Lentiviral vectors targeting lymphocytes

Abstract

Provided herein are lentiviral vectors comprising a mutated, heterologous envelope protein, a target protein, and at least one transgene for delivery to and expression by a cell characterized by the target protein. Also provided are methods and materials for producing the lentiviral vectors described herein, methods for transducing target cells, and cells transduced with lentiviral vectors according to the present disclosure.

Description

[Technical field]

[0001] Description of sequence listing The sequence listing associated with this application is provided in text format instead of paper and is incorporated herein by reference. The name of the text file containing the sequence listing is 930207_401WO_SEQUENCE_LISTING.txt. The text file is 158KB, was created on February 25, 2022, and was submitted electronically via EFS-Web. [Background technology]

[0002] Lentiviral vectors play an important role in gene-modified cell therapy, especially T cell therapy. Recently approved T cell therapies rely on retroviral vectors to transduce therapeutic molecules (e.g., chimeric antigen receptors (CARs)) into T lymphocytes. A risk associated with the production of CAR T cells is the introduction of transgenes into other cell types. The use of integrating vectors with broad cell tropism, e.g., lentiviral vectors pseudotyped with VSV-G envelope proteins, although rare, may pose a significant safety concern. [Brief description of the drawings]

[0003] [Figure 1] FIG. 1 is a schematic diagram of a helper plasmid suitable for use in a third generation LVV production system. [Figure 2A] Graph showing on-target and off-target entry of Jurkat T cells and Raji B cells by lentiviral vectors carrying mutant VSV-G envelopes (Trop-002, Trop-051, Trop-052, Trop-055, and Trop-061) to abolish LDL receptor binding and the T cell targeting protein CD80. The left bar for each sample is on-target entry and the right bar is off-target. Binding of the T cell targeting protein to its cognate ligand on the T cells results in entry of the lentiviral vector, followed by measurement of expression of the reporter green fluorescent protein (GFP). [Figure 2B] 1 shows FACS plots showing on-target and off-target entry of Jurkat T cells and Raji B cells by lentiviral vectors carrying mutant VSV-G envelopes (Trop-002, Trop-051, Trop-052, Trop-055, and Trop-061) to abolish LDL receptor binding and the T cell targeting protein CD80. Binding of the T cell targeting protein to its cognate ligand on the T cells results in entry of the lentiviral vector, after which expression of the reporter green fluorescent protein (GFP) is measured. [Figure 3A] Figure 1 shows that the T cell targeting protein CD80 expressed from a VSV-G packaging plasmid is expressed at relatively similar levels as mutant VSV-G on the surface of HEK293 producer cells. [Figure 3B] A diagram showing that LVV generated by this approach are capable of transducing target Jurkat T cells, but not Raji B cells. [Figure 4] (Top) A diagram showing the expression levels of CD80 and mutant VSV-G on the surface of HEK293T producer cells using LVV generated by cloning the CD80 target protein into the Rev packaging plasmid or into the mutant VSV-G packaging plasmid, and (bottom) a diagram showing that LVV generated by cloning the CD80 target protein into the Rev packaging plasmid or into the mutant VSV-G packaging plasmid transduce target Jurkat T cells. [Diagram 5] (Left) Expression of CD80 target protein on the surface of HEK293T producer cells as a function of CD80 plasmid concentration using the five plasmid packaging system. (Right) LVV generated by the five plasmid packaging system transduced target Jurkat T cells and transduction efficiency was related to the concentration of CD80 packaging plasmid. [Figure 6A]Shown is the titer of lymphocyte-targeting LVV produced in adherent HEK293 producer cells. LVV were harvested from the HEK293 adherent cell culture medium by centrifugation. [Figure 6B] Shown is the titer of lymphocyte-targeting LVV produced in adherent HEK293 producer cells. LVV were harvested from HEK293 adherent cell media by anion exchange chromatography followed by tangential flow filtration. [Figure 6C] Shown is the titer of lymphocyte-targeting LVV produced in suspension HEK293 producer cells. LVV was harvested from HEK293 suspension cell culture medium by anion exchange chromatography. [Figure 6D] Figure 1 shows the titers of lymphocyte-targeting LVV produced in adherent HEK293 or suspension HEK293 producer cells. Enrichment by AEX / TFF led to the preparation of LVV with high levels of purity and recovery. [Figure 7] Schematic diagram examining T cell transduction by LVV containing a BCMA CAR transgene or T cell-targeted LVV containing a BCMA CAR transgene (anti-CD3 and CD80) in PBMCs from healthy human donors. The graph shown on the bottom right shows that even at a low MOI, T cell-targeted LVV transduces T cells at higher levels than standard LVV, and that T cell-targeted LVV, in contrast to standard LVV, is able to transduce T cells in the absence of IL-2 and exogenous activating antibodies (anti-CD3 and anti-CD28). [Figure 8] Graph showing T cell proliferation from PBMCs obtained from three different donors and transduced using standard LVV or T cell redirected LVV (anti-CD3 and CD80) in the presence or absence of exogenous activating anti-CD3 and anti-CD28 antibodies. [Figure 9]A schematic diagram is shown examining T cell transduction by LVV containing a CD19 CAR transgene or T cell-targeted LVV (anti-CD3 and CD80) containing a CD19 CAR transgene in PBMCs from healthy human donors. The graph shown on the bottom right shows that T cell-targeted LVV transduces T cells at higher levels than standard LVV, and that T cell-targeted LVV, in contrast to standard LVV, is able to transduce T cells in the absence of exogenous activating antibodies (anti-CD3 and anti-CD28). [Figure 10] Graphs showing the transduction efficiency of T cells and the level of T cell activation by anti-CD3 targeting proteins (12F6 in VH-VL and VL-VH orientations) used to generate LVV targeted to T cells. [Figure 11] Graphs showing that BCMA CAR T cells exhibit increased expression of T cell effector cytokines (TNFα-left, TNFα and IFNγ-right) following culture with BCMA positive cell lines, which is not observed in BCMA negative cell lines, whether generated by standard or T cell redirected LVV (anti-CD3 and CD80). [Figure 12A] Graph showing in vivo delivery of transgenes using T cell-targeted LVV. T cell-targeted LVV (anti-CD3 and CD80) specifically transduces human T cells (CD3+) but not human B cells (CD20+) in a humanized mouse model (n=5). [Figure 12B] Graph showing in vivo delivery of transgenes using LVV targeted to T cells, which transduced both CD8+ and CD8- (CD4) T cells compared to standard LVV which did not. [Figure 13] 1 shows a graph demonstrating that CD80-targeted LVV enhances transduction of CD4 T cells compared to standard LVV. [Figure 14]Graphs showing that T cell targeted (anti-CD3 and CD80) LVV transduces target Jurkat T cells but does not transduce off-target tumor cells (Raji, Ramos, Jeko-1, and NALM-6) compared to standard LVV. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0004] Described herein are engineered lentiviral vectors. The lentiviral vectors comprise a mutant, heterologous envelope protein, a target protein, and at least one transgene for delivery to and expression by a cell characterized by the target protein. In some embodiments, the target protein is selected to target immune cells, including, for example, lymphocytes or T cells. In certain such embodiments, the lentiviral vectors described herein can selectively target and efficiently transduce resting lymphocytes, such as T cells. In some embodiments, the lentiviral vectors described herein are capable of transducing and / or activating T cells in the absence of exogenous T cell stimulants. In some embodiments, the lentiviral vectors described herein enhance transduction of CD4 T cells compared to standard lentiviral vectors.

[0005] In some embodiments, lentiviral vectors incorporating the mutant env and target proteins described herein are capable of producing higher titers of LVV product compared to standard LVV incorporating another fusogenic env protein (e.g., coccal env, paramyxovirus env, truncated VSV-G env).

[0006] Also provided are methods and materials for producing the lentiviral vectors described herein, methods for transducing target cells, and cells transduced with the lentiviral vectors according to the present disclosure. In some embodiments, the lentiviral vectors described herein and / or cells transduced with such vectors may be used to treat a disease or disorder responsive to the presence of cells expressing a transgene delivered by the vector.

[0007] definition Before setting forth the present disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms that are used herein.

[0008] Any concentration range, percentage range, ratio range, or integer range herein is understood to include any integer value within the recited range, and fractions thereof (e.g., 1 / 10 and 1 / 100 of an integer), as appropriate, unless otherwise specified. Also, any numerical range recited herein for any physical property, such as polymer subunits, size, or thickness, is understood to include any integer within the recited range, unless otherwise specified. As used herein, the term "about" means ±20% of the indicated range, value, or structure, unless otherwise specified. As used herein, the terms "a" and "an" are understood to refer to "one or more" of the recited components. The use of alternatives (e.g., "or") is understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include," "having," and "comprise" are used interchangeably, and it is intended that these terms and variations thereof be considered non-limiting.

[0009] Terms understood by those skilled in the art of antibody technology are given their respective art-acquired meanings unless expressly defined otherwise herein. The term "antibody" is used in the broadest sense and includes polyclonal and monoclonal antibodies. "Antibody" may refer to an intact antibody comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and an antigen-binding portion (or antigen-binding domain) of the intact antibody that has or retains the ability to bind to a target molecule. An antibody may be a naturally occurring, recombinantly produced, engineered, or modified form of immunoglobulin, such as an intrabody, a peptibody, a nanobody, a single domain antibody, a SMIP, a multispecific antibody (e.g., a bispecific antibody, a diabody, a triabody, a tetrabody, a tandem di-scFv, a tandem tri-scFv, an ADAPTIR). A monoclonal antibody or an antigen-binding portion thereof may be non-human, chimeric, humanized, or human, preferably humanized or human. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). The term "antigen-binding portion" or "antigen-binding domain" of an intact antibody is intended to encompass "antibody fragments," which refers to a portion of an intact antibody and refers to the antigen-determining variable region or complementarity-determining region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, Fab'-SH, F(ab')2, diabodies, linear antibodies, scFv antibodies, VH, and multispecific antibodies formed from antibody fragments. "Fab" (fragment antigen-binding) is the portion of an antibody that binds to an antigen and includes the variable region and CH1 of the heavy chain attached to the light chain via an interchain disulfide bond. The antibody can be of any class or subclass, including IgG and its subclasses (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.

[0010] The term "variable region" or "variable domain" in the context of an antibody refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains (or regions) of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three complementarity determining regions (CDR) (see, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated using the VH or VL domain of an antibody that binds to the antigen, and a library of complementary VL or VH domains, respectively, may be screened. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0011] The terms "complementarity determining region" and "CDR" are known in the art to be synonymous with "hypervariable region" or "HVR" and refer to noncontiguous sequences of amino acids within an antibody variable region that confer antigen specificity and / or binding affinity. Generally, each heavy chain variable region has three CDRs (HCDR1, HCDR2, HCDR3) and each light chain variable region has three CDRs (LCDR1, LCDR2, LCDR3).

[0012] As used herein, the terms "binding domain", "binding region" and "binding moiety" refer to a molecule, e.g., a peptide, oligopeptide, polypeptide, or protein, that has the ability to specifically and non-covalently bind, associate, combine with, recognize, or compound with a target molecule (e.g., a tumor antigen). Binding domains include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biomolecule or other target of interest. In some embodiments, the binding domain is an antigen binding domain, e.g., an antibody or a functional binding domain or antigen binding portion thereof. Exemplary binding domains include single chain antibody variable regions (e.g., domain antibodies, sFv, scFv, Fab), receptor ectodomains (e.g., TNF-α), ligands (e.g., cytokines, chemokines), or synthetic polypeptides selected for their ability to specifically bind to a biomolecule.

[0013] "Major histocompatibility molecules" (MHC molecules) refer to glycoproteins that deliver peptide antigens to the cell surface. MHC class I molecules are heterodimers composed of a transmembrane α chain (containing three α domains) and a non-covalently bound β2-microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, α and β, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides derived from the cytosol to the cell surface, where the peptide:MHC complex is detected by CD8 + MHC class II molecules deliver peptides from the vesicle system to the cell surface, where they are recognized by CD4 + Recognized by T cells. MHC molecules can be from a variety of animal species, including human, mouse, rat, or other mammals.

[0014] "Chimeric antigen receptor" (CAR) refers to a chimeric fusion protein that does not naturally occur in a host cell and contains two or more different domains linked together in a manner that can function as a receptor when expressed on the surface of the cell. CARs are generally composed of an extracellular domain that contains a binding domain that binds to a target antigen, an optional extracellular spacer domain, a transmembrane domain, and an intracellular signaling domain (e.g., containing an immunoreceptor tyrosine-based activation motif (ITAM)), and optionally an intracellular costimulatory domain. In certain embodiments, the intracellular signaling domain of a CAR has an ITAM (e.g., CD3ζ) that contains an intracellular signaling domain and an intracellular costimulatory domain (e.g., 4-1BB). In certain embodiments, a CAR is synthesized as a single polypeptide chain or encoded by a nucleic acid molecule as a single polypeptide chain.

[0015] A variety of assays are known for identifying binding domains of the present disclosure that specifically bind to a particular target, as well as for determining the affinity of the binding domains, such as Western blots, ELISAs, analytical ultracentrifugation, spectroscopy, surface plasmon resonance (BIACORE®) analysis, and MHC tetramer analysis (see, e.g., Scatchard et al., Ann. NY Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; Altman et al., Science 274:94-96, 1996; and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents). As used herein, "specifically binds" refers to a binding domain, or a fusion protein thereof, that specifically binds to a target molecule with greater than 10% specificity. 5 M -1 or higher affinity or K a (i.e., the equilibrium binding constant for a particular binding interaction in units of 1 / M) while not significantly binding or associating with any other molecules or components in the sample.

[0016] The terms "antigen" and "Ag" refer to a molecule capable of inducing an immune response. The induced immune response may include antibody production, activation of specific immunocompetent cells, or both. Macromolecules including proteins, glycoproteins, and glycolipids may function as antigens. Antigens may be derived from recombinant or genomic DNA. As contemplated herein, antigens need not be (i) encoded solely by the full-length nucleotide sequence of a gene, or (ii) encoded by a "gene" at all. Antigens may be generated or synthesized, or they may be obtained from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0017] The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant within an antigen to which a cognate immune binding molecule, such as an antibody or fragment thereof (e.g., scFv), T cell receptor (TCR), CAR, or other binding molecule, domain, or protein, specifically binds. Epitope determinants usually comprise chemically active surface groupings of molecules, such as amino acids or sugar side chains, and may have specific three-dimensional structural characteristics and specific charge characteristics. Epitopes may be linear or conformational epitopes.

[0018] As used herein, an "effector domain" is an intracellular portion of a fusion protein or chimeric receptor that can directly or indirectly promote a biological or physiological response in a cell expressing the effector domain upon receiving an appropriate signal. In certain embodiments, the effector domain is part of a protein or protein complex that receives a signal upon binding. In other embodiments, the effector domain is part of a protein or protein complex that directly binds to a target molecule that induces a signal from the effector domain. For example, in response to the binding of a CAR to a target molecule, the effector domain can transmit a signal to the inside of a host cell and induce an effector function. If the effector domain contains one or more signaling domains or motifs, the effector domain can directly promote a cellular response. In other embodiments, the effector domain indirectly promotes a cellular response by binding to one or more other proteins that directly promote the cellular response.

[0019] "Junction amino acid" or "junction amino acid residue" refers to one or more (e.g., about 2-20) amino acid residues between two adjacent motifs, regions, or domains of a polypeptide. Junction amino acids can result from the design of a chimeric protein (e.g., amino acid residues resulting from the use of a restriction enzyme site during construction of a nucleic acid molecule encoding a fusion protein).

[0020] "Disease" is the health condition of a subject that the subject is unable to maintain homeostasis, and if the disease is not improved, the health condition of the subject will continue to deteriorate.In contrast, "disorder" or "undesirable condition" in a subject is a health condition in which the subject can maintain homeostasis, but the health condition of the subject is less than that in the absence of the disorder or undesirable condition.If left untreated, the disorder or undesirable condition does not necessarily lead to further deterioration of the health condition of the subject.

[0021] "Nucleic acid molecules" and "polynucleotides" may be in the form of RNA or DNA, including cDNA, genomic DNA, and synthetic DNA. Nucleic acid molecules may be composed of naturally occurring nucleotides (e.g., deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., α-enantiomeric forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides may have modifications or substitutions in the sugar moiety, or in the pyrimidine or purine base moiety. Nucleic acid monomers may be linked by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester bonds include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidates, phosphoroamidates, and the like. Nucleic acid molecules may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding strand (antisense strand). The coding molecule may have a coding sequence identical to a coding sequence known in the art or may have a different coding sequence that is capable of encoding the same polypeptide as a result of redundancy or degeneracy in the genetic code or by splicing.

[0022] "Encoding" refers to the inherent property of certain polynucleotide sequences, e.g., DNA, cDNA, and mRNA sequences, to serve as templates for the synthesis of other polymers and macromolecules having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids in biological processes and the biological properties resulting therefrom. Thus, a polynucleotide encodes a protein when transcription and translation of the mRNA corresponding to that polynucleotide produces the protein in a cell or other biological system. Both the coding and non-coding strands can be said to encode proteins or other products of the polynucleotide. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence.

[0023] As used herein, the term "endogenous" or "native" refers to a gene, protein, compound, molecule, or activity that is normally present in a host or host cell, and includes naturally occurring variants of the gene, protein, compound, molecule, or activity.

[0024] As used herein, "homolog" or "homolog" refers to a molecule or activity from a host cell to which it is ancestrally linked to a second gene or activity, e.g., from the same host cell, from a different host cell, from a different organism, from a different strain, from a different species. For example, a heterologous molecule or a heterologous gene encoding the molecule may be homologous to a native host cell molecule or gene encoding the molecule, respectively, and may optionally have an altered structure, sequence, expression level, or any combination thereof.

[0025] As used herein, a "heterologous" nucleic acid molecule, construct, or sequence refers to a nucleic acid molecule or a portion of a nucleic acid molecule that is not native to a host cell, but can be homologous to a nucleic acid molecule or a portion of a nucleic acid molecule from the host cell. The source of the heterologous nucleic acid molecule, construct, or sequence can be from a different genus or species. In some embodiments, the heterologous nucleic acid molecule is not naturally occurring. In certain embodiments, the heterologous nucleic acid molecule is added to a host cell or host genome (i.e., not endogenous or native), for example, by conjugation, transformation, transfection, transduction, electroporation, etc., and the added molecule may be integrated into the genome of the host cell or may exist as extrachromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and may exist in multiple copies. Additionally, "heterologous" refers to a non-native enzyme, protein, or other activity that is encoded by a non-endogenous nucleic acid molecule introduced into the host cell, even if the host cell encodes the homologous protein or activity.

[0026] As used herein, the terms "engineered," "recombinant," "mutant," "modified," or "non-naturally occurring" refer to an organism, microorganism, cell, nucleic acid molecule, or vector that has been modified by the introduction of a heterologous nucleic acid molecule, or to a cell or microorganism that has been genetically engineered by human intervention, i.e., modified by the introduction of a heterologous nucleic acid molecule, or to a cell or microorganism that has been altered such that the expression of an endogenous nucleic acid molecule or gene is controlled, deregulated, or constitutive, where such alterations or modifications may be introduced by genetic engineering. Human-generated genetic changes may include, for example, modifications that introduce nucleic acid molecules (which may include expression control elements such as promoters) that code for one or more proteins, chimeric receptors, or enzymes, or the addition, deletion, substitution, or other functional disruption of or addition to the genetic material of a cell. Exemplary modifications include modifications from a reference or parent molecule in regions that code for heterologous or homologous polypeptides, or functional fragments thereof. Additional exemplary modifications include, for example, modifications in non-coding regulatory regions, where the modification alters the expression of a gene or operon.

[0027] As used herein, the term "transgene" refers to a gene or polynucleotide encoding a protein of interest (e.g., CAR) whose expression is desired in a host cell and which has been introduced into the cell by genetic engineering techniques. Transgenes can encode proteins of therapeutic interest as well as proteins such as reporters, tags, markers, suicide proteins, etc. Transgenes can be derived from natural sources, modifications of natural genes, or recombinant or synthetic molecules. In certain embodiments, the transgene is a component of a vector.

[0028] The term "overexpressed" antigen or "overexpression" of an antigen refers to an abnormally high level of antigen expression in a cell. Overexpressed antigens or overexpression of antigens are often associated with pathologies, such as in hematological malignancies and cells that form solid tumors in specific tissues or organs of a subject. Solid tumors or hematological malignancies characterized by overexpression of tumor antigens can be identified by standard assays known in the art.

[0029] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, and longer chains, which are commonly referred to in the art as proteins and of which there are many types. "Polypeptides" include, among others, biologically active fragments, substantially homogeneous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0030] As used herein, the term "mature polypeptide" or "mature protein" refers to a protein or polypeptide that is secreted or localized within the cell membrane or inside a particular cellular organelle (e.g., the endoplasmic reticulum, Golgi, or endosome) and that includes a partially cleaved N-terminal signal sequence (e.g., one or more amino acids of the signal sequence that remain, but less than the entire signal sequence) or that does not include an N-terminal signal sequence (i.e., the N-terminal signal sequence has been completely removed from the protein or polypeptide, e.g., by an endogenous cleavage process).

[0031] A "signal sequence", also called a "signal peptide", "leader sequence", "leader peptide", "localization signal" or "localization sequence", is a short peptide (usually 13-36 amino acids long) present at the N-terminus of a newly synthesized protein destined for the plasma membrane or secretory pathway. A signal sequence usually contains a short stretch of hydrophilic positively charged amino acids at the N-terminus, a central hydrophobic domain of 5-15 residues, and a C-terminal region with a signal sequence cleavage site. In eukaryotes, the signal sequence directs the translocation of the newly synthesized protein to the endoplasmic reticulum, where it is cleaved by a signal peptidase to generate the mature protein, which then proceeds to the appropriate destination. The variability in the length and amino acid composition of the signal sequence makes it difficult to accurately predict the cleavage site. In the disclosure of polypeptide sequences referred to in this specification, a signal sequence is also contemplated, which is absent or has a partial signal sequence.

