibodies generated via human B cell hybridoma technology.

[0104] In certain embodiments, the binding molecule or antigen binding domain may comprise a portion of a "recombinant human antibody", wherein the phrase includes human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from transgenic and / or transchromosomal animals (e.g., mice or cattle) of human immunoglobulin genes (see, e.g., Taylor, LD et al., Nucl. Acids Res., Vol. 20: pp. 6287-6295, 1992) or antibodies prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies may have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0105] In certain embodiments, the binding molecule or antigen binding domain may comprise a portion of a "monoclonal antibody", wherein the term as used herein refers to an antibody obtained from a substantially homogeneous antibody population, e.g., except for naturally occurring mutations that may be present in small amounts or well-known post-translational modifications such as amino acid phosphorylation or deamidation, methionine oxidation, or asparagine or glutamine deamidation, the individual antibodies that make up the population are identical, and each monoclonal antibody will generally recognize a single epitope on the antigen. In a specific embodiment, as used herein, a "monoclonal antibody" is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to any particular method for preparing an antibody. For example, monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma method first described by Kohler et al., Nature Vol. 256: p. 495, 1975, or may be prepared using recombinant DNA methods in bacteria or eukaryotic animals or plant cells (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques described in, for example, Clackson et al., Nature, Vol. 352: 624-628, 1991 and Marks et al., J. Mol. Biol., Vol. 222: 581-597, 1991. Other methods for preparing clonal cell lines and monoclonal antibodies expressed thereby are well known in the art. See, for example, Short Protocols in Molecular Biology (Ausubel et al., ed., 5th ed., 2002).

[0106] A typical 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the 4-chain unit is generally about 150,000 daltons. Each L chain is connected to the H chain by a covalent disulfide bond, and the two H chains are connected to each other by one or more disulfide bonds according to the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bonds. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) of each of the α chain and the γ chain and four CH domains of the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at its other end. VL is aligned with VH, and CL is aligned with the first constant domain (CH1) of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain and the 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, e.g., Basic and Clinical Immunology 71 (Stites et al., eds., 8th edition, 1994); and Immunobiology (Janeway et al., eds., 5th edition, 2001).

[0107] The term "Fab" or "Fab region" refers to the region of an antibody that binds to an antigen. Conventional IgGs typically include two Fab regions, each Fab region residing on one of the two arms of a Y-shaped IgG structure. Each Fab region is typically composed of a variable region and a constant region of each of a heavy chain and a light chain. More specifically, the variable region and constant region of the heavy chain in the Fab region are VH and CH1 regions, and the variable region and constant region of the light chain in the Fab region are VL and CL regions. The VH, CH1, VL, and CL in the Fab region can be arranged in various ways to impart antigen binding ability according to the present disclosure. For example, the VH and CH1 regions can be on one polypeptide, and the VL and CL regions can be on separate polypeptides, similar to the Fab regions of conventional IgGs. Alternatively, the VH, CH1, VL, and CL regions can all be on the same polypeptide and oriented in different orders, as described in more detail in the following section.

[0108] The term "variable region", "variable domain", "V region" or "V domain" refers to a portion of the light chain or heavy chain of an antibody, which is usually located at the amino terminus of the light chain or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and a length of about 100 to 110 amino acids in the light chain, and is used for the binding and specificity of each specific antibody to its specific antigen. The variable region of the heavy chain may be referred to as "VH". The variable region of the light chain may be referred to as "VL". The term "variable" refers to the fact that certain fragments of the variable region are widely different in sequence between antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, variability is not evenly distributed over the 110 amino acid span of the variable region. Instead, the V region consists of a stretch of about 15-30 amino acids called a framework region (FR) that is less variable (e.g., relatively unchanged), separated by shorter regions of greater variability (e.g., extreme variability) called "hypervariable regions", each of which is about 9-12 amino acids long. The heavy and light chain variable regions each contain four FRs, mainly in a β sheet configuration, connected by three hypervariable regions, which form loops connecting the β folded structure and in some cases form part of it. The hypervariable regions in each chain are held together closely together by the FRs and the hypervariable regions from the other chain, contributing to the formation of the antigen binding site of the antibody (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th edition, 1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions, such as antibody participation in antibody-dependent cellular toxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions between different antibodies are widely different in sequence. In a specific embodiment, the variable region is a human variable region.

[0109] The term "variable region residue numbering according to Kabat" or "amino acid position numbering as in Kabat" and variations thereof refer to the numbering system for heavy chain variable regions or light chain variable regions used for antibody compilation in Kabat et al. (supra). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of the FR or CDR of the variable domain. For example, the heavy chain variable domain may include 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, etc. according to Kabat). By comparing the homologous region of the antibody sequence with the "standard" Kabat numbering sequence, the Kabat residue numbering of a given antibody can be determined. When referring to the residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system (e.g., Kabat et al., supra) is generally used. When referring to residues in the constant region of an immunoglobulin heavy chain, the "EU numbering system" or "EU index" is generally used (e.g., the EU index as reported in Kabat et al., supra). The "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.

[0110] When used with reference to an antibody, the term "heavy chain" refers to a polypeptide chain of about 50 kDa-70 kDa, wherein the amino terminal portion includes a variable region of about 120 to 130 or more amino acids, and the carboxyl terminal portion includes a constant region. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types (e.g., isotypes), referred to as α (α), δ (δ), ε (ε), γ (γ), and μ (μ). Different heavy chains are of different sizes: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these different types of heavy chains produce antibodies of the five well-known classes (e.g., isotypes), namely IgA, IgD, IgE, IgG, and IgM, including the four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4.

[0111] When used in reference to an antibody, the term "light chain" refers to a polypeptide chain of about 25 kDa, wherein the amino terminal portion includes a variable region of about 100 to about 110 or more amino acids, and the carboxyl terminal portion includes 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.

[0112] 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 the VH β-sheet framework of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2 or L3) within the non-framework region of the VL β-sheet framework of an antibody. Thus, CDRs are variable region sequences interspersed within framework region sequences.

[0113] The CDR regions are well known to those skilled in the art and have been defined by well-known numbering systems. For example, the Kabat complementarity determining region (CDR) is based on sequence variability and is the most commonly used (see, e.g., Kabat et al., supra). "Chothia" refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. Vol. 19: pp. 901-917, 1987). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering, Vol. 2 (Kontermann and Dübel, ed., 2nd ed., 2010)). The "contact" hypervariable regions are based on 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., Vol. 27, No. 1: pp. 55-77, 2003). IMGT is an integrated information system dedicated to the study of immunoglobulins (IG), T cell receptors (TCR) and major histocompatibility complexes (MHC) of humans and other vertebrates. In this article, CDRs are referenced both according to the amino acid sequence and the position within the light chain or heavy chain. Since the "position" of CDRs within the structure of the immunoglobulin variable domain is conserved between species and is present in structures called loops, CDRs and framework residues are easily identified by using a numbering system that aligns the variable domain sequences according to structural features. This information can be used to transplant and replace CDR residues from immunoglobulins of one species into an acceptor framework, usually from a human antibody. Honegger and Plückthun developed an additional numbering system (AHon), J. Mol. Biol. No. 309: pp. 657-670, 2001. The correspondence between numbering systems, including, for example, Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art (see, for example, Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). Residues from each of these hypervariable regions or CDRs are exemplified in Table 1 below.

[0114] Table 1. Exemplary CDRs according to various numbering systems

[0115]

[0116] The boundary of a given CDR may vary according to the scheme used to identify it. Therefore, unless otherwise indicated, the terms "CDR" and "complementarity determining region" of a given antibody or its region (such as a variable region) and the individual CDRs (e.g., CDR-H1, CDR-H2) of an antibody or its region should be understood to encompass the complementary determining region defined by any known scheme as described above. In some cases, the scheme for identifying a specific CDR or CDR is specified, such as the CDR defined by the IMGT, Kabat, Chothia or Contact methods. In other cases, the specific amino acid sequence of CDR is given. It should be noted that the CDR region can also be defined by a combination of various numbering systems, such as a combination of Kabat and Chothia numbering systems or a combination of Kabat and IMGT numbering systems. Therefore, terms such as "CDRs set forth in a specific VH" include any CDR1 defined by the above-mentioned exemplary CDR numbering system, but are not limited thereto. Once a variable region (e.g., VH or VL) is given, it will be appreciated by those skilled in the art that the CDR in the region can be defined by different numbering systems or a combination thereof.

[0117] The hypervariable regions may include the following "extended hypervariable regions": 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.

[0118] The term "constant region" or "constant domain" refers to the carboxyl terminal portion of the light chain and the heavy chain, which is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions, such as interactions with Fc receptors. The term refers to the portion of the immunoglobulin molecule that has a more conserved amino acid sequence relative to the rest of the immunoglobulin (the variable region containing the antigen binding site). The constant region may include the CH1, CH2 and CH3 regions of the heavy chain and the CL region of the light chain.

[0119] The term "framework" or "FR" refers to those variable region residues that flank the CDRs. FR residues are present, for example, in chimeric, humanized, human domain antibodies (e.g., single domain antibodies), diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than hypervariable region residues or CDR residues.

[0120] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including, for example, a native sequence Fc region, a recombinant Fc region, and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl-terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during the production or purification of the antibody or by recombinant engineering of nucleic acids encoding the antibody heavy chain. Therefore, the composition of a complete antibody may include an antibody population with all K447 residues removed, an antibody population without the K447 residue removed, and an antibody population with and without a mixture of antibodies containing the K447 residue. A "functional Fc region" has an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors), etc. Such effector functions generally require an Fc region in combination with a binding region or binding domain (e.g., an antibody variable region or domain), and can be evaluated using various assays known to those skilled in the art. A "variant Fc region" includes an amino acid sequence that is different from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, the variant Fc region has at least one amino acid substitution compared to the Fc region of a native sequence Fc region or a parent polypeptide, for example, about one to about ten amino acid substitutions in the Fc region of a native sequence Fc region or a parent polypeptide, or about one to about five amino acid substitutions. The variant Fc region herein may have at least about 80% homology with the Fc region of a native sequence Fc region and / or a parent polypeptide, or at least about 90% homology therewith, for example, at least about 95% homology therewith.

[0121] As used herein, "epitope" is a term of the art and refers to a local 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 epitope or a conformational, non-linear or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be a continuous amino acid of a polypeptide (a "linear" epitope), or an epitope can comprise amino acids from two or more discontinuous regions of a polypeptide (a "conformational", "non-linear" or "discontinuous" epitope). Those skilled in the art will appreciate that, in general, a linear epitope may or may not depend on a secondary, tertiary or quaternary structure. For example, in some embodiments, a binding molecule binds to a group of amino acids, regardless of whether they are folded in a native three-dimensional protein structure. In other embodiments, the binding molecule requires that the amino acid residues constituting the epitope exhibit a specific conformation (e.g., bending, twisting, flipping or folding) in order to recognize and bind to the epitope.

[0122] "Percent (%) amino acid sequence identity" with respect to a peptide, polypeptide or antibody sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specified peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN. TM (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.

[0123] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to transplant one or more antigen-specifics to immune effector cells such as T cells or NK cells. In some embodiments, CAR comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain of NK cells and / or other receptors that are specific for one or more antigens (such as tumor antigens). "CAR-NK cells" refer to NK cells expressing CAR.

[0124] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interspersed with non-amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including but not limited to non-natural amino acids), as well as other modifications known in the art. It should be understood that because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, in certain embodiments, a "polypeptide" may appear as a single chain or as two or more related chains.

[0125] "Polynucleotide" or "nucleic acid" as used interchangeably herein refers to a polymer of nucleotides of any length and includes 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 may contain modified nucleotides, such as methylated nucleotides and their analogs. As used herein, "oligonucleotide" refers to a short, usually single-stranded synthetic polynucleotide, which is usually 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 and fully applicable to oligonucleotides. Cells that produce the binding molecules of the present disclosure may include parent hybridoma cells, as well as bacteria and eukaryotic host cells into which nucleic acids encoding antibodies have been introduced. Unless otherwise indicated, the left end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The 5' to 3' direction of addition of the nascent RNA transcript is called the transcription direction; the sequence region on the DNA chain having the same sequence as the RNA transcript, which is from the 5' to 5' end of the RNA transcript, is called the "upstream sequence"; the sequence region on the DNA chain having the same sequence as the RNA transcript, which is from the 3' to 3' end of the RNA transcript, is called the "downstream sequence".

[0126] "Isolated nucleic acid" is a nucleic acid, such as 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. "Isolated" nucleic acid molecules are nucleic acid molecules separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. In addition, "isolated" nucleic acid molecules, such as cDNA molecules, may be substantially free of other cell materials or culture media when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding a single domain antibody or an antibody as described herein are separated or purified. The term encompasses nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biosynthesized by heterologous systems. Substantially pure molecules may include isolated forms of the molecule. Specifically, the "isolated" nucleic acid molecule encoding the CAR described herein is a nucleic acid molecule identified and separated from at least one contaminant nucleic acid molecule, which is typically associated with a contaminant nucleic acid molecule in the environment in which it is produced.

[0127] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0128] As used herein, the term "operably linked" and similar phrases (e.g., gene fusion) when used with reference to nucleic acids or amino acids refer to the operational connection of nucleic acid sequences or amino acid sequences, respectively, 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 accurate production of nucleic acid molecules (e.g., RNA). In some embodiments, operably linked nucleic acid elements result in the transcription of open reading frames and ultimately produce polypeptides (i.e., expression of open reading frames). As another example, an operably linked peptide is one in which functional domains are at an appropriate distance from each other to confer the intended function of each domain.

[0129] The term "vector" refers to a material for carrying or containing a nucleic acid sequence, which includes, for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) as described herein, so that the nucleic acid sequence is introduced into a host cell. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include a selection sequence or a marker operable for stable integration into a host cell chromosome. In addition, the vector may include one or more selective marker genes and appropriate expression control sequences. The selective marker genes that may be included, for example, provide resistance to antibiotics or toxins, supplement nutritional deficiencies, or provide key nutrients that are not in the culture medium. The expression control sequence may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc. that are well known in the art. When two or more nucleic acid molecules are co-expressed (e.g., antibody heavy chain and light chain or antibody VH and VL), both nucleic acid molecules may be inserted into, for example, a single expression vector or a separate expression vector. For single vector expression, the encoding nucleic acid may be operably connected to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of nucleic acid molecules into a host cell may be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for gene product expression, or other suitable analytical methods to test the expression of the introduced nucleic acid sequence or its corresponding gene product. It will be understood by those skilled in the art that the nucleic acid molecule is expressed in an amount sufficient to produce the desired product, and it is further understood that the expression level can be optimized using methods well known in the art to obtain sufficient expression.

[0130] As used herein, the term "host" refers to an animal, such as a mammal (eg, a human).

