Methods for genetically modifying lymphocytes in blood or in enriched pbmc

Abstract

To provide methods and compositions for genetically modifying lymphocytes, for example, T cells and / or NK cells in a shorter time than before and / or in whole blood or a blood component.SOLUTION: The disclosure provides methods and compositions for genetically modifying lymphocytes, for example, T cells and / or NK cells in a shorter time than before, and / or in whole blood or in a blood component. In some embodiments, a lymphodepletion filter assembly is used before or after a reaction mixture is formed, and the lymphocytes are brought into contact with recombinant retrovirus particles in a closed system to genetically modify the lymphocytes.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This application is a continuation-in-part of International Application No. PCT / US2018 / 051392, filed Sep. 17, 2018; claims the benefit of U.S. Provisional Patent Application No. 62 / 726,293, filed Sep. 2, 2018; U.S. Provisional Patent Application No. 62 / 726,294, filed Sep. 2, 2018; U.S. Provisional Patent Application No. 62 / 728,056, filed Sep. 6, 2018; U.S. Provisional Patent Application No. 62 / 732,528, filed Sep. 17, 2018; U.S. Provisional Patent Application No. 62 / 821,434, filed Mar. 20, 2019; and U.S. Provisional Patent Application No. 62 / 894,853, filed Sep. 1, 2019; International Application No. PCT / US2018 / 051392 is a continuation-in-part of International Application No. PCT / US2018 / 020818, filed Mar. 3, 2018; claims the benefit of U.S. Provisional Patent Application No. 62 / 560,176, filed Sep. 18, 2017; U.S. Provisional Patent Application No. 62 / 564,253, filed Sep. 27, 2017; U.S. Provisional Patent Application No. 62 / 564,991, filed Sep. 28, 2017; and U.S. Provisional Patent Application No. 62 / 728,056, filed Sep. 6, 2018; International Application No. PCT / US2018 / 020818 is a continuation-in-part of International Application No. PCT / US2017 / 023112, filed Mar. 19, 2017; a continuation-in-part of International Application No. PCT / US2017 / 041277, filed Jul. 8, 2017; a continuation-in-part of U.S. Patent Application No. 15 / 462,855, filed Mar. 19, 2017; a continuation-in-part of U.S. Patent Application No. 15 / 644,778, filed Jul. 8, 2017; claims the benefit of U.S. Provisional Patent Application No. 62 / 467,039, filed Mar. 3, 2017; U.S. Provisional Patent Application No. 62 / 560,176, filed Sep. 18, 2017; U.S. Provisional Patent Application No. 62 / 564,253, filed Sep. 27, 2017; and U.S. Provisional Patent Application No. 62 / 564,991, filed Sep. 28, 2017; International Application No. PCT / US2017 / 023112 claims the benefit of U.S. Provisional Patent Application No. 62 / 390,093, filed Mar. 19, 2016; U.S. Provisional Patent Application No. 62 / 360,041, filed Jul. 8, 2016; and U.S. Provisional Patent Application No. 62 / 467,039, filed Mar. 3, 2017;International Application No. PCT / US2017 / 041277 claims the benefit of International Application No. PCT / US2017 / 023112, filed on March 19, 2017; U.S. Patent Application No. 15 / 462,855, filed on March 19, 2017; U.S. Provisional Patent Application No. 62 / 360,041, filed on July 8, 2016; and U.S. Provisional Patent Application No. 62 / 467,039, filed on March 3, 2017; U.S. Patent Application No. 15 / 462,855 claims the benefit of U.S. Provisional Patent Application No. 62 / 390,093, filed on March 19, 2016; U.S. Provisional Patent Application No. 62 / 360,041, filed on July 8, 2016; and U.S. Provisional Patent Application No. 62 / 467,039, filed on March 3, 2017; U.S. Patent Application No. 15 / 644,778 is a continuation-in-part of International Application No. PCT / US2017 / 023112, filed on March 19, 2017; a continuation-in-part of U.S. Patent Application No. 15 / 462,855, filed on March 19, 2017; and claims the benefit of U.S. Provisional Patent Application No. 62 / 360,041, filed on July 8, 2016 and U.S. Provisional Patent Application No. 62 / 467,039, filed on March 3, 2017. These applications are hereby incorporated by reference in their entirety.;

[0002] Sequence Listing This application incorporates by reference herein the materials of the electronic sequence listing submitted herewith. The materials of the electronic sequence listing were submitted as a text (.txt) file entitled "F1_001_WO_05_Sequence_Listing_September_02_2019.txt", created on September 2, 2019, with a file size of 450 KB, and the whole of which is incorporated by reference herein.;

[0003] This disclosure relates to the field of immunology, or more specifically, to the genetic modification of T lymphocytes or other immune cells, and methods of controlling the proliferation of such cells.; BACKGROUND OF THE DISCLOSURE

[0004] Lymphocytes isolated from a subject (e.g., a patient) can be activated in vitro, genetically modified, and made to express synthetic proteins that enable redirected engagement with other cells and the environment based on an incorporated gene program. Examples of such synthetic proteins include recombinant T cell receptors (TCRs) and chimeric antigen receptors (CARs). One CAR currently in use is a fusion of an extracellular recognition domain (e.g., an antigen-binding domain), a transmembrane domain, and one or more intracellular signaling domains encoded by a replication-incompetent recombinant retrovirus.

[0005] Replication-incompetent recombinant retroviruses have shown efficacy in infecting non-dividing cells, but resting CD4 and CD8 lymphocytes are resistant to gene transfer by these vectors. To overcome this difficulty, these cells are typically activated in vitro using a stimulatory reagent before gene modification by a CAR gene vector can occur. Following stimulation and transduction, the genetically modified cells are expanded in vitro and then reintroduced into a lymphocyte-depleted patient. Antigen engagement in vivo allows the intracellular signaling portion of the CAR to initiate activation-related responses and the release of cytolytic molecules in immune cells, inducing target cell death.

[0006] Such current methods require extensive manipulation and manufacture of T cells that grow outside the body prior to reinfusion into the patient, as well as lymphocyte depletion chemotherapy that releases cytokines and depletes competing receptors to promote T cell engraftment. Such CAR therapies are also unable to control the in vivo growth rate after being introduced into the body or safely direct to targets that are also expressed outside of tumors. As a result, CAR therapies today typically range from 1×10 5 ~1×10 8Injected from cells expanded ex vivo for 12 - 28 days using a dose in cells / kg, directed to a target, for example a tumor target, and its off tumor on target toxicity is generally acceptable. These relatively long ex vivo expansion times cause problems with cell viability and sterility, as well as sample identity, in addition to scalability issues. Thus, there is a strong need for safer, more effective, and scalable T cell or NK cell therapies.

[0007] Understanding the processes that promote lymphocyte transduction, proliferation, and survival is central to various potential commercial applications, including immunological processes, and thus there is a need for improved methods and compositions for studying lymphocytes. For example, it is useful to identify the manner in which lymphocytes can be genetically modified, as well as the methods and compositions that can be used to better characterize and understand the factors that affect lymphocyte survival and proliferation. Additionally, it is useful to identify compositions that promote lymphocyte proliferation and survival. Such compositions can be used to study the regulation of such processes. In addition to methods and compositions for studying lymphocytes, improved viral packaging cell lines and methods for making and using them are needed. For example, such cell lines and methods are useful in analyzing the different components of recombinant viruses, such as recombinant retroviral particles, and in methods of using packaging cell lines for the production of recombinant retroviral particles.

[0008] More recent methods have been developed that can be performed without pre - activation and ex vivo expansion. However, it would be highly desirable to further reduce the complexity and time required for such methods, especially when such methods involve collecting a subject's blood, for example, within an infusion center and then re - introducing it to the subject on the same day. Additionally, simpler and faster methods alone, or methods that require less specialized equipment, have the potential to generalize these cell therapy processes that are currently only regularly performed at highly specialized medical centers.

[0009] One group attempted to simplify ex vivo processing of cell therapy by eliminating ex vivo transduction, by intravenous injection of viral particles to transduce cells in vivo. However, such methods require large amounts of vector and carry the risk of inactivation of retroviral particles by clotting factors and / or other enzymes present in vivo. Finally, such methods carry the risk of high levels of transduction of non-target cells / organs. SUMMARY OF THE INVENTION

[0010] Provided herein are methods, compositions, and kits useful for overcoming problems associated with the efficacy and safety of methods for transducing and / or genetically modifying lymphocytes such as T cells and / or NK cells. Certain embodiments of such methods are useful for performing adoptive cell therapy using these cells. Thus, in some aspects, provided herein are methods, compositions, and kits for genetically modifying lymphocytes, particularly T cells and / or NK cells, and / or for modulating the activity of transduced and / or genetically modified T cells and / or NK cells. Such methods, compositions, and kits provide improved efficacy and safety over current techniques, particularly with respect to T cells and / or NK cells expressing recombinant T cell receptors (TCRs), chimeric antigen receptors (CARs), and in exemplary embodiments, microenvironment-restricted biological (''MRB'') CARs. Transduced and / or genetically modified T cells and / or NK cells produced and / or used by the methods provided herein include functionality and combinations of functionality, in exemplary embodiments delivered from a retroviral (e.g., lentiviral) genome via retroviral (e.g., lentiviral) particles, which provide improved functionality for such cells and methods of using such cells, such as research methods, commercial production methods, and adoptive cell therapy. For example, such cells can be generated in a shorter time ex vivo and have improved growth characteristics that can be better regulated.

[0011] In some embodiments, methods are provided for transducing and / or genetically modifying lymphocytes such as T cells and / or NK cells, and in exemplary embodiments, ex vivo methods are provided for transducing and / or genetically modifying resting T cells and / or NK cells. Some of these embodiments can be carried out much more rapidly than previous methods, facilitating more efficient research, more effective commercial production, and improved methods of patient care. The methods, compositions, and kits provided herein can be used as research tools, in commercial production, and in adoptive cell therapy with transduced and / or genetically modified T cells and / or NK cells that express a TCR or CAR.