[0032] The "percent identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced to optimize the alignment of the two or more sequences (i.e., % identity = number of identical positions / total number of positions x 100). Comparison of sequences and determination of percent identity between two or more sequences can be accomplished using mathematical algorithms, such as the BLAST and Gapped BLAST programs, using their default parameters (e.g., Altschul et al., J. Mol. Biol. 215:403, 1990; see also BLASTN at www.ncbi.nlm.nih.gov / BLAST).

[0033] "Conservative substitution" is recognized in the art as replacing one amino acid with another amino acid with similar properties.Exemplary conservative substitutions are well known in the art (see, for example, WO97 / 09433, published March 13, 1997, p. 10; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY: NY (1975), pp. 71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8).

[0034] The term "chimera" refers to any nucleic acid molecule or protein that contains a combination of sequences that are not endogenous and are not naturally found associated or linked to each other in nature. For example, a chimeric nucleic acid molecule can contain nucleic acids that encode various domains from multiple different genes. In another example, a chimeric nucleic acid molecule can contain regulatory and coding sequences that are derived from different sources, or regulatory and coding sequences that are derived from the same source but arranged in a manner different from that found in nature.

[0035] As used herein, the term "promoter" is defined as a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence.

[0036] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence is a core promoter sequence, and in other instances, this sequence may include an enhancer sequence and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0037] A "constitutive" promoter is a nucleotide sequence which, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0038] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only if an inducer corresponding to that promoter is present in the cell.

[0039] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoded by or specified by a gene, causes a gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0040] As used herein, the terms "under transcriptional control" or "operably linked" mean that the promoter is in the correct location and orientation with respect to the polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0041] A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. A vector can be, for example, a plasmid, a cosmid, a virus, or a phage. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the introduction of a nucleic acid into a cell. An "expression vector" is a vector that, when present in an appropriate environment, is capable of directing the expression of a protein encoded by one or more transgenes carried by the vector. The one or more transgenes expressed by the vectors described herein are encoded in an expression cassette.

[0042] A "lentiviral vector" is a vector derived from a lentivirus and contains one or more lentiviral packaging proteins and / or one or more lentiviral proteins necessary for expression of one or more genes carried by the vector. The abbreviation "LVV" is used herein to refer to lentiviral vector (singular) and lentiviral vectors (plural).

[0043] "Lentivirus" refers to a genus of retroviruses capable of infecting dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to, human immunodeficiency viruses, including HIV (HIV types 1 and 2), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0044] "Retrovirus" refers to an RNA virus that is a single-stranded positive-sense RNA molecule. Retroviruses contain reverse transcriptase and integrase enzymes. Once retroviruses enter a target cell, they use reverse transcriptase to transcribe their RNA molecule into a DNA molecule. The integrase enzyme is then used to integrate the DNA molecule into the genome of the host cell. Once integrated into the genome of the host cell, sequences derived from the retrovirus are called proviruses (e.g., proviral sequences or provisms sequences).

[0045] As used herein, the term "expression cassette" refers to a unique component of a vector nucleic acid that contains at least one transgene and regulatory sequences (e.g., promoter, 3'UTR) that control its expression in a host cell. A tandem expression cassette refers to a component of a vector nucleic acid that contains at least two transgenes under the control of the same set of regulatory sequences for tandem expression of the at least two transgenes. In certain embodiments, the tandem expression cassette contains at least two transgenes under the control of the same promoter. In certain embodiments, the first and second transgenes are separated by an internal ribosome entry site (IRES), a furin cleavage site, or a self-cleaving viral 2A peptide to allow for co-expression of two proteins from a single mRNA.

[0046] The term "immune system cell" or "immune cell" refers to any cell of the immune system that is derived from hematopoietic stem cells in the bone marrow. Hematopoietic stem cells give rise to two major lineages: myeloid progenitors (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitors (which give rise to lymphoid cells such as T cells, B cells, and natural killer (NK) cells). Examples of immune system cells include CD4+ T cells, CD8+ T cells, CD4-CD8- double negative T cells, γδ T cells, regulatory T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells are sometimes referred to as "antigen presenting cells" or "APCs," which are specialized cells that can activate T cells when major histocompatibility complex (MHC) receptors on the surface of APCs complexed with peptides interact with TCRs on the surface of T cells.

[0047] The term "lymphocyte" refers to immune cells of lymphoid origin, which are cells that display at least one phenotype characteristic of lymphocytes or their precursors or progenitors that distinguish them from cells of the erythroid or myeloid lineages. The term "lymphocyte" includes T cells, B cells, and natural killer (NK) cells.

[0048] The term "T cell" refers to a cell of the T cell lineage. A "cell of the T cell lineage" refers to a cell that displays at least one phenotype characteristic of a T cell or its precursor or progenitor, which distinguishes the cell from other cells of lymphoid origin and from cells of the erythroid or myeloid lineage. Such phenotypic characteristics include the expression of one or more proteins specific for T cells (e.g., CD3 + , CD4 + , CD8 + ), or physiological, morphological, functional, or immunological characteristics specific to T cells. For example, cells of the T cell lineage can be progenitor or precursor cells that are committed to the T cell lineage, CD25 + Immature and inactivated T cells, CD4 or CD8 lineage committed cells, CD4 + CD8 + Thymocyte precursors double positive, CD4 single positive + Or CD8 + , TCRαβ or TCRγδ, or mature functional or activated T cells. The term "T cells" refers to naive T cells (CD45RA+, CCR7+, CD62L+, CD27+, CD45RO-), central memory T cells (CD45RA-, CD45RO-), + , CD62L + , CCR7+, CD27+), effector memory T cells (CD45RA-, CD45RO+, CCR7-, CD62L-, CD27-), mucosal-associated invariant T (MAIT) cells, γδ T cells, Tregs, natural killer T cells, and tissue-resident T cells.

[0049] The term "natural killer cells" or "NK cells" refers to large granular lymphocytes (LGLs), which constitute a third type of cell differentiated from B and T lymphocytes that generate a common lymphoid precursor. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they enter the circulation. NK cells differ from natural killer T cells (NKTs) in phenotype, origin, and respective effector functions, and in many cases, the activity of NKT cells promotes the activity of NK cells by secreting IFNγ. In contrast to NKT cells, NK cells do not express T cell antigen receptors (TCRs), the pan-T marker CD3, or the surface immunoglobulin (Ig) B cell receptor, but in humans they usually express the surface markers CD16 (FcγRIII) and CD56, and in C57BL / 6 mice they express NK1.1 or NK1.2. Up to 80% of human NK cells also express CD8.

[0050] The term "B cell" refers to a cell of the B cell lineage. A "cell of the B cell lineage" refers to a cell that displays at least one phenotype characteristic of a B cell or its precursor or progenitor, which distinguishes the cell from other cells of lymphoid origin and from cells of the erythroid or myeloid lineages. Such phenotypic characteristics can include expression of one or more proteins specific for B cells (e.g., cells positive for one or more of CD19+, CD72+, CD24+, CD20+, CD21+, CD22+, CD38+, CD40+, CD72+, CD32b+, CD268+, CD269+, CD267+, CD86+, CD80+, CD40+, CD52+, CD138+, CD27+, CD28+, CD21+, CD23+, CD84+, CD257+, CD270+, CD37+, and CD74+), or physiological, morphological, functional, or immunological characteristics specific for B cells. For example, cells of the B cell lineage can be progenitor or precursor cells committed to the B cell lineage (e.g., prepro-B cells, pro-B cells, and pre-B cells), immature and inactivated B cells, or mature and functional or activated B cells. Thus, "B cells" encompass naive B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmocytoid cells, plasmablasts, and memory B cells (e.g., CD27+, IgD-).

[0051] The term "cytotoxic activity" in reference to a cell (e.g., T cell) expressing an immune receptor (e.g., CAR) on its surface, also referred to as "cytolytic activity", means that upon antigen-specific signaling (e.g., via the CAR), the cell induces the target cell to undergo apoptosis. In some embodiments, the cytotoxic cell can induce apoptosis in the target cell through the release of cytotoxins, such as perforin, granzymes, and granulysin, from granules. Perforin inserts into the target cell membrane, forming holes that allow water and salt to rapidly enter the target cell. Granzymes are serine proteases and induce apoptosis in the target cell. Granulysin can also form holes in the target cell membrane and is an inflammatory molecule. In some embodiments, the cytotoxic cell can induce apoptosis in the target cell through the interaction of Fas ligand, which is upregulated on T cells after antigen-specific signaling, with the Fas molecule expressed on the target cell. Fas is an apoptosis signaling receptor molecule on the surface of several different cells.

[0052] "Disease" is the health condition of a subject that the subject is unable to maintain homeostasis, and if the disease is not improved, the health condition of the subject will continue to deteriorate.In contrast, "disorder" or "undesirable condition" in a subject is a health condition in which the subject can maintain homeostasis, but the health condition of the subject is less than that in the absence of the disorder or undesirable condition.If left untreated, the disorder or undesirable condition does not necessarily lead to further deterioration of the health condition of the subject.

[0053] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled proliferation of abnormal cells. The abnormal cells may form solid tumors or may constitute hematological malignancies. Cancer cells may spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0054] The terms "subject," "patient," and "individual" are used interchangeably herein and are intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and transgenic species thereof.

[0055] "Adoptive cellular immunotherapy" or "adoptive immunotherapy" refers to the administration of naturally occurring or genetically engineered disease antigen-specific immune cells (e.g., T cells). Adoptive cellular immunotherapy can be autologous (immune cells are from the recipient), allogeneic (immune cells are from a donor of the same species) or syngeneic (immune cells are from a donor who is genetically identical to the recipient).

[0056] "Autologous" refers to a graft (eg, organ, tissue, cells) derived from the same subject that is subsequently reintroduced into the subject.

[0057] "Allogeneic" refers to a graft derived from a different subject of the same species.

[0058] A "therapeutically effective amount" or "effective amount" of a lentiviral vector or a cell transduced by a lentiviral vector (e.g., a T cell expressing a CAR encoded by a transgene of the lentiviral vector) described herein refers to the amount of lentiviral particles or cells sufficient to result in amelioration of one or more symptoms of the disease, disorder, or undesirable condition being treated.

[0059] "Treating" or "treatment" or "ameliorating" refers to the medical management of a disease, disorder, or undesirable condition of interest. In general, an appropriate dose or treatment regimen, including the lentiviral vector or cells expressing a CAR of the present disclosure, is administered in an amount sufficient to elicit a therapeutic or prophylactic effect. Therapeutic or prophylactic / preventive effects include improved clinical outcomes, alleviation or reduction of symptoms associated with a disease, disorder, or undesirable condition, reduced occurrence of symptoms, improved quality of life, prolonged disease-free state, reduced severity of a disease, disorder, or undesirable condition, stabilization of disease, delayed disease progression, remission, survival, prolonged survival, or any combination thereof.

[0060] The term "anti-tumor effect" refers to a biological effect that may be manifested by a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" may also be manifested by the prevention of hematological malignancies or tumor formation.

[0061] Further definitions are provided throughout this disclosure.

[0062] Lentiviral Vectors Lentiviruses are a genus of retroviruses that usually cause slowly progressive disease due to their ability to integrate into the host genome. Modified lentivirus genomes are useful as viral vectors for delivering nucleic acids to host cells.

[0063] The present disclosure provides a self-inactivating lentiviral vector ("LVV") comprising a viral envelope containing a mutated heterologous envelope protein and a molecule that characterizes tropism (also called a "targeting protein"). The LVV described herein further comprises a transgene, which is capable of specifically binding to a target immune cell and transducing the target immune cell such that the transgene is expressed by the immune cell. In some embodiments, the LVV may carry two or more transgenes. In a further specific embodiment, the transgene encodes a chimeric antigen receptor (CAR).

[0064] Mutant viral envelope proteins The lentiviral vectors described herein are pseudotyped with a mutant heterologous viral envelope protein that, in the absence of mutation, mediates both cell attachment and membrane fusion. In certain embodiments, the mutant envelope protein comprises at least one mutation that inhibits the ability of the envelope protein to bind to its natural target while preserving the fusion properties of the envelope protein. In certain embodiments, the heterologous envelope protein is a vesicular stomatitis virus G protein ("VSV-G", "VSV-G envelope protein" or "VSV-G protein") that comprises one or more mutations that inhibit the binding of VSV-G to low density lipoprotein receptor ("LDL-R") while preserving the fusion function of VSV-G protein.

[0065] When referring to the ability of the envelope protein to bind to its native target, the terms "inhibit" and "inhibits" encompass both a complete elimination of binding by the envelope protein to its native target and a significant reduction in the binding of the envelope protein to its native target. In certain embodiments, a "significant reduction" refers to a reduction selected from at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, and at least 10% reduction in binding to the native target.

[0066] The nucleic acid and amino acid sequences shown in Table 10 provide examples of reference sequences for wild-type VSV-G envelope proteins and mutant VSV-G envelope proteins according to the present disclosure. SEQ ID NO: 77 is a reference nucleic acid sequence encoding the VSV-G envelope protein, and SEQ ID NO: 78 is the amino acid sequence of the reference VSV-G envelope protein. SEQ ID NO: 87 is a nucleic acid sequence encoding the cleavable signal peptide of the reference VSV-G envelope protein. SEQ ID NO: 88 is the amino acid sequence of the cleavable signal peptide of the reference VSV-G envelope protein. SEQ ID NO: 89 is a reference nucleic acid sequence encoding the VSV-G envelope protein lacking the signal sequence. SEQ ID NO: 90 is the amino acid sequence of the reference VSV-G envelope protein lacking the sequence. The location and nature of the VSV-G mutations disclosed herein are described with reference to the nucleic acid and amino acid sequences shown, respectively, by SEQ ID NO: 77, lacking the N-terminal signal sequence of SEQ ID NO: 87, and SEQ ID NO: 78, lacking the N-terminal signal sequence of SEQ ID NO: 88. Thus, for purposes of identifying a mutation position, a reference to position 1 of the VSV-G env amino acid sequence refers to position 17, which is a lysine (K) in the wild-type VSV-G amino acid sequence of SEQ ID NO:78, or position 1 of the wild-type VSV-G amino acid sequence of SEQ ID NO:90.

[0067] In some embodiments, the mutant VSV-G envelope protein comprises mutations at amino acid positions H8, N9, Q10, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, T352, E353, R354, a TT insertion between N9 and Q10, a GGS insertion between H8 and N9, a GGS insertion between N9 and Q10, a TT insertion between N208 and Y209, a GGS insertion between P46 and K47, a GGS insertion between N208 and Y209, and / or a deletion of residues 1-8. In certain embodiments, the VSV-G envelope protein comprises two or more mutations at amino acid positions selected from H8, N9, Q10, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, T352, E353, R354, a TT insertion between N9 and Q10, a GGS insertion between H8 and N9, a GGS insertion between N9 and Q10, a TT insertion between N208 and Y209, a GGS insertion between P46 and K47, a GGS insertion between N208 and Y209, and a deletion of residues 1-8. In other embodiments, the VSV-G envelope protein comprises three or more mutations at amino acid positions selected from H8, N9, Q10, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, T352, E353, R354, a TT insertion between N9 and Q10, a GGS insertion between H8 and N9, a GGS insertion between N9 and Q10, a TT insertion between N208 and Y209, a GGS insertion between P46 and K47, a GGS insertion between N208 and Y209, and a deletion of residues 1-8. In other embodiments, the mutant VSV-G envelope protein comprises the mutations H8A, K47A, K47Q, Y209A, R354A, and / or R354Q.In yet other embodiments, the VSV-G envelope protein comprises one or more mutations selected from N9, Q10, K50, A51, S183, S179, N180, I182, M184, I347, T350, T352, and E353, a TT insertion between N9 and Q10, a GGS insertion between H8 and N9, a GGS insertion between N9 and Q10, a TT insertion between N208 and Y209, a GGS insertion between P46 and K47, a GGS insertion between N208 and Y209, and a deletion of residues 1-8. In yet other embodiments, the VSV-G envelope protein comprises two or more mutations selected from mutations at one or more of N9, Q10, K50, A51, S183, S179, N180, I182, M184, I347, T350, T352, and E353, a TT insertion between N9 and Q10, a GGS insertion between H8 and N9, a GGS insertion between N9 and Q10, a TT insertion between N208 and Y209, a GGS insertion between P46 and K47, a GGS insertion between N208 and Y209, and a deletion of residues 1-8. In some embodiments, the mutant VSV-G envelope protein is selected from one or more of the following mutations: H8A, N9A, Q10A, K47A, K47Q, N180A, I182A, Y209A, T352A, T352W, E353A, R354A, and R354Q. In some embodiments, the mutant VSV-G envelope protein is selected from one or more of the following mutations: H8A, K47A, K47Q, Y209A, R354A, and R354Q. In some embodiments, the mutant VSV-G envelope protein comprises a K47 mutation and a R354 mutation. In some embodiments, the mutant VSV-G envelope protein comprises a K47Q mutation and a R354A mutation. In some embodiments, the mutant VSV-G envelope protein comprises an N180 mutation, an I182 mutation, a T352 mutation, and an E353 mutation. In some embodiments, the mutant VSV-G envelope protein comprises an N180A mutation, an I182A mutation, a T352A mutation, and an E353A mutation. In some embodiments, the mutant VSV-G envelope protein comprises a T352 mutation and an E353 mutation.In further embodiments, the mutant VSV-G envelope protein comprises a T352W mutation and an E353A mutation. In some embodiments, the mutant VSV-G envelope protein comprises an N9 mutation, a Q10 mutation, and an N180 mutation. In some embodiments, the mutant VSV-G envelope protein comprises an N9A mutation, a Q10A mutation, and an N180A mutation. In some embodiments, the mutant VSV-G envelope protein comprises a GGS insertion between H8 and N9 and a GGS insertion between N9 and Q10. In some embodiments, the mutant VSV-G envelope protein comprises a TT insertion between N9 and Q10. In some embodiments, the mutant VSV-G envelope protein comprises a GGS insertion between P46 and K47. In other embodiments, the mutant VSV-G envelope protein is as described in Nikolic et al., "Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein." Nature Comm., 2018, 9:1029, the relevant disclosure of which is incorporated herein by reference.

[0068] In certain embodiments, the mutant VSV envelope protein comprises a K47Q mutation and a R354A mutation. In certain embodiments, the mutant VSV-G envelope protein has an amino acid sequence according to SEQ ID NO:74.

[0069] In certain embodiments, the mutant VSV envelope protein comprises an N180A mutation, an I182A mutation, a T352A mutation, and an E353A mutation. In certain embodiments, the mutant VSV-G envelope protein has an amino acid sequence according to SEQ ID NO:93.

[0070] In certain embodiments, the mutant VSV envelope protein comprises a T352W mutation and an E353A mutation. In certain embodiments, the mutant VSV-G envelope protein has an amino acid sequence according to SEQ ID NO:95.

[0071] In certain embodiments, the mutant VSV envelope protein comprises an N9A mutation, a Q10A mutation, and an N180A mutation. In certain embodiments, the mutant VSV-G envelope protein has an amino acid sequence according to SEQ ID NO:97.

[0072] In certain embodiments, the mutant VSV envelope protein comprises a TT insertion mutation between N9 and Q10. In certain embodiments, the mutant VSV-G envelope protein has an amino acid sequence according to SEQ ID NO:99.

[0073] In certain embodiments, the mutant VSV envelope protein comprises a GGS insertion mutation between P46 and K47. In certain embodiments, the mutant VSV-G envelope protein has an amino acid sequence according to SEQ ID NO: 101.

[0074] In certain embodiments, the mutant VSV envelope protein comprises a GGS insertion between H8 and N9 and a GGS insertion between N9 and Q10. In certain embodiments, the mutant VSV-G envelope protein has an amino acid sequence according to SEQ ID NO: 103.

[0075] Target Protein The target protein is a membrane-bound protein presented on the viral envelope, and includes an extracellular domain including a lymphocyte-targeting domain and a transmembrane domain. The lymphocyte-targeting domain is any protein or peptide having an amino acid sequence that is a binding partner of a target molecule or ligand (e.g., a cognate protein or ligand) on the target lymphocyte cell surface. In some embodiments, the lymphocyte-targeting domain is a T cell-targeting domain. In some embodiments, the lymphocyte-targeting domain is a NK cell-targeting domain. In some embodiments, the lymphocyte-targeting domain is a B cell-targeting domain. For example, in certain embodiments, the extracellular domain of the target protein includes a lymphocyte-targeting domain that specifically binds to a protein or ligand on the surface of a defined cell population, e.g., a population of lymphocytes that is characterized by the presence of the target protein or ligand on the cell surface. In some embodiments, the lymphocyte-targeting domain targets a specific type of lymphocyte, e.g., T cells, B cells, or natural killer (NK) cells. In some embodiments, the target protein further comprises an extracellular linker or hinge domain located between the transmembrane domain and the extracellular domain.

[0076] In some embodiments, the target protein is a full-length cell surface protein or receptor, with each component (e.g., each of the extracellular domain, transmembrane domain, and intracellular domain) being derived from a single protein or receptor. In other embodiments, the target protein is a chimeric protein, with at least two components being derived from different proteins or receptors. In some embodiments, the target protein is obtained from a mammalian species, including human, primate, cow, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, and pig. In some embodiments, the target protein is a fully human sequence. In some embodiments, the target protein is a chimeric sequence that includes a human sequence component(s) and a component(s) from another species or a synthetic component(s). In some embodiments, the chimeric target protein can be a fully synthetic protein. In still other embodiments, the chimeric target protein can include one or more of an extracellular domain, an extracellular binding domain, a transmembrane domain, an intracellular domain, a hinge domain, or a linker that is a fully synthetic protein.