[0131] As used herein, the term "host cell" refers to a particular subject cell that can be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may differ from the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in the progeny or due to integration of the nucleic acid molecule into the host cell genome.

[0132] As used herein, the term "autologous" means any material originating from the same individual that is later reintroduced into that individual.

[0133] As used herein, the term "allogeneic" refers to a transplant derived from a different individual of the same species.

[0134] As used herein, the term "transfected" or "transformed" or "transduced" refers to the process by which exogenous nucleic acids are transferred or introduced into host cells. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0135] As used herein, the term "separation" refers to the process of increasing the percentage of a substance in a composition. For example, separating one type of cell from a cell population refers to the process of producing a cell population in which the percentage of cells of this type is increased compared to the percentage of cells of this type in the original cell population. Therefore, when used in the context of one type of cell, the term "separated" does not mean that the isolated cell population contains 100% of this type of cell, but rather means that the percentage of this type of cell in the cell population is increased after the separation process.

[0136] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopeia, the European Pharmacopeia, or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

[0137] "Excipient" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, solvent or encapsulating material. Excipients include, for example, encapsulating materials or additives, such as absorption enhancers, antioxidants, binders, buffers, carriers, coatings, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, spices, preservatives, propellants, release agents, sterilants, sweeteners, solubilizers, wetting agents and mixtures thereof. The term "excipient" may also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete) or vehicle.

[0138] 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; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins such as 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 TM , polyethylene glycol (PEG) and PLURONICS TM Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed., 1990).

[0139] In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical formulation and suitable for use in contact with tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications 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, a pharmaceutically acceptable excipient is a pH buffered solution.

[0140] In some embodiments, excipient is a sterile liquid, such as water and oil, including those of petroleum, animal, plant or synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the composition (for example, pharmaceutical composition) is administered intravenously, water is an exemplary excipient. Saline solution and dextrose aqueous solution and glycerol solution can also be used as liquid excipients, especially for injection solution. Excipient can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. If necessary, the composition can also contain a small amount of wetting agent or emulsifier or pH buffer. The composition can be in the form of solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release preparation, etc. Oral composition, including preparation, can include standard excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0141] For example, a composition including a pharmaceutical compound may contain a binding molecule (eg, an antibody), eg, in isolated or purified form, together with a suitable amount of an excipient.

[0142] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of a single domain antibody or a therapeutic molecule or pharmaceutical composition comprising an agent and a single domain antibody provided herein sufficient to produce the desired result.

[0143] The terms "subject" and "patient" are used interchangeably. As used herein, in certain embodiments, the subject is a mammal, such as a non-primate or primate (e.g., a human). In specific embodiments, the subject is a human. In one embodiment, the subject is a mammal, such as a human, diagnosed with a disease or disorder. In another embodiment, the subject is a mammal, such as a human, at risk of developing a disease or disorder.

[0144] "Administering" refers to the act of injecting or otherwise physically delivering a substance present outside the body into a patient's body, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0145] As used herein, the term "treatment" refers to a reduction or improvement in the progression, severity and / or duration of a disease or condition resulting from the administration of one or more therapies. Treatment can be determined by assessing whether there has been a reduction, alleviation and / or relief of one or more symptoms associated with the underlying disorder, such that although the patient may still be afflicted with the underlying disorder, improvement in the patient is observed. The term "treatment" includes management and amelioration of a disease. The term "management" refers to the beneficial effects that a subject obtains from a therapy that does not necessarily result in a cure of the disease.

[0146] The terms "prevent" and "preventing" refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms (eg, diabetes or cancer).

[0147] The terms "about" and "approximately" mean 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.

[0148] As used in this disclosure and in the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0149] It should be understood that anywhere an embodiment is described herein with the term "comprising", other similar embodiments described according to "consisting of" and / or "consisting essentially of" are also provided. It should also be understood that anywhere an embodiment is described herein with the phrase "consisting essentially of", other similar embodiments described according to "consisting of" are also provided.

[0150] The term “between” as used in phrases such as “between A and B” or “between AB” refers to a range that includes both A and B.

[0151] The term "and / or" as used in phrases such as "A and / or B" is intended herein to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass 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).

[0152] 5.2. Methods for Preparing Immune Effector Cells for Transduction

[0153] In one aspect, provided herein are methods for preparing immune effector cells for transduction.

[0154] In some embodiments, the immune effector cell is a cytotoxic cell. In certain embodiments, the cytotoxic cell is a natural killer cell. In certain embodiments, the cytotoxic cell is a cytotoxic T lymphocyte. In certain embodiments, the immune effector cell is an αβT cell. In certain embodiments, the immune effector cell is a γδT cell.

[0155] In some embodiments, immune effector cells are contacted with one or more agents that increase the expression of low-density lipoprotein receptors (LDLR). In some embodiments, immune effector cells are contacted with one or more first agents that increase the expression of low-density lipoprotein receptors (LDLR). In some embodiments, immune effector cells are contacted with one or more second agents that suppress the intracellular antiviral defense mechanisms of immune effector cells. In some embodiments, immune effector cells are contacted with one or more third agents that enhance transduction.

[0156] In some embodiments, immune effector cells are contacted with one or more first agents that increase the expression of low-density lipoprotein receptors (LDLR) and one or more second agents that suppress the intracellular antiviral defense mechanism of immune effector cells. In certain embodiments, immune effector cells are contacted with the first agent before contacting with the second agent. In certain embodiments, immune effector cells are contacted with the first agent after contacting with the second agent. In certain embodiments, immune effector cells are contacted with the first agent while contacting with the second agent.

[0157] In some embodiments, immune effector cells are contacted with one or more first agents that increase the 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 while contacting with the third agent.

[0158] In some embodiments, immune effector cells are contacted with one or more second agents that suppress the intracellular antiviral defense mechanism of immune effector cells and one or more third agents that enhance transduction. In certain embodiments, immune effector cells are contacted with the second agent before contacting with the third agent. In certain embodiments, immune effector cells are contacted with the second agent after contacting with the third agent. In certain embodiments, immune effector cells are contacted with the second agent while contacting with the third agent.

[0159] In some embodiments, immune effector cells are contacted with one or more first agents that increase the expression of low-density lipoprotein receptors (LDLR), one or more second agents that suppress the intracellular antiviral defense mechanism of 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 with the second agent, and then with the third agent. In certain embodiments, immune effector cells are contacted with the first agent, then with the third agent, and then with the second agent. In certain embodiments, immune effector cells are contacted with the second agent, then with the first agent, and then with the third agent. In certain embodiments, immune effector cells are contacted with the second agent, then with the third agent, and then with the first agent. In certain embodiments, immune effector cells are contacted with the third agent, then with the first agent, and then with the second agent. In certain embodiments, immune effector cells are contacted with the third agent, then with the second agent, and then with the first agent. In certain embodiments, immune effector cells are contacted with the third agent, then with the second agent, and then with the first agent. In certain embodiments, immune effector cells are contacted with the first agent before contacting with the second agent and the third agent at the same time. In certain embodiments, the immune effector cell contacts the first agent after contacting the second agent and the third agent at the same time. In certain embodiments, the immune effector cell contacts the second agent before contacting the first agent and the third agent at the same time. In certain embodiments, the immune effector cell contacts the second agent after contacting the first agent and the third agent at the same time. In certain embodiments, the immune effector cell contacts the third agent before contacting the first agent and the second agent at the same time. In certain embodiments, the immune effector cell contacts the third agent after contacting the first agent and the second agent at the same time. In certain embodiments, the immune effector cell contacts the first agent, the second agent, and the third agent at the same time.

[0160] In certain embodiments, the first agent is a small molecule. In certain embodiments, the first agent is a macromolecule. 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.

[0161] In certain embodiments, the second agent is a 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor. In certain embodiments, the PDK1 inhibitor is a small molecule. In certain embodiments, the PDK1 inhibitor is a macromolecule. In certain embodiments, the PDK1 inhibitor is organic. In certain embodiments, the PDK1 inhibitor is inorganic. In certain embodiments, the PDK1 inhibitor is BX795.

[0162] 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 macromolecule. In certain embodiments, the third agent is organic. In certain embodiments, the third agent is inorganic. In certain embodiments, the third agent is Vectofusin. In certain embodiments, the third agent is Retronectin.

[0163] In certain embodiments, immune effector cells are contacted with a first agent that increases the expression of low-density lipoprotein receptors (LDLR), a second agent that suppresses the intracellular antiviral defense mechanism of immune effector cells, 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.

[0164] In some embodiments, the method further comprises transducing the nucleic acid into an immune effector cell. 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).

[0165] In certain embodiments, the nucleic acid comprises a promoter. In certain embodiments, the promoter is selected from the group consisting of a CAG promoter, a PGK promoter, an EF1a promoter, and an EFS promoter. 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.

[0166] 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.

[0167] 5.3. Methods for generating immune effector cells expressing exogenous functional receptors

[0168] On the other hand, provided herein is a method for producing immune effector cells expressing chimeric antigen receptors (CAR). The method for preparing immune effector cells for transducing nucleic acids encoding CAR is described in detail in Section 4.2 above. In some embodiments, immune effector cells are cytotoxic cells. In certain embodiments, cytotoxic cells are natural killer cells. In certain embodiments, cytotoxic cells are cytotoxic T lymphocytes.

[0169] 5.3.1. Chimeric Antigen Receptors

[0170] 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.

[0171] Signal peptide

[0172] In certain embodiments, the CAR provided herein may include a signal peptide (also referred to as a signal sequence) at the N-terminus of the polypeptide. In general, a signal peptide is a peptide sequence that targets a polypeptide to a desired site in a cell. In some embodiments, the signal peptide targets the secretory pathway of the effector molecule to the cell and will allow the effector molecule to be integrated and anchored into a lipid bilayer. For the use of compatible signal peptides in the CAR described herein, including signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences, it will be apparent to those skilled 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 chains.

[0173] Extracellular antigen binding domain

[0174] The extracellular antigen binding domain of CAR described herein includes one or more antigen binding domains. In some embodiments, the extracellular antigen binding domain of CAR provided herein is monospecific. In other embodiments, the extracellular antigen binding domain of CAR provided herein is multispecific. In some embodiments, the extracellular antigen binding domain includes two or more antigen binding domains, which are directly fused to each other via a peptide bond or via a peptide linker.

[0175] In some embodiments, the extracellular antigen binding domain constitutes an antibody or a fragment thereof. For example, the binding domain may be derived from a monoclonal antibody (including agonists, antagonists, neutralizing antibodies, full-length or complete monoclonal antibodies), antibodies with multi-epitope or mono-epitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed by at least two complete antibodies (e.g., bispecific antibodies, as long as they exhibit the desired biological activity), single-chain antibodies and fragments thereof. Antibodies may be human, humanized, chimeric and / or affinity mature, as well as antibodies from other species such as mice, rabbits, llamas, and the like. In some embodiments, antibodies include polypeptide products of B cells within the immunoglobulin polypeptide class, which are capable of binding to specific molecular antigens and are composed of two pairs of identical polypeptide chains, wherein each pair has a heavy chain (about 50 kDa-70 kDa) and a light chain (about 25 kDa), each amino terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxyl terminal portion of each chain includes a constant region. See, for example, Antibody Engineering (Borrebaeck, ed., 2nd ed., 1995); and Kuby, Immunology (3rd edition, 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies or humanized variants thereof, including those from camelid species (e.g., llamas or alpacas), intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen binding fragments) of any of the above, which refers 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 Fv (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. Specifically, the antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., antigen binding domains or molecules containing antigen binding sites that bind to antigens (e.g., one or more CDRs of antibodies). Such antibody fragments may 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, Vol. 22: pp. 189-224; Plückthun and Skerra, 1989, Meth. Enzymol. Vol. 178: pp. 497-515; and Day, Advanced Immunochemistry(2nd edition, 1990). The antibodies provided herein can be 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 antibodies can be agonistic antibodies or antagonistic antibodies. The antibodies may be neither agonistic nor antagonistic.

[0176] In a specific embodiment, the extracellular antigen binding domain of the CAR of the present invention comprises a single-chain Fv (sFv or scFv). ScFv is an antibody fragment comprising VH and VL antibody domains connected into a single polypeptide chain. Preferably, the scFv polypeptide also includes a polypeptide linker between the VH domain and the VL domain, which enables the sFv to form a structure required for antigen binding. See Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, ed., Springer-Verlag, New York, pp. 269-315 (1994).

[0177] In another specific embodiment, the extracellular antigen binding domain of the CAR of the present invention constitutes one or more single domain antibodies (sdAb). The sdAbs can be of the same or different sources and can be of the same or different sizes. Exemplary sdAbs include, but are not limited to, heavy chain variable domains from heavy chain-only antibodies (e.g., VHH or V NAR ), binding molecules naturally lacking light chains, single domains derived from conventional 4-chain antibodies (such as V H or V L ), humanized antibodies of only heavy chains, 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, including the single domain antibodies described above in the present disclosure, can be used to construct the CAR described herein. sdAb can be derived from any species, including but not limited to mice, rats, humans, camels, llamas, lampreys, fish, sharks, goats, rabbits, and cattle. The single domain antibodies considered herein also include naturally occurring single domain antibody molecules from species other than Camelidae and sharks.

[0178] In some embodiments, sdAbs are derived from naturally occurring single-domain antigen-binding molecules, which are called heavy chain antibodies lacking 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, Vol. 363: pp. 446-448, 1993. For the sake of clarity, the variable domains derived from heavy chain molecules that naturally lack light chains are referred to herein as VHH to distinguish them from the conventional V of four-chain immunoglobulins. H Distinguish. Such VHH can be derived from antibodies produced in Camelidae species, such as camels, llamas, llamas, dromedaries, alpacas and guanacos. Other species except Camelidae can produce heavy chain molecules that naturally lack light chains, and such VHH are within the scope of the present disclosure. In addition, humanized versions of VHH and other modifications and variants are also considered and within the scope of the present disclosure. In some embodiments, sdAb is derived from the variable region of immunoglobulins found in cartilaginous fish. For example, sdAb can be derived from an immunoglobulin isotype called a novel antigen receptor (NAR) found in shark serum. The method for producing a single domain molecule derived from a NAR variable region ("IgNAR") is described in WO 03 / 014161 and Streltsov, Protein Sci. Vol. 14: pp. 2901-2909, 2005.

[0179] In some embodiments, naturally occurring VHH domains for a particular antigen or target may be obtained from a (primary or immune) library of Camelidae VHH sequences. Such methods may or may not involve the use of the antigen or target, or at least a portion, fragment, antigenic determinant or epitope thereof, to screen such libraries 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, a synthetic or semisynthetic library derived from a modified (primary or immune) VHH library may be used, such as a VHH library obtained from a (primary or immune) VHH library by techniques such as random mutagenesis and / or CDR shuffling, as described, for example, in WO00 / 43507.