[0012] With respect to the methods, uses, and compositions provided herein related to the transduction of lymphocytes such as T cells and / or NK cells, methods are provided herein that include transduction reactions of enriched PBMCs or transduction reactions without prior PBMC enrichment in whole blood, etc., as well as related uses and compositions, which are simplified and rapid methods for performing ex vivo cell processing, such as CAR-T therapy, for example. Such methods require less specialized measurement and training. Further, such methods reduce the risk of non-target cell transduction as compared to in vivo transduction methods. Further provided herein are specific target inhibitory RNAs, polypeptide lymphoproliferative factors, and pseudotyping factors that, optionally in combination with any of the other embodiments provided herein, can provide powerful methods, uses, and compositions for promoting in vitro, ex vivo, and in vivo expansion of lymphocytes, particularly T cells and / or NK cells, including embodiments of the above methods, uses, and compositions.

[0013] Further details regarding aspects and embodiments of the present disclosure are provided throughout this patent application. Sections and section headings are for ease of reading and are not intended to limit the disclosure of methods, compositions, and kits or the functional elements therein across entire sections.

Brief Description of the Drawings

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[0015] Definitions As used herein, the terms "chimeric antigen receptor" or "CAR" or "CARs" refer to engineered receptors that confer antigen specificity to cells, such as T cells, NK cells, macrophages, and stem cells. The CARs of the present invention include at least one antigen-specific targeting region (ASTR), a transmembrane domain (TM), and an intracellular activation domain (IAD), and may include a stalk and one or more costimulatory domains (CSD). In another embodiment, the CAR is a bispecific CAR specific for two different antigens or epitopes. After the ASTR specifically binds to the target antigen, the IAD activates intracellular signaling. For example, the IAD can utilize the antigen-binding properties of an antibody to redirect the specificity and reactivity of T cells towards a selected target in an MHC-unrestricted manner. Due to MHC-unrestricted antigen recognition, T cells expressing CARs can recognize antigens independent of antigen processing, thus avoiding a major mechanism of tumor escape. Furthermore, when expressed in T cells, CARs do not readily dimerize with the endogenous T cell receptor (TCR) alpha and beta chains.

[0016] As used herein, the term "microenvironment" means any part or region of a tissue or body that has a definite or temporary, physical, or chemical difference from other regions of the tissue or body. For example, as used herein, the "tumor microenvironment" refers to the environment in which a tumor exists, which is the non-cellular region within the tumor and the region immediately outside the tumor tissue, but is not related to the intracellular compartment of the cancer cells themselves. The tumor microenvironment can refer to all conditions of the tumor environment, including conditions that create a structural and / or functional environment for the malignant process to survive and / or expand and / or spread. For example, the tumor microenvironment can include, but is not limited to, changes in conditions such as pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, concentration of electrolytes, concentration of glucose, concentration of hyaluronan, concentration of lactate or lactate salt, concentration of albumin, level of adenosine, level of R-2-hydroxyglutarate, concentration of pyruvate, concentration of oxygen, and / or presence of oxidizing agents, reducing agents, or cofactors, and other conditions understood by those skilled in the art.

[0017] As used interchangeably herein, the terms "polynucleotide" and "nucleic acid" refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0018] As used herein, the term "antibody" includes polyclonal and monoclonal antibodies, including intact antibodies and antibody fragments that retain specific binding to an antigen. Antibody fragments include, but are not limited to, fragment antigen binding (Fab) fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fab'-SH fragments, (Fab')2Fv fragments, Fd fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments such as single-chain variable fragments (scFv), bivalent scFv, trivalent scFv, and single-domain antibody fragments (e.g., sdAb, sdFv, nanobody). The term includes genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, single-chain antibodies, fully human antibodies, humanized antibodies, fusion proteins comprising antigen-specific targeting regions of antibodies and non-antibody proteins, heteroconjugate antibodies, multispecificities such as bispecific antibodies, diabodies, triabodies, and tetra-bodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise specified, the term "antibody" should be understood to include its functional antibody fragments. The term also includes intact or full-length antibodies, including any class or subclass of antibody, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0019] As used herein, the term "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments (each having a single antigen-binding site) and a residual "Fc" fragment (so-called because of its ability to easily crystallize). Pepsin treatment produces an F(ab')2 fragment having two antigen-binding sites capable of cross-linking antigens.

[0020] As used interchangeably herein, the terms "single-chain Fv", "scFv", or "sFv" antibody fragments refer to antibody V H and V L domains that are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further includes a polypeptide linker or spacer between the V H domain and the V L domain, thereby enabling the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0021] As used herein, "naturally occurring" VH and VL domains refer to VH and VL domains isolated from a host without further molecular evolution to alter their affinity when generated in scFv format under certain conditions as disclosed in U.S. Patent No. 8,709,755 B2 and Application WO / 2016 / 033331 A1.

[0022] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents and is expressed as the dissociation constant (Kd). The affinity is at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater, or more, than the affinity of the antibody for an irrelevant amino acid sequence. The affinity of the antibody for the target protein may be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM), or more. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation upon dilution. The terms "immunoreactive" and "preferentially binds" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0023] As used herein, the term "binding" refers to the direct association between two molecules by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bond interactions, including interactions such as salt bridges and hydrogen bridges. Nonspecific binding refers to binding at an affinity of less than about 10 -7 M, for example, binding at an affinity of 10 -6 M, 10 -5 M, 10 -4 M, etc.

[0024] As used herein, reference to a "cell surface expression system" or "cell surface display system" refers to the display or expression of a protein or a portion thereof on the surface of a cell. Typically, cells are generated that express a protein of interest fused to a cell surface protein. For example, the protein is expressed as a fusion protein with a transmembrane domain.

[0025] As used herein, the term "element" includes polypeptides, regions of polypeptides, and polypeptides comprising fusions of functional mutants or fragments thereof, as well as polynucleotides including microRNA and shRNA, and functional mutants or fragments thereof.

[0026] As used herein, the term "region" is any segment of a polypeptide or polynucleotide.

[0027] As used herein, a "domain" is a region of a polypeptide or polynucleotide having functional and / or structural characteristics.

[0028] As used herein, the term "stalk" or "stalk domain" refers to a flexible polypeptide connector region that provides structural flexibility and spacing to adjacent polypeptide regions and can consist of natural or synthetic polypeptides. The stalk can be derived from the hinge or hinge region of an immunoglobulin (e.g., IgG1), which is generally defined as extending from Glu216 to Pro230 of human IgG1 (Burton (1985) Molec. Immunol., 22:161-206). The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine residues that form the inter-heavy chain disulfide (S-S) bonds in the same positions. The stalk can be natural or non-natural, including but not limited to a modified hinge region as disclosed in U.S. Patent No. 5,677,425. The stalk can include the complete hinge region derived from any class or subclass of antibody. The stalk can also include regions derived from CD8, CD28, or other receptors that provide a similar function of providing flexibility and spacing to adjacent regions.

[0029] As used herein, the term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the materials that coexist in the natural system is isolated. Such a polynucleotide can be part of a vector, and / or such a polynucleotide or polypeptide can be part of a composition, and such a vector or composition can still be isolated in that it is not part of its natural environment.

[0030] As used herein, "polypeptide" is a single chain of amino acid residues linked by peptide bonds. The polypeptide is not folded into a fixed structure and has no post-translational modifications. "Protein" is a polypeptide that is folded into a fixed structure. "Polypeptide" and "protein" are used interchangeably herein.

[0031] As used herein, a polypeptide can be "purified" to remove contaminants in the natural environment of the polypeptide, such as materials that interfere with the diagnostic or therapeutic use of the polypeptide, e.g., enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. A polypeptide can be purified to (1) more than 90%, more than 95%, or more than 98%, e.g., up to more than 99% by weight of antibody as determined by the Lowry method, (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver staining.

[0032] As used herein, the term "immune cell" generally includes leukocytes (white blood cells) derived from hematopoietic stem cells (HSCs) produced in the bone marrow. "Immune cells" include, for example, lymphocytes (T cells, B cells, natural killer (NK) cells) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells).

[0033] As used herein, "T cell" includes all types of immune cells expressing CD3, including T helper cells (CD4 + cells), cytotoxic T cells (CD8 + cells), regulatory T cells (Tregs) and gamma-delta T cells.

[0034] As used herein, "cytotoxic cell" includes CD8 +T cells, natural killer (NK) cells, NK-T cells, γδ T cells, CD4 + subpopulations of cells, and neutrophils, which are cells capable of mediating a cytotoxic response, are included.

[0035] As used herein, the term "stem cell" generally includes pluripotent or multipotent stem cells. "Stem cells" include, for example, embryonic stem cells (ES); mesenchymal stem cells (MSC); induced pluripotent stem cells (iPS); committed progenitor cells (hematopoietic stem cells (HSC); bone marrow-derived cells, etc.).

[0036] As used herein, terms such as "treatment", "treating", etc. refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing the disease and / or the adverse effects resulting from the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, such as a human, and includes: (a) preventing the occurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., preventing its onset; and (c) alleviating the disease, i.e., causing regression of the disease.

[0037] As used interchangeably herein, the terms "individual", "subject", "host", and "patient" refer to mammals including, but not limited to, humans, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats).

[0038] As used herein, the terms "therapeutically effective amount" or "effective amount" refer to the amount of an agent, or the total amount of two agents, that is sufficient to have an impact on such treatment of a disease when administered to a mammal or other subject for treating the disease. A "therapeutically effective amount" varies depending on the agent, the disease and its severity, and the age, weight, etc. of the subject being treated.

[0039] As used herein, the terms "evolve" or "evolving" refer to generating different polynucleotides encoding different polypeptides that are themselves improved biomolecules and / or contribute to the generation of another improved biomolecule using one or more methods of mutagenesis. "Physiological" or "normal" or "normal physiological" conditions include, but are not limited to, conditions such as pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, concentration of electrolytes, concentration of glucose, concentration of hyaluronan, concentration of lactate or lactate salt, concentration of albumin, level of adenosine, level of R-2-hydroxyglutarate, concentration of pyruvate, concentration of oxygen, and / or the presence of an oxidizing agent, reducing agent, or cofactor, and other conditions that are considered to be within the normal range in the tissue or organ at the site of administration to the subject or the site of action.