[0077] In some embodiments, the lymphocyte-targeting domain comprises an antibody. In such embodiments, the extracellular targeting domain can be a full-length antibody, an antibody fragment, a nanobody, or a single chain Fv fragment (scFv).

[0078] In certain embodiments, the targeting molecule comprises a domain that targets T cells, which specifically binds to a T cell surface marker, including, for example, a T cell antigen, a T cell surface receptor, or any other protein present on the surface of the target T cell. In some embodiments, the target protein is CD3, CD28, CD80, 4-1BB, AhR, CD3, CD2, CD7, CD4, CD8, CD25, CD44, CD45RA, CD47, CD62L, CD69, CD94, CD95, CD127, CD161, CD183 (CXCR3), CD184 (CXCR4), CD185 (CXCR5), CD193 (CCR3), CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CCR12, CCR13, CCR14, CCR15, CCR16, CCR17, CCR18, CCR19, CCR18, CCR19, CCR19, CCR20, CCR21, CCR22, CCR23, CCR24, CCR25, CCR25, CCR26, CCR27, CCR28, CCR29, CCR30, CCR31, CCR32, CCR33, CCR34, CCR35, CCR36, CCR37, CCR38, CCR39, CCR40, CCR410, CCR311, CCR32, CCR33, CCR34, CCR35, CCR36, CCR37, CCR38, CCR39 ... The antibody specifically binds to a T cell marker selected from D194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), CCR10, PD-1, TCRa / b, CD5, CD27, CD45RO, CD45RB, CD57, CD103, CD122, P2RX7, TIGIT, LAG-3, TIM-3, IL6ST, and any combination thereof.

[0079] In a further embodiment, the target protein specifically binds to a γδ T cell marker selected from γδ TCR, Vdelta1, Vdelta2, NKG2D (KLRK1, CD314), and any combination thereof.

[0080] In other embodiments, the target protein specifically binds to an NK T cell marker selected from invariant TCR (Va24-Ja18), CD185 (CXCR5), CXCR6, IL-21R, and any combination thereof.

[0081] In other embodiments, the target protein specifically binds to a MAIT cell marker selected from Va7.2, Ja33, CXCR6, IL-18R, KLRB1 (CD161), VLA4 (alpha 4 beta 1 integrin), and any combination thereof.

[0082] In other embodiments, the target protein specifically binds to an NK cell marker selected from CD56, NKp46, CD16, KIR(s), NKG2 proteins (e.g., NKG2D (KLRK1, CD314)), KLRB1 (CD161), KLRD1 (cd94), IL2Rb (CD122), IL-21R, SLAMF6 (CD352), SLAMF7 (CD319), IL-18R, and any combination thereof.

[0083] In other embodiments, the target protein specifically binds to a B cell marker selected from CD19, CD20, CD21, CD22, CD24, CD38, CD40, CD72, CD32b, CD268, CD269, CD267, CD86, CD80, CD52, CD138, CD27, CD28, CD23, CD84, CD257, CD270, CD37, CD74, and CD269, and any combination thereof.

[0084] In embodiments, the target protein specifically binds to a lymphocyte marker (eg, B cells and T cells) selected from CD80, CD27, CD28, and any combination thereof.

[0085] In some embodiments of the targeting protein, a linker is located between the transmembrane domain and the lymphocyte-targeting domain. The linker is an amino acid linker, and may be a rigid linker, a flexible linker, or an oligomerization linker. A rigid linker is an amino acid sequence that lacks flexibility (e.g., may contain at least one proline). In some embodiments, the rigid linker comprises a platelet-derived growth factor receptor (PDGFR) stalk or a CD8a stalk. In some embodiments, the PDGFR stalk comprises an amino acid sequence comprising AVGQDTQEVIVVPHSLPFK (SEQ ID NO: 104). In some embodiments, the PDGFR stalk comprises an amino acid sequence comprising ASAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFAK (SEQ ID NO: 105).

[0086] A flexible linker is an amino acid sequence that has many degrees of freedom (e.g., it may include multiple amino acids with small side chains, such as glycine or alanine). In some embodiments, a flexible linker includes an amino acid sequence that includes GAPGAS. In some embodiments, a flexible linker includes an amino acid sequence consisting of GAPGSGGGGSGGGGSAS (SEQ ID NO: 106). In some embodiments, a flexible linker includes an amino acid sequence that includes GGGGS. In some embodiments, a flexible linker includes an amino acid sequence that includes GGGS. In some embodiments, a flexible linker includes an amino acid sequence that includes (GAPGAS) N , (G3S) N , or (G4S) N where N is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0087] An oligomerization linker is an amino acid that can be oligomerized to another related amino acid. In some embodiments, an oligomerization linker is an amino acid sequence that can form a dimer, trimer, or tetramer. In some embodiments, an oligomerization linker comprises an IgG4 hinge domain (e.g., ESKYGPPCPPCPAVGQDTQEVIVVPHSLPFK (SEQ ID NO: 107)). In some embodiments, an oligomerization linker comprises an amino acid sequence that can form a tetrameric coiled coil (e.g., ASGGGGSGELAAIKQELAAIKKELAAIKWELAAIKQGAG (SEQ ID NO: 108)). In some embodiments, an oligomerization linker comprises an amino acid sequence that can form a dimeric coiled coil (e.g., ASESKYGPPCPPCP (SEQ ID NO: 109)).

[0088] The target protein usually contains a signal sequence (also called a signal peptide of a localization sequence). The signal sequence may be located at the N-terminus or C-terminus of the target protein. The signal sequence functions to translocate the target protein to a membrane that functions as an envelope for the lentiviral vector. Non-limiting examples of signal sequences that may be included in the target proteins described herein include an Ig kappa leader sequence (e.g., a mouse Ig kappa leader sequence comprising METDTLLLWVLLLWVPGSTG (SEQ ID NO: 110)), a CD8a signal peptide sequence (e.g., a CD8a signal peptide comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 12)), and a B2M signal peptide sequence (e.g., a B2M signal peptide sequence comprising MSRSVALAVLALLSLSGLEA (SEQ ID NO: 123)). In certain embodiments, the mature target protein has a partially truncated signal sequence that retains one or more amino acids of the full-length signal sequence. In other embodiments, cleavage by a signal peptidase completely removes the signal sequence, resulting in a mature target protein that is completely devoid of a signal sequence.

[0089] In some embodiments, the target protein is a CD80 protein. When the target protein is a CD80 protein, it may comprise an extracellular domain according to SEQ ID NO:6. In such embodiments, the CD80 protein may comprise a signal peptide according to SEQ ID NO:4 and a transmembrane domain and an intracellular domain according to SEQ ID NO:8. In certain embodiments, the CD80 target protein comprised in the lentiviral vectors described herein comprises an amino acid sequence according to SEQ ID NO:2, or an amino acid sequence according to SEQ ID NO:2 lacking the signal peptide of SEQ ID NO:4.

[0090] In some embodiments, where the target protein is a CD80 protein, it may comprise an extracellular domain having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:6. In such embodiments, the CD80 protein may comprise a signal peptide having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:4, and / or transmembrane and intracellular domains having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:8. In certain embodiments, a CD80 target protein included in a lentiviral vector described herein comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:2, or to SEQ ID NO:2 lacking the signal peptide of SEQ ID NO:4. [Table 1-1] [Table 1-2]

[0091] In some embodiments, lentiviral vectors containing a CD80 targeting protein show higher transduction of CD4 T cells than CD8 T cells as a relative percentage of total CD4 and CD8 T cells compared to transduction with standard LVV. As used herein, "standard lentiviral vector" or "standard LVV" refers to a lentiviral vector that does not contain the mutant VSV-G envelope protein and lymphocyte targeting protein of the present disclosure. In some embodiments, standard LVV is produced using a third generation LVV packaging system. In some embodiments, lentiviral vectors containing a CD80 targeting protein enhance transduction of CD4 T cells over CD8 T cells by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more as a relative percentage of total CD4 and CD8 T cells compared to standard LVV.

[0092] In some embodiments, the target protein is an anti-CD3 target protein. In some embodiments, the anti-CD3 target protein is an antibody or a binding fragment thereof, including, for example, an scFv. When the target protein is an anti-CD3 target protein, it may comprise an anti-CD3 scFv having a light chain variable region according to SEQ ID NO: 14, a G3S linker according to SEQ ID NO: 16, and a heavy chain variable region according to SEQ ID NO: 18. In some embodiments, the anti-CD3 target protein may comprise an anti-CD3 scFv having a light chain variable region according to SEQ ID NO: 115, a G3S linker according to SEQ ID NO: 16, and a heavy chain variable region according to SEQ ID NO: 113. The described anti-CD3 target protein may comprise a signal peptide according to SEQ ID NO: 12, and a hinge and transmembrane domain according to SEQ ID NO: 20. In certain embodiments, the anti-CD3 target protein comprised in the lentiviral vector described herein comprises an amino acid sequence according to SEQ ID NO: 10, or an amino acid sequence according to SEQ ID NO: 10 lacking the signal peptide of SEQ ID NO: 12. In another specific embodiment, the anti-CD3 target protein comprised in the lentiviral vector described herein comprises an amino acid sequence according to SEQ ID NO:117, or an amino acid sequence according to SEQ ID NO:117 lacking the signal peptide of SEQ ID NO:12.

[0093] In some embodiments, when the target protein is an anti-CD3 target protein, it may comprise an anti-CD3 scFv having a light chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:14 and a heavy chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:18. The described anti-CD3 target proteins may comprise a signal peptide having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:12, and hinge and transmembrane domains having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:20. In certain embodiments, an anti-CD3 target protein included in a lentiviral vector described herein comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:10, or SEQ ID NO:10 lacking the signal peptide of SEQ ID NO:12.

[0094] In some embodiments, when the target protein is an anti-CD3 target protein, it may comprise an anti-CD3 scFv having a light chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:115, and a heavy chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:113. The described anti-CD3 target proteins may comprise a signal peptide having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:12, and hinge and transmembrane domains having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:20. In certain embodiments, an anti-CD3 target protein included in a lentiviral vector described herein comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:117, or SEQ ID NO:117 lacking the signal peptide of SEQ ID NO:12. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0095] In yet further embodiments, the lentiviral vectors according to the present specification may comprise multiple target proteins, or a single target protein with multiple different lymphocyte targeting domains. For example, the lentiviral vectors described herein may comprise at least two target proteins, each of which has a domain that targets NK cells that specifically bind to different targets. In another example, the lentiviral vectors described herein may comprise at least two target proteins, each of which has a domain that targets T cells that specifically bind to different targets. In another example, the lentiviral vectors described herein may comprise at least two target proteins, each of which has a domain that targets B cells that specifically bind to different targets. In yet another example, the lentiviral vectors described herein may comprise at least two target proteins, a first target protein having a domain that targets T cells, and a second target protein having a domain that targets NK cells. In yet another example, the lentiviral vectors described herein may comprise at least two target proteins, a first target protein having a domain that targets T cells, and a second target protein having a domain that targets B cells. In yet another example, the lentiviral vectors described herein can include at least two targeting proteins, a first targeting protein having a domain that targets B cells and a second targeting protein having a domain that targets NK cells. In alternative embodiments, the lentiviral vectors described herein include a single targeting protein with two or more different lymphocyte targeting domains, two or more different T cell targeting domains, two or more different NK cell targeting domains, or two or more different B cell targeting domains (e.g., bispecific or multispecific targeting proteins). In any such embodiment, the T lymphocyte targeting domains included in the targeting protein can be selected from any of those described herein.In yet another example, the lentiviral vectors described herein can include at least two targeting proteins, a first targeting protein having a domain that targets lymphocytes and a second targeting protein having a domain that targets T cells. In yet another example, the lentiviral vectors described herein can include at least two targeting proteins, a first targeting protein having a domain that targets lymphocytes and a second targeting protein having a domain that targets B cells.

[0096] In an exemplary embodiment, the lentiviral vector with multiple target proteins comprises a CD80 target protein and an anti-CD3 target protein, as detailed in Tables 1 and 2, respectively. In some embodiments, the CD80 target protein comprises an amino acid sequence according to SEQ ID NO:2, or an amino acid sequence according to SEQ ID NO:2 lacking the signal peptide of SEQ ID NO:4. In some embodiments, the anti-CD3 target protein comprises an amino acid sequence according to SEQ ID NO:10, or an amino acid sequence according to SEQ ID NO:10 lacking the signal peptide of SEQ ID NO:12. In some embodiments, the anti-CD3 target protein comprises an amino acid sequence according to SEQ ID NO:117, or an amino acid sequence according to SEQ ID NO:117 lacking the signal peptide of SEQ ID NO:12. When the lentiviral vector comprises a CD80 target protein and an anti-CD3 target protein, the target proteins can be encoded on separate expression vectors used for the production of the lentiviral vector. Alternatively, both the CD80 target protein and the anti-CD3 target protein can be encoded on a tandem expression cassette, and a single expression cassette is provided within a single expression vector that provides expression of both target proteins for the production of the lentiviral vector. Exemplary embodiments of the nucleic acid sequence and corresponding amino acid sequence of a CD80 targeting protein and an anti-CD3 targeting protein expressed from the same expression cassette are shown in Table 3. In such embodiments, the two targeting proteins may be linked by a P2A self-cleaving peptide. Table 3 provides exemplary nucleic acids that may be used in a tandem expression cassette comprising a CD80 binding protein linked to an anti-CD3 binding protein via a P2A sequence. In certain embodiments, the P2A sequence is encoded by a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:29, and the amino acid sequence of the P2A sequence has an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:30.In some embodiments, a tandem expression cassette comprising a CD80 binding protein linked to an anti-CD3 binding protein comprises the nucleic acid sequence of SEQ ID NO:21 or has at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:21. In some embodiments, a tandem expression cassette comprising a CD80 binding protein linked to an anti-CD3 binding protein encodes the amino acid sequence of SEQ ID NO:22 or encodes the amino acid sequence of SEQ ID NO:22 lacking the signal peptide of SEQ ID NO:24. In some embodiments, a tandem expression cassette comprising a CD80 binding protein linked to an anti-CD3 binding protein encodes an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:22, or to SEQ ID NO:22 lacking the signal peptide of SEQ ID NO:24. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0097] Transgene The lentiviral vectors described herein contain a transgene encoding one or more proteins that are delivered to and expressed by lymphocytes targeted by the vector (e.g., T cells, B cells, or NK cells). In certain embodiments, the transgene encodes a chimeric antigen receptor (CAR), which comprises an extracellular domain comprising a binding domain that specifically binds to a target molecule, an intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM), and a transmembrane domain connecting the extracellular domain and the intracellular signaling domain.

[0098] Binding domains suitable for use in the CAR of the present disclosure include any antigen-binding polypeptide. Binding domains may include antibodies or antigen-binding fragments thereof, including, for example, full-length heavy chains, Fab fragments, Fab', F(ab')2, sFv, VH domains, VL domains, dAbs, VHHs, CDRs, and scFvs specific for a target disease antigen. In certain embodiments, the CAR binding domain is murine, chimeric, human, or humanized. In further embodiments, the CAR binding domain is a human or humanized VFv. H and V L In a further embodiment, the CAR binding domain is an scFv having the V H and V L Combine regions, N=1-10 (GGGS) N Linker or (GGGGS) N In other embodiments, the CAR binding domain is an scFv with a linker. H and V L It is an scFv with a linker comprising SEQ ID NO:64 connecting the domains.

[0099] The target molecule bound by the extracellular domain of the CAR of the present disclosure may be found on or associated with a target cell.Exemplary target cells include cancer cells, cells associated with autoimmune disease or disorder, neurodegenerative disease, or inflammatory disease or disorder, infectious microorganisms (e.g., bacteria, viruses, or fungi) and infected cells (e.g., virus-infected cells).Cells of infectious organisms, such as mammalian parasites, are also contemplated as target cells.

[0100] The extracellular domain contained in the CAR described herein may include a binding domain that targets one or more of various target molecules. In certain embodiments, the CAR includes a binding domain that specifically binds to a tumor antigen. In some such embodiments, the CAR binding domain is selected from the group consisting of CD19, BCMA, alpha folate receptor, 5T4, Ab integrin, B7-H3, B7-H6, CAIX, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD52, CD70, CD79a, CD79b, CD80, CD123, CD138, CD171, CEA, CSPG4, EGFR, ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EpCAM, FAP, fetal AchR, FLT3, Fra, GD2, GD3, glypican-3 (GPC3), HLA-A Specifically binds to one or more of: 1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, HLADR, IL-11Ralpha, IL-13Ralpha2, lambda, Lewis-Y, kappa, mesothelin, Muc1, Muc16, NCAM, NKG2d ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM, VEGFR2, BAFF-R, claudin 18.2, CD86, FcRL5, GPRC5, and TACI.

[0101] In certain embodiments, the extracellular domain of the CAR encoded by the transgene of the lentiviral vector described in the present disclosure optionally includes an extracellular, non-signaling spacer or linker domain between the binding domain and the transmembrane domain. If included, such a spacer or linker domain may position the binding domain away from the host cell surface and further allow for proper cell-to-cell contact, binding, and activation. The extracellular spacer domain is generally located between the extracellular binding domain and the transmembrane domain of the CAR. The length of the extracellular spacer may be varied to optimize binding of the target molecule based on the selected target molecule, the selected binding epitope, the size and affinity of the binding domain (see, e.g., Guest et al., J. Immunother. 28:203-11, 2005, PCT Publication No. WO 2014 / 031687). In certain embodiments, the extracellular spacer domain is an immunoglobulin hinge region (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgD). The immunoglobulin hinge region can be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. The modified IgG4 hinge region is described in PCT Publication No. WO2014 / 031687, which is incorporated herein by reference in its entirety. In some embodiments, the extracellular spacer domain comprises a modified IgG4 hinge region having the amino acid sequence ESKYGPPCPPCP (SEQ ID NO: 118).

[0102] Other examples of hinge regions that may be used in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8a, CD4, CD28, and CD7, which may be wild type or variants thereof. In some embodiments, the extracellular spacer domain comprises all or a portion of an immunoglobulin Fc domain selected from a CH1 domain, a CH2 domain, a CH3 domain, or a combination thereof (see, e.g., PCT Publication No. WO2014 / 031687, which are incorporated herein by reference in their entirety). In further embodiments, the extracellular spacer domain may comprise the stalk region of a type II C-lectin (the extracellular domain located between the C-type lectin domain and the transmembrane domain). Type II C-lectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D.

[0103] The CAR of the present disclosure comprises a transmembrane domain that connects the extracellular domain and the intracellular signaling domain and is disposed between the extracellular domain and the intracellular signaling domain. The transmembrane domain typically ranges from about 15 amino acids to about 30 amino acids in length. The transmembrane domain is a hydrophobic alpha helix that crosses the host cell membrane and anchors the CAR within the host cell membrane. The transmembrane domain can be fused directly to the binding domain or to the extracellular spacer domain, if present. In certain embodiments, the transmembrane domain is derived from an integral membrane protein (e.g., a receptor, a cluster of differentiation (CD) molecule, an enzyme, a transporter, a cell adhesion molecule, etc.). The transmembrane domain can be selected from the same molecule as the extracellular domain or the intracellular signaling domain. In some embodiments, the transmembrane domain and the extracellular domain are each selected from different molecules. In some embodiments, the transmembrane domain and the intracellular signaling domain are each selected from different molecules. In still other embodiments, the transmembrane domain, the extracellular domain, and the intracellular signaling domain are each selected from different molecules.

[0104] Exemplary transmembrane domains for use in the CARs of the present disclosure include CD28, CD2, CD4, CD8a, CD5, CD3ε, CD3δ, CD3ζ, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD95(Fas), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD152(CTLA4), CD154(CD40L), CD200R, CD223(LAG3), CD270(HVEM). , CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5, and Zap70. An exemplary CD8a transmembrane domain with a CD8a hinge region comprises the amino acid sequence of SEQ ID NO:52.

[0105] The intracellular signaling domain of the CAR is an intracellular effector domain that can transmit a functional signal to a cell in response to binding of the extracellular domain of the CAR to a target molecule and activates at least one of the normal effector functions or responses of an immune cell, such as a T cell engineered to express the CAR. In some embodiments, the CAR induces a T cell function, such as cytotoxic activity or T helper activity, such as secretion of a cytokine or other factor. The intracellular signaling domain can be any portion of an intracellular signaling molecule that retains sufficient signaling activity. In some embodiments, the intracellular signaling domain is obtained from an antigen receptor component (e.g., TCR) or a costimulatory molecule. In some embodiments, the full-length intracellular signaling domain of an antigen receptor or costimulatory molecule is used. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor or costimulatory molecule is used, provided that the truncated portion retains sufficient signaling activity. In further embodiments, the intracellular signaling domain is a variant of a full-length or truncated portion of the intracellular signaling domain of an antigen receptor costimulatory molecule, provided that the variant retains full signaling activity (i.e., is a functional variant).

[0106] In certain embodiments, the intracellular signaling domain of the CAR comprises an immunoreceptor tyrosine-based activation motif (ITAM) containing signaling domain. An ITAM-containing signaling domain generally comprises at least one (1, 2, 3, 4, or more) ITAM, which is represented by the general formula: YXXL / IX 6-8ITAM-containing signaling domains can initiate T cell activation signaling after antigen binding or ligand engagement. ITAM-signaling domains include, for example, the intracellular signaling domains of CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD278 (ICOS), DAP10, DAP12, FcRγ, and CD66d. An exemplary CD3ζ signaling domain that can be used in the CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO:56 or SEQ ID NO:122.

[0107] The intracellular signaling domain of the CAR optionally includes a costimulatory signaling domain that, when activated in conjunction with a primary or classical (e.g., ITAM-driven) activation signal, promotes or enhances a T cell response, e.g., T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or a combination thereof. Costimulatory signaling domains for use in CARs include, for example, CD27, CD28, CD40L, GITR, NKG2C, CARD1, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, LFA-1, LIGHT, NKG2C, NKD2C, SLP76, TRIM, ZAP70, or combinations thereof. In certain embodiments, the costimulatory signaling domain comprises a signaling domain of OX40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), or 4-1BB (CD137). An exemplary 4-1BB costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 54. In certain embodiments, the CAR comprises one, two or more costimulatory signaling domains.