[0180] In some embodiments, sdAb is recombinant, CDR-grafted, humanized, camelized, de-immunized and / or produced in vitro (e.g., selected by phage display). In some embodiments, the amino acid sequence of the framework region can be changed by "camelization" of specific amino acid residues in the framework region. Camelization refers to one or more amino acid residues in the amino acid sequence of the (naturally occurring) VH domain of a conventional 4-chain antibody being replaced or substituted by one or more amino acid residues appearing in the corresponding position in the VHH domain of a heavy chain antibody. This can be performed in a manner known in the art, which is clear to those skilled in the art. Such "camelizing" substitutions are preferably inserted and / or present at amino acid positions that form and / or occur at the VH-VL interface and / or at the so-called Camelidae Hallmark residues, as defined herein (see, e.g., WO 94 / 04678, Davies and Riechmann FEBS Letters Vol. 339: pp. 285-290, 1994; Davies and Riechmann, Protein Engineering Vol. 9 No. 6: pp. 531-537, 1996; Riechmann, J. Mol. Biol. Vol. 259: pp. 957-969, 1996; and Riechmann and Muyldermans, J. Immunol. Meth. Vol. 231: pp. 25-38, 1999).

[0181] In some embodiments, the sdAb is a human single domain antibody produced by a transgenic mouse or rat expressing a human heavy chain segment. See, e.g., US20090307787, U.S. Pat. No. 8,754,287, US20150289489, US20100122358, and WO2004049794.

[0182] In some embodiments, a single domain antibody is generated from a conventional four-chain antibody. See, e.g., EP 0 368684; Ward et al., Nature, Vol. 341, No. 6242: 544-546, 1989; Holt et al., Trends Biotechnol., Vol. 21, No. 11: 484-490, 2003; WO 06 / 030220; and WO 06 / 003388.

[0183] In some embodiments, the extracellular antigen binding domain comprises a humanized antibody or fragment thereof. A humanized antibody may comprise a human framework region and a human constant region sequence.

[0184] Humanized antibodies can be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Biology, 1993; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089). Immunology, Vol. 28, No. 4 / 5: pp. 489-498; Studnicka et al., 1994, Protein Engineering, Vol. 7, No. 6: pp. 805-814; and Roguska et al., 1994, PNAS, Vol. 91: pp. 969-973), chain shuffling (U.S. Pat. No. 5,565,332), and techniques disclosed in, for example, the following references: U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, WO 93 / 17105, Tan et al., J. Immunol., Vol. 169: pp. 1119-1125, 2002; Caldas et al., Protein Eng., Vol. 13, No. 5: pp. 353-360, 2000; Morea et al., Methods, Vol. 20, No. 3: pp. 267-279, 2000; Baca et al., J. Biol. Chem. Vol. 272, No. 16: pp. 10678-10684, 1997; Roguska et al., Protein Eng., Vol. 9, No. 10: pp. 895-904, 1996; Couto et al., Cancer Res., Vol. 55 (23 Suppl.): pp. 5973s-5977s, 1995; Couto et al., Cancer Res., Vol. 55, No. 8: pp. 1717-1722, 1995; Sandhu JS, Gene, Vol. 150 No. 2: pp. 409-410, 1994; and Pedersen et al., J. Mol. Biol. Vol. 235 No. 3: pp. 959-973, 1994. See also U.S. Patent Publication No. US 2005 / 0042664 A1 (February 24, 2005), each of which is incorporated herein by reference in its entirety.

[0185] Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are generally referred to as "import" residues, which are generally taken from the "import" variable domain. Humanization can be performed, for example, by replacing the corresponding sequence of a human antibody with a hypervariable region sequence according to Jones et al., 1986, Nature Vol. 321: pp. 522-525; Riechmann et al., 1988, Nature Vol. 332: pp. 323-327; and Verhoeyen et al., 1988, Science Vol. 239: pp. 1534-1536).

[0186] In some cases, humanized antibodies are constructed by CDR transplantation, wherein the amino acid sequence of six CDRs of a parent non-human (e.g., rodent) antibody is transplanted onto a human antibody framework. For example, Padlan et al. determined that only about one-third of the residues in the CDR actually contact the antigen, and referred to these residues as "specificity determining residues" or SDRs (Padlan et al., 1995, FASEB J. Vol. 9: pp. 133-139). In SDR grafting technology, only SDR residues are grafted onto a human antibody framework (see, e.g., Kashmiri et al., 2005, Methods Vol. 36: pp. 25-34).

[0187] The selection of human variable domains (both light and heavy chains) for preparing humanized antibodies is important for reducing antigenicity. For example, according to the so-called "best fit" method, the sequence of the variable domains of non-human (e.g., rodent) antibodies is screened for the entire library of known human variable domain sequences. The human sequence closest to rodents can be selected as the human framework of humanized antibodies (Sims et al., 1993, J.Immunol. Vol. 151: pp. 2296-2308; and Chothia et al., 1987, J.Mol.Biol. Vol. 196: pp. 901-917). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a specific light chain or heavy chain subgroup. The same framework can be used for several different humanized antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA Vol. 89: pp. 4285-4289; and Presta et al., 1993, J. Immunol. Vol. 151: pp. 2623-2632). In some cases, the framework is derived from the consensus sequence of the most abundant human subclass VL6 subgroup I (VL6I) ​​and VH subgroup III (VHIII). In another approach, human germline genes are used as the source of the framework region.

[0188] In an alternative paradigm based on CDR comparison, called superhumanization, FR homology is irrelevant. The method consists of a comparison of non-human sequences with a functional human germline genomic library. Then select those genes that encode the same or closely related canonical structures for the mouse sequence. Next, among the genes that share a canonical structure with non-human antibodies, select the gene with the highest homology in the CDR as a FR donor. Finally, non-human CDRs are grafted onto these FRs (see, for example, Tan et al., 2002, J. Immunol. Vol. 169: pp. 1119-1125).

[0189] It is also generally desired that the antibody be humanized while retaining its affinity for the antigen and other favorable biological properties. To achieve this goal, according to a method, humanized antibodies are prepared using a three-dimensional model of the parent and humanized sequences by the analytical process of the parent sequence and various conceptual humanized products. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. These include, for example, WAM (Whitelegg and Rees, 2000, Protein Eng. Vol. 13: pp. 819-824), Modeller (Sali and Blundell, 1993, J. Mol. Biol. Vol. 234: pp. 779-815) and Swiss PDB Viewer (Guex and Peitsch, 1997, Electrophoresis Vol. 18: pp. 2714-2723). Examination of these displays enables analysis of the likely role of the residues in the functioning 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 characteristics, such as increased affinity for the target antigen, can be achieved. In general, the hypervariable region residues are directly and substantially most involved in influencing antigen binding.

[0190] Another method for antibody humanization is based on a measure of antibody humanization called human chain content (HSC). The method compares the mouse sequence with the repertoire of human germline genes, and the difference is scored as HSC. The target sequence is then humanized by maximizing its HSC rather than using global identity measurements to generate a variety of different humanized variants (Lazar et al., 2007, Mol. Immunol., Vol. 44: pp. 1986-1998).

[0191] In addition to the above methods, empirical methods can also be used to generate and select humanized antibodies. These methods include methods based on large gene libraries for generating humanized variants and using enrichment techniques or high-throughput screening techniques to select the best clones. Antibody variants can be separated from phage, ribosome and yeast display libraries and separated by bacterial colony screening (see, for example, Hoogenboom, 2005, Nat. Biotechnol. Vol. 23: pp. 1105-1116; Dufner et al., 2006, Trends Biotechnol. Vol. 24: pp. 523-529; Feldhaus et al., 2003, Nat. Biotechnol. Vol. 21: pp. 163-170; and Schlapschy et al., 2004, Protein Eng. Des. Sel., Vol. 17: pp. 847-860).

[0192] In the FR library method, a collection of residue variants is introduced at a specific position in the FR, and the library is subsequently screened to select the FR that best supports the transplanted CDR. The residues to be replaced may include some or all of the "Vernier" residues identified as potentially contributing to the CDR structure (see, e.g., Foote and Winter, 1992, J. Mol. Biol. Vol. 224: pp. 487-499), or a more limited set of target residues identified by Baca et al. (1997, J. Biol. Chem. Vol. 272: pp. 10678-10684).

[0193] In FR shuffling, the complete FR is combined with a non-human CDR, rather than generating a combinatorial library of selected residue variants (see, e.g., Dall'Acqua et al., 2005, Methods Vol. 36: pp. 43-60). A two-step method can be used: a first step of humanizing VL, and then screening the library for binding with humanized VH. Alternatively, a one-step FR shuffling method can be used. This method has been shown to be more effective than two-step screening because 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., Vol. 44: pp. 3049-3060).

[0194] The "human source transformation" method is based on the experimental identification of the necessary minimum specific determinant (MSD), and is based on the sequential replacement of non-human fragments into the human FR library and the evaluation of binding. It starts from the CDR3 region of the non-human VH chain and the VL chain, and the other regions of the non-human antibody are gradually replaced with human FRs, including CDR1 and CDR2 of both VH and VL. The method generally results in epitope retention and identification of antibodies with multiple subclasses of different human V-segment CDRs. Human source transformation allows the separation of antibodies with 91%-96% homology to human germline gene antibodies (see, e.g., Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007).

[0195] "Human engineering" methods involve changing non-human antibodies or antibody fragments such as mice or chimeric antibodies or antibody fragments by making specific changes to the amino acid sequence of the antibody, so as to produce modified antibodies with reduced immunogenicity in humans, which still retain the desired binding properties of the original non-human antibody. Typically, the technology involves classifying the amino acid residues of non-human (e.g., mouse) antibodies as "low risk", "medium risk" or "high risk" residues. Classification is performed using an overall risk / reward calculation that evaluates the predicted benefit of a specific substitution (e.g., for immunogenicity in humans) for the risk that substitution will affect the folding of the resulting antibody. Specific human amino acid residues to be substituted at a given position (e.g., low or medium risk) of a non-human (e.g., mouse) antibody sequence can be selected by comparing the amino acid sequence from the variable region of a non-human antibody with the corresponding region of a specific or shared human antibody sequence. The amino acid residues at low or medium risk positions in the non-human sequence can replace the corresponding residues in the human antibody sequence according to the comparison. Techniques for making human engineered proteins are described in more detail in Studnicka et al., 1994, Protein Engineering, Vol. 7: pp. 805-814; U.S. Pat. Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619; and PCT Publication WO 93 / 11794.

[0196] For example, composite human antibodies can be used TM Composite human antibodies are produced using the Antitope technology (Antitope Ltd., Cambridge, United Kingdom). In order to produce composite human antibodies, variable region sequences are designed from fragments of multiple human antibody variable region sequences in a manner that avoids T cell epitopes, thereby minimizing the immunogenicity of the resulting antibodies. Such antibodies may include human constant region sequences, such as human light chain and / or heavy chain constant regions.

[0197] Deimmunized antibodies are antibodies in which T cell epitopes have been removed. Methods for preparing deimmunized antibodies have been described. See, for example, Jones et al., Methods Mol Biol. 2009; Vol. 525: pp. 405-423, xiv, and De Groot et al., Cell. Immunol., Vol. 244: pp. 148-153, 2006). Deimmunized antibodies contain variable regions and human constant regions depleted of T cell epitopes. In short, the VH and VL of the antibody are cloned, and then T cell epitopes are identified by testing overlapping peptides derived from the VH and VL of the antibody in a T cell proliferation assay. T cell epitopes are identified via computer methods to identify peptides that bind to human MHC class II. Mutations are introduced into VH and VL to eliminate binding to human MHC class II. Mutated VH and VL are then used to generate deimmunized antibodies.

[0198] In some embodiments, the extracellular antigen binding domain comprises multiple binding domains. In some embodiments, the extracellular antigen binding domain constitutes a multispecific antibody or a fragment thereof. In other embodiments, the extracellular antigen binding domain constitutes a multivalent antibody or a fragment thereof. The term "specificity" refers to the selective recognition of a specific epitope of an antigen by an antigen-binding protein. The term "multispecific" as used herein means that an antigen-binding protein has two or more antigen binding sites, wherein at least two antigen binding sites bind to different antigens. The term "valence" as used herein means that there is a specified number of binding sites in an antigen-binding protein. A full-length antibody has two binding sites and is divalent. Therefore, the terms "trivalent", "tetravalent", "pentavalent" and "hexavalent" respectively represent that there are two binding sites, three binding sites, four binding sites, five binding sites and six binding sites in an antigen-binding protein.

[0199] Multispecific antibodies such as bispecific antibodies are antibodies with binding specificity to at least two different antigens. Methods for preparing multispecific antibodies are known in the art, such as by co-expression of two immunoglobulin heavy chain-light chain pairs, wherein the two heavy chains have different specificities (see, e.g., Milstein and Cuello, 1983, Nature Vol. 305: pp. 537-540). For detailed information on generating multispecific antibodies (e.g., bispecific antibodies), see, e.g., Bispecific Antibodies (Kontermann ed., 2011).

[0200] The antibodies disclosed herein may be multivalent antibodies (e.g., tetravalent antibodies) having two or more antigen binding sites, which can be easily produced by recombinantly expressing nucleic acids encoding polypeptide chains of antibodies. In certain embodiments, the multivalent antibody comprises (or consists of) three to about eight antigen binding sites. In one such embodiment, the multivalent antibody comprises (or consists of) four antigen binding sites. The 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, wherein VD1 is the first variable domain, VD2 is the second variable domain, Fc is a 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: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein may also comprise at least two (e.g., four) light chain variable domain polypeptides. The multivalent antibodies herein may, for example, comprise 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.

[0201] There are multiple binding domains in the extracellular antigen binding domain of the CAR of the present invention. Different domains can be fused to each other via a peptide linker. In some embodiments, the domains are directly fused to each other without any peptide linker. The peptide linker may be the same or different. Each peptide linker may have the same or different length and / or sequence, depending on the structural features and / or functional features of different domains. Each peptide linker can be selected and optimized independently. The length, flexibility and / or other characteristics of the peptide linker used in CAR may have a certain influence on the characteristics including but not limited to affinity, specificity or avidity for one or more specific antigens or epitopes. In some embodiments, the peptide linker comprises flexible residues (such as glycine and serine) so that adjacent domains move freely relative to each other. For example, a glycine-serine doublet may be a suitable peptide linker.

[0202] The peptide linker can have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody can be used as a linker. See, for example, WO1996 / 34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n 、(GSGGS) n 、(GGGS) n and (GGGGS) n, wherein 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, such as WO2016014789, WO2015158671, WO2016102965, US20150299317, WO2018067992, US7741465, Colcher et al., J.Nat.Cancer Inst. Vol. 82: pp. 1191-1197, 1990 and Bird et al., Science Vol. 242: pp. 423-426, 1988 are also included in the CAR provided herein, and the disclosure of each of the above references is incorporated herein by reference.