[0040] As used herein, a "genetically modified cell" is a cell that contains exogenous nucleic acid, regardless of whether the exogenous nucleic acid is integrated into the genome of the cell. As used herein, a "transduced cell" is a cell that contains exogenous nucleic acid that is integrated into the genome of the cell.

[0041] As used herein, "polypeptide" can include a part or the whole of a protein molecule, as well as post-translational or other modifications.

[0042] The pseudotyping elements used herein can include a "binding polypeptide" that includes one or more polypeptides, typically glycoproteins, that identify and bind to a target host cell, and one or more "fusogenic polypeptides" that mediate the fusion of the retrovirus with the target host cell membrane, thereby enabling the retroviral genome to enter the target host cell. As used herein, a "binding polypeptide" may also be referred to as a "T cell and / or NK cell binding polypeptide" or a "target engagement element", and a "fusogenic polypeptide" may also be referred to as a "fusogenic element".

[0043] "Quiescent" lymphocytes, such as quiescent T cells, are lymphocytes in the G0 phase of the cell cycle that do not express activation markers such as Ki-67. Quiescent lymphocytes include naive T cells that have not encountered a specific antigen and memory T cells that have been altered by a previous encounter with an antigen. "Quiescent" lymphocytes may also be referred to as "resting" lymphocytes.

[0044] As used herein, "lymphocyte depletion" includes, for example, methods of reducing the number of lymphocytes in a subject by administration of a lymphocyte depleting agent. Lymphocyte depletion can also be achieved by partial or whole body fractionated radiotherapy. A lymphocyte depleting agent may be a compound or composition that can reduce the number of functional lymphocytes in a mammal when administered to the mammal. An example of such an agent is one or more chemotherapeutic agents. Such agents and dosages are known and can be selected by the treating physician depending on the subject being treated. Examples of lymphocyte depleting agents include, but are not limited to, fludarabine, cyclophosphamide, cladribine, denileukin diftitox, or combinations thereof.

[0045] RNA interference (RNAi) is a biological process by which RNA molecules inhibit gene expression or translation by neutralizing target RNA molecules. The RNA target may be mRNA or other RNAs that are susceptible to functional inhibition by RNAi. As used herein, an "inhibitory RNA molecule" refers to an RNA molecule that, by its presence in a cell, brings about RNAi and results in a decrease in the expression of the transcript targeted by the inhibitory RNA molecule. The inhibitory RNA molecules used herein have a 5' stem and a 3' stem that can form an RNA duplex. The inhibitory RNA molecule may be, for example, an miRNA (either endogenous or artificial) or shRNA, a precursor of an miRNA (i.e., Pri-miRNA or Pre-miRNA) or shRNA, or a dsRNA that is directly transcribed or introduced as an isolated nucleic acid into a cell or a subject.

[0046] As used herein, the term "double-stranded RNA" or "dsRNA" or "RNA duplex" refers to an RNA molecule composed of two strands. Double-stranded molecules include two RNA strands that hybridize to form a double-stranded RNA structure, or a single-stranded RNA strand that itself folds back on itself to form a double-stranded structure. In most, but not necessarily all, double-stranded regions, the bases are base-paired. The double-stranded region contains a sequence complementary to the target RNA. The sequence complementary to the target RNA is the antisense sequence, often 18 - 29, 19 - 29, 19 - 21, or 25 - 28 nucleotides in length, or in some embodiments between 18, 19, 20, 21, 22, 23, 24, 25 at the lower end and 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 at the upper end, with the lower end of a particular range always being lower than the upper end. Such structures typically include a 5' stem, a loop, and a 3' stem adjacent to each stem and connected by a loop that is not part of the double-stranded portion. In certain embodiments, the loop contains at least 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In other embodiments, the loop contains between 2 - 40, 3 - 40, 3 - 21, or 19 - 21 nucleotides, or in some embodiments between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 at the lower end and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 at the upper end, with the lower end of a particular range always being lower than the upper end.

[0047] As used herein, the term "microRNA flanking sequence" refers to a nucleotide sequence that includes a microRNA processing element. A microRNA processing element is the minimal nucleic acid sequence that contributes to the generation of mature microRNA from precursor microRNA. Often, these elements are within a 40 nucleotide sequence adjacent to the microRNA stem-loop structure. In some cases, the microRNA processing element is found within a stretch of a nucleotide sequence 5 - 4,000 nucleotides in length adjacent to the microRNA stem-loop structure.

[0048] The term "linker", when used with respect to multi-inhibitory RNA molecules, refers to a connecting means that binds two inhibitory RNA molecules.

[0049] As used herein, "recombinant retrovirus" refers to a non-replicable or "replication-incompetent" retrovirus, unless expressly described as a replicable retrovirus. The terms "recombinant retrovirus" and "recombinant retrovirus particle" are used interchangeably herein. Such retroviruses / retrovirus particles can be any type of retrovirus particle, including, for example, gamma retroviruses, and in exemplary embodiments, lentiviruses. As is known, such retrovirus particles, such as lentivirus particles, are typically formed in packaging cells by transfecting the packaging cells with a plasmid containing packaging components such as Gag, Pol and Rev, an envelope or pseudotyping plasmid encoding a pseudotyping element, and a transfer, genomic, or retrovirus (e.g., lentivirus) expression vector, which is typically a plasmid encoding a gene of interest or other coding sequence. Thus, a retrovirus (e.g., lentivirus) expression vector contains sequences (e.g., 5' LTR and 3' LTR adjacent to psi packaging elements and target heterologous coding sequences, etc.) that facilitate expression and packaging after transfection into cells. The terms "lentivirus" and "lentivirus particle" are used interchangeably herein.

[0050] The "framework" of miRNA consists of the "5'microRNA flanking sequence" and / or the "3'microRNA flanking sequence" surrounding the miRNA, and in some cases, a loop sequence that separates the stems of the stem-loop structures within the miRNA. In some examples, the "framework" is derived from a naturally occurring miRNA such as miR-155, for example. The terms "5'microRNA flanking sequence" and "5'arm" are used interchangeably herein. The terms "3'microRNA flanking sequence" and "3'arm" are used interchangeably herein.

[0051] As used herein, the term "miRNA precursor" refers to any length of RNA molecule that can be enzymatically processed into a miRNA, such as a primary RNA transcript, pri-miRNA, or pre-miRNA.

[0052] As used herein, the term "construct" refers to an isolated polypeptide or an isolated polynucleotide encoding a polypeptide. A polynucleotide construct can encode a polypeptide, such as a lymphoproliferative factor. One of ordinary skill in the art will understand whether the construct refers to an isolated polynucleotide or an isolated polypeptide, depending on the context.

[0053] As used herein, "MOI" refers to the multiplicity of infection ratio, where MOI is equal to the ratio of the number of viral particles used for infection per cell number. Functional titration of the number of viral particles can be performed using FACS and reporter expression.

[0054] "Peripheral blood mononuclear cells" (PBMCs) include peripheral blood cells with round nuclei and include lymphocytes (such as T cells, NK cells, and B cells) as well as monocytes. Some blood cell types that are not PBMCs include red blood cells, platelets, and granulocytes (i.e., neutrophils, eosinophils, and basophils).

[0055] It should be understood that the present disclosure, as well as the aspects and embodiments provided herein, are not limited to the specific examples disclosed and may thus naturally vary. It should also be understood that the terms used herein are for the purpose of disclosing only specific examples and embodiments, and are not intended to be limiting, as the scope of the present disclosure is limited only by the appended claims.

[0056] Where a range of values is provided, unless the context clearly dictates otherwise, up to one tenth of the unit of the lower limit, each intervening value between the upper and lower limits of that range, and any other stated or intervening value within the stated range are included in the present disclosure. It is understood that the upper and lower limits of these smaller ranges may independently be included in a smaller range and are likewise encompassed by the present invention, subject to any specifically excluded limits within the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed by the present invention. Where multiple lower values and multiple higher values are given for overlapping ranges, those skilled in the art will recognize that the selected ranges will include lower values that are smaller than the higher values. All headings in this application are for the convenience of the reader and are not limiting.

[0057] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, but the preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference for the purpose of disclosing and describing the methods and / or materials with respect to which the publication is cited.

[0058] As used in this specification and the appended claims, it should be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a chimeric antigen receptor" includes plural such chimeric antigen receptors and their equivalents known to those skilled in the art, and the like. It is further noted that the claims may be drafted to exclude optional elements. Thus, this description is intended to serve as a basis for using exclusive terms such as "solely", "only", etc. in connection with the recitation of claim elements, or for using "negative" limitations.

[0059] For clarity, it is understood that certain features of the invention that are described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, the various features of the invention that are described in the context of a single embodiment may also be provided separately or in any suitable sub-combination. All combinations of embodiments related to the invention are specifically embraced by the invention and are disclosed herein as if each combination were individually and explicitly disclosed. Further, all sub-combinations of the various embodiments and their elements are also specifically embraced by the invention and are disclosed herein as if each such sub-combination were individually and explicitly disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The present disclosure overcomes the problems of the prior art by providing improved methods and compositions for genetically modifying lymphocytes, such as NK cells, and in exemplary embodiments, T cells. Some of the methods and compositions herein provide a simplified and faster process for transducing lymphocytes, avoiding some steps that require special devices. Further, any such treatment is performed ex vivo, thus enabling the option of removing various unwanted cells, and the method provides better control of the post-transduction treatment. Thus, the method provides an important step towards the generalization of cell therapies.