[0108] In some embodiments, the CAR of the present disclosure is a first generation CAR, a second generation CAR, or a third generation CAR. A first generation CAR generally has an intracellular signaling domain including an intracellular signaling domain of CD3ζ, FcγRI or other ITAM-containing activation domain, and provides a T cell activation signal. A second generation CAR further includes a costimulatory signaling domain (e.g., a costimulatory signaling domain from an endogenous T cell costimulatory receptor such as CD28, 4-1BB, or ICOS). A third generation CAR includes an ITAM-containing activation domain, a first costimulatory signaling domain, and a second costimulatory signaling domain.

[0109] In some embodiments, one or more of the extracellular domain, the binding domain, the linker, the transmembrane domain, the intracellular signaling domain, or the costimulatory domain comprises a junction amino acid. A "junction amino acid" or "junction amino acid residue" refers to one or more (e.g., about 2-20) amino acid residues between two adjacent domains, motifs, regions, modules, or fragments of a protein, e.g., between a binding domain and an adjacent linker, between a transmembrane domain and an adjacent extracellular or intracellular domain, or at one or both ends of a linker connecting two domains, motifs, regions, modules, or fragments (e.g., between a linker and an adjacent binding domain, or between a linker and an adjacent hinge). Junction amino acids can result from the design of the fusion protein's construction (e.g., amino acid residues resulting from the use of a restriction enzyme site or a self-cleaving peptide sequence during construction of a polynucleotide encoding a fusion protein). For example, a transmembrane domain of a fusion protein can have one or more junction amino acids at the amino terminus, carboxy terminus, or both.

[0110] In certain embodiments, the transgene encodes an anti-CD19 CAR molecule. In such embodiments, the extracellular binding domain may comprise an scFv as detailed in Table 4, having an anti-CD19 light chain variable region of SEQ ID NO: 46, a G4S linker of SEQ ID NO: 48, and an anti-CD19 heavy chain variable region of SEQ ID NO: 50. An anti-CD19 CAR useful in the context of the present specification may comprise a CD8a hinge and transmembrane domain of SEQ ID NO: 52. In certain embodiments, an anti-CD19 CAR comprises a 4-1BB costimulatory domain according to SEQ ID NO: 54 and a CD3 zeta effector domain according to SEQ ID NO: 56. In certain embodiments, the anti-CD19 CAR encoded by the transgene has an amino acid sequence according to SEQ ID NO: 42, or an amino acid sequence according to SEQ ID NO: 42 lacking the signal peptide of SEQ ID NO: 44.

[0111] In other embodiments, the transgene encodes an anti-CD19 CAR molecule, and the extracellular binding domain may comprise an scFv having an anti-CD19 light chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:46, and an anti-CD19 heavy chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:50. An anti-CD19 CAR useful in the context herein may comprise a hinge and transmembrane domain of CD8a having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:52. In certain embodiments, the anti-CD19 CAR comprises a 4-1BB costimulatory domain having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:54, and a CD3 zeta effector domain having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:56. In certain embodiments, the anti-CD19 CAR encoded by the transgene has an amino acid sequence with at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 42, or to SEQ ID NO: 42 lacking the signal peptide of SEQ ID NO: 44. [Table 4-1] [Table 4-2] [Table 4-3]

[0112] In other embodiments, the transgene encodes an anti-BCMA CAR molecule. In such embodiments, the extracellular binding domain may comprise an scFv as detailed in Table 5, having an anti-BCMA light chain variable region of SEQ ID NO: 62, a linker of SEQ ID NO: 64, and an anti-BCMA heavy chain variable region of SEQ ID NO: 66. An anti-BCMA CAR useful in the context of the present specification may comprise a CD8a hinge and transmembrane domain of SEQ ID NO: 52. In certain embodiments, an anti-BCMA CAR comprises a 4-1BB costimulatory domain according to SEQ ID NO: 54 and a CD3 zeta effector domain according to SEQ ID NO: 122. In certain embodiments, the anti-BCMA CAR encoded by the transgene has an amino acid sequence according to SEQ ID NO: 58, or an amino acid sequence according to SEQ ID NO: 58 lacking the signal peptide of SEQ ID NO: 60.

[0113] In some embodiments, the transgene encodes an anti-BCMA CAR molecule. In such embodiments, the extracellular binding domain may comprise an scFv having an anti-BCMA light chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:62 and an anti-BCMA heavy chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:66. An anti-BCMA CAR useful in the context of the present specification may comprise a hinge and transmembrane domain of CD8a having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to SEQ ID NO:52. In certain embodiments, the anti-BCMA CAR comprises a 4-1BB costimulatory domain having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:54, and a CD3 zeta effector domain having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:122. In certain embodiments, the anti-BCMA CAR encoded by the transgene has an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:58, or to SEQ ID NO:58 lacking the signal peptide of SEQ ID NO:60. [Table 5-1] [Table 5-2] [Table 5-3]

[0114] The CAR of the present disclosure may comprise a polynucleotide sequence derived from any mammalian species, including human, primate, cow, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, pig, transgenic species thereof, or any combination thereof. In some embodiments, the chimeric antigen receptor is murine, chimeric, human, or humanized.

[0115] Methods for producing lentiviral vectors The present disclosure provides a method for producing LVV. LVV was developed based on the genome of retrovirus by combining its components into a recombinant plasmid DNA vector. The plasmid DNA vector can then be transfected into a producer cell line to introduce the genes required for lentiviral particle production. The LVV packaging system generally includes a transfer plasmid encoding the transgene of interest, an envelope plasmid (e.g., VSV-G), and a packaging plasmid(s). The second generation LVV packaging system includes a single packaging plasmid encoding Gag, Pol, Rev, and Tat genes, as well as a separate Env plasmid. In some embodiments, the method described herein utilizes a third generation vector system to produce LVV. The third generation vector production system improves the safety of the second generation LVV packaging system and is usually a four plasmid system including a transgene plasmid combined with three packaging plasmids, VSV-G, GagPol, and Rev. Thus, Rev and GagPol are separated into two plasmids. Tat is also removed from the third generation LVV packaging system by adding a chimeric 5'LTR fused to a heterologous promoter (e.g., CMV or RSV promoter) on a transfer plasmid. The gag gene encodes the Gag polyprotein precursor, which contains lentiviral structural proteins, including matrix, capsid, and nucleocapsid. The pol gene encodes the Pol polyprotein precursor, which provides lentiviral enzymatic functions essential for replication, including protease, reverse transcriptase, and integrase. The rev gene encodes the Rev protein, which binds to the Rev response element (RRE), allowing nuclear export of unspliced ​​and singly spliced ​​HIV RNA during viral replication. The Gag and Pol polyprotein precursors are cleaved during the production of viral particles.

[0116] Producer cells that can be used to generate the LVV of the present disclosure include human embryonic kidney (HEK) 293 cells and derivatives thereof. The producer cells can be an adherent cell line, e.g., HEK293T producer cells, or a suspension cell line, e.g., HEK293T / 17 SF producer cells.

[0117] The transgene plasmid is combined with three packaging plasmids, GagPol plasmid, VSV-G env plasmid, and Rev plasmid, and used to transfect producer cells, e.g., HEK293 producer cells. In some embodiments, the producer cells are transfected with a defined ratio of the transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid. In some embodiments, the ratio of the packaging plasmids is determined by mass. In some embodiments, the mass of each of the transgene plasmid and GagPol plasmid is higher than the mass of each of the VSV-G env plasmid and Rev plasmid. In some embodiments, the defined ratio of the transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid is about 1:1:1:1 to about 5:4:1:1. In some embodiments, the producer cells are transfected with the transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid in a defined ratio of about 2:1:1:1 to about 5:4:1:1. In some embodiments, the producer cells are transfected with the transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid in a defined ratio of about 2:1:1:1. In some embodiments, the producer cells are transfected with the transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid in a defined ratio of about 3:1:1:1. In some embodiments, the producer cells are transfected with the transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid in a defined ratio of about 3.125:3.125:2.5:1.25. In some embodiments, the producer cells are transfected with the transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid in a defined ratio of about 4:2:1:1.In some embodiments, the producer cells are transfected with the transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid in a defined ratio of about 5:4:1:1. Productive lentiviral particles are harvested from the producer cell culture medium. Exemplary materials and methods for producing LVV particles are described in Production of Lentiviral Vectors, Merten et al., Molecular Therapy - Methods & Clinical Development (2016), 3, 16017, the contents of which are incorporated herein by reference. Third generation production systems are used for research and development and clinical purposes. A schematic diagram of a helper plasmid suitable for use in a third generation LVV production system is shown in Figure 1.

[0118] The transgene plasmid encodes the only genetic material to be introduced into lymphocytes (e.g., T cells or NK cells) targeted by the resulting LVV and comprises a lentiviral backbone containing a transgene expression cassette flanked by cis-acting elements for encapsidation, reverse transcription and integration. In certain embodiments, the transgene plasmid contains a deletion of the U3 element of the 3'LTR, which causes a loss of transcriptional capacity of the viral LTR upon transfer into a target cell. The expression cassette contained in the transgene plasmid may encode a single heterologous protein (e.g., a single CAR as described herein) or multiple heterologous proteins (e.g., multiple CARs as described herein) for introduction into and expression by the target cell.

[0119] In some embodiments of the four plasmid LVV system, the VSV-G env plasmid comprises a tandem expression cassette encoding a mutant VSV-G envelope protein disclosed herein and a target protein. In certain embodiments, the tandem expression cassette contained in the VSV-G env plasmid comprises a polynucleotide encoding a first signal peptide, a polynucleotide encoding a target protein, a polynucleotide encoding an internal ribosome entry site (IRES), a furin cleavage site, or one of the viral 2A peptides, a polynucleotide encoding a second signal peptide, and a polynucleotide encoding a mutant VSV-G envelope protein. In certain embodiments, the polynucleotide encoding the mutant VSV-G envelope protein is located 5' to the polynucleotide encoding the target protein. In other embodiments, the polynucleotide encoding the mutant VSV-G envelope protein is located 3' to the polynucleotide encoding the target protein. The polynucleotide encoding the target protein and the polynucleotide encoding the mutant VSV-G are separated in a tandem cassette by an IRES, a furin cleavage site, or a polynucleotide encoding a viral 2A peptide, which allows for co-expression of two proteins from a single mRNA. In certain embodiments, the viral 2A peptide is Porcine Teschovirus-1 (P2A), Thosea asigna virus (T2A), Equine rhinitis A virus (E2A), Foot and Mouth Disease virus (F2A), or variants thereof. Specific examples of the amino acid and nucleotide sequences of the viral 2A peptide are shown in Table 7. [Table 7]

[0120] In certain embodiments, the present disclosure provides a method of producing a lymphocyte-targeting lentiviral vector according to any one of the preceding claims, the method comprising: transfecting a producer cell with a GagPol plasmid, a Rev plasmid, a transgene plasmid, and a VSV-G env plasmid, wherein the GagPol plasmid comprises one or more polynucleotides encoding a lentiviral gag gene and a lentiviral pol gene and is capable of expressing lentiviral gag polyproteins and lentiviral pol polyproteins in the producer cell, the Rev plasmid comprises a polynucleotide encoding a lentiviral rev gene and is capable of expressing lentiviral rev proteins in the producer cell, the transgene plasmid comprises an expression cassette comprising a polynucleotide encoding a CAR, the VSV-G env plasmid comprises a tandem expression cassette, the tandem expression cassette comprises a polynucleotide encoding a mutant VSV-G envelope protein disclosed herein and a polynucleotide encoding a lymphocyte-targeting protein disclosed herein ... The env plasmid is capable of expressing the mutant VSV-G envelope protein and a protein that targets the lymphocyte in the producer cell, the producer cell is cultured in a culture medium, and the lentiviral vector is harvested from the culture medium.

[0121] In some embodiments, the producer cells are transfected with a defined ratio of the transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid. In some embodiments, the defined ratio of the transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid is about 1:1:1:1 to about 5:4:1:1. In some embodiments, the producer cells are transfected with a defined ratio of the transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid of about 2:1:1:1 to about 5:4:1:1. In some embodiments, the producer cells are transfected with a defined ratio of the transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid of about 2:1:1:1. In some embodiments, the producer cells are transfected with the transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid in a defined ratio of about 3:1:1:1. In some embodiments, the producer cells are transfected with the transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid in a defined ratio of about 3.125:3.125:2.5:1.25. In some embodiments, the producer cells are transfected with the transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid in a defined ratio of about 4:2:1:1. In some embodiments, the producer cells are transfected with the transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid in a defined ratio of about 5:4:1:1.

[0122] In one embodiment, the tandem expression cassette of the VSV-G env plasmid encodes an anti-CD3 target protein and a mutant VSV-G envelope protein. For example, in certain embodiments, the tandem expression cassette may comprise a polynucleotide having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:67, which encodes an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:68. Such embodiments are described in more detail in Table 8. In another embodiment, the tandem expression cassette may comprise a polynucleotide having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 67 lacking the signal peptide of SEQ ID NO: 31, which encodes an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 68 lacking the signal peptide of SEQ ID NO: 32. As shown in Table 8, the anti-CD3 target protein is separated from the mutant VSV-G envelope protein via a P2A self-cleaving peptide. In certain embodiments, the P2A peptide may be encoded by a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:69, and the P2A peptide may have an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:70. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6]

[0123] In another embodiment, the tandem expression cassette of the VSV-G env plasmid encodes a CD80 target protein and a mutant VSV-G envelope protein. For example, in a particular embodiment, the tandem expression cassette comprises a polynucleotide according to SEQ ID NO: 75, which encodes the amino acid sequence of SEQ ID NO: 76. Such an embodiment is described in more detail in Table 9. As shown in Table 9, the CD80 target protein is encoded by SEQ ID NO: 69 and is separated from the mutant VSV-G envelope protein via the P2A self-cleaving peptide having an amino acid sequence according to SEQ ID NO: 70.

[0124] In some embodiments in which the tandem expression cassette of the VSV-G env plasmid encodes a CD80 target protein and a mutant VSV-G envelope protein, the tandem expression cassette may comprise a polynucleotide having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:75, which encodes an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:76. In certain embodiments, the CD80 target protein is separated from the mutant VSV-G envelope protein via a P2A self-cleaving peptide encoded by a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:69, and the P2A self-cleaving peptide comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:70. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5]

[0125] In another embodiment, the tandem expression cassette of the VSV-G env plasmid encodes a CD80 target protein, an anti-CD3 target protein, and a mutant VSV-G envelope protein. For example, in certain embodiments, the tandem expression cassette may comprise a CD80 target polynucleotide according to SEQ ID NO: 1, which encodes the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the tandem expression cassette may comprise an anti-CD3 target polynucleotide according to SEQ ID NO: 9, which encodes the amino acid sequence of SEQ ID NO: 10. Such embodiments are described in more detail in Table 3. As shown in Table 3, the CD80 target protein is encoded by SEQ ID NO: 29 and is separated from the anti-CD3 target protein via a P2A self-cleaving peptide having an amino acid sequence according to SEQ ID NO: 30.

[0126] In some embodiments in which the tandem expression cassette of the VSV-G env plasmid encodes a CD80 target protein, an anti-CD3 target protein, and a mutant VSV-G envelope protein, the tandem expression cassette may comprise a polynucleotide encoding the CD80 target protein and an anti-CD3 target protein having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:21, which encodes an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:22. In certain embodiments, the CD80 target protein is separated from the anti-CD3 target protein via a P2A self-cleaving peptide encoded by a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:29, and the P2A self-cleaving peptide comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:30.

[0127] In some embodiments, in which the tandem expression cassette of the VSV-G env plasmid encodes a CD80 targeting protein, an anti-CD3 targeting protein, and a mutant VSV-G envelope protein, the CD80 targeting protein and the anti-CD3 targeting protein are separated from the mutant VSV-G envelope protein via a 2A self-cleaving peptide.

[0128] In some embodiments of the four-plasmid LVV system, the GagPol plasmid comprises a tandem expression cassette encoding Gag and Pol polyprotein precursors and a target protein as disclosed herein. In certain embodiments, the tandem expression cassette contained in the GagPol plasmid comprises a polynucleotide encoding a first signal peptide, a polynucleotide encoding a target protein, a polynucleotide encoding one of an internal ribosome entry site (IRES), a furin cleavage site, or a viral 2A peptide, a polynucleotide encoding a second signal peptide, and a polynucleotide encoding Gag and Pol polyprotein precursors. In certain embodiments, the polynucleotides encoding the Gag and Pol polyprotein precursors are located 5' to the polynucleotide encoding the target protein. In other embodiments, the polynucleotides encoding the Gag and Pol polyprotein precursors are located 3' to the polynucleotide encoding the target protein. The polynucleotides encoding the target protein and the polynucleotides encoding the Gag and Pol polyprotein precursors are separated in a tandem cassette by a polynucleotide encoding an IRES, a furin cleavage site, or a viral 2A peptide, which allows for co-expression of the Gag and Pol polyprotein precursors and the target protein from a single mRNA. In certain embodiments, the viral 2A peptide is porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot and mouth disease virus (F2A), or variants thereof. Specific examples of the amino acid and nucleotide sequences of the viral 2A peptides are shown in Table 7.

[0129] In some embodiments, the GagPol plasmid comprising the Gag and Pol precursor polyproteins and the tandem expression cassettes encoding the target protein is transfected into producer cells at a higher molar concentration than the VSV-G env plasmid. In some embodiments, the GagPol plasmid comprising the Gag and Pol precursor polyproteins and the tandem expression cassettes encoding the target protein is transfected into producer cells at a lower molar concentration than the VSV-G env plasmid.

[0130] In one embodiment, the tandem expression cassette of the GagPol plasmid encodes an anti-CD3 target protein and the Gag and Pol precursor polyproteins, hi some embodiments, the anti-CD3 target protein is separated from the Gag and Pol precursor polyproteins via the P2A self-cleaving peptide.

[0131] In another embodiment, the tandem expression cassettes of the GagPol plasmid encode a CD80 target protein and the Gag and Pol precursor polyproteins, hi some embodiments, the CD80 target protein is separated from the Gag and Pol precursor polyproteins via the P2A self-cleaving peptide.

[0132] In some embodiments of the four-plasmid LVV system, the Rev plasmid comprises a tandem expression cassette encoding the Rev protein and a target protein disclosed herein. In certain embodiments, the tandem expression cassette contained in the Rev plasmid comprises a polynucleotide encoding a first signal peptide, a polynucleotide encoding a target protein, a polynucleotide encoding one of an internal ribosome entry site (IRES), a furin cleavage site, or a viral 2A peptide, a polynucleotide encoding a second signal peptide, and a polynucleotide encoding a Rev protein. In certain embodiments, the polynucleotide encoding the Rev protein is located 5' to the polynucleotide encoding the target protein. In other embodiments, the polynucleotide encoding the Rev protein is located 3' to the polynucleotide encoding the target protein. The polynucleotide encoding the target protein and the polynucleotide encoding the Rev protein are separated in the tandem cassette by a polynucleotide encoding an IRES, a furin cleavage site, or a viral 2A peptide that allows for co-expression of the Rev protein and the target protein from a single mRNA. In certain embodiments, the viral 2A peptide is porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot and mouth disease virus (F2A), or variants thereof. Exemplary amino acid and nucleotide sequences of the viral 2A peptides are shown in Table 7.

[0133] In some embodiments, the Rev plasmid comprising a tandem expression cassette encoding the Rev protein and the target protein is transfected into producer cells at a higher molar concentration than the VSV-G env plasmid, hi some embodiments, the Rev plasmid comprising a tandem expression cassette encoding the Rev protein and the target protein is transfected into producer cells at a lower molar concentration than the VSV-G env plasmid.

[0134] In one embodiment, the tandem expression cassette of the Rev plasmid encodes an anti-CD3 target protein and a Rev protein, hi some embodiments, the anti-CD3 target protein is separated from the Rev protein via a P2A self-cleaving peptide.

[0135] In another embodiment, the tandem expression cassette of the GagPol plasmid encodes a CD80 targeting protein and a Rev protein, hi some embodiments, the CD80 targeting protein is separated from the Rev protein via a P2A self-cleaving peptide.

[0136] Examples of mutant VSV-G sequences that can be used in the four plasmid, third generation lentiviral vector system of the present disclosure are shown in Table 10. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7] [Table 10-8] [Table 10-9] [Table 10-10] [Table 10-11] [Table 10-12] [Table 10-13] [Table 10-14]

[0137] In other embodiments, the LVV production method according to the present disclosure may utilize a five plasmid system. When a five plasmid system is used, the LVV production system utilizes four plasmids from a third generation vector system (i.e., a transgene plasmid, a GagPol plasmid, an Env plasmid, and a VSV-G env plasmid). In addition, the five plasmid system described herein includes a fifth plasmid ("target protein plasmid") that encodes a target protein. The GagPol and Env packaging plasmids included in the five plasmid system may be standard packaging plasmids described in Production of Lentiviral Vectors, Merten et al., Molecular Therapy-Methods & Clinical Development (2016), 3, 16017. The transgene plasmid includes an expression cassette encoding one or more CAR transgenes according to the present disclosure, and the VSV-G env plasmid includes an expression cassette encoding a mutant VSV-G described herein.

[0138] In some embodiments, the fifth plasmid comprises an expression cassette encoding a CD80 target protein. In some embodiments, the fifth plasmid comprises an expression cassette encoding an anti-CD3 target protein. In some embodiments, the fifth plasmid comprises an expression cassette encoding a CD80 target protein and an anti-CD3 target protein. Examples of CD80 target proteins and anti-CD3 target proteins that can be used in the fifth plasmid are shown in Tables 1-3.