[0203] 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 associated with a special disease state on a target cell. In some embodiments, the antigen is a tumor antigen. Tumors express many proteins that can be used as target antigens for immune responses, especially T cell-mediated immune responses. The antigen targeted by CAR can be an antigen on a single diseased cell or an antigen expressed on different cells that each contributes to the disease. The antigen targeted by CAR can be directly or indirectly involved in the disease.

[0204] Tumor antigens are proteins produced by tumor cells that can trigger an immune response, particularly a T cell-mediated immune response. Exemplary tumor antigens include, but are not limited to, glioma-associated antigen, 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, mut hsp70-2, M-CSF, prostate enzymes, 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, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0205] In some embodiments, tumor antigens include one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express many proteins that can be used 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 oncogene HER2 / Neu / ErbB-2. Another group of target antigens is carcinoembryonic antigens, such as carcinoembryonic antigen (CEA).

[0206] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and do not appear on other cells in the body. TAA-related antigens are not unique to tumor cells, but are also expressed on normal cells under conditions that fail to induce an immune tolerance state to the antigen. The expression of antigens on tumors can occur under conditions that enable 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 cannot respond, or they can be antigens that are usually present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0207] Non-limiting examples of TSA or TAA antigens include: differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER2 / neu; unique tumor antigens generated by chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7.

[0208] 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, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-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, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP and TPS.

[0209] Hinge area

[0210] In some embodiments, the CAR provided herein comprises a hinge domain between an extracellular antigen binding domain and a transmembrane domain. The hinge domain is an amino acid segment usually found between two domains of a protein, and can allow the flexibility of the protein and one or two 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.

[0211] The hinge domain of an antibody (such as an IgG, IgA, IgM, IgE or IgD antibody) is compatible for use in a pH-dependent chimeric receptor system as described herein. In some embodiments, the hinge domain is a hinge domain that connects the constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge domain is a hinge domain of an antibody and comprises the hinge domain of an antibody and one or more constant regions of an antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of an antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH2 constant region and CH3 constant region of an antibody. 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 the hinge region and the CH2 constant region and CH3 constant region of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and CH3 constant region of an IgG1 antibody.

[0212] Non-naturally occurring peptides can also be used as hinge domains of the chimeric receptors described herein. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand binding domain of the Fc receptor and the N-terminus of the transmembrane domain is a peptide linker, such as a (GxS)n linker, wherein x and n can independently be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or greater.

[0213] The hinge domain may contain about 10-100 amino acids, such as about 15-75 amino acids, 20-50 amino acids, or any one of 30-60 amino acids. In some embodiments, the hinge domain may be 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.

[0214] 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 compatible 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 and confers flexibility to the chimeric receptor.

[0215] Transmembrane domain

[0216] The CAR of the present disclosure comprises a transmembrane domain that can be fused directly or indirectly to an extracellular antigen binding domain. The transmembrane domain can be derived from a natural or synthetic source. As used herein, a "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. A transmembrane domain compatible with use in the CAR described herein can be obtained from a naturally occurring protein. Alternatively, it can be a synthetic, non-naturally occurring protein segment, such as a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0217] The membrane-spanning domain is classified based on the three-dimensional structure of the membrane-spanning domain. For example, the membrane-spanning domain can form an alpha helix, a complex of more than one alpha helix, a β-barrel or any other stable structure that can cross the phospholipid bilayer of the cell. In addition, the membrane-spanning domain can also or alternatively be classified based on the membrane-spanning domain topology (including the number of times the membrane-spanning domain passes through the membrane and the orientation of the protein). For example, a single membrane-spanning protein passes through the cell membrane once, and multiple membrane-spanning proteins pass through the cell membrane at least twice (for example, 2, 3, 4, 5, 6, 7 times or more times). Membrane proteins can be defined as type I, type II or type III, depending on their ends and the topological structure of the membrane-spanning segment relative to the inside and outside of the cell. Type I membrane proteins have a single membrane-spanning region and are oriented so that the N-terminal of the protein is present in the cell extracellular side of the lipid bilayer of the cell, and the C-terminal of the protein is present in the cytoplasm side. Type II membrane proteins also have a single membrane-spanning region, but are oriented so that the C-terminal of the protein is present in the cell extracellular side of the lipid bilayer of the cell, and the N-terminal of the protein is present in the cytoplasm side. Type III membrane proteins have multiple transmembrane segments and can be further subdivided based on the number of transmembrane segments and the location of the N- and C-termini.

[0218] In some embodiments, the transmembrane domain of CAR described herein is derived from a single transmembrane protein of type I. In some embodiments, the transmembrane domain from multiple transmembrane proteins is also compatible for use in CAR described herein. Multiple transmembrane proteins may include composite (at least 2, 3, 4, 5, 6, 7 or more) alpha helices or beta folded structures. In some embodiments, the N-terminal and C-terminal of multiple transmembrane proteins are present on opposite sides of the lipid bilayer, such as the N-terminal of the protein is present on the cytoplasmic side of the lipid bilayer, and the C-terminal of the protein is present on the extracellular side.

[0219] The transmembrane domain used in the CAR described herein may also include 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 fold. In some embodiments, the protein segment is about 15-100 amino acids. In some embodiments, the protein segment is at least about 20 amino acids, for example, 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, for example, in U.S. Patent No. 7,052,906 and PCT Publication No. WO 2000 / 032776, the relevant disclosures of which are incorporated herein by reference.

[0220] The membrane-spanning domain provided herein may include a membrane-spanning region and a cytoplasmic region located at the C-terminal side of the membrane-spanning domain. The cytoplasmic region of the membrane-spanning domain may include three or more amino acids, and in some embodiments, helps to orient the membrane-spanning domain in the lipid bilayer. In some embodiments, one or more cysteine ​​residues are present in the membrane-spanning region of the membrane-spanning domain. In some embodiments, one or more cysteine ​​residues are present in the cytoplasmic region of the membrane-spanning domain. In some embodiments, the cytoplasmic region of the membrane-spanning domain includes positively charged amino acids. In some embodiments, the cytoplasmic region of the membrane-spanning domain includes amino acids, i.e., arginine, serine, and lysine.

[0221] In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane domain of the CAR provided herein comprises an artificial hydrophobic sequence. For example, phenylalanine, tryptophan and valine triplets may be present at the C-terminus of the transmembrane domain. In some embodiments, the transmembrane region mainly comprises hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a polyleucine-alanine sequence. The hydrophilicity or hydrophobic or hydrophilic properties of a protein or protein segment can be assessed by any method known in the art, such as Kyte and Doolittle hydrophilicity analysis.

[0222] In some embodiments, the transmembrane domain of the CAR comprises a transmembrane domain selected from the group consisting of the alpha, beta or zeta chain of a 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 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL-2Rβ, IL-2Rγ, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD15 0, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or NKG2C.

[0223] Intracellular signaling domain

[0224] The intracellular signaling domain in the CAR provided herein is responsible for activating at least one normal effector function of the immune effector cell expressing CAR. The term "effector function" refers to a specialized function of a cell. The effector function of NK cells can be, for example, cytolytic activity, including the secretion of cytokines. Therefore, the term "cytoplasmic signaling domain" refers to a portion of a protein that transduces effector function signals and instructs cells to perform specialized functions. Although the entire cytoplasmic signaling domain can generally be used, in many cases, it is not necessary to use the entire chain. In terms of using a truncated portion of a cytoplasmic signaling domain, such a truncated portion can be used instead of a complete chain, as long as it transduces an effector function signal. Therefore, the term cytoplasmic signaling domain is intended to include any truncated portion of a cytoplasmic signaling domain sufficient to transduce an effector function signal.

[0225] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell. "Primary intracellular signaling domain" refers to a cytoplasmic signaling sequence that acts in a stimulating manner to induce immune effector function. In some embodiments, the primary intracellular signaling domain contains a signaling motif known as an activation motif based on immunoreceptor tyrosine or ITAM. As used herein, "ITAM" is a conserved protein motif that is typically present in the tail of a signaling molecule expressed in many immune cells. The motif may include two repeats of an amino acid sequence YxxL / I separated by 6-8 amino acids, wherein each x is independently any amino acid, producing a conserved motif YxxL / Ix(6-8)YxxL / I. ITAMs within signaling molecules are important for intracellular signal transduction, which is mediated at least in part by phosphorylation of tyrosine residues in ITAMs after activation of signaling molecules. ITAMs can also act as docking sites for other proteins involved in signaling pathways. Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3ze, FcRγ (FCER1G), FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0226] co-stimulatory signaling domain

[0227] In some embodiments, CAR includes at least one co-stimulatory signaling domain. As used herein, the term "co-stimulatory 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). In addition to stimulating antigen-specific signals, many immune effector cells require co-stimulation to promote cell proliferation, differentiation and survival, and to activate the effector functions of cells.

[0228] The costimulatory signaling domain of the chimeric receptor described herein can be a cytoplasmic signaling domain from a costimulatory protein that transduces signals 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 a NK cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the immune cell, such as, but not limited to, proliferation and survival.

[0229] 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 any one of about 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 primary intracellular signaling domain (such as a cytoplasmic signaling domain of CD3z) and one or more costimulatory signaling domains. In some embodiments, the one or more costimulatory signaling domains and the primary intracellular signaling domain (such as a cytoplasmic signaling domain of CD3z) are fused to each other via an optional peptide linker. The primary intracellular signaling domain and the one or more costimulatory signaling domains can be arranged in any suitable order. In some embodiments, the one or more co-stimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3z). Multiple co-stimulatory signaling domains can provide additive or synergistic stimulation.

[0230] The activation of the costimulatory signaling domain in the host cell (e.g., immune cell) can induce cells to increase or reduce the production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival and / or cytotoxicity. The costimulatory signaling domain of any costimulatory molecule can be compatible for use in CAR described herein. The type of costimulatory signaling domain is selected based on factors such as the following: the type of immune effector cell (e.g., T cell, NK cell, macrophage, neutrophil or eosinophil) and the desired immune effector function (e.g., ADCC effect) in which the effector molecule will express.Examples of costimulatory signaling domains used in CARs can be cytoplasmic signaling domains of costimulatory proteins, including but not limited to 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 / TNFSF8, 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 any other co-stimulatory molecules, such as 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 co-stimulatory signaling domains are selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

[0231] In some embodiments, the intracellular signaling domain in the CAR of the present disclosure includes a costimulatory signaling domain (i.e., 4-1BB) derived from CD137. In some embodiments, the intracellular signaling domain includes 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 costimulatory signaling domain described herein, so that the costimulatory signaling domain can regulate the immune response of immune cells. In some embodiments, the costimulatory signaling domain comprises up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8) compared to the wild-type counterpart. Such costimulatory signaling domains comprising one or more amino acid variations may be referred to as variants. Relative to a costimulatory signaling domain that does not comprise a mutation, a mutation in the amino acid residues of the costimulatory signaling domain may result in an increase in signal transduction and an enhanced stimulation of the immune response. Mutation of amino acid residues in the costimulatory signaling domain can result in reduced signal transduction and reduced stimulation of an immune response relative to a costimulatory signaling domain that does not contain the mutation.

[0232] Exemplary CARs

[0233] In some embodiments, a CAR provided herein comprises the amino acid sequence of any one of the CARs exemplified below in Section 6. In some embodiments, a CAR provided herein comprises the amino acid sequence of any one of the CARs known to those skilled in the art.

[0234] In some embodiments, the CAR provided herein comprises an amino acid sequence with a certain percentage of identity relative to any of the CARs exemplified in Section 6 below. In some embodiments, the CAR provided herein comprises an extracellular domain or consists of an extracellular domain with 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 of the CARs exemplified in Section 6 below. In some embodiments, the CAR provided herein comprises an amino acid sequence with a certain percentage of identity to any of the CARs known to those skilled in the art. In some embodiments, the 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 those skilled in the art.

[0235] The determination of the 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 for comparing two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, Vol. 87: pp. 2264-2268, 1990, such as the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, Vol. 90: pp. 5873-5877, 1993. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. Vol. 215: pp. 403, 1990. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameter set, for example, for score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameter set, e.g., for a score of 50, wordlength = 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. Vol. 25: pp. 3389-3402, 1997. Alternatively, PSI BLAST can be used to perform iterated searches that detect distance relationships (Id.) between molecules. When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, CABIOS Vol. 4: pp. 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 to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0236] In some embodiments, it is envisioned that the amino acid sequence of the CAR described herein is modified. 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, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity or solubility. Therefore, in addition to the extracellular domain described herein, it is envisioned that variants of the domain described herein may be prepared. For example, scFv variants may 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 who understand amino acid changes may change the post-translational process of the antibody.

[0237] The variation can be a substitution, deletion or insertion of one or more codons encoding a polypeptide, which results in a change in the amino acid sequence compared to the original antibody or polypeptide. Interested sites for substitution mutagenesis include CDRs and FRs.

[0238] Amino acid substitution can be the result of replacing another amino acid with an amino acid having similar structure and / or chemical properties, such as replacing leucine with serine, for example, conservative amino acid substitution. Standard techniques known to those skilled in the art can be used to introduce mutations in the nucleotide sequence encoding the molecule provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis that cause amino acid substitutions. Insertion or deletion can be optionally within the range of about 1 to 5 amino acids. In certain embodiments, substitution, deletion or insertion include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions or less than 2 amino acid substitutions relative to the original molecule. In a specific embodiment, substitution is a conservative amino acid substitution carried out at one or more predicted non-essential amino acid residues. The allowed variants can be determined by systematically inserting, deleting or replacing the amino acid in the sequence and testing the obtained variants for the activity shown by the parent antibody.

[0239] Antibodies generated by conservative amino acid substitutions are included in the present disclosure. In conservative amino acid substitutions, amino acid residues are replaced by amino acid residues with side chains with similar charges. As mentioned above, families of amino acid residues with side chains with similar charges have been defined in the art. 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be randomly introduced along all or part of the coding sequence such as by saturation mutagenesis, and the biological activity of the resulting mutants can be screened to identify the mutants retaining activity. After mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined. Conservative substitutions can be made (e.g., within a group of amino acids with similar properties and / or side chains) so as to maintain or not significantly change the properties.

[0240] Amino acids can be grouped according to the similarity of the properties of their side chains (see, e.g., Lehninger, Biochemistry , pp. 73-75 (2nd 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 several 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 affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. For example, any cysteine ​​residue that is not involved in maintaining the correct conformation of the antibody can also be substituted, for example, with another amino acid such as alanine or serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Non-conservative substitutions will require exchanging a member of one of these categories for another.

[0241] One type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, the resulting variant selected for further study will have changes (e.g., improvements) in certain biological properties relative to the parent antibody (e.g., increased affinity, reduced immunogenicity) and / or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity mature antibodies, which can be conveniently generated, for example, using affinity maturation techniques based on phage display, such as those described herein. In short, one or more CDR residues are mutated and the variant antibody is displayed on a phage and screened for a specific biological activity (e.g., binding affinity).