[0061] Exemplary methods and compositions for genetically modifying lymphocytes, such as NK cells and in exemplary embodiments, T cells, are performed in a shorter time than previous methods. Further, compositions having multiple uses are provided, including use in these improved methods. Some of these compositions are genetically modified lymphocytes with improved quality of growth and survival, including, for example, in vitro culture in the absence of growth factors. Such genetically modified lymphocytes can be used, for example, as research tools to better understand factors affecting T cell growth and survival, and also for commercial production, for example, of specific factors such as growth factors and immunomodulatory agents that are collected and tested, or used in commercial products.

[0062] Methods for transducing and / or genetically modifying lymphocytes In certain embodiments, a method of transducing and / or genetically modifying lymphocytes, such as peripheral blood mononuclear cells (PBMCs), typically T cells and / or NK cells, and in certain exemplary embodiments resting T cells and / or resting NK cells (typically a population thereof), comprising contacting the lymphocytes with replication-incompetent recombinant retroviral particles (typically a population thereof), wherein the replication-incompetent recombinant retroviral particles typically comprise a pseudotyping element on their surface, and wherein said contacting (and incubation under contacting conditions) promotes membrane binding, membrane fusion, and optionally transduction of the resting T cells and / or NK cells by the replication-incompetent recombinant retroviral particles, thereby producing genetically modified T cells and / or NK cells. In an exemplary embodiment, pre-activation of the T cells and / or NK cells is not required, and an activation element, which can be any activation element provided herein, is present in the reaction mixture in which the contacting is performed. In a further exemplary embodiment, the activation element is present on the surface of the replication-incompetent recombinant retroviral particles. In an exemplary embodiment, the activation element is an anti-CD3, such as anti-CD3 scFv or anti-CD3 scFvFc.

[0063] In some embodiments, in the methods provided herein for transducing and / or genetically modifying PBMCs or lymphocytes, typically T cells and / or NK cells, the contacting step and any subsequent optional incubation that includes removing retroviral particles not associated with the cells can be performed (or can occur) within 72 hours, 48 hours, or 24 hours, or within any of the contacting time ranges provided herein. However, in exemplary embodiments, the contacting is performed for less than 2 hours, less than 1 hour, less than 30 minutes, or less than 15 minutes, but in each case there is at least an initial contacting step where the retroviral particles and the cells are contacted in suspension in the transduction reaction mixture. This contacting typically includes the first step where retroviral particles not associated with the cells of the reaction mixture are separated from the cells and then further processed. Such a suspension can include allowing the cells and retroviral particles to sediment, or applying a force such as centrifugal force to the bottom of the container or chamber to cause such sedimentation, as discussed in more detail herein. In exemplary embodiments, such g-forces are lower than those successfully used in spinoculation procedures. Further discussion of contact times, as well as contact and any incubation, is discussed further herein. In further exemplary embodiments, the contacting is performed during only the initial contacting step without further incubation in the reaction mixture (without further incubation in the reaction mixture that includes free retroviral particles in suspension and cells in suspension), or is performed with an incubation in the reaction mixture for 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 1 hour, which may be a step of separating free retroviral particles in the reaction mixture from particles associated with the cells.

[0064] Various embodiments of this method, as well as other aspects such as NK cells and T cells produced by such methods, are disclosed in detail herein. Further, various elements or steps of aspects of such methods for transducing and / or genetically modifying PBMCs, lymphocytes, T cells and / or NK cells are provided herein, for example in this section and in the section of exemplary embodiments, and such methods are provided throughout this specification as will be further discussed herein, including embodiments. For example, any embodiment of an aspect for transducing and / or genetically modifying PBMCs or lymphocytes provided in this section and in the section of exemplary embodiments, such as NK cells or in the exemplary embodiments T cells, can include any of the embodiments of replication-incompetent recombinant retroviral particles provided herein that include one or more lymphocyte proliferative elements, CARs, pseudotyping elements, riboswitches, activation elements, membrane-bound cytokines, miRNAs, Kozak-like sequences, WPRE elements, triple stop codons, and / or other elements disclosed herein, and can be combined with the methods herein for generating retroviral particles using packaging cells. In certain exemplary embodiments, the retroviral particles are lentiviral particles. Such methods for genetically modifying and / or transducing PBMCs or lymphocytes such as T cells and / or NK cells can be performed in vitro or ex vivo. One of ordinary skill in the art will recognize that the details provided herein for transducing and / or genetically modifying PBMCs or lymphocytes such as T cells and / or NK cells can be applied to any aspect that includes such steps.

[0065] In certain exemplary embodiments, the cells are genetically modified and / or transduced in vivo, in vitro, or ex vivo without the need for prior activation or stimulation. In certain exemplary embodiments, the cells are activated during contact and not at all, or for more than 15 minutes, 30 minutes, 1, 2, 4, or 8 hours prior to contact. In certain exemplary embodiments, activation by elements not present on the surface of the retroviral particles is not required to genetically modify and / or transduce the cells. Thus, such activation or stimulatory elements are not required outside of the retroviral particles prior to, during, or after contact. Thus, these exemplary embodiments that do not require prior activation or stimulation provide the ability to rapidly conduct in vitro experiments aimed at better understanding T cells and the biological mechanisms therein. Further, such methods provide for much more efficient commercial production of biological products produced using PBMCs, lymphocytes, T cells, or NK cells, and the development of such commercial production methods. Finally, such methods provide for more rapid ex vivo processing of PBMCs for adoptive cell therapy, and fundamentally simplify the provision of such therapies, for example, by providing point-of-care methods.

[0066] Compositions and methods for lymphocyte transduction in whole blood lymphocytes in whole blood In certain embodiments, provided herein is a method of transducing and / or genetically modifying peripheral blood mononuclear cells (PBMCs) or lymphocytes, typically T cells and / or NK cells, and in certain exemplary embodiments resting T cells and / or resting NK cells, in a reaction mixture comprising blood or a component thereof, and / or an anticoagulant, the method comprising contacting the lymphocytes with replication-incompetent recombinant retroviral particles in a reaction mixture representative of a separate embodiment provided herein. The reaction mixture in an exemplary embodiment comprises lymphocytes and replication-incompetent recombinant retroviral particles, a T cell activation element, and in an exemplary embodiment, one or more additional blood components as shown below that are present because the reaction mixture comprises at least 10% whole blood, and the replication-incompetent recombinant retroviral particles typically comprise a pseudotyping element on their surface. In such methods, the contacting (and incubation under contacting conditions) promotes the association of the lymphocytes with the replication-incompetent recombinant retroviral particles, and the recombinant retroviral particles genetically modify and / or transduce the lymphocytes. The reaction mixture of this embodiment comprises at least 10% whole blood (e.g., at least 10%, 20%, 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% whole blood) and optionally, an effective amount of an anticoagulant, or the reaction mixture further comprises at least one additional blood or blood preparation component other than PBMCs, e.g., the reaction mixture comprises an effective amount of an anticoagulant and one or more blood preparation components other than PBMCs. In an exemplary embodiment, such blood or blood preparation components other than PBMCs are one or more (e.g., at least 1, 2, 3, 4, or 5) or all of the following additional components. a) Red blood cells (wherein the red blood cells occupy from 1 to 60% of the volume of the reaction mixture), b) Neutrophils (wherein the neutrophils occupy at least 10% of the white blood cells in the reaction mixture or the reaction mixture comprises at least 10% as many neutrophils as T cells), c) Basophils (wherein the basophils occupy at least 0.05% of the white blood cells in the reaction mixture), d) Acidophil (wherein the reaction mixture contains at least 0.1% of white blood cells in the reaction mixture), e) Plasma (wherein the plasma occupies at least 1% of the volume of the reaction mixture), and f) Anticoagulant (Such blood or blood preparation components a - f above are referred to herein as ( "notable non - PBMC blood or blood preparation components")).

[0067] One or more additional blood components are present in certain exemplary embodiments of the reaction mixture (including related uses, genetically modified T cells or NK cells, or methods for genetically modifying the T cell and / or NK cell embodiments provided herein), but in these exemplary embodiments, the reaction mixture contains at least 10% whole blood, and in certain exemplary embodiments at least 25%, 50%, 75%, 90%, or 95% whole blood, or for example 25% - 95% whole blood. In these exemplary embodiments, such a reaction mixture is formed by combining whole blood with an anticoagulant (e.g., collecting whole blood into a blood collection tube containing an anticoagulant) and adding a solution of recombinant retrovirus to the blood together with the anticoagulant. Thus, in exemplary embodiments, the reaction mixture contains an anticoagulant as described in more detail herein. In some embodiments, the whole blood is not umbilical cord blood or does not contain umbilical cord blood.

[0068] The reaction mixtures in these embodiments typically do not include the PBMC enrichment procedure before the transduction reaction mixture is formed. Thus, typically, such reaction mixtures contain the additional components listed in a) - f) above that are not PBMCs. Further, in an exemplary embodiment, since the reaction mixture consists essentially of whole blood or contains whole blood, the reaction mixture contains all of the additional components listed in a) - e) above. "Consisting essentially of whole blood" means blood that has been separated from an individual, has not been subjected to a PBMC enrichment procedure, and has been diluted by less than 50% with another solution. For example, this dilution can be due to the addition of an anticoagulant and the addition of an amount of liquid containing retroviral particles. Further embodiments of reaction mixtures for methods and compositions related to the transduction of lymphocytes in whole blood are provided herein.

[0069] In another aspect, genetically modified lymphocytes produced by the above-described method of transducing and / or genetically modifying lymphocytes in whole blood are provided herein, and in exemplary embodiments, genetically modified T cells and / or NK cells. In yet another aspect, the use of replication-incompetent recombinant retroviral particles in the manufacture of a kit for genetically modifying lymphocytes of a subject, in exemplary embodiments T cells and / or NK cells, is provided herein, and use of the kit includes the above-described method of transducing and / or genetically modifying lymphocytes in whole blood. In another aspect, a method for administering genetically modified lymphocytes to a subject is provided herein, wherein the genetically modified lymphocytes are generated by the above-described method of transducing and / or genetically modifying lymphocytes in whole blood. Aspects provided herein that include such methods of transducing and / or genetically modifying lymphocytes in whole blood, such use of such methods in the manufacture of a kit, reaction mixtures formed by such methods, genetically modified lymphocytes produced by such methods, and methods for administering genetically modified lymphocytes produced by such methods are referred to herein as "Aspects of Compositions and Methods for Transducing Lymphocytes in Whole Blood". Exemplary embodiments of such aspects include contacting T cells and / or NK cells with retroviral particles in whole blood, but it should be noted that such aspects also include other embodiments in which one or more of the above additional components a-f are present in the transduction reaction mixture at a concentration higher than the typical concentration after the PBMC enrichment procedure.