[0139] In certain embodiments, the present disclosure provides a method of producing a lymphocyte-targeting lentiviral vector according to any one of the preceding claims, the method comprising: transfecting a producer cell with a GagPol plasmid, a Rev plasmid, a transgene plasmid, a VSV-G env plasmid, and a lymphocyte-targeting protein plasmid, wherein the GagPol plasmid comprises one or more polynucleotides encoding a lentiviral gag gene and a lentiviral pol gene and is capable of expressing lentiviral gag proteins and lentiviral pol proteins in the producer cell, the Rev plasmid comprises a polynucleotide encoding a lentiviral rev gene and is capable of expressing lentiviral rev proteins in the producer cell, the transgene plasmid comprises an expression cassette comprising a polynucleotide encoding a CAR, and the VSV-G env plasmid is capable of expressing a lentiviral rev protein in the producer cell. the env plasmid comprises a polynucleotide encoding a mutant VSV-G envelope protein described herein and is capable of expressing the mutant VSV-G envelope protein in the producer cell; the lymphocyte targeting protein plasmid comprises a polynucleotide encoding a lymphocyte targeting protein described herein and is capable of expressing the lymphocyte targeting protein in the producer cell; culturing the producer cell in a medium; and harvesting the lentiviral vector from the medium.

[0140] In some embodiments, the producer cells are transfected with defined ratios of transgene plasmid, GagPol plasmid, VSV-G env plasmid, Rev plasmid, and lymphocyte-targeting plasmid. The lymphocyte-targeting plasmid can be at a ratio (by weight) of about 0.25 to about 5 relative to the VSV-G env plasmid. For example, the ratio of lymphocyte-targeting protein plasmid to the VSV-G env plasmid can be adjusted using defined ratios for the four packaging plasmid systems described herein (transgene plasmid, GagPol plasmid, VSV-G env plasmid, Rev plasmid). For example, the lymphocyte-targeting plasmid can be at a ratio of about 0.25:1, 0.5:1, 1:1, 1.25:1, 1.5:1, 1.75:1, 2:1, 2.25:1, 2.5:1, 2.75:1, 3:1, 3.25:1, 3.5:1, 3.75:1, 4:1, 4.25:1, 4.5:1, 4.75:1 or 5:1 to the VSV-G env plasmid.

[0141] In an embodiment of the method of producing LVV utilizing a combination of five plasmids, the fifth plasmid is a target protein plasmid and includes an expression cassette encoding one or more target proteins. For example, the target protein plasmid may include an expression cassette including a polynucleotide encoding a CD80 target protein, an anti-CD3 target protein, or both a CD80 target protein and an anti-CD3 target protein. In certain embodiments, the target protein vector plasmid includes an expression cassette including a polynucleotide according to SEQ ID NO: 1 that encodes a CD80 target protein according to SEQ ID NO: 2. In other embodiments, the target protein plasmid includes an expression cassette including a polynucleotide according to SEQ ID NO: 9 that encodes an anti-CD3 target protein according to SEQ ID NO: 10. In other embodiments, the target protein plasmid includes an expression cassette including a polynucleotide according to SEQ ID NO: 116 that encodes an anti-CD3 target protein according to SEQ ID NO: 117. In other embodiments, the target protein plasmid includes a tandem expression cassette including a polynucleotide according to SEQ ID NO: 21 that encodes a polypeptide according to SEQ ID NO: 22. Examples of mutant VSV-G sequences that can be used in the five plasmid, third generation lentiviral vector system of the present disclosure are shown in Table 10.

[0142] In the four plasmid packaging system, the concentration of lymphocyte targeting protein on the surface of transduced cells can be related to the concentration of the plasmid containing the targeting protein. For example, if the lymphocyte targeting protein plasmid is contained in the VSV-G env plasmid, the concentration of the lymphocyte targeting protein on the surface of the transduced cells can be related to the concentration of the VSV-G env plasmid. In another example, if the lymphocyte targeting protein plasmid is contained in the GagPol plasmid, the concentration of the lymphocyte targeting protein on the surface of the transduced cells can be related to the concentration of the GagPol plasmid. In another example, if the lymphocyte targeting protein plasmid is contained in the Rev plasmid, the concentration of the lymphocyte targeting protein on the surface of the transduced cells can be related to the concentration of the Rev plasmid. In the five plasmid packaging system, the concentration of lymphocyte targeting protein on the surface of transduced cells can be related to the concentration of the plasmid containing the targeting protein.

[0143] In the four plasmid packaging system, the transduction efficiency can be related to the concentration of the plasmid containing the target protein. For example, when the protein plasmid plasmid that targets lymphocytes is contained in the VSV-G env plasmid, the transduction efficiency can be related to the concentration of the VSV-G env plasmid. In another example, when the protein plasmid plasmid that targets lymphocytes is contained in the GagPol plasmid, the transduction efficiency can be related to the concentration of the GagPol plasmid. In another example, when the protein plasmid plasmid that targets lymphocytes is contained in the Rev plasmid, the transduction efficiency can be related to the concentration of the Rev plasmid. In the five plasmid packaging system, the transduction efficiency can be related to the concentration of the protein plasmid that targets lymphocytes.

[0144] Following transfection of producer cells, the lentiviral particle product may be harvested from cell supernatant or culture medium. Downstream processing of LVV to maximize recovery of LVV while minimizing components that may adversely affect efficacy or safety is known in the art. A typical process involves a series of purification steps, including removal of cells and their debris, followed by concentration of the LVV, and removal of host cell or serum proteins, nucleic acids, and lipids. The LVV product may be further concentrated before being exchanged into a formulation buffer suitable for stability, and then finally sterile filtered and stored or applied. The harvested LVV supernatant may first undergo a clarification step to remove large impurities, such as aggregates and cell debris. In some embodiments, the clarification step includes centrifugation and / or conventional flow filtration. In some embodiments, the clarification step includes a nuclease digestion step. In some embodiments, the LVV product is further subjected to a purification step, including, for example, ion exchange chromatography (e.g., anion exchange chromatography). The LVV may also be concentrated, for example, by tangential flow filtration or ultrafiltration / diafiltration. In some embodiments, harvesting the LVV from the medium comprises centrifugation. In some embodiments, harvesting the LVV from the medium comprises anion exchange chromatography. In some embodiments, harvesting the LVV from the medium comprises anion exchange chromatography and tangential flow filtration.

[0145] In some embodiments, lentiviral vectors described herein incorporating a mutant VSV-G env and one or more lymphocyte-targeting proteins are capable of producing higher titers of LVV product compared to standard LVV incorporating another fusogenic env protein (e.g., coccal env, paramyxovirus env, truncated VSV-G env). As used herein, viral titer refers to infectious viral particle titer measured in transducing units (TU) per mL. In some embodiments, the titer of the LVV described herein is at least 1e7TU / mL, at least 1e8TU / mL, at least 1e9TU / mL, at least 1e10TU / mL, at least 1e11TU / mL, or at least 1e12TU / mL as measured in concentrated LVV product. In some embodiments, the titer of the LVV described herein is about 1e7TU / mL to about 1e12TU / mL in concentrated LVV product.

[0146] Engineered lymphocytes The LVV described herein can be used to modify target lymphocytes (e.g., T cells, B cells, or NK cells) to express a CAR encoded by a transgene carried by the LVV. In certain embodiments, the engineered lymphocytes are transduced by an LVV according to the present disclosure by contacting the lymphocytes with the LVV of the present disclosure. In such embodiments, the engineered lymphocytes (e.g., T cells, B cells, or NK cells) express a CAR encoded by a transgene carried by the LVV. These lymphocytes are also referred to herein as "CAR-modified lymphocytes." In certain embodiments, the engineered T cells are referred to herein as "CAR-modified T cells." In other specific embodiments, the engineered NK cells are referred to herein as "CAR-modified NK cells." In certain embodiments, the engineered T cells are referred to herein as "CAR-modified B cells." In certain embodiments, the CAR-modified T cells express a CAR encoded by a transgene carried by the LVV described herein and are capable of expressing naive T cells (CD45RA+, CCR7+, CD62L+, CD27+, CD45RO-), central memory T cells (CD45RA-, CD45RO-), or a combination of both. + , CD62L + , CCR7+, CD27+), effector memory T cells (CD45RA-, CD45RO+, CCR7-, CD62L-, CD27-), γδ T cells, mucosal-associated invariant T (MAIT) cells, Tregs, natural killer T cells, and tissue-resident T cells.

[0147] Transduction of target lymphocytes (eg, T cells, B cells, or NK cells) with the LVV described herein can be performed ex vivo or in vivo.

[0148] In certain embodiments, the lymphocytes (e.g., T cells, B cells, or NK cells) can be primary cells or cell lines derived from humans, mice, rats, or other mammals. When obtained from mammals, lymphocytes can be obtained from a number of sources, including blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. Lymphocyte compositions (e.g., T cell compositions, B cell compositions, or NK cell compositions) can be enriched or purified. T cell lines are well known in the art, some of which are described in Sandberg et al., Leukemia 21:230, 2000. In certain embodiments, the T cells lack endogenous expression of the TCRα gene, the TCRβ gene, or both. Such T cells may naturally lack endogenous expression of the TCR α and β chains, or may be modified to block expression of the TCR α chain, TCR β chain, or both genes (e.g., T cells from transgenic mice that do not express the TCR α and β chains or cells engineered to inhibit expression of the TCR α and β chains) or to knock out the TCR α chain, TCR β chain, or both genes.

[0149] In some embodiments, a source of lymphocytes (e.g., T cells or NK cells) may be obtained from a subject (e.g., whole blood, peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue) prior to genetically modifying the lymphocytes (e.g., T cells, B cells, or NK cells) with a polynucleotide encoding a CAR molecule. Lymphocytes, T cells, B cells, or NK cells can be enriched or isolated from a sample taken from the subject using methods known in the art. For example, T cells may be enriched in a whole blood sample taken from a subject using known methods for hypotonic lysis of red blood cells, or T cells may be isolated from a sample taken from a subject using known gradient sedimentation, e.g., Ficoll® reduction techniques. Alternatively, T cells may be isolated from whole blood using antibody-labeled magnetic beads followed by column separation. Specific host cell subsets can be collected according to known techniques and enriched or depleted by known techniques, such as affinity binding to antibodies, flow cytometry and / or immunomagnetic selection. In some embodiments, after the enrichment and / or depletion step, the T cells are placed in contact with the disclosed LVV such that the T cells obtained from the subject are transduced by the LVV and express the CAR encoded by the transgene carried by the LVV. In some embodiments, after the enrichment and / or depletion step, the NK cells are placed in contact with the disclosed LVV such that the NK cells obtained from the subject are transduced by the LVV and express the CAR encoded by the transgene carried by the LVV. In some embodiments, after the enrichment and / or depletion step, the B cells are placed in contact with the disclosed LVV such that the B cells obtained from the subject are transduced by the LVV and express the CAR encoded by the transgene carried by the LVV.

[0150] Generally, resting T cells, such as resting CD4 and CD8 lymphocytes, resting B cells, and resting NK cells, are ineffective for gene transfer by lentiviral vectors. Resting T cells, also known as resting T cells or naive T cells, refer to T cells that are not mitotically active or have never been exposed to alloantigens presented on antigen-presenting cells, such as macrophages or dendritic cells. An example of a marker for resting T cells is CD28. Alternatively, markers that are expressed on activated T cells but not on resting T cells include, for example, 4-1BB, PD-1, and HLA-DR. Similarly, resting B cells and resting NK cells refer to B cells and NK cells, respectively, that are not mitotically active or have never been exposed to alloantigens. Examples of markers for resting B cells include CD21 and CD23, and the absence of CD80, CD86, CD95, or CD25. CD137 and GITR, which are expressed on activated NK cells, are not present on resting NK cells. As a result, to facilitate the transduction of T cells, B cells, or NK cells with lentiviral vectors, the T cells, B cells, or NK cells are typically activated in vitro with a stimulatory agent, and then genetically modified via the lentiviral vector. After stimulation and transduction, the genetically modified cells are then typically expanded in vitro and then reintroduced into the patient. However, the LVV described herein is capable of transducing resting T cells, B cells, and / or resting NK cells. In certain embodiments, a method of transducing T cells with an LVV according to the present invention includes placing the LVV in contact with a population of T cells, where the T cells are not activated during the transduction process and the T cells are not exposed to exogenous stimulatory agents. In some such embodiments, methods of transducing T cells, where the T cells are not activated during the transduction process and the T cells are not exposed to exogenous stimuli, include transducing T cells with an LVV that includes a CD80 targeting protein, an anti-CD3 targeting protein, or both a CD80 targeting protein and an anti-CD3 targeting protein.Examples of exogenous T cell stimulants include an anti-CD3 antibody or an antigen-binding fragment thereof (i.e., an anti-CD3 antibody or an antigen-binding fragment thereof that is not part of the LVV), an anti-CD28 antibody or an antigen-binding fragment thereof (i.e., an anti-CD28 antibody or an antigen-binding fragment thereof that is not part of the LVV), an anti-CD2 antibody or an antigen-binding fragment thereof, IL-2, IL-7, IL-15, PHA, or any combination thereof. The exogenous stimulant may be contacted with the T cells in a soluble form or may be immobilized on a solid substrate, such as a bead or a cell culture plate, and contacted with the T cells. In some embodiments, an anti-CD3 antibody or an antigen-binding fragment thereof, an anti-CD28 antibody or an antigen-binding fragment thereof, or both an anti-CD3 antibody and an anti-CD28 antibody or an antigen-binding fragment thereof, are contacted with the T cells in a soluble form or immobilized on a solid substrate. Examples of anti-CD3 antibodies include OKT3, UCTH1, and BW264 / 56. An example of an anti-CD2 antibody is LT2. An example of an anti-CD28 antibody is 15E8. In certain embodiments, a method of transducing NK cells with an LVV according to the present disclosure includes placing the LVV in contact with a population of NK cells, where the NK cells are not activated during the transduction process and the NK cells are not exposed to an exogenous stimulant. Examples of exogenous NK cell stimulants include an anti-CD2 antibody or antigen-binding fragment thereof, an anti-CD335 antibody or antigen-binding fragment thereof, IL-2, IL-15, IL-12, IL-18, IL-21, or a combination thereof. In certain embodiments, a method of transducing B cells with an LVV according to the present disclosure includes placing the LVV in contact with a population of B cells, where the B cells are not activated during the transduction process and the B cells are not exposed to an exogenous stimulant. Examples of exogenous B cell stimulants include CD154 and mixed Ig F(ab)2.

[0151] In some embodiments, lentiviral vectors carrying a CD80 targeting protein and an anti-CD3 targeting protein can efficiently transduce both CD4 and CD8 T cells compared to standard LVV.

[0152] In some embodiments, lentiviral vectors bearing CD80 targeting protein and anti-CD3 targeting protein are capable of activating T cells to a level comparable to T cells transduced with standard LVV treated with exogenous stimuli without exogenous stimuli (e.g., T cell stimulants). Examples of exogenous T cell stimulants include an anti-CD3 antibody or antigen-binding fragment thereof (i.e., an anti-CD3 antibody or antigen-binding fragment thereof that is not part of LVV), an anti-CD28 antibody or antigen-binding fragment thereof (i.e., an anti-CD28 antibody or antigen-binding fragment thereof that is not part of LVV), an anti-CD2 antibody or antigen-binding fragment thereof, IL-2, IL-7, IL-15, PHA, or any combination thereof.

[0153] In certain embodiments, methods of transducing T cells with an LVV according to the present specification show improved transduction of CD4 T cells compared to standard LVV. In some such embodiments, a method of enhancing transduction of CD4 T cells in a mixed population of T cells (e.g., composed of CD4 and CD8 T cells) comprises transducing the mixed population of T cells with an LVV according to the present specification that contains a CD80-targeting protein.

[0154] In certain embodiments, methods of transducing T cells with LVV according to the present disclosure efficiently transduce both CD4 and CD8 T cells. In some such embodiments, methods of transducing T cells in which both CD4 and CD8 T cells are efficiently transduced include transducing T cells with LVV comprising a CD80 targeting protein and an anti-CD3 targeting protein.

[0155] In certain embodiments, the LVV in accordance with the present specification can transduce T cells under "stress" conditions, e.g., at a low multiplicity of infection (MOI) or without treatment with exogenous IL-2 during the transduction process. In some embodiments, the LVV in accordance with the present specification can transduce T cells at about 5 to about 25 times lower LVV concentrations than LVV that does not target lymphocytes.

[0156] Expression of the CAR molecule on the host cell can be assessed by methods known in the art, such as quantitative PCR or flow cytometry after staining with a fluorescently labeled antigen for the CAR binding domain.

[0157] Expression of the CAR molecule on the host cell can be functionally characterized according to any of a number of art-accepted methods for assaying the activity of host T cells, including measuring T cell binding, activation or induction, as well as measuring antigen-specific T cell responses. Examples include T cell proliferation, T cell cytokine release, antigen-specific T cell stimulation, CTL activity (e.g., from pre-loaded target cells), and the like. 51These include detection of Cr or europium release, induction of caspase activity in target cells, extracellular release of lactate dehydrogenase by target cells), changes in T cell phenotypic marker expression, and measurement of other measures of T cell function. Similar methods can also be used to assay host NK cell activity. Procedures for performing these and similar assays can be found, for example, in Lefkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998). See also Current Protocols in Immunology; Weir, Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA (1986); Mishell and Shigii (eds.) Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA (1979); Green and Reed, Science 281:1309 (1998) and references cited therein. Cytokine levels may be measured according to methods known in the art, including, for example, ELISA, ELISPOT, intracellular cytokine staining, flow cytometry, and any combination thereof (e.g., intracellular cytokine staining and flow cytometry). Proliferation and clonal expansion of immune cells due to antigen-specific induction or stimulation of an immune response may be determined by isolating lymphocytes, such as circulating lymphocytes, in a sample of peripheral blood cells or lymph node cells, stimulating the cells with an antigen, and measuring cytokine production, cell proliferation and / or cell viability, such as by tritiated thymidine incorporation or non-radioactive assays, such as the MTT assay.

[0158] Therapeutic Methods and Compositions In one aspect, the disclosure provides a method of treating a disease in a subject, the method comprising administering to the subject an effective amount of an LVV described herein, a CAR-modified lymphocyte (e.g., a T cell, a B cell, or an NK cell) described herein, or a pharmaceutical composition thereof. In another aspect, the method of treating a disease in a subject according to the present disclosure comprises administering to the subject an effective amount of an LVV described herein, a CAR-modified lymphocyte (e.g., a T cell, a B cell, or an NK cell) described herein, or a pharmaceutical composition thereof, in combination with one or more additional therapeutic agents.

[0159] Diseases that may be treated with the LVV or CAR-modified lymphocytes (e.g., T cells, B cells, or NK cells) according to the present specification include cancer. Adoptive immunotherapy and gene therapy are promising treatments for various types of cancer (Morgan et al., Science 314:126, 2006; Schmitt et al., Hum. Gene Ther. 20:1240, 2009; June, J. Clin. Invest. 117:1466, 2007) and infectious diseases (Kitchen et al., PLoS One 4:38208, 2009; Rossi et al., Nat. Biotechnol. 25:1444, 2007; Zhang et al., PLoS Pathog. 6:e1001018, 2010; Luo et al., J. Mol. Med. 89:903, 2011).

[0160] A wide range of cancers, including solid tumors and leukemias, are suitable for the compositions and methods disclosed herein. Exemplary cancers that may be treated using the receptors, modified host cells, and compositions described herein include adenocarcinomas of the breast, prostate, and colon, all forms of bronchial carcinoma of the lung, myeloid leukemia, melanoma, hepatoma, neuroblastoma, papilloma, apdoma, stratoma, branchiocarcinoma, malignant carcinoid syndrome, carcinoid heart disease, and carcinomas (e.g., Walker, basal cell, basosquamous, Brown-Pierce, ductal, Ehrlich tumor, Krebs 2, Merkel cell, mucinous, non-small cell lung, oat cell, papillary, sclerotic, bronchiolar, bronchogenic, squamous cell, and transitional). Additional types of cancer that may be treated using the receptors, modified host cells, and compositions described herein include histiocytic disorders, malignant histiocytosis, leukemia, Hodgkin's disease, immunoproliferative small, non-Hodgkin's lymphoma, plasmacytoma, multiple myeloma, chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), plasmacytoma, reticuloendotheliosis, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumor, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, chordoma, craniopharyngioma, dysgerminoma, hamartoma, mesenchymoma, mesonephroma, sarcoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma, trophoblastic tumor. Additionally, the following types of cancer are contemplated to be suitable for treatment using the receptors, modified host cells, and compositions described herein: adenoma, cholangiomas, cholesteatomas, cystadenocarcinomas, cystadenomas, granulosa cell tumors, male germinomas, hepatocarcinomas, sweat adenomas, pancreatic islet cell tumors, Leydig cell tumors, papillomas, Sertoli cell tumors, thecal cell tumors, leiomyomas, leiomyosarcomas, myoblastomas, myomas, sarcomas, rhabdomyomas, rhabdomyosarcomas, ependymomas, ganglioneuromas, gliomas, medulloblastomas, meningiomas, schwannoma, neuroblastomas, neuroepithelioma, neurofibromas, neuromas, paragangliomas, and achromaffin paragangliomas.Types of cancer that may be treated also include angiokeratoma, eosinophilic angiolymphocytosis, sclerosing hemangioma, hemangiomatosis, glomus hemangiomatosis, hemangioendothelioma, hemangioma, hemangiopericytoma, angiosarcoma, lymphangioma, lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma, angiosarcoma, leiomyosarcoma, leukemia sarcoma, liposarcoma, lymphangiosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcoma, neoplasm, neurofibromatosis, cervical dysplasia, and peritoneal carcinoma.