[0242] Changes (e.g., substitutions) may be made in the CDRs, for example to improve antibody affinity. Such changes may be made in CDR "hot spots", residues encoded by codons that undergo mutations at high frequency during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. Vol. 207: pp. 179-196, 2008) and / or SDRs (a-CDRs), and the resulting variant antibodies or fragments thereof are tested for binding affinity. For example, Hoogenboom et al., Methods in Molecular Biology, Vol. 178: pp. 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), have described affinity maturation by constructing a secondary library and reselecting from the secondary library. 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 includes a CDR-directed approach in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues that participate in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling.

[0243] In some embodiments, substitutions, insertions or deletions may occur within one or more CDRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be produced in the CDRs. In some embodiments, each CDR is unchanged, or contains no more than one, two or three amino acid substitutions.

[0244] A useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham and Wells, Science, Vol. 244: pp. 1081-1085, 1989. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced 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. Additional substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitutions.

[0245] Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

[0246] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N-terminus or C-terminus of an antibody with an enzyme (e.g., for ADEPT) or a polypeptide, which prolongs the serum half-life of the antibody.

[0247] Can use method known in the art to change, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning and PCR mutagenesis.Site-directed mutagenesis (see, for example, Carter, Biochem J., Vol. 237: pp. 1-7, 1986; and Zoller et al., Nucl. Acids Res., Vol. 10: pp. 6487-6500, 1982), cassette mutagenesis (see, for example, Wells et al., Gene, Vol. 34: pp. 315-323, 1985) or other known techniques can be performed on cloned DNA to produce antibody variant DNA.

[0248] In addition, in the context of CAR, all of the above descriptions of the present disclosure are also applicable to engineered T cell receptors (TCR) other than CAR. Therefore, on the other hand, provided herein is a method for producing immune effector cells expressing TCR. 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.

[0249] TCR provided herein includes recombinant or engineered TCR, as used herein, which is included as a functional exogenous receptor provided herein, and refers to a peptide expressed in an immune cell. The function of recombinant or engineered TCR may include, for example, the immune activity of cells (such as cancer and infected cells with specific markers on their surface) redirecting immune cells for the desired type. It can replace endogenous TCR or co-express with endogenous TCR. In some embodiments, such recombinant TCR is a single-chain TCR comprising an open reading frame, wherein variable Vα domains and Vβ domains are paired with protein joints. This involves molecular cloning of TCR genes known to be specific for selected antigens. These chains are then generally introduced into T cells using retroviral vectors. Therefore, the expression of cloned TCRα genes and TCRβ genes gives transduced T cells functional specificity determined by the pairing of these new genes. The components of recombinant or engineered TCR are any functional subunits of TCR, such as recombinant TCRα and TCRβ, which are encoded by exogenous polynucleotide sequences introduced into cells.

[0250] 5.3.2. Polynucleotides

[0251] In certain embodiments, the present disclosure provides polynucleotides encoding CAR provided herein. The polynucleotides disclosed herein can be in RNA form or DNA form. DNA includes cDNA, genomic DNA and synthetic DNA; and can be double-stranded or single-stranded, and if single-stranded, can be a coding strand or a non-coding (antisense) strand. In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0252] The present disclosure also relates to variants of the polynucleotides described herein, wherein the variant encodes, for example, fragments, analogs and / or derivatives of antibodies or CARs disclosed herein. In certain embodiments, the present disclosure provides a polynucleotide comprising a polynucleotide having a nucleotide sequence that is 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 at least about 96%, 97%, 98% or 99% identical to a polynucleotide encoding a CAR disclosed herein. As used herein, the phrase "a polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence" is intended to mean that the nucleotide sequence of the polynucleotide is the same as the reference sequence, except that the polynucleotide sequence may include up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, in order to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced by another nucleotide, or up to 5% of the number of nucleotides of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, either interspersed individually among the nucleotides of the reference sequence or in one or more contiguous groups within the reference sequence.

[0253] Polynucleotide variants may contain changes in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain changes that produce silent substitutions, additions, or deletions but do not change the properties or activity of the encoded polypeptide. In some embodiments, polynucleotide variants include silent substitutions that do not result in changes in the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants may be produced for a variety of reasons, for example, in order to optimize the codon expression of a specific host (i.e., the codons in human mRNA are changed to preferred codons of bacterial hosts such as Escherichia coli (E. coli)). In some embodiments, polynucleotide variants include at least one silent mutation in the non-coding region or coding region of the sequence.

[0254] In some embodiments, polynucleotide variants are produced to regulate or change the expression (or expression level) of the encoded polypeptide. In some embodiments, polynucleotide variants are produced to increase the expression of the encoded polypeptide. In some embodiments, polynucleotide variants are produced to reduce the expression of the encoded polypeptide. In some embodiments, the polynucleotide variants have an increased expression of the encoded polypeptide compared to the parental polynucleotide sequence. In some embodiments, the polynucleotide variants have a reduced expression of the encoded polypeptide compared to the parental polynucleotide sequence.

[0255] 5.3.3. Carrier

[0256] Also provided are media comprising the polynucleotides described herein.In one embodiment, the nucleic acid molecules may be incorporated into a recombinant expression vector.

[0257] The present disclosure provides a vector for cloning and expressing any CAR 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 virus vectors and their derivatives. 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.

[0258] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to engineered mammalian cells in vitro or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many 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 immunomodulators (such as immune checkpoint inhibitors) coding sequences and / or self-inactivating lentiviral vectors carrying chimeric antigen receptors can be packaged with schemes known in the art. The resulting lentiviral vector 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 transgenes and their proliferation in daughter cells. Slow-virus vectors also have low immunogenicity and can transduce non-proliferating cells.

[0259] In some embodiments, the vector comprises any one of the nucleic acids encoding CAR described herein. The nucleic acid can be cloned into a vector using any molecular cloning method known in the art, including, for example, using restriction endonuclease sites and one or more selective markers. In some embodiments, the nucleic acid is operably connected to a promoter. A variety of promoters have been explored for gene expression in mammalian cells, and any promoter known in the art can be used in the present disclosure. Promoters can be roughly classified as constitutive promoters or regulated promoters, such as inducible promoters.

[0260] In some embodiments, the nucleic acid encoding CAR is operably connected to a constitutive promoter. Constitutive promoters allow heterologous genes (also referred to as transgenics) to be constitutively expressed in host cells. Exemplary constitutive promoters contemplated herein include, but are not limited to, cytomegalovirus (CMV) promoters, human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerol kinase promoters (PGK), simian virus 40 early promoters (SV40) and chicken beta-actin promoters (CAGG) coupled to CMV early enhancers. Such constitutive promoters have been extensively compared in a large number of studies in terms of their efficiency in driving transgenic expression.

[0261] In some embodiments, the nucleic acid encoding CAR is operably connected to an inducible promoter. Inducible promoters belong to the category of regulated promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of engineered immune effector cells or the physiological state of engineered immune effector cells, inducing factors (ie, inducers) or combinations thereof.

[0262] In some embodiments, the inducing conditions do not induce expression of endogenous genes in engineered mammalian cells and / or in subjects receiving the pharmaceutical composition. In some embodiments, the inducing conditions are selected from the group consisting of: an inducing factor, radiation (such as ionizing radiation, light), temperature (such as heat), redox state, tumor environment, and activation state of engineered mammalian cells.

[0263] In some embodiments, the vector also contains a selective marker gene or a reporter gene to select cells expressing CAR from a host cell population transfected by a lentiviral vector. Both the selective marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in host cells. For example, the vector may contain transcription and translation terminators, initiation sequences, and promoters for regulating the expression of nucleic acid sequences.

[0264] 5.4. Immune effector cells expressing chimeric antigen receptors

[0265] In another aspect, provided herein are immune effector cells expressing CAR produced according to the methods provided herein, e.g., as described in Sections 4.2 and 4.3 above.

[0266] In some embodiments, the immune effector cell is a cytotoxic cell. In certain embodiments, the cytotoxic cell is a natural killer cell. In certain embodiments, the cytotoxic cell is a cytotoxic T lymphocyte.

[0267] In some embodiments, the CAR expressed in the immune effector cells of the present invention comprises: an extracellular antigen binding domain; a transmembrane domain; and an intracellular signaling domain. In some embodiments, CAR also comprises one or more additional regions / domains, such as a signal peptide, a hinge region, a co-stimulatory signaling domain, a joint, etc., each of which can be as described in Section 4.3.1 above.

[0268] Specifically, in certain embodiments, the CAR provided herein may include a signal peptide located 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 will allow the effector molecule to be integrated and anchored into the lipid bilayer. For the use of compatible signal peptides in the CAR described herein, including signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences, it will be apparent to those skilled 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 chains.

[0269] The extracellular antigen binding domain of CAR described herein comprises one or more antigen binding domains. In some embodiments, the extracellular antigen binding domain constitutes an antibody or a fragment thereof. In a specific embodiment, the extracellular antigen binding domain of CAR of the present 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.

[0270] In some embodiments, the extracellular antigen binding domain comprises multiple binding domains. In some embodiments, the extracellular antigen binding domain constitutes a multispecific antibody or a fragment thereof. In other embodiments, the extracellular antigen binding domain constitutes a multivalent antibody or a fragment thereof. There are multiple binding domains in the extracellular antigen binding domain of the CAR of the present invention. Different domains can be fused to each other via a peptide linker. In some embodiments, the domains are directly fused 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 features and / or functional features of the different domains. Each peptide linker can be selected and optimized independently.

[0271] 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 associated with a special disease state on a target cell. In some embodiments, the antigen is a tumor antigen. Tumors express many proteins that can be used as target antigens for immune responses. The antigen targeted by CAR can be an antigen on a single diseased cell or an antigen expressed on different cells that each contributes to the disease. The antigen targeted by CAR can be directly or indirectly involved in the disease.

[0272] Tumor antigens are proteins produced by tumor cells that can trigger an immune response, particularly a T cell-mediated immune response. Exemplary tumor antigens include, but are not limited to, glioma-associated antigen, 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, mut hsp70-2, M-CSF, prostate enzymes, 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, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0273] In some embodiments, tumor antigens include one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express many proteins that can be used 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 oncogene HER2 / Neu / ErbB-2. Another group of target antigens is carcinoembryonic antigens, such as carcinoembryonic antigen (CEA).

[0274] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and do not appear on other cells in the body. TAA-related antigens are not unique to tumor cells, but are also expressed on normal cells under conditions that fail to induce an immune tolerance state to the antigen. The expression of antigens on tumors can occur under conditions that enable 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 cannot respond, or they can be antigens that are usually present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0275] Non-limiting examples of TSA or TAA antigens include: differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER2 / neu; unique tumor antigens generated by chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7.

[0276] 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, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-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, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP and TPS.

[0277] In some embodiments, the CAR provided herein comprises a hinge domain between the extracellular antigen binding domain and the transmembrane domain. 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 comprising a hinge domain is compatible for use in a chimeric receptor described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and imparts chimeric receptor flexibility.

[0278] The CAR of the present disclosure comprises a transmembrane domain that can be fused directly or indirectly to an extracellular antigen binding domain. The transmembrane domain can be derived from a natural or synthetic source. A transmembrane domain compatible with use in the CAR described herein can be obtained from a naturally occurring protein. Alternatively, it can be a synthetic, non-naturally occurring protein segment, such as a hydrophobic protein segment that is thermodynamically stable in a cell membrane. In some embodiments, the transmembrane domain is derived from type I, type II, or type III membrane proteins. In some embodiments, the transmembrane domain of the CAR described herein is derived from a type I single-pass membrane protein. In some embodiments, the transmembrane domain from multiple transmembrane proteins is also compatible with use in the CAR described herein. The transmembrane domain used in the CAR described herein may also include 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 fold. In some embodiments, a protein segment is at least about 20 amino acids, such as at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids.

[0279] The transmembrane domain provided herein may comprise a transmembrane region and a cytoplasmic region located at the C-terminal side of the transmembrane domain. In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues.

[0280] In some embodiments, the transmembrane domain of the CAR comprises a transmembrane domain selected from the group consisting of the alpha, beta or zeta chain of a 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 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL-2Rβ, IL-2Rγ, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD15 0, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or NKG2C.

[0281] The intracellular signaling domain in the CAR provided herein is responsible for activating at least one normal effector function of the immune effector cell expressing CAR. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell. In some embodiments, the primary intracellular signaling domain contains a signaling motif referred to as an activation motif based on immunoreceptor tyrosine or ITAM. Exemplary primary cytoplasmic signaling sequences containing ITAM include those derived from CD3z, FcRγ (FCER1G), FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0282] In some embodiments, CAR comprises at least one co-stimulatory signaling domain. The co-stimulatory signaling domain of the chimeric receptor described herein can be a cytoplasmic signaling domain from a co-stimulatory protein, which transduces signals and regulates responses mediated by immune cells. In some embodiments, the intracellular signaling domain includes a single co-stimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (such as any one of about 2, 3, 4 or more) co-stimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more identical co-stimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more co-stimulatory signaling domains from different co-stimulatory proteins, such as any two or more co-stimulatory proteins described herein. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (such as a cytoplasmic signaling domain of CD3z) and one or more co-stimulatory signaling domains. In some embodiments, the one or more costimulatory signaling domains and the primary 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 the 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 primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3z). Multiple costimulatory signaling domains can provide additive or synergistic stimulation.

[0283] The costimulatory signaling domain of any costimulatory molecule may be compatible for use in the CARs described herein.Examples of costimulatory signaling domains used in CARs can be cytoplasmic signaling domains of costimulatory proteins, including but not limited to 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 / TNFSF8, 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 any other co-stimulatory molecules, such as 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 co-stimulatory signaling domains are selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

[0284] In some embodiments, a CAR provided herein comprises the amino acid sequence of any one of the CARs exemplified below in Section 6. In some embodiments, a CAR provided herein comprises the amino acid sequence of any one of the CARs known to those skilled in the art.

[0285] In some embodiments, the CAR provided herein comprises an amino acid sequence with a certain percentage of identity relative to any of the CARs exemplified in Section 6 below. In some embodiments, the CAR provided herein comprises an extracellular domain or consists of an extracellular domain with 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 of the CARs exemplified in Section 6 below. In some embodiments, the CAR provided herein comprises an amino acid sequence with a certain percentage of identity to any of the CARs known to those skilled in the art. In some embodiments, the 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 those skilled in the art.

[0286] 5.5. Pharmaceutical Compositions

[0287] In one aspect, the present disclosure also provides a pharmaceutical composition comprising an engineered immune effector cell (e.g., NK cell, αβT cell or γδT cell) expressing 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., NK cell, αβT cell or γδT cell) of the present disclosure and a pharmaceutically acceptable excipient.