[0070] Various elements or steps of such methods for transducing lymphocytes in whole blood are provided herein, e.g., in this section and the section of exemplary embodiments, and such methods, as further discussed herein, include embodiments provided throughout this specification. One of ordinary skill in the art will recognize that many of the embodiments provided anywhere in this specification can be applied to either embodiments of compositions or embodiments of methods for transducing lymphocytes in whole blood. For example, any of the embodiments of compositions and methods for transducing lymphocytes in whole blood provided in this section and / or the section of exemplary embodiments, etc., can include any of the embodiments of replication-defective recombinant retroviral particles provided herein that include one or more polypeptide lymphoproliferative elements, inhibitory RNAs, CARs, pseudotyping elements, riboswitches, activating elements, membrane-bound cytokines, miRNAs, Kozak-type sequences, WPRE elements, triple stop codons, and / or other elements disclosed herein, and can be combined with the methods herein for generating retroviral particles using packaging cells.

[0071] As a non-limiting example of an embodiment that can be used in many aspects of this specification, as discussed in more detail herein, a pseudotyping element typically binds to lymphocytes (e.g., T cells and / or NK cells), in exemplary embodiments resting T cells and / or resting NK cells, and can promote membrane fusion either alone or in combination with other proteins of replication-defective recombinant retroviral particles. In certain exemplary embodiments, the retroviral particles are lentiviral particles. Such methods for genetically modifying lymphocytes such as T cells and / or NK cells in whole blood can be performed in vitro or ex vivo.

[0072] An anticoagulant is included in the reaction mixture for certain embodiments of aspects of the compositions and methods for transducing lymphocytes in whole blood provided herein. In some exemplary embodiments, blood is collected using, for example, standard blood collection protocols known in the art, with the anticoagulant present within a collection container (e.g., a tube or bag). Anticoagulants that can be used in aspects of the compositions and methods for transducing lymphocytes in whole blood provided herein include compounds or biological agents that block or limit the thrombin blood coagulation cascade. Anticoagulants include metal chelating agents, preferably calcium ion chelating agents such as citrate (e.g., including free citrate ions), which include citric acid, sodium citrate, phosphoric acid, adenine and mono- or polysaccharides, such as dextrose, oxalate, and EDTA; heparin and heparin analogs, such as unfractionated heparin, low molecular weight heparin, and other synthetic saccharides; and vitamin K antagonists, such as coumarin, including solutions of citrate containing one or more of these components. Exemplary citrate compositions include acid citrate dextrose (ACD) (also called anticoagulant citrate dextrose solution A and solution B (U.S. Pharmacopeia 26, 2002, page 158)); and citrate phosphate dextrose (CPD) solution (which can also be prepared as CPD-A1, as is known in the art). Thus, the anticoagulant composition can also include phosphate ions or monobasic phosphate ions, adenine, and monosaccharides or polysaccharides.

[0073] Such anticoagulants can be present in the reaction mixture at a concentration (i.e., an effective amount) effective to prevent blood clotting, as is known in the art, or, for example, at a concentration that is 2 times, 1.5 times, 1.25 times, 1.2 times, 1.1 times, or 9 / 10, 4 / 5, 7 / 10, 3 / 5, 1 / 2, 2 / 5, 3 / 10, 1 / 5, or 1 / 10 of the effective concentration. The effective concentrations of many different anticoagulants are known and can be readily determined empirically by analyzing different concentrations for their ability to prevent physically observable blood clotting. A number of coagulometers for measuring clotting are commercially available, and various sensor technologies such as QCM sensors can be used (see, for example, Yao et al., “Blood Coagulation Testing Smartphone Platform Using Quartz Crystal Microbalance Dissipation Method,” Sensors (Basel). 2018 Sep;18(9):3073). The effective concentration includes the concentration of any commercially available anticoagulant in a commercially available tube or bag after the anticoagulant has been diluted with the volume of blood for the tube or bag. For example, in certain embodiments of the compositions and methods for transducing lymphocytes in whole blood provided herein, the concentration of acid citrate dextrose (ACD) in the reaction mixture can be between 0.1 and 5 times, or between 0.25 and 2.5 times, between 0.5 and 2 times, between 0.75 and 1.5 times, between 0.8 and 1.2 times, between 0.9 and 1.1 times, about 1 time, or 1 time the concentration of ACD in a commercially available ACD blood collection tube or bag. For example, in a standard process, blood can be collected in a tube or bag containing 3.2% (109 mM) sodium citrate (109 mM) at a ratio of 9 parts blood and 1 part anticoagulant. Thus, in certain exemplary embodiments using a reaction mixture prepared by adding 1-2 parts of a retroviral particle solution to this mixture of 1 part anticoagulant to 9 parts blood, the citrate concentration can be, for example, between 0.25% and 0.4%, or between 0.30% and 0.35%. In an exemplary standard blood collection embodiment, 15 ml of ACD solution A is present in a blood bag for collecting 100 mL of blood.ACD before blood addition contains citric acid (anhydrous) 7.3 g / L (0.73%), sodium citrate (dihydrate) 22.0 g / L (2.2%), and dextrose (monohydrate) 24.5 g / L [USP] (2.4%). After adding 100 ml of blood to the bag containing ACD, for example, a quantity of 5 - 20 ml of genetically modified retroviral particles is added. Thus, in some embodiments, the concentration of the ACD components in the reaction mixture may be 0.05 - 0.1%, or 0.06 - 0.08% citric acid (anhydrous), 0.17 - 0.27, or 0.20 - 0.24 sodium citrate (dihydrate), 0.2 - 0.3, or 0.20 - 0.28, or 0.22 and 0.26% dextrose (monohydrate). In certain embodiments, sodium citrate is used at a concentration of 0.001 - 0.02 M in the reaction mixture.

[0074] In some embodiments, heparin is present in the reaction mixture at a concentration that is between 0.1 - 5 times, or between 0.25 - 2.5 times, 0.5 - 2 times, 0.75 - 1.5 times, 0.8 - 1.2 times, 0.9 - 1.1 times, about 1 time, or 1 time the concentration of heparin in a commercially available heparin blood collection tube, for example. Heparin is a glycosaminoglycan anticoagulant having a molecular weight in the range of 5,000 - 30,000 daltons. In some embodiments, heparin is used at a concentration of about 1.5 - 45, 5 - 30, 10 - 20, or 15 USP units / ml of the reaction mixture. In some embodiments, the effective concentration of EDTA, for example as K2EDTA, in the reaction mixture herein may be between 0.15 - 5 mg / ml, between 1 - 3 mg / ml, between 1.5 - 2.2 mg / ml, or between 1 - 2 mg / ml, or about 1.5 mg / ml. The reaction mixture in aspects of the compositions and methods for transducing lymphocytes in whole blood provided herein may contain two or more anticoagulants whose combined effective dose prevents the coagulation of the blood before the formation of the reaction mixture and / or the reaction mixture itself.

[0075] In some embodiments, the anticoagulant may be administered to the subject prior to collection of blood from the subject for ex vivo transduction, such that coagulation of the blood when collected is inhibited, at least in part and through at least the contacting step and any subsequent optional incubation period. In such embodiments, for example, acid citrate dextrose may be administered to the subject at between 80 mg / kg / day and 5 mg / kg / day (mg refers to mg of citrate and kg is applicable to the mammal being treated). Heparin may be delivered, for example, at a dosage between 5 units / kg / hour and 30 units / kg / hour.

[0076] In addition to, or in place of, the anticoagulant, embodiments of the compositions and methods for transducing lymphocytes in whole blood provided herein can include at least one additional component selected from one or more of the following components: a) red blood cells (wherein the red blood cells occupy from 0.1% to 75% of the volume of the reaction mixture), b) neutrophils (wherein the neutrophils occupy at least 10% of the white blood cells in the reaction mixture or the reaction mixture contains at least 10% as many neutrophils as T cells), c) basophils (wherein the basophils occupy at least 0.05% of the white blood cells in the reaction mixture), d) eosinophils (wherein the reaction mixture contains at least 0.1% of the white blood cells in the reaction mixture), e) plasma (wherein the plasma occupies at least 1% of the volume of the reaction mixture), and f) platelets (wherein the platelets contain at least 1×10 6 platelets per liter of reaction mixture).

[0077] Regarding red blood cells, in some embodiments, the red blood cells may occupy between 0.1, 0.5, 1, 5, 10, 25, 35, or 40% of the volume of the reaction mixture at the lower limit of the range and 25, 50, 60, or 75% of the volume of the reaction mixture at the upper limit of the range. In exemplary embodiments, the red blood cells occupy between 1% and 60%, between 10% and 60%, between 20% and 60%, between 30% and 60%, between 40% and 60%, between 40% and 50%, between 42% and 48%, between 44% and 46%, about 45% or 45%.

[0078] Regarding neutrophils, in some embodiments, the neutrophils may occupy between 0.1, 0.5, 1, 5, 10, 20, 25, 35, or 40% of the white blood cells in the reaction mixture at the lower limit of the range and 25, 50, 60, 70, 75, and 80% of the white blood cells in the reaction mixture at the upper limit of the range, for example, between 25% and 70% of the white blood cells in the reaction mixture, or between 30% and 60%, or between 40% and 60%. In some embodiments, there are more neutrophils in the reaction mixture herein than T cells and / or NK cells.