[0161] Examples of hyperproliferative disorders that are amenable to treatment using the receptors, modified host cells, and compositions described herein include B cell cancers (B cell malignancies), including B cell lymphomas (e.g., various forms of Hodgkin's disease, non-Hodgkin's lymphoma (NHL) or central nervous system lymphoma), leukemias (e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, the B cell blastic phenomenon of chronic myeloid leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia, and myelomas (e.g., multiple myeloma). Additional B cell cancers that may be obtained include small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, isolated bone plasmacytoma, extraosseous plasmacytoma, extranodal marginal zone B-cell lymphoma of mucosal intralymphoid tissue (MALT), extranodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma / leukemia, B-cell proliferation of uncertain grade, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.

[0162] The CAR-modified lymphocytes contained in the compositions disclosed herein and administered to a subject may include CAR-modified T cells, such as CD4+ T cells, CD8+ T cells, natural killer cells, gamma delta T cells, or MAIT cells, CAR-modified B cells, or CAR-modified NK cells. In certain embodiments, the method of treating a subject includes administering an effective amount of the LVV described herein or CAR-modified lymphocytes (i.e., recombinant cells expressing one or more CARs) according to the present disclosure. In some embodiments, CAR-modified T cells are administered to the subject. In some embodiments, CAR-modified NK cells are administered to the subject. In some embodiments, CAR-modified B cells are administered to the subject. The CAR-modified lymphocytes (e.g., T cells, B cells, or NK cells) may be xenogeneic, syngeneic, allogeneic, or autologous to the subject.

[0163] The pharmaceutical composition comprising the LVV or CAR engineered lymphocytes can be administered in a manner appropriate to the disease or condition to be treated (or prevented), as determined by those skilled in the medical arts. The appropriate dose, appropriate duration of administration, and frequency of administration of the composition are determined by factors such as the patient's condition, size, weight, body surface area, age, sex, type and severity of disease, the specific therapy administered, the specific form of the active ingredient, the time and method of administration, and other drugs administered concomitantly. The present disclosure provides a pharmaceutical composition comprising the LVV or CAR modified lymphocytes and a pharma- ceutically acceptable carrier, diluent or excipient. Suitable excipients include water, saline, dextrose, glycerol, and the like, and combinations thereof. Other suitable infusion vehicles can be any isotonic vehicle formulation, including saline, Normosol R (Abbott), Plasma-Lyte A (Baxter), 5% dextrose in water, or lactated Ringer's solution.

[0164] The therapeutically effective amount of engineered lymphocytes (e.g., T cells, B cells, or NK cells) included in the pharmaceutical composition is at least one cell (e.g., one CAR-modified T cell), and more typically at least 10 2 More than one, for example up to 10 6 pieces, maximum 107 pieces, maximum 10 8 pieces, maximum 10 9 pieces, maximum 10 10 pcs or up to 10 11 In certain embodiments, the cells are about 10 6 ~about 10 10 pieces / m 2 , preferably about 10 7 ~about 10 9 cells / m 2 The number of cells will depend on the intended end use of the composition as well as the type of cells contained therein. For example, a composition comprising T cells modified to contain a CAR will comprise a T cell population comprising from about 5% to about 95% or more of such cells. In certain embodiments, a composition comprising a CAR modified T cell will comprise a cell population comprising at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells. In certain embodiments, a composition comprising CAR modified NK cells comprises a cell population comprising at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells. In certain embodiments, a composition comprising CAR modified B cells comprises a cell population comprising at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells. For the uses provided herein, the lymphocytes (e.g., T cells, B cells, or NK cells) are generally in a volume of 1 liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. Therefore, the desired cell density is usually 10 4 Higher than 10 cells / ml, generally 7 Higher than 10 cells / ml, typically 8The lymphocytes (e.g., T cells, B cells, or NK cells) may be administered as a single infusion or in multiple infusions over a period of time. Repeated infusions of CAR-modified lymphocytes (e.g., T cells, B cells, or NK cells) may be spaced days, weeks, months, or even years apart if there is a relapse of disease or disease activity. A clinically meaningful number of immune cells may be administered in a cumulative total of 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 A preferred dose for administration of host cells containing a recombinant expression vector described herein is about 10 7 cells / m 2 , about 5x10 7 cells / m 2 , about 10 8 cells / m 2 , about 5x10 8 cells / m 2 , about 10 9 cells / m 2 , about 5x10 9 cells / m 2 , about 10 10 cells / m 2 , about 5x10 10 cells / m 2 , or about 10 11 cells / m 2 It is.

[0165] The LVV and / or CAR-modified lymphocyte (e.g., T cell, B cell, or NK cell) compositions described herein can be administered to a subject intravenously, intraperitoneally, intratumorally, intrabone marrow (e.g., intraosseous administration), intralymph node (intranodal), and / or intracerebrospinal fluid.

[0166] The LVV vector and / or CAR modified lymphocyte (e.g., T cell, B cell, or NK cell) composition may be administered to a subject in combination with one or more additional therapeutic agents. Examples of therapeutic agents that may be administered in combination with the LVV vector or CAR modified lymphocyte (e.g., T cell, B cell, or NK cell) according to the present specification include radiation therapy, adoptive cellular immunotherapy (e.g., recombinant TCR, affinity-enhanced TCR, CAR, TCR-CAR, scTCR fusion protein, dendritic cell vaccine), antibody therapy, immune checkpoint molecule inhibitor therapy, ultraviolet light therapy, electric pulse therapy, high intensity focused ultrasound therapy, oncolytic virus therapy, or drug therapy, such as chemotherapeutic drugs, therapeutic peptides, hormones, aptamers, antibiotics, antiviral agents, antifungal agents, anti-inflammatory agents, small molecule therapy, or any combination thereof.

[0167] Radiation therapy includes external beam radiation therapy (e.g., conventional external beam radiation therapy, stereotactic radiation therapy, three-dimensional conformal radiation therapy, intensity-modulated radiation therapy, intensity-modulated arc therapy, particle beam therapy, proton beam therapy, and Auger therapy), brachytherapy, total body radioisotope therapy, intraoperative radiation therapy, or any combination thereof.

[0168] Exemplary antibodies that may be used in combination with the LVV or CAR-modified lymphocyte (e.g., T cell, B cell, or NK cell) compositions described herein include rituximab, pertuzumab, trastuzumab, alemtuzumab, ibritumomab tiuxetan, brentuximab vedotin, cetuximab, bevacizumab, abciximab, adalimumab, alefacept, basilizimab, belimumab, bezlotoxumab, canakinumab, certolizumab pegol, daclizumab, denosumab, efalizumab, golimumab, olaratumab, palivizumab, panitumumab, and tocilizumab.

[0169] Exemplary immune checkpoint molecule inhibitors that may be used in combination with the LVV or CAR-modified lymphocyte (e.g., T cell, B cell, or NK cell) compositions described herein include checkpoint inhibitors that target PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244 / 2B4, CD160, TIGIT, LAIR-1, PVRIG / CD112R, or any combination thereof. In certain embodiments, the immune checkpoint inhibitor may be an antibody, peptide, RNAi agent, or small molecule. The antibody specific for CTLA-4 may be ipilimumab or tremelimumab. The antibody specific for PD-1 can be pidilizumab, nivolumab, or pembrolizumab.The antibody specific for PD-L1 can be durvalumab, atezolizumab, or avelumab.

[0170] Exemplary chemotherapeutic agents that may be used in combination with the LVV or CAR-modified lymphocyte (e.g., T cell, B cell, or NK cell) compositions described herein may include alkylating agents, platinum-based agents, cytotoxic agents, inhibitors of chromatin function, topoisomerase inhibitors, microtubule inhibitors, DNA damaging agents, antimetabolites (e.g., folate antagonists, pyrimidine analogs, purine analogs, sugar-modifying analogs), DNA synthesis inhibitors, DNA interacting agents (e.g., intercalating agents), and DNA repair inhibitors.

[0171] Chemotherapy as referred to herein includes non-specific cytotoxic agents that inhibit mitosis or cell division, as well as molecular targeted therapies that block the growth and spread of cancer cells by targeting specific molecules (e.g., oncogenes) involved in tumor growth, progression, and metastasis. Exemplary non-specific chemotherapeutic agents that may be used in combination with the expression cassette compositions described herein may include alkylating agents, platinum-based agents, cytotoxic drugs, inhibitors of chromatin function, topoisomerase inhibitors, microtubule inhibitors, DNA damaging agents, antimetabolites (e.g., folate antagonists, pyrimidine analogs, purine analogs, sugar-modifying analogs), DNA synthesis inhibitors, DNA interacting agents (e.g., intercalating agents), hypomethylating agents, and DNA repair inhibitors.

[0172] Examples of chemotherapeutic agents contemplated for use in the combination therapy contemplated herein include vemurafenib, dabrafenib, trametinib, cobimetinib, anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paragon®), rifampicin (Renvoy ... aplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (ActinomycinD, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposomal injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®), (registered trademark), Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan ( Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), Mylotarg, paclitaxel (Taxol®), Phoenix (Yttrium 90 / MX-DTPA), pentostatin, polyphenoprosan 20 and carmustine implants (Gliadel®), FDA-approved tamoxifen citrate (Nolv adex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), injectable topotecan hydrochloride (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), ibrutinib, venetoclax, crizotinib, alectinib, brigatinib, ceritinib, and vinorelbine (Navelbine®).

[0173] Exemplary alkylating agents for use in the combination therapies contemplated herein include nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes, uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard, etc. Mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), chlorambucil (Leukeran®), pipobromobromide (Panama®), These include mannose (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and dacarbazine (DTIC-Dome®).Further exemplary alkylating agents for use in the combination therapies contemplated herein include oxaliplatin (Eloxatin®), temozolomide (Temodar® and Temodal®), dactinomycin (also known as actinomycin-D, Cosmegen®), melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®), altretamine (also known as hexamethylmelamine (HMM), HMM, HMM), dactinomycin (also known as actinomycin-D, Cosmegen® ... exalen®), carmustine (BiCNU®), bendamustine (Treanda®), busulfan (Busulfex® and Myleran®), carboplatin (Paraplatin®), lomustine (also known as CCNU, CeeNU®), cisplatin (also known as CDDP, Platinol® and Platinol®-AQ), chlorambucil (Leukeran®), cyclophosphamide (cyclophosphamide ... Sulfamide (Cytoxan® and Neosar®), dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-Dome®), altretamine (also known as hexamethylmelamine (HMM), Hexalen®), ifosfamide (Ifex®), prednumustine, procarbazine (Matulane®), mechlorethamine (also known as nitrogen mustard, mustine and salts), These include, but are not limited to, mechlorethamine hydrochloride, Mustargen®), streptozocin (Zanosar®), thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex®), cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®), and bendamustine HCl (Treanda®).

[0174] Exemplary platinum-based agents for use in the combination therapies contemplated herein include carboplatin, cisplatin, oxaliplatin, nedaplatin, picoplatin, satraplatin, phenanthriplatin, and triplatin tetranitrate.

[0175] Exemplary hypomethylating agents for use in combination therapy include azacitidine and decitabine.

[0176] Exemplary molecular targeted inhibitors for use in the combination therapies contemplated herein include small molecules that target molecules involved in cancer cell proliferation and survival, including, for example, receptor tyrosine kinase inhibitors, RAF inhibitors, BCL-2 inhibitors, ABL inhibitors, TRK inhibitors, c-KIT inhibitors, c-MET inhibitors, CDK4 / 6 inhibitors, FAK inhibitors, FGFR inhibitors, FLT3 inhibitors, IDH1 inhibitors, IDH2 inhibitors, PDGFRA inhibitors, and RET inhibitors.

[0177] Exemplary molecular targeted therapies include hormone antagonists, signal transduction inhibitors, gene expression inhibitors (e.g., translation inhibitors), apoptosis inducers, angiogenesis inhibitors (e.g., VEGF pathway inhibitors), tyrosine kinase inhibitors (e.g., EGF / EGFR pathway inhibitors), growth factor inhibitors, GTPase inhibitors, serine / threonine kinase inhibitors, transcription factor inhibitors, inhibitors of driver mutations associated with cancer, B-Raf inhibitors, RAF inhibitors, MEK inhibitors, mTOR inhibitors, adenosine pathway inhibitors, EGFR inhibitors agents, PI3K inhibitors, BCL2 inhibitors, VEGFR inhibitors, MET inhibitors, MYC inhibitors, BCR-ABL inhibitors, ABL inhibitors, HER2 inhibitors, H-RAS inhibitors, K-RAS inhibitors, PDGFR inhibitors, ALK inhibitors, ROS1 inhibitors, BTK inhibitors, TRK inhibitors, c-KIT inhibitors, c-MET inhibitors, CDK4 / 6 inhibitors, FAK inhibitors, FGFR inhibitors, FLT3 inhibitors, IDH1 inhibitors, IDH2 inhibitors, PARP inhibitors, PARP inhibitors, PDGFRA inhibitors, and RET inhibitors.

[0178] Exemplary angiogenesis inhibitors include A6 (Angstrom Pharmaceuticals), ABT-510 (Abbott Laboratories), ABT-627 (Atrasentan) (Abbott Laboratories / Xinlay), ABT-869 (Abbott Laboratories), Actimid (CC4047, pomalidomide) (Celgene Corporation), AdGVPEDF.11D (GenVec), ADH-1 (Exherin) (Adherex Technologies), AEE788 (Novartis), AG-013736 (Axitinib) (Pfizer), AG3340 (Prinomastat) (Agouron Pharmaceuticals), AGX1053 (AngioGenex), AGX51 (AngioGenex), ALN-VSP (ALN-VSP O2) (Alnylam Pharmaceuticals), AMG 386 (Amgen), AMG706 (Amgen), Apatinib (YN968D1) (Jiangsu Hengrui Medicine), AP23573 (Ridaforolimus / MK8669) (Ariad Pharmaceuticals), AQ4N (Novavea), ARQ 197 (ArQule), ASA404 (Novartis / Antisoma), Atiprimod (Callisto) Pharmaceuticals), ATN-161 (Attenuon), AV-412 (Aveo Pharmaceuticals), AV-951 (Aveo Pharmaceuticals), Avastin (bevacizumab) (Genentech), AZD2171 (cediranib / Recentin) (AstraZeneca), BAY 57-9352 (teratinib) (Bayer), BEZ235 (Novartis), BIBF1120 (Boehringer Ingelheim Pharmaceuticals), BIBW 2992 (Boehringer Ingelheim Pharmaceuticals), BMS-275291 (Bristol-Myers Squibb), BMS-582664 (brivanib) (Bristol-MyersSquibb), BMS-690514 (Bristol-Myers Squibb), calcitriol, CCI-779 (Torisel) (Wyeth), CDP-791 (ImClone Systems), ceflatonin (homoharringtonine / HHT) (ChemGenex Therapeutics), Celebrex (celecoxib) (Pfizer), CEP-7055 (Cephalon / Sanofi), CHIR-265 (Chiron Corporation), NGR-TNF, COL-3 (Metastat) (Collagenex Pharmaceuticals), combretastatin (Oxigene), CP-751,871 (figitumumab) (Pfizer), CP-547,632 (Pfizer), CS-7017 (Daiichi Sankyo) Pharma), CT-322 (Angiocept) (Adnexus), Curcumin, Dalteparin (Fragmin) (Pfizer)), Disulfiram (Antabuse), E7820 (Eisai Limited), E7080 (Eisai Limited), EMD 121974 (Cilengitide) (EMD Pharmaceuticals), ENMD-1198 (EntreMed), ENMD-2076 (EntreMed), Endostar (Simcere), Erbitux (ImClone / Bristol-Myers Squibb), EZN-2208 (Enzon Pharmaceuticals), EZN-2968 (Enzon Pharmaceuticals), GC1008 (Genzyme), genistein, GSK1363089 (foretinib) (GlaxoSmithKline), GW786034 (pazopanib) (GlaxoSmithKline), GT-111 (Vascular Biogenics Ltd.), IMC-1121B (ramucirumab) (ImClone Systems), IMC-18F1 (ImClone Systems), IMC-3G3 (ImClone LLC), INCB007839 (Incyte Corporation), INGN 241 (IntrogenTherapeutics), Iressa (ZD1839 / Gefitinib), LBH589 (Faridak / Panobinostst) (Novartis), Lucentis (ranibizumab) (Genentech / Novartis), LY317615 (enzastaurin) (Eli Lilly and Company), Macugen (pegaptanib) (Pfizer), MEDI522 (Abegrin) (MedImmune), MLN518 (tanzutinib) (Millennium), Neovastat (AE941 / Benefin) (Aeterna Zentaris), Nexavar (Bayer / Onyx), NM-3 (Genzyme Corporation), noscapine (Cougar Biotechnology), NPI-2358 (Nereus Pharmaceuticals), OSI-930 (OSI), Palomid 529 (Paloma Pharmaceuticals,Inc.), Panzem Capsules (2ME2) ​​(EntreMed), Panzem NCD (2ME2) ​​(EntreMed), PF-02341066 (Pfizer), PF-04554878 (Pfizer), PI-88 (Progen Industries / Medigen Biotechnology), PKC412 (Novartis), Polyphenon E (green tea extract) (Polypheno E International,Inc.), PPI-2458 (Praecis Pharmaceuticals), PTC299 (PTC Therapeutics), PTK787 (vatalanib) (Novartis), PXD101 (belinostat) (CuraGen Corporation), RAD001 (everolimus) (Novartis), RAF265 (Novartis), regorafenib (BAY73-4506) (Bayer), Revlimid (Celgene), Retaane (AlconResearch), SN38 (Liposomal) (Neopharm), SNS-032 (BMS-387032) (Sunesis), SOM230 (pasireotide) (Novartis), squalamine (Genaera), suramin, Sutent (Pfizer), Tarceva (Genentech), TB-403 (Thrombogenics), Tempostatin (Collard Biopharmaceuticals), tetrathiomolybdate (Sigma-Aldrich), TG100801 (TargeGen), thalidomide (Celgene Corporation), tinzaparin sodium, TKI258 (Novartis), TRC093 (Tracon Pharmaceuticals Inc.), VEGF Trap (aflibercept) (Regeneron Pharmaceuticals), VEGF Trap-Eye (Regeneron Pharmaceuticals), Veglin (VasGene These include, but are not limited to, bortezomib (Millennium), XL184 (Exelixis), XL647 (Exelixis), XL784 (Exelixis), XL820 (Exelixis), XL999 (Exelixis), ZD6474 (AstraZeneca), vorinostat (Merck), and ZSTK474.

[0179] Exemplary B-Raf inhibitors include vemurafenib, dabrafenib, and encorafenib.

[0180] Exemplary MEK inhibitors include binimetinib, cobimetinib, refametinib, selumetinib, and trametinib.

[0181] Exemplary BTK inhibitors include ibrutinib, Loxo-305, tirabrutinib, GDC-0853, acalabrutinib, ONO-4059, spebrutinib, BGB-3111, HM71224, and M7583.

[0182] Exemplary TRK inhibitors include entrectinib, larotrectinib, CH7057288, ONO-7579, LOXO-101, lestaurtinib, and LOXO-195.

[0183] Exemplary c-KIT inhibitors include imatinib, sunitinib, and ponatinib.

[0184] Exemplary c-MET inhibitors include capmatinib, crizotinib, tivantinib, onartuzumab, INCB28060, AMG-458, savolitinib, and tepotinib.

[0185] Exemplary CDK4 / 6 inhibitors include palbociclib, ribociclib, abemaciclib, and trilaciclib.

[0186] Exemplary FAK inhibitors include defactinib, GSK2256098, BI853520, and PF-00562271.

[0187] Exemplary FGFR inhibitors include erdafitinib, pemigatinib, infigratinib, rogaratinib, AZD4547, BGJ398, FP-1039, and ARQ087.

[0188] Exemplary FLT-3 inhibitors include quizartinib, crenolanib, gilteritinib, midostaurin, and lestaurtinib.

[0189] Exemplary IDH1 inhibitors include ivosidenib, BAY-1436032, and AGI-5198.

[0190] Exemplary IDH2 inhibitors include enasidenib.

[0191] Exemplary PARP inhibitors include talazoparib, niraparib, rucaparib, olaparib, veliparib, CEP 9722, E7016, AG014699, MK4827, BMN-673, and Pamiparib (BGB-290).

[0192] Exemplary PDGFRA inhibitors include imatinib, regorafenib, crenolanib, and olaratumab.

[0193] Exemplary pan-RAF inhibitors include belvarafenib, LXH254, LY3009120, INU-152, and HM95573.

[0194] Exemplary RET inhibitors include lenvatinib, alectinib, vandetanib, cabozantinib, BLU-667, and LOXO-292.

[0195] Exemplary ROS1 inhibitors include ceritinib, lorlatinib, entrectinib, crizotinib, TPX-0005, and DS-6051b.

[0196] Exemplary vascular endothelial growth factor (VEGF) receptor inhibitors include bevacizumab (Avastin®), axitinib (Inlyta®), brivanib alaninate (BMS-582664, (S)-((R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl) 2-aminopropanoate), sorafenib (Nexavar®), pazopanib (Votrient®), sunitinib malate (Sutent®), cediranib (AZD2171, CAS 288383-20-1), Vargatef (BIBF1120, CAS 928326-83-4), foretinib (GSK1363089), telatinib (BAY57-9352, CAS 332012-40-5), apatinib (YN968D1, CAS 811803-05-1), imatinib (Gleevec®), ponatinib (AP24534, CAS 943319-70-8), tivozanib (AV951, CAS 475108-18-0), regorafenib (BAY73-4506, CAS 755037-03-7), vatalanib dihydrochloride (PTK787, CAS 212141-51-0), brivanib (BMS-540215, CAS 649735-46-6), vandetanib (Caprelsa® or AZD6474), motesanib diphosphate (AMG706, CAS 857876-30-3, N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide, described in PCT Publication No. WO02 / 066470), dovitinib dilactate (TKI258, CAS 852433-84-2), linfanib (ABT869, CAS 796967-16-3), cabozantinib (XL184, CAS 849217-68-1), lestaurtinib (CAS 111358-88-4), N-[5-[[[5-(1,1-Dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide (BMS38703, CAS 345627-80-7), (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514), N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine (XL647, CAS 781613-23-8), 4-methyl-3-[[1-methyl-6-(3-pyridinyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]amino]-N-[3-(trifluoromethyl)phenyl]-benzamide (BHG712, CAS 940310-85-0), and aflibercept (Eylea®).