[0288] In a specific embodiment, the term "excipient" may also refer to a diluent, an adjuvant (e.g., Freund's adjuvant (complete or incomplete)), a carrier or a vehicle. Pharmaceutical excipients may be sterile liquids, such as water and oils, including those derived from oils of petroleum, animals, plants or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions and aqueous dextrose solutions and glycerol solutions may also be used 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, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. If desired, the composition may also contain a small amount of a wetting agent or emulsifier or a pH buffer. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations, etc. Examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Such compositions will contain a preventive or therapeutically effective amount of an active ingredient provided herein, such as a purified form, and an appropriate amount of an excipient to provide a form suitable for administration to a patient. The formulation should be compatible with the mode of administration.

[0289] In some embodiments, the choice of excipient is determined in part by the particular cell and / or method of administration.Thus, there are a variety of suitable formulations.

[0290] Generally, acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants (including ascorbic acid, methionine, vitamin E, sodium metabisulfite); preservatives, isotonic agents, stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents such as EDTA and / or nonionic surfactants.

[0291] Buffers can be used to control pH within a range that optimizes therapeutic effectiveness, especially if stability is pH dependent. Suitable buffers for use with the present disclosure include organic and inorganic acids and their salts. For example, citrates, phosphates, succinates, tartrates, fumarates, gluconates, oxalates, lactates, acetates. Additionally, the buffer may include histidine and trimethylamine salts such as Tris.

[0292] Preservatives may be added to retard microbial growth. Suitable preservatives for use with the present disclosure include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl alcohol or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol and m-cresol.

[0293] Tonicity agents (sometimes referred to 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, they are often referred to as "stabilizers" because they can interact with the charged groups of the amino acid side chains, thereby reducing the possibility of intermolecular and intramolecular interactions. Exemplary tonicity agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0294] Additional exemplary excipients include: (1) fillers, (2) solubilizers, (3) stabilizers, and (4) agents to prevent denaturation or adhesion to the container walls. Such excipients include: polyhydroxy sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbitol, xylose, ribose, ribitol, myoinisitose, inositol, galactose, dulcitol, glycerol, cyclitols (e.g., , cyclohexanehexol), polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioacetate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0295] Nonionic surfactants or detergents (also called "wetting agents") may be present to help dissolve the therapeutic agent and protect the therapeutic protein from aggregation caused by agitation, which also allows the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Suitable nonionic surfactants include, for example, polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), Polyols, Polyoxyethylene sorbitan monoether ( -20, -80, etc.), lauromacrogol 400, polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. 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.

[0296] In order to make the pharmaceutical compositions for in vivo administration, they are preferably sterile. The 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, such as an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle.

[0297] The route of administration is according to known and accepted methods, such as by single or multiple boluses or infusion over a long period of time in a suitable manner, for example, injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical application, inhalation or by sustained release or long-term release means.

[0298] 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 release or sustained release (see, e.g., Sefton, Crit. Ref. Biomed. Eng. Vol. 14: pp. 201-240, 1987; Buchwald et al., Surgery Vol. 88: pp. 507-516, 1980; and Saudek et al., N. Engl. J. Med. Vol. 321: pp. 569-574, 1989). In another embodiment, a polymeric material can be used to achieve controlled or sustained release of a preventive or therapeutic agent (e.g., a fusion protein as described herein) or a composition provided herein (see, e.g., Medical Applications of Controlled Release (Langer and Wise, ed., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball, eds., 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. Vol. 23: 61-126, 1983; Levy et al., Science Vol. 228: 190-192, 1985; During et al., Ann. Neurol., Vol. 25: 351-356, 1989; Howard et al., J. Neurosurg. Vol. 71: 105-112, 1989; U.S. Pat. Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; and 5,128,326; PCT Publication Nos. 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-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained release formulation is inert, free of leachable impurities, storage stable, sterile, and biodegradable.In another embodiment, a controlled or sustained release system can be placed near a specific target tissue (e.g., nasal passages or lungs), thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release, Vol. 2, pp. 115-138, 1984). Controlled release systems are discussed, for example, by Langer, Science, Vol. 249: pp. 1527-1533, 1990. Any technique known to those skilled in the art can be used to produce sustained-release formulations comprising one or more agents as described herein (see, e.g., U.S. Pat. No. 4,526,938, PCT Publication Nos. WO 91 / 05548 and WO 96 / 20698, Ning et al., Radiotherapy & Oncology Vol. 39: pp. 179-189, 1996; Song et al., PDA J. of Pharma. Sci. & Tech., Vol. 50: pp. 372-397, 1995; Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater., Vol. 24: pp. 853-854, 1997; and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater., Vol. 24: pp. 759-760, 1997).

[0299] The pharmaceutical compositions described herein may also contain more than one active compound or agent, which is necessary for the specific indication being treated. Alternatively or in addition, the composition may include a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, or a growth inhibitory agent. Such molecules are suitable for being present in combination in an amount effective for the intended purpose.

[0300] The active ingredient can also be embedded in microcapsules, prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition.

[0301] Various 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, recombinant cells capable of expressing the single domain antibodies or therapeutic molecules provided herein, construction of nucleic acids as part of a retrovirus or other vector, and the like.

[0302] In some embodiments, the pharmaceutical composition provided herein contains binding molecules and / or cells of the amount (e.g., therapeutically effective amount or preventive effective amount) of an effective treatment or prevention disease or disorder. In some embodiments, the subject of treatment is monitored for treatment or prevention efficacy by regular assessment. For repeated administration for several days or longer, according to the disease, repeated treatment is performed until the disease symptoms of expectation are suppressed. However, other dosage regimens are also useful and can be determined.

[0303] 5.6. Methods and Purpose

[0304] In another aspect, provided herein are methods for using engineered immune effector cells (e.g., NK cells) expressing CAR and uses of the engineered immune effector cells, such as those described in Section 4.4 above.

[0305] In some embodiments, the engineered immune effector cells (e.g., NK cells) expressing CAR provided herein can be used as allogeneic CAR-NK cell therapy. In some embodiments, the CAR-NK cell therapy of the present invention has more safety features that traditional autologous CAR-NK therapy does not have, such as no cytokine storm or low cytokine storm, no regulatory T cell stimulation, autologous tissue damage reduction, autoimmune induction reduction, graft-versus-host disease reduction, etc.

[0306] Such methods and uses include methods of treatment and uses, for example, involving administering these cells or compositions comprising these cells to a subject suffering from a disease or disorder. In some embodiments, the cells are administered in an effective amount to achieve treatment of the disease or disorder. Uses include the use of cells in such methods and treatments, and in the preparation of a medicament in order to perform such methods of treatment. In some embodiments, these methods are performed by administering these cells or compositions comprising these cells to a subject suffering from or suspected of suffering from the disease or disorder. In some embodiments, these methods thereby treat the disease or disorder of the subject.

[0307] In some embodiments, the treatment provided herein causes a disease or disorder, or a symptom, adverse effect or result, or a complete or partial improvement or alleviation of a phenotype associated therewith. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. These terms include but are not limited to completely curing the disease or completely eliminating any symptom or the impact on all symptoms or results.

[0308] As used herein, in some embodiments, the treatment provided herein delays the development of a disease or disorder, for example, postpones, hinders, slows down, delays, stabilizes, inhibits and / or postpones the development of a disease (such as cancer). This delay can be of different lengths of time, depending on the medical history of the disease and / or the individual treated. As will be apparent to those skilled in the art, sufficient or significant delays can actually encompass prevention, because the individual does not develop the disease or disorder. For example, advanced cancer, such as the development of metastasis, can be delayed. In other embodiments, the methods or uses provided herein prevent disease or disorder.

[0309] In some embodiments, the CAR-NK cell therapy of the present invention is used to treat solid tumor cancer. In other embodiments, the CAR-NK cell therapy of the present invention is used to treat blood cancer. In other embodiments, the disease or disorder is autoimmune disease and inflammatory disease.

[0310] In some embodiments, these methods include applying these cells or compositions containing these cells to a subject, tissue or cell, such as suffering from a disease or disorder, being in a disease or disorder risk or suspecting a subject, tissue or cell suffering from a disease or disorder. In some embodiments, these cells, colonies and compositions are applied to a subject suffering from a specific disease to be treated (for example, via adoptive cell therapy, such as adoptive NK cell therapy treatment) or disorder. In some embodiments, these cells or compositions are applied to a subject, such as suffering from a disease or disorder or being in a disease or disorder risk. In some embodiments, these methods are thus treated, for example, to improve one or more symptoms of a disease or disorder.

[0311] Methods of administering cells for adoptive cell therapy are known, as described in, e.g., U.S. Patent Application Publication No. 2003 / 0170238; U.S. Patent No. 4,690,915; Rosenberg, Nat Rev Clin Oncol. Vol. 8, No. 10: pp. 577-585, 2011; Themeli et al., Nat Biotechnol. Vol. 31, No. 10: pp. 928-933, 2013; Tsukahara et al., Biochem Biophys Res Commun Vol. 438, No. 1: pp. 84-89, 2013; and Davila et al., PLoS ONE Vol. 8, No. 4: p. e61338, 2013. These methods can be used together with the methods and compositions provided herein.

[0312] In some embodiments, cell therapy (e.g., adoptive NK cell therapy) is carried out by autologous transfer, wherein cells are separated and / or otherwise prepared from a subject to be treated with cell therapy or from a sample derived from such subject. Therefore, in some aspects, these cells are derived from a subject in need of treatment, and these cells are applied to the same subject after separation and processing. In other embodiments, cell therapy (e.g., adoptive NK cell therapy) is carried out by allogeneic transfer, wherein cells are separated and / or otherwise prepared from a subject other than a subject (e.g., a first subject) to be treated or to eventually be treated with cell therapy. In such embodiments, the cells are then applied to different subjects of the same species, such as 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.

[0313] In some embodiments, the subject to which the cells, cell populations, or compositions are administered is a primate, such as a human. The subject can be male or female, and can be of any suitable age, including infants, teenagers, young adults, adults, and elderly subjects. In some examples, the subject is a validated animal model for disease, adoptive cell therapy, and / or for evaluating toxicity outcomes.

[0314] The compositions provided herein can be used by any suitable means, for example, by injection, for example, intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, injection through the septum, subscleral injection, intrachoroidal injection, intracameral injection, subconjunctival (subconjectval) injection, subconjunctival (subconjuntival) injection, subfascial capsule injection, retrobulbar injection, peribulbar injection or posterior scleral parade delivery. In some embodiments, the compositions are administered parenterally, intrapulmonary and intranasally, and if local treatment needs, administered in the lesion. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

[0315] The amount of the preventive or therapeutic agent for effectively preventing and / or treating a disease or condition can be determined by standard clinical techniques. The effective dose can be derived by a dose response curve derived from an in vitro or animal model test system. For disease prevention or treatment, the appropriate dose of the binding molecule or cell can depend on the type of the disease to be treated or the type of disorder, the type of the binding molecule, the severity and process of the disease or disorder, whether the therapeutic agent is used for prevention or treatment purposes, previous therapy, the patient's clinical history and the reaction to the agent and the judgment of the attending physician. In some embodiments, the composition, molecule and cell are applied to the patient in a disposable or series of treatments. Multiple doses can be applied intermittently. An initially higher initial loading dose can be applied, followed by one or more lower doses.

[0316] In the case of genetically engineered cells, in some embodiments, a range of about one million to about 100 billion cells and / or an amount of cells per kilogram of body weight may be administered to a subject. In some embodiments, wherein the pharmaceutical composition comprises any of the engineered immune cells described herein, the pharmaceutical composition is administered at a dose of at least about 104, 105, 106, 107, 108, or 109 cells / kg of individual body weight. The dosage may vary depending on the specific attributes of the disease or disorder and / or patient and / or other treatments.

[0317] In some embodiments, the pharmaceutical composition is used once. In some embodiments, the pharmaceutical composition is used multiple times (such as 2 times, 3 times, 4 times, 5 times, 6 times or more times any one). In some embodiments, the pharmaceutical composition is used once or multiple times during the administration cycle. The administration cycle can be, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more weeks, or 1 month, 2 months, 3 months, 4 months, 5 months or more months. The optimal dose and treatment regimen for a specific patient can be determined by the technicians in the medical field by monitoring the disease signs of the patient and adjusting the treatment accordingly.

[0318] In some embodiments, the compositions provided herein are administered as part of a combination therapy, such as simultaneously or sequentially in any order with another therapeutic intervention (such as another antibody or engineered cell or receptor or agent, such as a cytotoxic agent or a therapeutic agent).

[0319] In some embodiments, the compositions provided herein are co-administered with one or more additional therapeutic agents or in combination with another therapeutic intervention simultaneously or in any order one after another. In some embodiments, cells are co-administered with another therapy close enough in time so that the cell population enhances the effect of 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.

[0320] In certain embodiments, once the cell is applied to a mammal (e.g., people), the biological activity of the engineered cell population is measured by any of many known methods. The parameters assessed include the specific binding of engineered or natural NK cells or other immune cells to antigens, in vivo, for example, by imaging, or in vitro, for example, by ELISA or flow cytometry. In certain embodiments, the ability of engineered cells to destroy target cells can be measured using any suitable method known in the art, such as described in, for example, Kochenderfer et al., J.Immunotherapy, Vol. 32, No. 7: Pages 689-702, 2009 and Herman et al. J.Immunological Methods, Vol. 285, No. 1: Pages 25-40, 2004 in cytotoxicity assays. In certain embodiments, the biological activity of cells can also be measured by measuring 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 clinical results (such as the reduction of tumor load or load).

[0321] 5.7. Kits and Articles of Manufacture

[0322] Kits, unit doses, and articles of manufacture comprising any of the engineered immune effector cells described herein are also provided. In some embodiments, a kit is provided, the kit containing any of the pharmaceutical compositions described herein and preferably providing instructions for use thereof.

[0323] The kit of the present application is in a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (e.g., sealed Mylar or plastic bags), etc. The kit may optionally provide additional components, such as buffer and explanatory information. Therefore, the present application also provides manufactured products, which include vials (such as sealed vials), bottles, jars, flexible packages, etc.

[0324] The manufactured article may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed of a variety of materials, such as glass or plastic. Typically, the container holds a composition that effectively treats a disease or disorder described herein (such as cancer), and may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The label or package insert indicates that the composition is used to treat a specific condition of an individual. The label or package insert will further include instructions for applying the composition to an individual. The label may indicate guidance for reconstruction and / or use. The container holding the pharmaceutical composition may be a vial that is used multiple times, and the vial that is used multiple times allows for repeated administration (e.g., 2 to 6 administrations) of the reconstructed formulation. The package insert refers to instructions that are typically included in the commercial packaging of a therapeutic product, which contains information about indications, usage, dosage, administration, contraindications, and / or warnings for use of such therapeutic products. In addition, the article of manufacture may further comprise a second container comprising 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 also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0325] The kit or article of manufacture can include multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies.