[0079] Regarding eosinophils, in some embodiments, the eosinophils may occupy between 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8% of the white blood cells in the reaction mixture at the lower limit of the range and 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.5, 4, 5, 6, 8, and 10% of the white blood cells in the reaction mixture at the upper limit of the range. In exemplary embodiments, the eosinophils occupy between 0.05% and 10.0% of the white blood cells in the reaction mixture, between 0.1% and 9%, between 0.2% and 8%, between 0.2% and 6%, between 0.5% and 4%, between 0.8% and 4%, or between 1% and 4%.

[0080] Regarding basophils, in some embodiments, the basophils may occupy between 0.05, 0.1, 0.2, 0.4, 0.45 and 0.5% of the white blood cells in the reaction mixture at the lower limit of the range and 0.8, 0.9, 1.0, 1.1, 1.2, 1.5, and 2.0% of the white blood cells in the reaction mixture at the upper limit of the range. In an exemplary embodiment, the basophils occupy between 0.05 - 1.4%, 0.1 - 1.4%, 0.2 - 1.4%, 0.3 - 1.4%, 0.4 - 1.4%, 0.5 - 1.4%, 0.5 - 1.2%, 0.5 - 1.1%, or 0.5 - 1.0% of the white blood cells in the reaction mixture.

[0081] Regarding plasma, in some embodiments, the plasma may occupy between 0.1, 0.5, 1, 5, 10, 25, 35 or 45% of the volume of the reaction mixture at the lower limit of the range and 25, 50, 60, 70, and 80% of the volume of the reaction mixture at the upper limit of the range. In an exemplary embodiment, the plasma occupies between 0.1 - 80%, 1 - 80%, 5 - 80%, 10 - 80%, 30 - 80%, 40 - 80%, 45 - 70%, 50 - 60%, 52 - 58%, 54 - 56%, about 55% or 55% of the reaction mixture.

[0082] Regarding platelets, in some embodiments, the platelets are from 1×10 5 , 1×10 6 , 1×10 7 , or 1×10 8 platelets per 1 mL of the reaction mixture at the lower limit of the range to 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 2×10 13 , or 2×10 14 platelets per 1 mL of the reaction mixture at the upper limit of the range. In an exemplary embodiment, the platelets are between 1×10 5 - 1×10 12 platelets per 1 ml of the reaction mixture, between 1×10 6 - 1×10 11 platelets per 1 ml of the reaction mixture, between 1×10 7 - 1×10 10platelets between, 1×10 per mL 8 ~1×10 9 platelets between, or 1×10 8 ~5×10 8 platelets between.

[0083] Exemplary cell processing methods for genetically modifying T cells and / or NK cells in the presence of blood or its components It is worth noting that some embodiments of the methods for genetic modification provided herein do not include the step of collecting blood from a subject. However, as shown in FIG. 1, some methods provided herein include the step (110) of collecting blood from a subject. As discussed in more detail herein, blood can be collected or obtained from a subject by any suitable method known in the art. For example, blood can be collected by venipuncture or any other blood collection method by which a sample of blood is taken. In some embodiments, the amount of blood collected is 25 ml to 250 ml, such as 25 ml to 60 ml, 50 ml to 90 ml, 75 ml to 125 ml, or 90 ml to 120 ml, or 95 to 110 ml.

[0084] In any of the aspects of the methods provided herein for genetically modifying lymphocytes (such as T cells and / or NK cells), regardless of whether blood is collected from a subject, the lymphocytes are contacted with replication-incompetent retroviral particles in a reaction mixture. In an exemplary embodiment, this contact, and the reaction mixture in which the contact occurs, are performed within a closed cell processing system, as discussed in more detail herein. In conventional closed cell processing methods, including ex vivo genetic modification and / or transduction of lymphocytes, particularly methods of autologous cell therapy, many steps such as PBMC enrichment, washing, cell activation, transduction, expansion, collection, and optionally reintroduction, etc. are performed over several days. In more recent methods (see Figure 1A), some of the steps and time involved in this ex vivo cell processing have been shortened (see, for example, WO2019 / 055946). These more recent methods (and the further improved cell processing methods provided herein) further utilize a rapid ex vivo transduction process that does not include, or minimally includes, pre-activation (such as contacting lymphocytes, such as T cells and / or NK cells, with an activator for less than 30, 15, 10, or 5 minutes before contacting with retroviral particles). In certain embodiments of such methods, T cell and / or NK cell activation elements are present in the reaction mixture in which the contact step occurs. In an exemplary embodiment, the T cell and / or NK cell activation elements are associated with the surface of the retroviral particles present in the reaction mixture. In an exemplary embodiment, such methods are used in point-of-care autologous cell therapy. However, such more recent methods still include a PBMC enrichment step / procedure (120), which typically takes at least about 1 hour within a closed system, followed by cell counting, transfer, and addition of medium, which takes at least about 45 minutes before the lymphocytes contact the retroviral particles to form a transduction reaction mixture (130A).As discussed in detail herein, following the "viral transduction" step, which is typically a contact step involving incubation, the lymphocytes are typically washed away (140A) from the retroviral particles remaining in suspension, for example using Sepax, collected (150A), and the final product is typically placed in an infusion bag for reinfusion or a cryopreservation vial for storage (160A). As discussed in further detail herein, conventional PBMC enrichment procedures typically involve a ficoll density gradient and centrifugal force (e.g., centrifugation) or centripetal force (e.g., Sepax), or use leukapheresis to enrich PBMCs.

[0085] As shown in the examples provided herein, surprisingly, lymphocytes (e.g., T cells and / or NK cells) can contact replication-incompetent retroviral particles in a reaction mixture of whole blood containing an anticoagulant, and it has been found that a significant proportion of the lymphocytes can be genetically modified and transduced. Thus, it has been discovered that effective genetic modification of lymphocytes by recombinant retroviral particles can be performed in the presence of blood components and blood cells in addition to PBMCs. Further, based on the surprising discovery discussed above regarding effective genetic modification of T cells and optionally NK cells by retroviral particles, in the exemplary embodiments herein, even when the contact is performed with whole blood, replication-incompetent retroviral particles are directly added to the whole blood to form a reaction mixture (130B) such that the lymphocytes are genetically modified and / or transduced, and a further simplified method is provided wherein the cells in the whole blood are contacted with the replication-incompetent retroviral particles with optional incubation provided herein. Thus, such a further improved method in this exemplary embodiment typically does not include a lymphocyte enrichment step before the lymphocytes in whole blood containing an anticoagulant contact the retroviral particles. This further improved method, like other cell processing methods herein, is typically performed within a closed cell processing system and may or may minimally include pre-activation before the lymphocytes contact the retroviral particles. In these further simplified methods, the lymphocytes in the whole blood can be directly contacted with the retroviral particles within a blood bag. After the contact step (130B) in such a method, the lymphocytes that have contacted the retroviral particles are washed and concentrated to a high concentration using a PBMC concentration procedure (135B), thereby reducing neutrophils and facilitating reintroduction into the subject. Thus, in such embodiments, typically no PBMC concentration procedure and lymphocyte enrichment filtration are performed before the cells in whole blood containing an anticoagulant contact the recombinant retroviral particles. However, in the embodiment of FIG. 1B, such a PBMC concentration method is performed using Sepax with a Ficoll gradient (135B), for example, after contact (130B) is performed with optional incubation.After PBMC enrichment, the lymphocytes are optionally further washed away from residual retroviral particles, for example, using Sepax (140B), collected (150B), and the final product is typically placed in an infusion bag for reinfusion or a cryopreservation vial for storage (160B).

[0086] Figure 2 provides a non-limiting, exemplary example of a cell processing leukocyte depletion filtration assembly (200) that can be used as a leukocyte depletion filter in the method of FIG. 1 to concentrate nucleated cells. In an exemplary embodiment, the exemplary leukocyte depletion filtration assembly (200), which is a single-use filtration assembly, includes a leukocyte depletion medium (e.g., a filter set) within a filter enclosure (210) having an inlet (225) and an outlet (226), as well as a configuration of bags, valves, and / or channels / tubes that provides the ability to concentrate, enrich, wash, and collect retained leukocytes or nucleated red blood cells using perfusion and reverse perfusion (see, e.g., EP2602315A1, which is hereby incorporated by reference in its entirety). In an exemplary embodiment, the leukocyte depletion filtration assembly (200) is a commercially available HemaTrate filter (Cook Regenetec, Indianapolis, IN). The leukocyte depletion filtration assembly can be used to concentrate total nucleated cells (TNC) including granulocytes, which are removed in the PBMC enrichment procedure of a closed cell processing system. Since filter assemblies with leukocyte depletion media such as the HemaTrate filter of EP2602315A1 and the exemplary leukocyte depletion filter assembly of FIG. 2 do not remove granulocytes, they are not considered PBMC enrichment assemblies or filters herein, and the methods of incorporating them are not considered PBMC enrichment procedures or steps herein.

[0087] The leukocyte removal filter assembly (200) of FIG. 2 is a single-use sterile assembly that includes various tubes and valves, typically needleless valves, that enable the isolation of leukocytes from whole blood and blood cell preparations containing leukocytes, as well as the rapid washing and concentration of leukocytes. In this exemplary assembly, after the reaction mixture has been subjected to a contacting step with optional incubation as disclosed in detail herein, a blood bag (215), e.g., a 500 ml PVC bag containing approximately 120 ml of a transduction / contact reaction mixture containing whole blood, an anticoagulant, and retroviral particles, is connected to the assembly (200) at the first assembly opening (217) of the inlet tube (255). Lymphocytes containing some T cells and / or NK cells with associated retroviral particles, as well as some lymphocytes that may be genetically modified at this point, as well as other blood cells and components in the whole blood reaction mixture, and the anticoagulant, enter the inlet tube (255) through the first assembly opening (217) by gravity when the clamp on the first inlet tube (255) is released. Genetically modified T cells and / or NK cells pass through the inlet valve (247) and the collection valve (245), enter the filter enclosure (210) through the filter enclosure inlet (225), and contact a leukocyte removal IV filter set (e.g., SKU J1472A Jorgensen Labs) within the filter enclosure (210). Nucleated red blood cells containing leukocytes are retained by the filter, while other blood components pass through the filter, enter the outlet tube (256) from the filter enclosure outlet (226), then pass through the outlet valve (247), and are collected in a waste collection bag (216), which may be, for example, a 2L PVC waste collection bag.