[0197] Exemplary EGF pathway inhibitors include tyrophostin 46, EKB-569, erlotinib (Tarceva®), gefitinib (Iressa®), erbitux, nimotuzumab, lapatinib (Tykerb®), cetuximab (anti-EGFR mAb), 188These include, but are not limited to, Re-labeled nimotuzumab (anti-EGFR mAb), as well as compounds generally and specifically disclosed in WO 97 / 02266, EP 0 564 409, WO 99 / 03854, EP 0 520 722, EP 0 566 226, EP 0 787 722, EP 0 837 063, U.S. Pat. No. 5,747,498, WO 98 / 10767, WO 97 / 30034, WO 97 / 49688, WO 97 / 38983, and WO 96 / 33980. Exemplary EGFR antibodies include, but are not limited to, cetuximab (Erbitux®), panitumumab (Vectibix®), matuzumab (EMD-72000), trastuzumab (Herceptin®), nimotuzumab (hR3), zalutumumab, TheraCIM h-R3, MDX0447 (CAS 339151-96-1), and ch806 (mAb-806, CAS 946414-09-1).Exemplary epidermal growth factor receptor (EGFR) inhibitors include erlotinib hydrochloride (Tarceva®), ceritinib, brigatinib, osimeritinib, icotinib, gefitinib (Iressa®), N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3“S”)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide, Tovok®), vandetanib (C aprelsa®), lapatinib (Tykerb®), (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514), canertinib dihydrochloride (CI-1033), 6-[4-[(4-ethyl-1-piperazinyl)methyl]phenyl]-N-[(1R)-1-phenylethyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine (AEE788, CAS 497839-62-0), Mubritinib (TAK165), Peritinib (EKB569), Afatinib (BIBW2992), Neratinib (HKI-272), N-[4-[[1-[(3-fluorophenyl)methyl]-1H-indazol-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]-carbamic acid, (3S)-3-morpholinylmethyl ester (BMS599626), N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine (XL647, CAS 781613-23-8), 4-[4-[[(1R)-1-phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol (PKI166, CAS 187724-61-4), and rocelitinib.

[0198] Exemplary mTOR inhibitors include rapamycin (Rapamune®), as well as its analogs and derivatives, SDZ-RAD, temsirolimus (Torisel®, also known as CCI-779), ridaforolimus (formerly known as deferolimus, (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0 4,9 ]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, described in PCT Publication No. WO03 / 064383), everolimus (Afinitor® or RAD001), rapamycin (AY22989, Sirolimus®), Simapimod (CAS 164301-51-3), (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055), 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-1-one (PF04691502, CAS 1013101-36-4), and N 2 -[1,4-dioxo-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartyl-L-serine-, inner salt (SF1126, CAS 936487-67-1).

[0199] Exemplary phosphoinositide 3-kinase (PI3K) inhibitors include duvelisib, idelalisib, 4-[2-(1H-indazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine (also known as GDC 0941, described in PCT Publication Nos. WO 09 / 036082 and WO 09 / 055730), 2-methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile (also known as BEZ 235 or NVP-BEZ 235, described in PCT Publication No. WO06 / 122806), 4-(trifluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridin-2-amine (also known as BKM120 or NVP-BKM120, described in PCT Publication No. WO2007 / 084786), Tozasertib (VX680 or MK-0457, CAS 639089-54-6), (5Z)-5-[[4-(4-pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidinedione (GSK1059615, CAS 639089-54-6), 958852-01-2), (1E,4S,4aR,5R,6aS,9aR)-5-(acetyloxy)-1-[(di-2-propenylamino)methylene]-4,4a,5,6,6a,8,9,9a-octahydro-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-cyclopenta[5,6]naphtho[1,2-c]pyran-2,7,10(1H)-trione (PX866, CAS 502632-66-8), and 8-phenyl-2-(morpholin-4-yl)-chromen-4-one (LY294002, CAS 154447-36-6).

[0200] Exemplary protein kinase B (PKB) or AKT inhibitors include 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f][1,6]naphthyridin-3(2H)-one (MK-2206, CAS 1032349-93-1), Perifosine (KRX0401), 4-dodecyl-N-1,3,4-thiadiazol-2-yl-benzenesulfonamide (PHT-427, CAS 1191951-57-1), 4-[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-7-[(3S)-3-piperidinylmethoxy]-1H-imidazo[4,5-c]pyridin-4-yl]-2-methyl-3-butyn-2-ol (GSK690693, CAS 937174-76-0), 8-(1-hydroxyethyl)-2-methoxy-3-[(4-methoxyphenyl)methoxy]-6H-dibenzo[b,d]pyran-6-one (palomid 529, P529, or SG-00529), Tricirbine (6-amino-4-methyl-8-(β-D-ribofuranosyl)-4H,8H-pyrrolo[4,3,2-de]pyrimido[4,5-c]pyridazine), (αS)-α-[[[5-(3-methyl-1H-indazol-5-yl)-3-pyridinyl]oxy]methyl]-benzeneethanamine (A674563, CAS 552325-73-2), 4-[(4-chlorophenyl)methyl]-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidinamine (CCT128930, CAS 885499-61-6), 4-(4-chlorophenyl)-4-[4-(1H-pyrazol-4-yl)phenyl]-piperidine (AT7867, CAS 857531-00-1), and Archexin (RX-0201, CAS 663232-27-7).

[0201] In certain embodiments, the tyrosine kinase inhibitor used in combination with the LVV or CAR modified T cells is an anaplastic lymphoma kinase (ALK) inhibitor. Exemplary ALK inhibitors include crizotinib, ceritinib, alectinib, brigatinib, dalantercept, entrectinib, and loratinib.

[0202] In certain embodiments in which the LVV or CAR-modified lymphocytes (e.g., T cells or NK cells) are administered in combination with one or more additional therapies, the LVV, CAR-modified lymphocytes (e.g., T cells or NK cells), or one or more additional therapies may be administered at a dose that would be considered subtherapeutic if administered as a monotherapy. Combination therapy includes administration of an LVV or CAR-modified lymphocyte (e.g., T cell or NK cell) composition described herein prior to the additional therapy (e.g., 1-30 days or earlier before the additional therapy), concurrently with (the same day as) the additional therapy, or subsequent to the additional therapy (e.g., 1-30 days or later after the additional therapy). In certain embodiments, the LVV or CAR-modified lymphocytes (e.g., T cells or NK cells) are administered concurrently with the one or more additional therapies. In further embodiments, the LVV or CAR-modified lymphocytes (e.g., T cells or NK cells) are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days before or after administration of the one or more additional therapies. In yet further embodiments, the LVV or CAR-modified lymphocytes (e.g., T cells or NK cells) are administered within 4 weeks, 3 weeks, 2 weeks, or 1 week before or after administration of the one or more additional therapies. When the one or more additional therapies include multiple administrations, the LVV or CAR-modified lymphocytes (e.g., T cells or NK cells) can be administered before or after the first administration of the one or more additional therapies, after the last administration of the one or more additional therapies, or between multiple administrations of the one or more additional therapies.

[0203] Subjects that may be treated by the compositions and methods of the present disclosure include animals, such as humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, or pigs. The subjects may be male or female and of any suitable age, including juvenile, adolescent, adult, and geriatric subjects. EXAMPLES

[0204] Example 1: Methods for generating lentiviral vectors with defined tropism The self-inactivating lentiviral vectors (LVV) described herein were produced using a third generation production system. An expression plasmid containing the gene sequences required for delivery by LVV was combined with three packaging plasmids, VSV-G, GagPol, and Rev, in defined ratios and used to transfect HEK293 producer cells. After 2-3 days, productive viral particles were harvested from the HEK293 medium.

[0205] The tropism of the resulting LVV was examined to determine whether it could be redirected using the non-viral target proteins described herein expressed from one of the three packaging plasmids or by the addition of a fifth plasmid to the transfection mix. The LVV vector contained a mutant VSV-G envelope protein that contained a mutation that abolished the binding of the mutant VSV-G to its natural receptor, and viral entry occurred via the target protein that defines the non-viral tropism. It was found that a mutation in the LDL receptor binding site of VSV-G abolished its binding to its natural receptor. VSV-G with various mutations (Trop-002 (SEQ ID NO: 74), Trop-051 (SEQ ID NO: 93), Trop-052 (SEQ ID NO: 95), Trop-055 (SEQ ID NO: 97), or Trop-061 (SEQ ID NO: 103)) was expressed on LVV together with CD80 (SEQ ID NO: 2). CD80 is the natural ligand for the T cell surface receptors, CD28 and CTLA4, and confers tropism to T cells but not B cells. Binding of VSV-G to its natural receptors was assessed by transduction of Raji B cells. Several mutations in VSV-G abrogate the transduction of Raji B cells, but maintain transduction of Jurkat T cells (Figures 2A-2B).

[0206] In the first method, a CD80-derived tropism targeting protein (SEQ ID NO: 76) was expressed from a VSV-G packaging plasmid. CD80 is the natural ligand for the T cell surface receptors, CD28 and CTLA4, and confers tropism to T cells. A codon-optimized CD80 molecule (encoding SEQ ID NO: 2) was cloned downstream of a mutant VSV-G (Trop-002, encoding SEQ ID NO: 74) containing a mutation that abolishes LDL receptor binding and used to package LVV expressing green fluorescent protein (GFP). CD80 and VSV-G were expressed at relatively equal levels on the surface of HEK293 producer cells, suggesting equal decoration on LVV particles (Figure 3A). LVV generated by this approach were able to transduce T cells, as assessed by GFP expression (Figure 3B).

[0207] In the second method, the tropism-defining CD80 target protein (encoding SEQ ID NO: 2) was cloned into the Rev packaging plasmid and transfected into the VSV-G env (Trop-002 (SEQ ID NO: 74)) plasmid at a lower molar concentration. This modified plasmid was used to package GFP-expressing LVV. Evaluation of VSV-G and CD80 expression on HEK-293 producer cells showed trends in relative expression levels depending on the molar concentration of the packaging plasmid. LVV generated by these packaging approaches were able to transduce T cells, as assessed by GFP expression (Figure 4). Reduced expression of CD80 was observed in LVV packaged with the CD80 target protein contained in the Rev packaging plasmid, due to the lower ratio of Rev plasmid.

[0208] In the third method, the tropism-defining CD80 targeting protein (encoding SEQ ID NO:2) was added as an additional plasmid during the transfection of HEK293 cells to produce LVV (Trop-002 VSV-G env). The amount of CD80 plasmid used during LVV production correlated with its expression in HEK293 producer cells, presumably representing decoration of LVV particles. LVV generated with this packaging approach were capable of transducing T cells, assessed by GFP expression, and transduction efficiency correlated with CD80 levels on the LVV particles (Figure 5).

[0209] Regardless of the method selected, high titers of tropism-redirected LVVs can be produced in adherent or suspension HEK293 producer cells and concentrated by centrifugation or anion exchange chromatography (AEX) followed by tangential flow filtration (TFF) (Figure 6A-6C). Concentration by AEX / TFF led to the preparation of LVVs with high levels of purity and recovery (Figure 6D).

[0210] Example 2: CD4 T cells enriched by CD80LVV The targeting molecule that defines the tropism precisely defines the specific cell type that is transduced with this LVV. CD28, the ligand for CD80, is naturally expressed on T cells. However, in human peripheral blood, CD4 T cells express CD28 more frequently than CD8 T cells (80% vs. 50%). When LVV was packaged to decorate the Trop-002 VSV-G envelope with the CD80 targeting protein (SEQ ID NO: 76) and used to transduce human PBMCs, a higher composition of transduced CD4 T cells was observed compared to transduced CD8 T cells, which were maintained in culture for up to one week (Figure 13).

[0211] Example 3: A method for ex vitro generation of tumor-reactive T cells engineered to express a chimeric antigen receptor (CAR) using lentiviral vectors containing T cell specific tropism Compared to standard lentiviral vectors (LVV) using common third generation packaging systems, the LVV packaged with redirected tropism provided herein may have improved transduction efficiency and specificity. The LVV particles are not taken up by other undesired cell types, and therefore the level of available viral particles is likely to be increased when transducing specific cell types as part of a heterogeneous mixture. We evaluated whether improved transduction of T cells by CAR molecules is possible in heterogeneous peripheral blood mononuclear cell (PBMC) mixtures. A representative anti-BCMA CAR (SEQ ID NO: 58) used to treat multiple myeloma or a representative anti-CD19 CAR (SEQ ID NO: 42) used to treat non-Hodgkin's lymphoma was used. A GFP reporter was used as a control. LVV was packaged to modify the envelope with anti-CD3 targeting protein and CD80 targeting protein and redirect tropism to T cell surface molecules, CD3, CD28 and CTLA4. The CD80 target protein was cloned into the Trop-002 VSV-G env packaging plasmid (SEQ ID NO:76) and the anti-CD3 target protein (encoding SEQ ID NO:10) was expressed from a fifth packaging plasmid.

[0212] We investigated the transduction efficiency of T cells in PBMCs obtained from leukapheresis of healthy donors after informed consent. PBMCs were enriched using red blood cell lysis and the corresponding donors were transduced with standard LVV or with LVV with T cell-redirected targeting (anti-CD3 and CD80) at LVV concentrations 5-25 times lower than the LVV concentrations typically used for CAR T cell transduction. Anti-BCMA CAR expression was assessed by flow cytometry after staining with fluorescently labeled BCMA molecules to specifically detect anti-BCMA CAR T cells. Even at extremely low virus levels, T cells transduced using T cell-redirected LVV showed higher levels of transduction compared to standard LVV (Figure 7).

[0213] In the same study, we evaluated whether anti-BCMA CAR T cells could be generated without the use of T cell-activating antibodies. T cell-activating antibodies (e.g., anti-CD28 and CD3) are usually required for transduction using standard LVV during CAR T cell generation. It was confirmed that standard LVV indeed requires activation with antibodies to transduce normal human primary T cells (Figure 7). In contrast, T cell-redirected LVV (anti-CD3 and CD80) can cause transduction even in the absence of prior T cell activation (in the absence of IL-2 and the exogenous activating antibodies anti-CD3 and anti-CD28) (Figure 7). When cultured in medium containing IL-2, T cell activation results in subsequent cell proliferation. PBMCs transduced with standard or T cell-redirected LVV (anti-CD3 and CD80) in the presence or absence of exogenous activating anti-CD3 and anti-CD28 antibodies showed that T cell-redirected LVV can specifically proliferate T cells even in the absence of antibody activation (Figure 8). In another example, transduction of anti-CD19 CAR was also achieved without prior T cell activation when using T cell redirected LVV (anti-CD3 and CD80) (Figure 9).

[0214] T cell activation and transduction efficiency can be determined by the choice of anti-CD3 antibody used to generate the T cell redirected LVV. Examples of envelope-bound anti-CD3 binding proteins with 12F6 binding domains in VH(SEQ ID NO:113)-VL(SEQ ID NO:115) and VL(SEQ ID NO:115)-VH(SEQ ID NO:113) orientations have been shown to induce variable amounts of T cell transduction and T cell activation (Figure 10).

[0215] Example 4: T cell specificity of lentiviral vectors targeting T cells The T cell specificity of LVV (Trop-002 VSV-G env) packaged to decorate the envelope with anti-CD3 targeting protein (SEQ ID NO: 10) and CD80 targeting protein (SEQ ID NO: 2) was confirmed by transduction of a panel of T and B cell lines (Raji, Ramos, Jeko-1, NALM-6). Transduction of these cell lines, assessed by flow cytometry of the GFP transgene, was compared to standard LVV. Both standard LVV and T cell-targeted LVV efficiently transduced the T cell line (Jurkat). Standard LVV efficiently transduced all these B cell lines, confirming their susceptibility to LVV transduction, whereas substantial transduction by T cell-targeted LVV was not detected in any of the B cell lines (Figure 14).

[0216] Example 5: Tumor-specific T cells generated using lentiviral vectors targeting T cells Using the methods described herein, T cells expressing anti-BCMA CAR (SEQ ID NO: 58) were generated by transducing PBMCs of healthy donors with LVV carrying Trop-002 VSV-G env and CD80 and anti-CD3 targeting proteins. The CD80 targeting protein was cloned into the Trop-002 VSV-G env packaging plasmid (SEQ ID NO: 76) and the anti-CD3 targeting protein (encoding SEQ ID NO: 10) was expressed from a fifth packaging plasmid to produce LVV targeted to T cells. This T cell transduction was performed in the absence of T cell stimulation (exogenous anti-CD3 and anti-CD28) that is normally required for efficient LVV-mediated gene transfer.

[0217] First, we investigated the activity of anti-BCMA CAR T cells generated by T cell-redirected LVV compared to standard LVV. Anti-tumor activity was assessed by intracellular cytokine staining for interferon gamma and tumor necrosis factor alpha after co-culture with BCMA-negative (Nalm-6) or BCMA-positive (RPMI-8826) cell lines. Anti-BCMA CAR T cells showed increased expression of T cell effector cytokines after culture with BCMA-positive cell lines, which was not observed in BCMA-negative cell lines, whether generated by standard or T cell-redirected LVV (Figure 11).

[0218] Example 6: A method for T cell-specific in vivo delivery of genetic material using T cell-tropic lentiviral vectors Efficient and specific transduction of T cells by T cell-targeted LVV may allow safe and effective delivery of genetic material to T cells in vivo. This hypothesis was tested using a humanized mouse model in which T cell-targeted LVV expressing GFP was injected intravenously. LVV carrying Trop-002 mutant VSV-G and coated with anti-CD3 and CD80 targeting proteins were produced. The CD80 targeting protein was cloned into the Trop-002 VSV-G env packaging plasmid (SEQ ID NO: 76) and the anti-CD3 targeting protein (encoding SEQ ID NO: 10) was expressed from a fifth packaging plasmid to produce T cell-targeted LVV. Immunodeficient NCG mice were humanized by intravenous injection of human PBMCs and supported by daily intraperitoneal administration of recombinant IL-2 for 4 days. One day after PBMC administration, the mice were intravenously administered T cell-targeted lentiviral vectors. Peripheral blood and spleen cells were collected on day 7, and T cell specificity was assessed by flow cytometry. GFP-positive cells were detected only in human T cells (CD3+), but not in human B cells (CD20+), confirming the T cell specificity of LVV transduction (Figure 12A). GFP was detected in both CD8+ and CD8- (CD4) T cells, indicating that T cell-specific LVV transduced both CD4 and CD8 T cells in vivo (Figure 12B).

[0219] Specific T cell transduction has important safety advantages over other in vivo CAR delivery approaches by minimizing the risk of transducing tumor cells. T cell transduction without tumor cell transduction is evaluated using PBMCs from chronic lymphocytic leukemia patients with detectable CD19 tumor cells. PBMCs are transduced with LVV targeting T cells, and transduced cells are evaluated using flow cytometry.

[0220] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the Application Data Sheet, including but not limited to U.S. Provisional Patent Application No. 63 / 154,639, filed February 26, 2021, are incorporated herein by reference in their entirety. Aspects of these embodiments can be modified, if necessary, to use concepts from various patents, applications, and publications to provide further embodiments.

[0221] These and other changes can be made to the embodiments in light of the above description. In general, the terms used in the following claims should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but rather to include all possible embodiments and the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims

1. A lentiviral vector particle comprising: (a) a viral envelope, (i) a heterologous lymphocyte-targeting protein, the lymphocyte-targeting protein comprising: an extracellular domain that comprises a lymphocyte-targeting domain; and a lymphocyte-targeting protein comprising a transmembrane domain; and (ii) Mutant VSV-G envelope proteins that exhibit inhibition of binding to LDL-R but mediate membrane fusion The viral envelope comprising: (b) an expression cassette containing a heterologous transgene.