[0326] For the sake of brevity, certain abbreviations are used herein. One example is the single-letter abbreviations representing amino acid residues. The amino acids and their corresponding three-letter and single-letter abbreviations are as follows:

[0327] Alanine Ala (A)

[0328] Arginine (R)

[0329] Asparagine Asn (N)

[0330] Aspartic acid Asp (D)

[0331] Cysteine ​​(C)

[0332] Glutamate (E)

[0333] Glutamine Gln (Q)

[0334] Glycine (G)

[0335] Histidine (H)

[0336] Isoleucine Ile (I)

[0337] Leucine (L)

[0338] Lysine (K)

[0339] MethionineMet (M)

[0340] Phenylalanine Phe (F)

[0341] Proline (P)

[0342] Serine (S)

[0343] Threonine (T)

[0344] Tryptophan Trp (W)

[0345] Tyrosine Tyr (Y)

[0346] Valine (V)

[0347] Affirmative language is used herein to describe multiple embodiments and generally disclose the present disclosure. The present disclosure also specifically includes embodiments in which specific themes such as substances or materials, method steps and conditions, schemes, procedures, determinations or analyses are excluded in whole or in part. Therefore, even if the present disclosure is not usually expressed in accordance with the content not included in the present disclosure, aspects that are not clearly included in the present disclosure are still disclosed herein.

[0348] Several embodiments of the present disclosure have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the present disclosure.

[0349] 6. Implementation Plan

[0350] The present disclosure includes the following non-limiting embodiments.

[0351] In one set of embodiments (Group A):

[0352] A1. A method for preparing immune effector cells for transduction, the method comprising:

[0353] i. a first step for increasing the expression of low-density lipoprotein receptor (LDLR); and ii. a second step for inhibiting the intracellular antiviral defense mechanism of the immune effector cells;

[0354] The first step may be performed before, simultaneously with or after the second step.

[0355] A2. A method according to embodiment A1, wherein the immune effector cells are autologous or allogeneic.

[0356] A3. A method according to embodiment A1, wherein the immune effector cell is a cytotoxic cell, and wherein, optionally the cytotoxic cell is a natural killer cell.

[0357] A4. The method of any one of embodiments A1 to A3, wherein the method further comprises providing one or more transduction enhancers.

[0358] A5. The method according to embodiment A4, wherein the transduction enhancer is Vectofusin. A6. The method according to embodiment A4, wherein the transduction enhancer is Retronectin.

[0359] A7. A method according to any one of embodiments A1 to A6, wherein the method further comprises introducing a nucleic acid into the immune effector cell.

[0360] A8. A method according to embodiment A7, wherein the nucleic acid comprises a promoter.

[0361] A9. The method according to embodiment A8, wherein the promoter is selected from the group consisting of CAG promoter, PGK promoter, EF1a promoter and EFS promoter.

[0362] A10. The method of any one of embodiments A7 to A9, wherein the nucleic acid is delivered via a lentiviral particle.

[0363] A11. A method according to embodiment A10, wherein the lentiviral particles are pseudotyped with vesicular stomatitis virus G (VSV-G).

[0364] A12. The method of any one of embodiments A7 to A11, wherein the nucleic acid encodes an exogenous functional receptor.

[0365] A13. A method according to embodiment A12, wherein the exogenous functional receptor is a chimeric antigen receptor (CAR) or TCR.

[0366] A14. The method of any one of embodiments A1 to A13, wherein the first step comprises contacting the immune effector cells with a statin.

[0367] A15. The method according to embodiment A14, wherein the statin is rosuvastatin.

[0368] A16. The method according to any one of embodiments A1 to A15, wherein the second step comprises contacting the immune effector cells with a 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor.

[0369] A17. The method according to embodiment A16, wherein the PDK1 inhibitor is BX795. In another group of embodiments (Group B):

[0370] B1. A method for preparing immune effector cells for transduction, the method comprising:

[0371] i. contacting the immune effector cells with a first agent that increases expression of the low-density lipoprotein receptor (LDLR);

[0372] ii. contacting the immune effector cells with a second agent that inhibits the intracellular antiviral defense mechanisms of the immune effector cells; and

[0373] iii. contacting the immune effector cells with a third agent that enhances transduction.

[0374] B2. A method according to embodiment B1, wherein the immune effector cells are autologous or allogeneic.

[0375] B3. A method according to embodiment B1, wherein the immune effector cell is a cytotoxic cell, and wherein, optionally the cytotoxic cell is a natural killer cell.

[0376] B4. A method according to any one of embodiments B1 to B3, wherein the first agent is a statin.

[0377] B5. The method according to embodiment B4, wherein the statin is rosuvastatin.

[0378] B6. The method according to any one of embodiments B1 to B5, wherein the second agent is a 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor.

[0379] B7. The method according to embodiment B6, wherein the PDK1 inhibitor is BX795. B8. The method according to any one of embodiments B1 to B7, wherein the third agent is Vectofusin and / or Retronectin.

[0380] B9. A method according to any one of embodiments B1 to B8, wherein the method further comprises transducing a nucleic acid into the immune effector cell.

[0381] B10. A method according to embodiment B9, wherein the nucleic acid comprises a promoter.

[0382] B11. The method according to embodiment B10, wherein the promoter is selected from the group consisting of CAG promoter, PGK promoter, EF1a promoter and EFS promoter.

[0383] B12. The method of any one of embodiments B9 to B11, wherein the nucleic acid is delivered via a lentiviral particle.

[0384] B13. The method of embodiment B12, wherein the lentiviral particles are pseudotyped with vesicular stomatitis virus G (VSV-G).

[0385] B14. A method according to any one of embodiments B9 to B13, wherein the nucleic acid encodes an exogenous functional receptor.

[0386] B15. A method according to claim B14, wherein the exogenous functional receptor is a chimeric antigen receptor (CAR) or TCR.

[0387] In another set of embodiments (Group C):

[0388] C1. A method comprising:

[0389] i. contacting the immune effector cells with a first agent that increases expression of the low-density lipoprotein receptor (LDLR);

[0390] ii. contacting the immune effector cells with a second agent that inhibits the intracellular antiviral defense mechanisms of the immune effector cells;

[0391] iii. contacting the immune effector cells with a third agent that enhances transduction; and

[0392] iv. Introduction of nucleic acids into immune effector cells.

[0393] C2. A method according to embodiment C1, wherein the immune effector cells are autologous or allogeneic.

[0394] C3. A method according to embodiment C1, wherein the immune effector cell is a cytotoxic cell, and wherein, optionally the cytotoxic cell is a natural killer cell.

[0395] C4. A method according to any one of embodiments C1 to C3, wherein the first agent is a statin.

[0396] C5. The method according to embodiment C2, wherein the statin is rosuvastatin.

[0397] C6. The method according to any one of embodiments C1 to C5, wherein the second agent is a 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor.

[0398] C7. The method according to embodiment C6, wherein the PDK1 inhibitor is BX795. C8. The method according to any one of embodiments C1 to C7, wherein the third agent is Vectofusin and / or Retronectin.

[0399] C9. The method of any one of embodiments C1 to C8, wherein the nucleic acid comprises a promoter.

[0400] C10. A method according to embodiment C9, wherein the promoter is selected from the group consisting of CAG promoter, PGK promoter, EF1a promoter and EFS promoter.

[0401] C11. The method of any one of embodiments C1 to C10, wherein the nucleic acid is delivered via a lentiviral particle.

[0402] C12. The method of embodiment C11, wherein the lentiviral particles are pseudotyped with vesicular stomatitis virus G (VSV-G).

[0403] C13. A method according to any one of embodiments C1 to C12, wherein the nucleic acid encodes an exogenous functional receptor.

[0404] C14. A method according to claim C13, wherein the exogenous functional receptor is a chimeric antigen receptor (CAR) or TCR.

[0405] In another set of embodiments (Group D):

[0406] D1. A method for preparing immune effector cells for transduction, the method comprising: a step for increasing the expression of low-density lipoprotein receptor (LDLR).

[0407] D2. A method according to embodiment D1, wherein the immune effector cells are autologous or allogeneic.

[0408] D3. A method according to embodiment D1, wherein the immune effector cells are cytotoxic cells, and wherein, optionally the cytotoxic cells are natural killer cells.

[0409] D4. A method according to embodiment D1, wherein the immune effector cells are γδ T cells.

[0410] D5. A method according to embodiment D1, wherein the immune effector cells are αβT cells.

[0411] D6. A method according to embodiment D1, wherein the step for increasing the expression of low-density lipoprotein receptor (LDLR) comprises: contacting the immune effector cells with a statin.

[0412] D7. The method according to embodiment D6, wherein the statin is rosuvastatin.

[0413] D8. The method according to embodiment D6, wherein the statin is atorvastatin.

[0414] D9. A method according to any one of embodiments D1 to D8, further comprising a step for inhibiting the intracellular antiviral defense mechanism of the immune effector cells.

[0415] D10. A method according to embodiment D9, 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.

[0416] D11. The method according to embodiment D10, wherein the PDK1 inhibitor is BX795. D12. The method according to any one of embodiments D1 to D11, wherein the method further comprises providing one or more transduction enhancers.

[0417] D13. The method according to embodiment D4, wherein the transduction enhancer is Vectofusin. D14. The method according to embodiment D4, wherein the transduction enhancer is Retronectin.

[0418] D15. A method according to any one of embodiments D1 to D14, wherein the method further comprises introducing a nucleic acid into the immune effector cell.

[0419] D16. A method according to embodiment D15, wherein the nucleic acid comprises a promoter.

[0420] D17. A method according to embodiment D16, wherein the promoter is selected from the group consisting of CAG promoter, PGK promoter, EF1a promoter and EFS promoter.

[0421] D18. The method of any one of embodiments D15 to D17, wherein the nucleic acid is delivered via a lentiviral particle.

[0422] D19. A method according to embodiment D18, wherein the lentiviral particles are pseudotyped with vesicular stomatitis virus G (VSV-G).

[0423] D20. A method according to any one of embodiments D15 to D19, wherein the nucleic acid encodes an exogenous functional receptor.

[0424] D21. A method according to embodiment D20, wherein the exogenous functional receptor is a chimeric antigen receptor (CAR) or TCR.

[0425] In another group of embodiments (Group E):

[0426] E1. A method for preparing immune effector cells for transduction, the method comprising contacting the immune effector cells with an agent that increases the expression of low-density lipoprotein receptor (LDLR).

[0427] E2. A method according to embodiment E1, wherein the immune effector cells are autologous or allogeneic.

[0428] E3. A method according to embodiment E1, wherein the immune effector cell is a cytotoxic cell, and wherein, optionally the cytotoxic cell is a natural killer cell.

[0429] E4. A method according to embodiment E1, wherein the immune effector cells are γδ T cells.

[0430] E5. A method according to embodiment E1, wherein the immune effector cells are αβ T cells.

[0431] E6. A method according to embodiment E1, wherein the step for increasing the expression of low-density lipoprotein receptor (LDLR) comprises: contacting the immune effector cells with a statin.

[0432] E7. The method according to embodiment E6, wherein the statin is rosuvastatin.

[0433] E8. The method according to embodiment E6, wherein the statin is atorvastatin.

[0434] E9. The method according to any one of embodiments E1 to E8, further comprising a step for inhibiting the intracellular antiviral defense mechanism of the immune effector cells.

[0435] E10. A method according to embodiment E9, 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.

[0436] E11. The method according to embodiment E10, wherein the PDK1 inhibitor is BX795.

[0437] E12. The method according to any one of embodiments E1 to E11, wherein the method further comprises providing one or more transduction enhancers.

[0438] E13. A method according to embodiment E12, wherein the transduction enhancer is Vectofusin.

[0439] E14. A method according to embodiment E12, wherein the transduction enhancer is Retronectin.

[0440] E15. The method according to any one of embodiments E1 to E14, wherein the method further comprises introducing a nucleic acid into the immune effector cell.

[0441] E16. A method according to embodiment E15, wherein the nucleic acid comprises a promoter.

[0442] E17. The method according to embodiment E16, wherein the promoter is selected from the group consisting of CAG promoter, PGK promoter, EF1a promoter and EFS promoter.

[0443] E18. The method according to any one of embodiments E15 to E17, wherein the nucleic acid is delivered via a lentiviral particle.

[0444] E19. The method according to embodiment E18, wherein the lentiviral particles are pseudotyped with vesicular stomatitis virus G (VSV-G).

[0445] E20. The method according to any one of embodiments E15 to 17, wherein the nucleic acid encodes an exogenous functional receptor.

[0446] E21. A method according to embodiment E20, wherein the exogenous functional receptor is a chimeric antigen receptor (CAR) or TCR.

[0447] 7. Examples

[0448] The following is a description of various methods and materials used in the studies, and is presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and is not intended to limit the scope of what the inventors consider to be their disclosure, nor is it intended to represent that the following experiments were performed and are all experiments that can be performed. It should be understood that the exemplary descriptions written in the present tense are not necessarily performed, but rather can be performed to generate data associated with the teachings of the present disclosure, etc. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, percentages, etc.), but some experimental errors and deviations should be accounted for.

[0449] 7.1. Example 1 - Generation of Natural Killer CAR (CAR-NK) Cells

[0450] NK cells were first activated in preparation for transduction. On day 0, 0.5 × 10 6 to 1×10 6 NK cells were distributed in 24-well plates.

[0451] The anti-biotin loaded MACSiBead particles for NK cell activation (Miltenyi Biotech, catalog number 130-094-483) were thoroughly resuspended and diluted to 1×10 6 5 μL (5×10 5 The amount of the anti-biotin MACSiBead particles (loaded) is transferred to a suitable tube. 100 μL culture medium is added to the anti-biotin MACSiBead particles of the load. The mixture is centrifuged at 300 × g for 5 minutes to wash the beads before being added to the NK cells of the plate. The supernatant is aspirated and the anti-biotin MACSiBead particles of the load are resuspended in 5 μL to 10 μL of fresh NK MACS culture medium (Miltenyi Biotech, catalog number 130-114-429) with NK MACS supplements. The prepared anti-biotin MACSiBead particles are added to the NK cells of the plate and gently mixed by pipetting up and down 2 to 3 times. The cells are incubated overnight at 37 ° C and 5% CO2.

[0452] Perform lentiviral transduction on day 5 or day 6. Resuspend the NK cells to determine if the cell number has changed from day 0. Thaw the lentiviral particles on ice. Once thawed, remove the lentiviral particles from the ice and equilibrate to room temperature before adding to the activated NK cells. The amount of virus required to transduce the cells is determined based on the desired multiplicity of infection (MOI). Once the NK cells are transduced with the lentiviral particles, they are returned to the incubator at 37°C and 5% CO2.