[0088] An optional buffer wash step can be performed by switching the inlet valve (247) to the wash position. In this optional wash step, a buffer bag (219), e.g., a 500 ml saline wash bag, is connected to the second assembly opening (218) of the inlet tube (255). When the clamp on the inlet tube (255) is released, the buffer moves by gravity through the second assembly opening (218) into the inlet tube (255). The buffer passes through the inlet valve (247) and the collection valve (245), enters the filter enclosure (210) through the filter enclosure inlet (225), passes through the leukocyte removal filter set within the filter enclosure (210), and rinses the retained lymphocytes. Filter. The buffer moves from the filter enclosure outlet (226) to the outlet tube (256), then passes through the outlet valve (247) and is collected in a waste collection bag (216) that may be the same waste collection bag used to collect the reaction mixture components that passed through the filter in the previous step, or a new waste collection bag replaced in place of the first waste collection bag, and then the buffer enters the second assembly opening (218). The optional wash step can be optionally performed multiple times by repeating the above process with additional buffer.

[0089] When all or substantially all of the reaction mixture within the blood bag (215) has passed through the filter (210) and an optional washing step is optionally performed, a reverse perfusion process is initiated to move fluid in the opposite direction of the assembly (200) for collecting lymphocytes retained by the filter set within the filter enclosure (210). Exemplary embodiments of the leukocyte removal filter assembly herein are adaptable for reperfusion. Before initiating the reverse perfusion process in the exemplary assembly (200), the outlet valve (247) is switched to the reperfusion position and the collection valve (245) is switched to the collection position. To initiate reperfusion, a buffer solution (e.g., PBS) (which may be, for example, in a 25 ml syringe) within the syringe (266) is passed into the outlet tube (256) by injection using the syringe (266). The buffer solution then enters the filter enclosure (210) through the filter enclosure outlet (226), and the lymphocytes retained by the filter set are moved from the filter enclosure (210) through the filter enclosure inlet (225) into the inlet tube (255). Lymphocytes, some of which may be gene modified and / or transduced at this point, and which contain some T cells and / or NK cells with associated retroviral particles, are then collected in a cell sample collection bag (265), which may be a 25 ml cryopreservation bag, after passing through the collection valve (245).

[0090] In some embodiments, kits for genetically modifying NK cells and / or, in exemplary embodiments, T cells are provided herein. The kit includes a leukapheresis filter assembly and any of the embodiments of the replication-incompetent retroviral vectors disclosed herein, typically contained in a tube or vial. The leukapheresis filtration assembly of such a kit typically includes a leukapheresis filter or set of leukapheresis filters within a filter enclosure, as exemplified by the exemplary assembly of FIG. 2, and a plurality of connected sterile tubes and a plurality of valves connected thereto, adapted for use in a single-use closed blood treatment system. Such a kit includes, in exemplary embodiments, an anticoagulant, a blood treatment buffer bag, a blood treatment waste collection bag, a blood treatment cell sample collection bag, and a sterile syringe, and optionally a blood collection bag. In exemplary embodiments, the kit includes a T cell activation element, such as anti-CD3, as disclosed in detail herein. Such activation elements can be provided in solution within a tube or vial containing retroviral particles, or in a separate tube or vial. In exemplary embodiments, the activation element is anti-CD3 associated with the surface of replication-incompetent retroviral particles. In exemplary embodiments, the replication-incompetent recombinant retroviral particles within the kit include a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and / or NK cells, and the one or more transcription units encode a first polypeptide comprising a chimeric antigen receptor (CAR) and optionally a lymphoproliferative element, according to any of the embodiments provided herein.

[0091] Steps and reaction mixtures for methods for genetically modifying lymphocytes Some embodiments of any method used in any aspect provided herein, which is a method for genetically modifying typically lymphocytes, PBMCs, and in exemplary embodiments NK cells and / or in further exemplary embodiments T cells, may include the step of collecting blood from a subject. The blood includes blood components including blood cells such as lymphocytes (e.g., T cells and NK cells) that can be used in the methods and compositions provided herein. In certain exemplary embodiments, the subject is a human subject suffering from cancer (i.e., a human cancer subject). It is noted that certain embodiments do not include such a step. However, in embodiments that include collecting blood from a subject, the blood can be collected or obtained from the subject by any suitable method known in the art, as discussed in more detail herein. For example, the blood can be collected by venipuncture or any other blood collection method from which a sample of blood is taken. In some embodiments, the amount of blood collected is from 50 ml to 250 ml, such as from 75 ml to 125 ml, or from 90 ml to 120 ml, or from 95 to 110 ml. In some embodiments, the amount of blood collected can be from 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 600, 700, 800, or 900 ml of the lower limit of the range to 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 600, 700, 800, or 900 ml, or up to 1 L of the upper limit of the range. In some embodiments, lymphocytes (e.g., T cells and / or NK cells) can be obtained by apheresis.In some embodiments, the amount of blood collected and processed during apheresis may be between 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.25, or 1.5 times the total blood volume of a subject at the lower limit of the range to 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, or 2.5 times the total blood volume of a subject at the upper limit of the range. The total blood volume of a human is typically in the range of 4.5 - 6 L, and thus, much more blood is collected and processed during apheresis than if blood were collected and then the lymphocytes therein were genetically modified and / or transduced, as in the exemplary embodiments herein.

[0092] In any of the aspects of the methods provided herein for genetically modifying lymphocytes (such as T cells and / or NK cells), regardless of whether blood is drawn from a control, the lymphocytes are contacted with replication-incompetent retroviral particles in a reaction mixture. The contact in any of the embodiments provided herein can be carried out, for example, within a blood bag or within a chamber of a closed system adapted for the processing of blood cells, as discussed in more detail herein. The transduction reaction mixture can include one or more buffers, ions, and media. With respect to retroviral particles and, in exemplary embodiments, with respect to lentiviral particles, in the specific exemplary reaction mixtures provided herein, the multiplicity of infection (MOI) is between 0.1 and 50, 0.5 and 50, 0.5 and 20, 0.5 and 10, 1 and 25, 1 and 15, 1 and 10, 1 and 5, 2 and 15, 2 and 10, 2 and 7, 2 and 3, 3 and 10, 3 and 15, or 5 and 15; or an MOI of at least 1 and less than 6, 11, or 51; or in some embodiments, there are replication-incompetent recombinant retroviral particles with an MOI between 5 and 10 units. In some embodiments, the MOI can be at least 0.1, 0.5, 1, 2, 2.5, 3, 5, 10, or 15. With respect to compositions and methods for transducing lymphocytes in blood, in certain embodiments, a higher MOI can be used than in methods where PBMCs are isolated and used in the reaction mixture. For example, exemplary embodiments of compositions and methods for transducing lymphocytes in whole blood assume 1×10 6 PBMCs / ml of blood and can use retroviral particles having an MOI between 1 and 50, 2 and 25, 2.5 and 20, 2.5 and 10, 4 and 6, or about 5, and in some embodiments between 5 and 20, 5 and 15, 10 and 20, or 10 and 15.

[0093] In an exemplary embodiment, this contact, and the reaction mixture in which the contact occurs, are carried out within a closed cell processing system, as discussed in more detail herein. Packaging cells, and in an exemplary embodiment a packaging cell line, and in particular an exemplary embodiment the packaging cells provided in certain aspects of this specification, can be used to generate replication-incompetent recombinant retroviral particles. The lymphocytes in the reaction mixture may be PBMCs, or in aspects of this specification that provide compositions and methods for transducing lymphocytes in whole blood, may be additional blood components including an anticoagulant and / or additional types of blood cells other than PBMCs. Indeed, in an exemplary embodiment of aspects of these compositions and methods for transducing lymphocytes in whole blood, the reaction mixture may be essentially whole blood, and typically an anticoagulant, retroviral particles, and a small amount of solution in which the retroviral particles are delivered to the whole blood.

[0094] In some of the reaction mixtures provided herein, T cells can be present between, for example, 10, 20, 30, or 40% of the lymphocytes in the reaction mixture at the lower limit of the range to 40, 50, 60, 70, 80, or 90% of the lymphocytes in the reaction mixture at the upper limit of the range. In an exemplary embodiment, T cells occupy between 10-90%, 20-90%, 30-90%, 40-90%, 40-80%, 45%-75% of the lymphocytes. In such embodiments, for example, NK cells can be present between 1, 2, 3, 4, or 5% of the lymphocytes in the reaction mixture at the lower limit of the range to 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14% of the lymphocytes in the reaction mixture at the upper limit of the range. In an exemplary embodiment, T cells occupy between 1-14%, 2-14%, 3-14%, 4-14%, 5-14%, 5-13%, 5-12%, 5-11%, or 5-10% of the lymphocytes in the reaction mixture.

[0095] In reaction mixtures related to aspects of compositions and methods for genetically modifying lymphocytes in whole blood provided herein, lymphocytes, including NK cells and T cells, can be present in a lower percentage of blood cells and a lower percentage of white blood cells in the reaction mixture than in methods that include a PBMC enrichment procedure prior to forming the reaction mixture. For example, in some embodiments of these aspects, more granulocytes or neutrophils than NK cells or even T cells are present in the reaction mixture. Details regarding the composition of the anticoagulant and one or more additional blood components present in the reaction mixture for aspects of genetically modifying lymphocytes in whole blood are provided in detail in other sections of this specification.

[0096] As disclosed herein, aspects of compositions and methods for transducing lymphocytes in whole blood typically do not include a PBMC enrichment step of the blood sample before the lymphocytes from the blood sample come into contact with retroviral particles in the reaction mixture disclosed herein for those aspects. However, in some embodiments, neutrophils / granulocytes are separated from other blood cells before the cells are contacted with replication-incompetent recombinant retroviral particles. In some embodiments, peripheral blood mononuclear cells (PBMCs) comprising peripheral blood lymphocytes (PBLs) such as T cells and / or NK cells are isolated from other components of the blood sample, for example using a PBMC enrichment procedure, before being combined with retroviral particles in the reaction mixture.