2. The heterologous lymphocyte targeting protein is (i) a heterologous T cell-targeting protein comprising an extracellular domain that comprises a T cell-targeting domain; (ii) a heterologous natural killer (NK) cell-targeting protein that comprises an extracellular domain that includes a NK cell-targeting domain; or (iii) a heterologous B cell-targeting protein comprising an extracellular domain that includes a B cell-targeting domain. The lentiviral vector particle of claim 1 , (a) the xenogeneic lymphocyte-targeting protein is a xenogeneic T cell-targeting protein that specifically binds to: (i) CD3, CD28, CD80, 4-1BB, AhR, CD3, CD2, CD7, CD4, CD8, CD25, CD44, CD45RA, CD47, CD62L, CD69, CD94, CD95, CD127, CD161, CD183 (CXCR3), CD184 (CXCR4), CD185 (CXCR5), CD193 (CCR3), CD194 (CC R4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), CCR10, PD-1, TCRa / b, CD5, CD27, CD45RO, CD45RB, CD57, CD103, CD122, P2RX7, TIGIT, LAG-3, TIM-3, and IL6ST, or any combination thereof; (ii) a γδ T cell marker selected from γδ TCR, Vdelta1, Vdelta2, and NKG2D (KLRK1, CD314); (iii) an NK T cell marker selected from invariant TCR (Va24-Ja18), CD185 (CXCR5), CXCR6, and IL-21R, or any combination thereof; or (iv) MAIT cell markers selected from Va7.2, Ja33, CXCR6, IL-18R, KLRB1 (CD161), and VLA4 (alpha4beta1 integrin), or any combination thereof; (b) the heterologous lymphocyte-targeting protein is a heterologous T cell-targeting protein that comprises an extracellular domain that includes a T cell-targeting domain that specifically binds to CD3 protein or CD28 protein; (c) the heterologous lymphocyte-targeting protein is a heterologous NK cell-targeting protein that specifically binds to an NK cell marker selected from CD56, NKp46, CD16, KIR(s), NKG2 proteins (e.g., NKG2D (KLRK1, CD314)), KLRB1 (CD161), KLRD1 (cd94), IL2Rb (CD122), IL-21R, SLAMF6 (CD352), SLAMF7 (CD319), and IL-18R, or any combination thereof; (d) the heterologous lymphocyte-targeting protein is a B cell-targeting protein that specifically binds to a B cell marker selected from CD19, CD20, CD21, CD22, CD24, CD38, CD40, CD72, CD32b, CD268, CD269, CD267, CD86, CD80, CD52, CD138, CD27, CD28, CD23, CD84, CD257, CD270, CD37, CD74, and CD269, or any combination thereof; (e) the target protein comprises a CD80 protein comprising an extracellular domain having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:6; (f) the target protein comprises a CD80 protein comprising a transmembrane and intracellular domain having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:8; (g) the target protein comprises a CD80 protein comprising SEQ ID NO:2 or comprising SEQ ID NO:2 lacking the signal peptide of SEQ ID NO:4; or (h) the targeting protein comprises an anti-CD3 scFv having a light chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 14 and a heavy chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 18, and optionally the targeting protein comprises an anti-CD3 targeting protein comprising (i) SEQ ID NO: 10, or SEQ ID NO: 10 lacking the signal peptide of SEQ ID NO: 12, or (ii) SEQ ID NO: 117, or SEQ ID NO: 117 lacking the signal peptide of SEQ ID NO:

12. The lentiviral vector particle of claim 1. (a) the target protein expressed in a host cell comprises a signal sequence; and / or (b) the mature targeting protein does not include a signal sequence or the mature targeting protein includes at least one amino acid from a signal sequence. (a) the mutant VSV-G envelope protein comprises a mutation selected from one or more, or two or more, of the following: Mutations at H8, N9, Q10, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, T352, E353, and R354; an insertion of TT between N9 and Q10, an insertion of GGS between H8 and N9, an insertion of GGS between N9 and Q10, an insertion of TT between N208 and Y209, an insertion of GGS between P46 and K47, and an insertion of GGS between N208 and Y209; Deletion of residues 1-8; (b) the mutant VSV-G envelope protein comprises a mutation selected from one or more of H8A, N9A, Q10A, K47A, K47Q, N180A, I182A, Y209A, T352A, T352W, E353A, R354A, and R354Q; (c) the mutant VSV-G envelope protein comprises a mutation selected from one or more of H8A, K47A, K47Q, Y209A, R354A, and R354Q; (d) the mutant VSV-G envelope protein comprises: (i) a mutation at K47 and a mutation at R354, (ii) a mutation at N180, a mutation at I182, a mutation at T352, and a mutation at E353; (iii) a T352 mutation and an E353 mutation, (iv) a mutation at N9, a mutation at Q10, and a mutation at N180, or (v) insertion of GGS between H8 and N9, and insertion of GGS between N9 and Q10; (vi) insertion of TT between N9 and Q10, or (vii) insertion of GGS between P46 and K47; Optionally, the mutant VSV-G envelope protein comprises: (i) a K47Q mutation and a R354A mutation, (ii) an N180A mutation, an I182A mutation, a T352A mutation, and an E353A mutation; (iii) a T352W mutation and an E353A mutation, or (iv) an N9A mutation, a Q10A mutation, and an N180A mutation; and / or (e) the mutant VSV-G envelope protein comprises an amino acid sequence of any one of SEQ ID NOs: 74, 93, 95, 97, 99, and 103; The lentiviral vector particle of claim 1.

6. 2. The lentiviral vector particle of claim 1, wherein the heterologous transgene encodes a chimeric antigen receptor (CAR), the CAR comprising an extracellular domain comprising a binding domain that specifically binds to a target molecule, an intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM) and optionally a costimulatory signaling domain, and a transmembrane domain connecting the extracellular and intracellular signaling domains.

7. The lentiviral vector particle of claim 6, wherein the CAR encoded by the transgene comprises: (a) CD19, BCMA, alpha folate receptor, 5T4, Ab integrin, B7-H3, B7-H6, CAIX, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD52, CD70, CD79a, CD79b, CD80, CD123, CD138, CD171, CEA, CSPG4, EGFR, ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EpCAM, FAP, fetal AchR, FLT3, Fra, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+ a binding domain that specifically binds to one or more of MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, HLADR, IL-11Ralpha, IL-13Ralpha2, lambda, Lewis-Y, kappa, mesothelin, Mucl, Mucl6, NCAM, NKG2d ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM, VEGFR2, BAFF-R, claudin 18.2, CD86, FcRL5, GPRC5, and TACI; (b) CD28, CD2, CD4, CD8a, CD5, CD3ε, CD3δ, CD3ζ, CD9, CD16, CD22, CD25 , CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD95 (F as), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD 154 (CD40L), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 a transmembrane domain selected from the transmembrane domains of (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5, and Zap70; (c) an intracellular signaling domain selected from a CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD278 (ICOS), DAP10, DAP12, FcRγ, and CD66d domain; (d) a costimulatory domain selected from one or more of CD27, CD28, CD40L, GITR, NKG2C, CARD1, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, LFA-1, LIGHT, NKG2C, NKD2C, SLP76, TRIM, and ZAP70 domains; and (e) optionally, between the binding domain and the transmembrane domain, an extracellular, non-signaling linker domain selected from: (i) an immunoglobulin hinge region selected from a wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge region; (ii) a hinge region selected from wild-type or modified CD8a, CD4, CD28, and CD7 hinge regions; (iii) all or a portion of an Fc domain selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain; and (iv) The stalk regions of type II C-lectins are derived from the stalk regions of CD23, CD69, CD72, CD94, NKG2A and NKG2D.

8. The lentiviral vector particle of claim 7, comprising: (a) the CAR encoded by the transgene is an anti-CD19 CAR comprising: a binding domain comprising an anti-CD19 scFv comprising a light chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to SEQ ID NO:46 and a heavy chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to SEQ ID NO:50; a hinge and transmembrane domain of CD8a having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:52; a CD3ζ effector domain having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:56; and a 4-1BB costimulatory domain having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:54; Optionally, the CAR encoded by the transgene comprises: (i) SEQ ID NO: 42, or SEQ ID NO: 42 lacking the signal peptide of SEQ ID NO: 44, or (ii) SEQ ID NO: 58, or SEQ ID NO: 58 lacking the signal peptide of SEQ ID NO: 60 Contains; (b) the CAR encoded by the transgene is an anti-BCMA CAR comprising: a binding domain comprising an anti-BCMA scFv comprising a light chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:62, and a heavy chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:66; a hinge and transmembrane domain of CD8a having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:52; a CD3ζ effector domain having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 122; and A 4-1BB costimulatory domain having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:

54.

9. A method for producing a lymphocyte-targeting lentiviral vector particle according to any one of claims 1 to 8, comprising: (a) transfecting producer cells with a GagPol plasmid, a Rev plasmid, a transgene plasmid, and a VSV-G env plasmid, (i) the GagPol plasmid comprises one or more polynucleotides encoding lentiviral gag and lentiviral pol genes and is capable of expressing lentiviral Gag and lentiviral Pol polyproteins in the producer cell; (ii) the Rev plasmid comprises a polynucleotide encoding a lentiviral rev gene and is capable of expressing a lentiviral Rev protein in the producer cell; (iii) the transgene plasmid comprises an expression cassette comprising a polynucleotide encoding a CAR; (iv) the VSV-G env plasmid comprises a tandem expression cassette, the tandem expression cassette comprising a polynucleotide encoding a mutant VSV-G envelope protein and a polynucleotide encoding a lymphocyte-targeting protein, the VSV-G env plasmid being capable of expressing the mutant VSV-G envelope protein and the lymphocyte-targeting protein in the producer cell; (b) culturing the producer cells in a medium; and (c) recovering the lentiviral vector from the medium.

10. 9. A method for producing lymphocyte-targeting lentiviral vector particles according to any one of claims 1 to 8, comprising: (a) transfecting producer cells with a GagPol plasmid, a Rev plasmid, a transgene plasmid, and a VSV-G env plasmid, (i) the GagPol plasmid comprises tandem expression cassettes encoding Gag and Pol polyprotein precursors and a lymphocyte targeting protein, the GagPol plasmid being capable of expressing lentiviral Gag polyprotein, lentiviral Pol polyprotein, and the lymphocyte targeting protein in the producer cell; (ii) the Rev plasmid comprises a polynucleotide encoding a lentiviral rev gene and is capable of expressing a lentiviral Rev protein in the producer cell; (iii) the transgene plasmid comprises an expression cassette comprising a polynucleotide encoding a CAR; (iv) the VSV-G env plasmid comprises a polynucleotide encoding a mutant VSV-G envelope protein, and the VSV-G env plasmid is capable of expressing the mutant VSV-G envelope protein in the producer cell. (b) culturing the producer cells in a medium; and (c) recovering the lentiviral vector from the medium.

11. 9. A method for producing lymphocyte-targeting lentiviral vector particles according to any one of claims 1 to 8, comprising: (a) transfecting producer cells with a GagPol plasmid, a Rev plasmid, a transgene plasmid, and a VSV-G env plasmid, (i) the GagPol plasmid comprises one or more polynucleotides encoding lentiviral gag and lentiviral pol genes and is capable of expressing lentiviral Gag and lentiviral Pol polyproteins in the producer cell; (ii) the Rev plasmid comprises a tandem expression cassette, the tandem expression cassette comprising a polynucleotide encoding a lentiviral rev gene and a polynucleotide encoding a lymphocyte targeting protein, the Rev plasmid being capable of expressing the lentiviral Rev protein and the lymphocyte targeting protein in the producer cell; (iii) the transgene plasmid comprises an expression cassette comprising a polynucleotide encoding a CAR; (iv) the VSV-G env plasmid comprises a polynucleotide encoding a mutant VSV-G envelope protein, and the VSV-G env plasmid is capable of expressing the mutant VSV-G envelope protein in the producer cell; (b) culturing the producer cells in a medium; and (c) recovering the lentiviral vector from the medium.

12. 9. A method for producing lymphocyte-targeting lentiviral vector particles according to any one of claims 1 to 8, comprising: (a) transfecting producer cells with GagPol plasmid, Rev plasmid, transgene plasmid, VSV-G env plasmid, and lymphocyte-targeting protein plasmid; (i) the GagPol plasmid comprises one or more polynucleotides encoding lentiviral gag and lentiviral pol genes and is capable of expressing lentiviral Gag and lentiviral Pol polyproteins in the producer cell; (ii) the Rev plasmid comprises a polynucleotide encoding a lentiviral rev gene and is capable of expressing a lentiviral Rev protein in the producer cell; (iii) the transgene plasmid comprises an expression cassette comprising a polynucleotide encoding a CAR; (iv) the VSV-G env plasmid comprises a polynucleotide encoding a mutant VSV-G envelope protein and is capable of expressing the mutant VSV-G envelope protein in the producer cell; (v) the lymphocyte targeting protein plasmid comprises a polynucleotide encoding a lymphocyte targeting protein and is capable of expressing the lymphocyte targeting protein in the producer cell; (b) culturing the producer cells in a medium; and (c) recovering the lentiviral vector from the medium.

13. (a) the lymphocyte-targeting protein encoded by the lymphocyte-targeting protein plasmid is a T cell-targeting protein; (b) the lymphocyte-targeting protein encoded by the lymphocyte-targeting protein plasmid is a NK cell-targeting protein; or 10. The method of claim 9, wherein (c) the lymphocyte-targeting protein encoded by the lymphocyte-targeting protein plasmid is a B cell-targeting protein. (a) the lymphocyte-targeting protein encoded by the lymphocyte-targeting protein plasmid is a T cell-targeting protein that specifically binds to: (i) CD3, CD28, CD80, 4-1BB, AhR, CD3, CD2, CD7, CD4, CD8, CD25, CD44, CD45RA, CD47, CD62L, CD69, CD94, CD95, CD127, CD161, CD183 (CXCR3), CD184 (CXCR4), CD185 (CXCR5), CD193 (CCR3), CD194 (CC R4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), CCR10, PD-1, TCRa / b, CD5, CD27, CD45RO, CD45RB, CD57, CD103, CD122, P2RX7, TIGIT, LAG-3, TIM-3, and IL6ST, or any combination thereof; (ii) a γδ T cell marker selected from γδ TCR, Vdelta1, Vdelta2, and NKG2D (KLRK1, CD314), or any combination thereof; (iii) an NK T cell marker selected from invariant TCR (Va24-Ja18), CD185 (CXCR5), CXCR6, and IL-21R, or any combination thereof; or (iv) MAIT cell markers selected from Va7.2, Ja33, CXCR6, IL-18R, KLRB1 (CD161), and VLA4 (alpha4beta1 integrin), or any combination thereof; (b) the target protein comprises a CD80 protein comprising an extracellular domain having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:6; (c) the target protein comprises a CD80 protein comprising a transmembrane and intracellular domain having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:8; (d) the target protein comprises a CD80 protein comprising SEQ ID NO:2 or comprising SEQ ID NO:2 lacking the signal peptide of SEQ ID NO:4; (e) the target protein is an anti-CD3 antibody having (i) a light chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 14, and a heavy chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:

18. scFv, or (ii) an anti-CD3 scFv having a light chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:115, and a heavy chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:113; (f) the targeting protein comprises an anti-CD3 targeting protein comprising (i) SEQ ID NO:10, or SEQ ID NO:10 lacking the signal peptide of SEQ ID NO:12, or (ii) SEQ ID NO:117, or SEQ ID NO:117 lacking the signal peptide of SEQ ID NO:12; (g) the lymphocyte-targeting protein encoded by the lymphocyte-targeting protein plasmid is an NK cell-targeting protein that specifically binds to an NK cell marker selected from CD56, NKp46, CD16, KIR(s), NKG2 protein (e.g., NKG2D (KLRK1, CD314)), KLRB1 (CD161), KLRD1 (cd94), IL2Rb (CD122), IL-21R, SLAMF6 (CD352), SLAMF7 (CD319), and IL-18R, or any combination thereof; or 10. The method of claim 9, wherein (h) the lymphocyte-targeting protein encoded by the lymphocyte-targeting protein plasmid is a B cell-targeting protein that specifically binds to a B cell marker selected from CD19, CD20, CD21, CD22, CD24, CD38, CD40, CD72, CD32b, CD268, CD269, CD267, CD86, CD80, CD52, CD138, CD27, CD28, CD23, CD84, CD257, CD270, CD37, CD74, and CD269, or any combination thereof. (a) the mutant VSV-G envelope protein encoded by the VSV-G env plasmid comprises a mutation selected from one or more or two or more of the following: Mutations at H8, N9, Q10, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, T350, T352, E353, and R354; an insertion of TT between N9 and Q10, an insertion of GGS between H8 and N9, an insertion of GGS between N9 and Q10, an insertion of TT between N208 and Y209, an insertion of GGS between P46 and K47, and an insertion of GGS between N208 and Y209; Deletion of residues 1-8; (b) the mutant VSV-G envelope protein comprises a mutation selected from one or more of H8A, N9A, Q10A, K47A, K47Q, N180A, I182A, Y209A, T352A, T352W, E353A, R354A, and R354Q; (c) the mutant VSV-G envelope protein encoded by the VSV-G env plasmid comprises a mutation selected from one or more of H8A, K47A, K47Q, Y209A, R354A, and R354Q; (d) the mutant VSV-G envelope protein comprises: (i) a mutation at K47 and a mutation at R354, (ii) a mutation at N180, a mutation at I182, a mutation at T352, and a mutation at E353; (iii) a T352 mutation and an E353 mutation, (iv) a mutation at N9, a mutation at Q10, and a mutation at N180, or (v) insertion of GGS between H8 and N9, and insertion of GGS between N9 and Q10; (vi) insertion of TT between N9 and Q10, or (vii) insertion of GGS between P46 and K47; Optionally, the mutant VSV-G envelope protein comprises: (a) a K47Q mutation and a R354A mutation, (b) an N180A mutation, an I182A mutation, a T352A mutation, and an E353A mutation; (c) a T352W mutation and an E353A mutation, or (d) an N9A mutation, a Q10A mutation, and an N180A mutation; (e) the mutant VSV-G envelope protein comprises the amino acid sequence of any one of SEQ ID NOs: 74, 93, 95, 97, 99, and 103; and / or (f) the CAR encoded by the transgene plasmid comprises an extracellular domain comprising a binding domain that specifically binds to a target molecule, an intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM) and optionally a costimulatory signaling domain, and a transmembrane domain connecting the extracellular and intracellular signaling domains.

10. The method of claim 9.

16. 10. The method of claim 9, wherein the CAR encoded by the transgene plasmid comprises: (a) CD19, BCMA, alpha folate receptor, 5T4, Ab integrin, B7-H3, B7-H6, CAIX, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD52, CD70, CD79a, CD79b, CD80, CD123, CD138, CD171, CEA, CSPG4, EGFR, ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EpCAM, FAP, fetal AchR, FLT3, Fra, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+ a binding domain that specifically binds to one or more of MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, HLADR, IL-11Ralpha, IL-13Ralpha2, lambda, Lewis-Y, kappa, mesothelin, Mucl, Mucl6, NCAM, NKG2d ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM, VEGFR2, BAFF-R, claudin 18.2, CD86, FcRL5, GPRC5, and TACI; (b) CD28, CD2, CD4, CD8a, CD5, CD3ε, CD3δ, CD3ζ, CD9, CD16, CD22, CD25 , CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD95 (F as), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD 154 (CD40L), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 a transmembrane domain selected from the transmembrane domains of (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5, and Zap70; (c) an intracellular signaling domain selected from a CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD278 (ICOS), DAP10, DAP12, FcRγ, and CD66d domain; (d) a costimulatory domain selected from one or more of CD27, CD28, CD40L, GITR, NKG2C, CARD1, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, LFA-1, LIGHT, NKG2C, NKD2C, SLP76, TRIM, and ZAP70 domains; and (e) optionally, between the binding domain and the transmembrane domain, an extracellular, non-signaling linker domain selected from: (i) an immunoglobulin hinge region selected from a wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge region; (ii) a hinge region selected from wild-type or modified CD8a, CD4, CD28, and CD7 hinge regions; (iii) all or a portion of an Fc domain selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain; and (iv) The stalk regions of type II C-lectins are derived from the stalk regions of CD23, CD69, CD72, CD94, NKG2A and NKG2D. (i) The CAR encoded by the transgene plasmid is an anti-CD19 CAR comprising: (a) a binding domain comprising an anti-CD19 scFv comprising a light chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to SEQ ID NO: 46, and a heavy chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to SEQ ID NO: 50; (b) a hinge and transmembrane domain of CD8a having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:52; (c) a CD3ζ effector domain having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:56; and (d) a 4-1BB costimulatory domain having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:54; Optionally, the CAR encoded by the transgene comprises SEQ ID NO:42, or SEQ ID NO:42 lacking the signal peptide of SEQ ID NO:44; or (ii) The method of any one of claims 30 to 53, wherein the CAR encoded by the transgene plasmid is an anti-BCMA CAR comprising: (a) a binding domain comprising an anti-BCMA scFv comprising a light chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:62, and a heavy chain variable region having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:66; (b) a hinge and transmembrane domain of CD8a having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:52; (c) a CD3ζ effector domain having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 122; and (d) a 4-1BB costimulatory domain having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:54; 10. The method of claim 9, optionally wherein the CAR encoded by the transgene comprises SEQ ID NO:58, or SEQ ID NO:58 lacking the signal peptide of SEQ ID NO:

60.

18. (a) the producer cell is transduced with a defined ratio of transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid, optionally wherein the defined ratios of transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid, respectively, are from about 1:1:1:1 to about 5:4:1:1; optionally wherein the producer cell is transduced with a defined ratio of transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid of about 4:2:1:1, or wherein the producer cell is transduced with a defined ratio of transgene plasmid, GagPol plasmid, VSV-G env plasmid, and Rev plasmid of about 3.125:3.125:2.5:1.25; (b) harvesting the lentiviral vector from the medium comprises centrifugation, anion exchange, chromatography, or any combination thereof; and / or 10. The method of claim 9, wherein (c) the LVV product harvested from the culture medium has a viral titer of about 1e7 TU / mL to about 1e12 TU / mL in a concentrated LVV product.

19. 10. A composition for use in a method of producing T cells, NK cells or B cells expressing a heterologous transgene or a CAR transgene, said composition comprising one or more lentiviral vector particles according to any one of claims 1 to 8, said method comprising transducing said T cells, said NK cells or said B cells with said one or more lentiviral vector particles, wherein transduction of said T cells, said NK cells or said B cells results in expression of said heterologous transgene or said CAR transgene by said T cells, said NK cells or said B cells.

20. (a) the T cells are resting T cells, the NK cells are resting NK cells, or the B cells are resting B cells; (b) the transduction of said T cells, said NK cells or said B cells is performed in vivo or ex vivo; (c) the one or more lentiviral vector particles are administered intravenously, intraperitoneally, intratumorally, intraosseously, or intranodal to the subject; or transducing the T cells, NK cells, or B cells comprises contacting T cells, NK cells, or B cells in whole blood or peripheral blood mononuclear cells (PBMCs) obtained from the subject with the one or more lentiviral vector particles; and / or (d) transduction is achieved without the use of exogenous T cell, NK cell or B cell stimulators; 20. The composition of claim 19, optionally wherein the exogenous T cell stimulator comprises an anti-CD3 antibody or an antigen-binding fragment thereof, an anti-CD28 antibody or an antigen-binding fragment thereof, or both.

21. A composition for use in the treatment of cancer, comprising a lentiviral vector particle described in any one of claims 1 to 8.

22. Use of a lentiviral vector particle described in any one of claims 1 to 8 in the manufacture of a medicament for the treatment of cancer.

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