[0453] Monitor cells for surface CAR expression as early as day 3 to 4 after transduction. Determine the cell number of NK cells. Collect 1 × 10 5 NK cells were added and transferred to the wells of a microtiter plate. The cells in each well of the microtiter plate were washed twice with 200 μL FACS buffer each time. The wells were stained with 100 μL staining buffer containing the appropriate CAR detection antibody. The plate was incubated at 4 ° C in the dark for at least 30 minutes. The cells were then washed twice with 150 μL FACS buffer each time and centrifuged at 400 × g for 4 minutes at room temperature. CAR expression on NK cells was then measured using flow cytometry analysis.

[0454] To evaluate 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. Anti-myc tag AF647 antibody (Cell Signaling, catalog number 2233, 1:200) was used to monitor surface antigen-1 CAR expression at multiple time points after transduction.

[0455] like Figures 1A to 1C As shown, NK-like cell lines (NK-92 and NK-L) were efficiently transduced at early time points (day 3) compared to the low expression observed in primary NK cells. However, in both NK-like cell lines (NK-92 and NK-L) and primary NK cells, antigen-1 CAR expression was downregulated over time.

[0456] 7.2. Example 2 - Optimization of gene transfer

[0457] To improve CAR transduction efficiency and surface CAR stability, the following additional steps were performed prior to or simultaneously with the addition of lentiviral particles to cells.

[0458] 7.2.1.3-Phosphoinositide-dependent kinase 1 (PDK1) inhibitors

[0459] NK cells have been selected during evolution to have resistance to viral infection.PDK1 inhibitors can block pattern recognition receptors that are naturally used by immune cells (such as NK cells) during viral infection.

[0460] To test whether PDK1 inhibitors can improve CAR transduction efficiency or surface CAR stability, primary NK cells were activated and transduced with antigen-1 CAR in the presence or absence of PDK1 inhibitor BX795 (InvivoGen, catalog number tlrl-bx7) at concentrations ranging from 0.5 μM to 7.5 μM. BX795 was incorporated into the transduction process by adding it to the cells containing viral supernatant and gently mixing. Surface CAR expression was monitored at regular intervals using anti-myc tag AF647 antibody (Cell Signaling, catalog number 2233, 1:200). Figure 2A A schematic schedule of the experiment is shown in .

[0461] like Figure 2B to Figure 2C As shown, primary NK cells transduced with BX795 showed improved surface CAR stability in a dose-dependent manner compared to cells without any BX795 transduction (n=2).

[0462] 7.2.2. Statins

[0463] For the lentiviral pseudotyped vesicular stomatitis virus G (VSV-G), the low-density lipoprotein receptor (LDLR) serves as the major entry receptor during internalization. Statins are commonly used to upregulate the expression of LDLR.

[0464] 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 before transduction. Then, activated primary NK cells were transduced with antigen-2CAR at an MOI of 5. Cells pretreated with DMSO were included as negative controls.

[0465] like Figure 3A As shown, incubation of primary NK cells with rosuvastatin during activation resulted in an increase in the level of LDLR expression on the cells (n=3). Figure 3B As shown, increased levels of LDLR expression on primary NK cells correlated with increased antigen-2 CAR expression.

[0466] 7.2.3. Transduction enhancers

[0467] Retronectin is a recombinant human fibronectin fragment that enhances lentiviral and retroviral mediated gene transduction by aiding the colocalization of target cells and viral particles. Vectofusin is a fully synthetic, non-toxic, cationic amphipathic peptide with viral transduction enhancing ability.

[0468] To test whether transduction enhancers could improve CAR transduction efficiency or surface CAR stability, primary NK cells were activated and transduced in the presence or absence of Retronectin and Vectofusin.

[0469] One day before plating primary NK cells, Retronectin (Takara Bio Inc., catalog number T202) was diluted from a 1 μg / μL stock solution in sterile PBS to prepare a 20 μg / mL working solution. The Retronectin working solution was added to the wells and evenly distributed in the wells. The plates were kept overnight at 4°C before plating the day. Retronectin was removed from the wells after overnight incubation. PBS containing 2% BSA was added to each well. The plates were incubated at room temperature for 30 minutes and washed twice with PBS. The NK cells were then plated and activated as described in Section 6.1 above.

[0470] Primary NK cells activated by transduction with antigen-2CAR at an MOI of 5. Vectofucin (Miltenyi, catalog number 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 to 15 minutes. The mixture was equilibrated to room temperature, then added to the NK cells and mixed with the cell suspension by pipetting up and down. The cells were then cultured at 37 ° C and 5% CO2. CAR expression in primary NK cells was assessed on the 10th day.

[0471] like Figure 4 As shown, the presence of transduction enhancers Retronectin and Vectofusin correlated with increased Antigen-3 CAR expression during lentiviral transduction of primary NK cells (n=3).

[0472] 7.2.4.Join

[0473] In order to solve the challenges of lentiviral transduction in primary NK cells, primary NK cells were transduced with a combination of PDK1 inhibitors, statins and transduction enhancers. Activated NK cells were transduced with antigen-3CAR at an MOI of 5. The cells were transduced in a dish coated with Retronectin together with BX795, rosuvastatin and Vectofucin. The method of incorporating BX795, rosuvastatin and Vectofucin was the same as described above in Section 6.2. After transduction, the plate was centrifuged at 850 × g for 2 hours at 37 ° C. Once the rotation was completed, the plate was returned to an incubator at 37 ° C and 5% CO2. At different time points after transduction, CAR expression was monitored with anti-antigen-3CAR recombinant protein AF647 antibody (2 μg / mL).

[0474] like FIG. 5A to FIG. 5D As shown, primary NK cells transduced with a combination of BX795, rosuvastatin, and Vectofucin showed higher expression and stability of surface CAR compared to lentivirus alone (n=3).

[0475] In order to solve the challenge of lentiviral transduction in primary T cells, primary αβT cells were transduced with a combination of PDK1 inhibitors, statins and transduction enhancers. Activated αβT cells were transduced with antigen-3CAR (inside) at an MOI of 5. The cells were transduced in a dish coated with Retronectin together with BX795 (3 μM), rosuvastatin (1 μM) and Vectofucin. The method of incorporating BX795, rosuvastatin and Vectofucin was the same as described above in Section 6.2. After transduction, the plate was centrifuged at 850 × g for 2 hours at 37 ° C. Once the rotation was completed, the plate was returned to an incubator at 37 ° C and 5% CO2. At different time points after transduction, CAR expression was monitored with anti-antigen-3CAR recombinant protein AF647 antibody (2 μg / mL).

[0476] like Figure 6 As shown, primary αβ T cells transduced with a combination of BX795, rosuvastatin, Retronectin, and Vectofucin showed higher CAR expression compared to lentivirus alone (n=3).

[0477] 7.3. Example 3 - Optimization of gene transfer in γδ T cells.

[0478] Lentiviral transduction of primary γδ T cells using VSV-G pseudotyped lentiviral vectors has been associated with low levels of CAR expression, limiting CAR screening capabilities. Lentiviral transduction of primary γδ T cells in the presence of compounds targeting LDLR (statins), antiviral pathways (BX795), and vectofusin was evaluated.

[0479] Four primary γδT cell samples from healthy donors were expanded for five days with zoledronic acid (ZOL), transduced in the presence or absence of enhancers (rosuvastatin, BX795 and Vectofusin), and CAR expression was evaluated. In short, frozen PBMC cells were thawed and resuspended with 1E6 cells / ml. PBMC cells were activated with 10 μM zoledronic acid and expanded 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 lentiviral vector at an MOI of 5, including the following steps: (1) pre-incubating the lentiviral vector encoding the antigen-4 specific CAR with statin, vectofusin-1 (10ug / ml) on ice for 10 minutes before adding to the cells; (2) adding 5μM BX795 to the cells and gently mixing; (3) spinning the cells at 850g for 2 hours at 32°C; and (4) returning the cells to the incubator at 37°C. Cells were analyzed for CAR expression by flow cytometry on days 3, 6, and 10.

[0480] Lentiviral transduction of primary γδ T cells in the presence of enhancers (rosuvastatin, BX795, and Vectofusin) reduced cell viability compared to lentiviral transduction without enhancers ( Figure 7 ). Lentiviral transduction of primary γδ T cells in the presence of the enhancer increased CAR levels in primary γδ T cells compared to lentiviral vector alone ( Figure 8 ).

[0481] Conclusions: Although lentiviral transduction of primary γδ T cells in the presence of enhancers (rosuvastatin, BX795, and Vectofusin) increased transduction, it was also associated with increased cell death, thus making practical implementation of this protocol challenging.

[0482] Next, lentiviral transduction of primary γδ T cells was assessed using VSV-G pseudotyped lentiviral vectors in the presence of an enhancer targeting LDLR.

[0483] Three primary γδT cell samples from healthy donors were expanded with zoledronic acid (ZOL) for five days, 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 evaluated. In brief, frozen PBMC cells were thawed and resuspended at 1E6 cells / ml. PBMC cells were activated with 10 μM zoledronic acid and expanded 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 an antigen-4-specific CAR at an MOI of 5, comprising the following steps: (1) pre-incubating the lentiviral vector encoding the antigen-4 CAR with statins on ice for 10 minutes and then adding it to the cells; (2) the cells were inoculated at 850g 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.

[0484] Lentiviral transduction of primary γδ T cells in the presence of statins was associated with higher CAR expression compared to cells transduced with lentiviral vector alone ( 9A to 9F The new protocol also achieved increased CAR expression on primary γδ T cells ( 9A to 9F ), enabling CAR screening for future experiments.

[0485] Next, the effect of pretreatment with statins in lentiviral transduction of primary γδ T cells on LDLR expression was evaluated. Three primary γδ T cell samples from healthy donors were expanded with Zol for five days, pretreated in the presence or absence of different concentrations of rosuvastatin and atorvastatin as described above, and evaluated for LDLR expression.

[0486] Compared with cells transduced with lentiviral vector alone, cells transduced with statins, atorvastatin ( Fig. 10A ) or rosuvastatin ( Fig. 10B ) pretreatment of primary γδT cells increased LDLR expression.

[0487] Next, we evaluated the effect of pretreatment with statins in 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 expanded with Zol for five days, pretreated in the presence or absence of different concentrations of rosuvastatin or atorvastatin, as described above, and evaluated for Vγ9Vδ2 expression using flow cytometry.

[0488] Compared with cells transduced with lentiviral vector alone, cells transduced with statins, atorvastatin ( Fig.11A ) or rosuvastatin ( Fig. 11B ) Pretreatment of primary γδT cells at 0 statin concentration did not change the frequency of Vγ9Vδ2 cells.

[0489] *****

[0490] It will be appreciated by those skilled in the art that modifications may be made to the above embodiments without departing from the broad inventive concept of the present invention. Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the present invention as defined in this specification.

[0491] Various publications, articles, and patents are cited or described in the background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, etc. included in this specification is intended to provide a context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

Claims

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

2. The method of claim 1, wherein the immune effector cells are autologous or allogeneic.

3. The method of claim 1, wherein the immune effector cell is a cytotoxic cell, and wherein optionally the cytotoxic cell is a natural killer cell. The method of claim 1 , wherein the immune effector cells are γδ T cells. The method of claim 1 , wherein the immune effector cells are αβ T cells.

6. The method of claim 1, wherein the step for increasing the expression of the low-density lipoprotein receptor (LDLR) comprises: The immune effector cells are contacted with a statin.

7. The method of claim 6, wherein the statin is rosuvastatin.

8. The method of claim 6, wherein the statin is atorvastatin.

9. The method according to any one of claims 1 to 8, further comprising a step for inhibiting the intracellular antiviral defense mechanisms of the immune effector cells.

10. The method of claim 9, wherein the step for inhibiting the intracellular antiviral defense mechanism of the immune effector cells comprises: The immune effector cells are contacted with a 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor.

11. The method of claim 10, wherein the PDK1 inhibitor is BX795.

12. The method according to any one of claims 1 to 11, wherein the method further comprises providing one or more transduction enhancers.

13. The method of claim 12, wherein the transduction enhancer is Vectofusin.

14. The method of claim 12, wherein the transduction enhancer is Retronectin.

15. The method of any one of claims 1 to 14, wherein the method further comprises introducing a nucleic acid into the immune effector cell.

16. The method of claim 15, wherein the nucleic acid comprises a promoter. 17 . The method according to claim 16 , wherein the promoter is selected from the group consisting of CAG promoter, PGK promoter, EF1a promoter and EFS promoter.

18. The method of any one of claims 15 to 17, wherein the nucleic acid is delivered via a lentiviral particle.

19. The method of claim 18, wherein the lentiviral particles are pseudotyped with vesicular stomatitis virus G (VSV-G).

20. The method according to any one of claims 15 to 19, wherein the nucleic acid encodes an exogenous functional receptor.

21. The method of claim 20, wherein the exogenous functional receptor is a chimeric antigen receptor (CAR) or TCR.

22. A method for preparing immune effector cells for transduction, the method comprising contacting the immune effector cells with an agent that increases expression of the low-density lipoprotein receptor (LDLR).

23. The method of claim 22, wherein the immune effector cells are autologous or allogeneic.

24. The method of claim 22, wherein the immune effector cell is a cytotoxic cell, and wherein optionally the cytotoxic cell is a natural killer cell.

25. The method of claim 22, wherein the immune effector cells are γδ T cells.

26. The method of claim 22, wherein the immune effector cells are αβ T cells.

27. The method of claim 22, wherein the step for increasing the expression of the low-density lipoprotein receptor (LDLR) comprises: The immune effector cells are contacted with a statin.

28. The method of claim 27, wherein the statin is rosuvastatin.

29. The method of claim 27, wherein the statin is atorvastatin.

30. The method according to any one of claims 22 to 29, further comprising a step for inhibiting the intracellular antiviral defense mechanisms of the immune effector cells.

31. The method of claim 30, 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.

32. The method of claim 31 , wherein the PDK1 inhibitor is BX795.

33. The method of any one of claims 22 to 32, wherein the method further comprises providing one or more transduction enhancers.

34. The method of claim 33, wherein the transduction enhancer is Vectofusin.

35. The method of claim 33, wherein the transduction enhancer is Retronectin.

36. The method of any one of claims 22 to 35, wherein the method further comprises introducing a nucleic acid into the immune effector cell.

37. The method of claim 36, wherein the nucleic acid comprises a promoter.

38. The method of claim 37, wherein the promoter is selected from the group consisting of CAG promoter, PGK promoter, EF1a promoter and EFS promoter.

39. The method of any one of claims 36 to 38, wherein the nucleic acid is delivered via a lentiviral particle.

40. The method of claim 39, wherein the lentiviral particles are pseudotyped with vesicular stomatitis virus G (VSV-G).

41. The method of any one of claims 36 to 38, wherein the nucleic acid encodes an exogenous functional receptor.

42. The method of claim 41, wherein the exogenous functional receptor is a chimeric antigen receptor (CAR) or a TCR.

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