[0097] The PBMC enrichment procedure is a procedure in which PBMCs are enriched at least 25-fold, typically at least 50-fold, from other blood cell types. For example, PBMCs are thought to constitute less than 1% of the blood cells in whole blood. After the PBMC enrichment procedure, at least 30%, and in some cases up to 70%, of the cells isolated in the PBMC fraction are PBMCs. Higher enrichment of PBMCs may be achieved using some PBMC enrichment procedures. A variety of different PBMC enrichment procedures are known in the art. For example, the PBMC enrichment procedure is a Ficoll density gradient centrifugation process that separates major cell populations such as lymphocytes, monocytes, granulocytes, erythrocytes, etc. throughout a density gradient medium. In such a method, the aqueous medium contains Ficoll, a hydrophilic polysaccharide that forms a high-density solution. After overlaying whole blood on top of or below the density medium without mixing the two layers and then centrifuging, the cells are dispersed according to density and the PBMC fraction forms a thin white layer at the interface of the plasma and the density gradient medium (see, for example, Panda and Ravindran (2013) Isolation of Human PBMCs. BioProtoc. Vol. 3(3)). Additionally, the centrifugal force can be used to separate PBMCs from other blood components with Ficoll using the rotational force of the Sepax cell processing system.

[0098] In another PBMC enrichment method, an automated leukapheresis collection system (SPECTRA OPTIA® APHERESIS SYSTEM from TERUMO BCT, INC., Lakewood CO 80215, USA) is used to separate the inflow of whole blood from the target PBMC fraction using high-speed centrifugation, typically while returning effluent substances such as plasma, erythrocytes, and granulocytes to the donor, although in the methods provided herein, returning is optional. Further processing may be required to remove residual erythrocytes and granulocytes. Both methods involve time-consuming purification of PBMCs, and in the leukapheresis method, the presence and participation of the patient are required during the PBMC enrichment step.

[0099] As a further non-limiting example of the PBMC enrichment procedure, in some embodiments of the methods of transduction or genetic modification herein, PBMCs are isolated using a Sepax or Sepax 2 cell processing system (BioSafe). In some embodiments, PBMCs are isolated using a CliniMACS Prodigy cell processor (Miltenyi Biotec). In some embodiments, an automated apheresis separator is used to collect blood from a subject and pass the blood through a device that sorts specific cell types (such as PBMCs, etc.) and returns the remainder to the subject. Density gradient centrifugation can be performed after apheresis. In some embodiments, PBMCs are isolated using a leukocyte removal filter assembly. In some embodiments, magnetic bead activated cell sorting is then used to purify specific cell populations from PBMCs, such as PBLs or subsets thereof, according to cell phenotype (i.e., positive selection), and then use them in the reaction mixtures herein.

[0100] Other purification methods can also be used, such as substrate adhesion that utilizes a substrate that mimics the environment that T cells encounter during mobilization to purify T cells prior to addition to the reaction mixture, or negative selection where unwanted cells are targeted for removal using antibody conjugates that target unwanted cells prior to the formation of the reaction mixture of the contacting step. In some embodiments, erythrocyte rosetting can be used to remove erythrocytes prior to forming the reaction mixture. In other embodiments, hematopoietic stem cells can be removed prior to the contacting step, and thus in these embodiments hematopoietic stem cells are not present during the contacting step. In some embodiments herein, particularly for compositions and methods for transducing lymphocytes in whole blood, ABC transporter inhibitors and / or substrates are not present (i.e., not present in the reaction mixture in which contact occurs) either with or without optional incubation before, during, or both before and during contact, or at any step of the method.

[0101] In certain exemplary embodiments of any aspect provided herein, lymphocytes are genetically modified and / or transduced in vivo, in vitro, or ex vivo, with or without prior activation or stimulation; and / or further, in some embodiments, without ex vivo or in vitro activation or stimulation after the first contact with or without optional incubation, or without requiring ex vivo or in vitro activation or stimulation after the first contact with or without optional incubation. Thus, in exemplary embodiments, some, most, at least 25%, 50%, 60%, 70%, 75%, 80%, 90%, at least 95%, at least 99%, or all of the lymphocytes are resting when they are combined with retroviral particles to form a reaction mixture, and typically are resting when they contact the retroviral particles in the reaction mixture. In methods for genetically modifying lymphocytes such as T cells and / or NK cells in blood or its components, the lymphocytes can be contacted in their typical resting state as they exist in blood collected in vivo immediately prior to collection. In some embodiments, the T cells and / or NK cells consist of 95 - 100% resting cells (KI-67 -) ). In some embodiments, the T cells and / or NK cells contacted by replication-incompetent recombinant retroviral particles contain from 90, 91, 92, 93, 94, and 95% resting cells at the lower limit of the range to 96, 97, 98, 99, or 100% resting cells at the upper limit of the range. In some embodiments, the T cells and / or NK cells include naive cells. In some exemplary embodiments, the sub-embodiments of this paragraph are included in aspects of compositions and methods for transducing lymphocytes in whole blood.

[0102] Contact between T cells and / or NK cells and replication-incompetent recombinant retroviral particles can promote the transduction of T cells and / or NK cells by the replication-incompetent recombinant retroviral particles. Without being limited by theory, during the period of contact, the replication-incompetent recombinant retroviral particles recognize and bind to T cells and / or NK cells at the point when the retrovirus and the host cell membrane begin to fuse. Then, as the next step of the transduction process, the genetic material from the replication-incompetent recombinant retroviral particles enters the T cells and / or NK cells, and at that point the T cells and / or NK cells are "genetically modified" as the term is used herein. It is noteworthy that such a process can occur several hours or days after contact is initiated, and even after unrelated retroviral particles have been washed away. The genetic material is then typically integrated into the genomic DNA of the T cells and / or NK cells, and at that point the T cells and / or NK cells are "transduced" as the term is used herein. Thus, in an exemplary embodiment, any method for genetically modifying the lymphocytes (e.g., T cells and / or NK cells) herein is a method for transducing the lymphocytes (e.g., T cells and / or NK cells). By day 6 in vivo or ex vivo after contact is initiated, it is believed that the majority of the genetically modified cells have been transduced. Methods for transducing lentiviruses are known. Exemplary methods are described, for example, in Wang et al. (2012) J. Immunother. 35(9):689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506:97-114; and Cavalieri et al. (2003) Blood. 102(2):497-505. Throughout the present disclosure, transduced T cells and / or NK cells include the progeny of ex vivo transduced cells that retain at least some of the nucleic acids or polynucleotides that are integrated into the genome of the cells during ex vivo transduction.In the methods of the present specification that enumerate "reintroducing" the transfected cells, it is understood that such cells are typically not in a converted state when collected from the blood of a subject.

[0103] Many of the methods provided herein involve genetic modification and transfection of T cells and / or NK cells. Methods for genetically modifying and transfecting T cells and / or NK cells ex vivo using replication-incompetent recombinant retroviral particles, such as replication-incompetent recombinant lentiviral particles, are known in the art. The methods provided herein do not require ex vivo stimulation or activation in exemplary embodiments. Thus, this common step in previous methods can be avoided in the present method, although ex vivo stimulating molecules such as anti-CD3 and / or anti-CD28 beads may be present during subsequent contact and optional incubation. However, ex vivo stimulation is not required in the exemplary methods provided herein.

[0104] In certain exemplary embodiments of any aspect of the present specification, blood cells such as lymphocytes, particularly T cells and / or NK cells, are activated during contact or subsequent optional incubation and are not activated at all or for a period exceeding 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, or 8 hours prior to contact. In certain exemplary embodiments, activation by elements not present on the surface of retroviral particles is not required to genetically modify lymphocytes. Thus, such activation or stimulating elements are not required other than retroviral particles before, during, or after contact. Thus, these exemplary embodiments that do not require pre-activation or stimulation, as discussed in more detail herein, provide the ability to rapidly conduct in vitro experiments aimed at better understanding T cells and the biological mechanisms therein. Further, such methods provide for much more efficient commercial production of biological products produced using PBMCs, lymphocytes, T cells, or NK cells, and the development of such commercial production methods. Finally, such methods provide for more rapid ex vivo processing of lymphocytes (e.g., NK cells and particularly T cells) for adoptive cell therapy, fundamentally simplifying the provision of such therapies, for example, by providing point-of-care methods.

[0105] In an exemplary embodiment, the T cells and / or NK cells are not activated prior to contacting with the recombinant retrovirus in the methods herein, but the T cell activation element in the exemplary embodiment is present in the reaction mixture where the first contact between the recombinant retrovirus and the lymphocytes occurs. For example, such a T cell activation element may be in solution in the reaction mixture. For example, soluble anti-CD3 antibody can be present in the reaction mixture at 25-200, 50-150, 75-125, or 100 ng / ml during contact and any subsequent incubation. In an exemplary embodiment, the T cell activation element is associated with the retrovirus surface. The T cell activation element may be any T cell activation element provided herein. In an exemplary embodiment, the T cell activation element may be anti-CD3 such as anti-CD3 scFv or anti-CD3 scFvFc. Thus, in some embodiments, the replication-incompetent recombinant retrovirus particles can further comprise a T cell activation element, which, in a further exemplary example, is associated with the outer side of the retrovirus surface.

[0106] The contacting step of the methods provided herein for transducing lymphocytes in whole blood and / or for genetically modifying typically involves a population of retrovirus particles, typically while forming a transduction reaction mixture by suspending in a liquid buffer and / or medium, contacting with a population of blood cells, typically a population of blood cells including additional blood components other than PBMCs that are not present after an anticoagulant and / or PBMC enrichment procedure. Following this contact, an optional incubation period may be involved in this reaction mixture containing blood cells including retrovirus particles and lymphocytes (e.g., T cells and / or NK cells) in suspension, similar to other aspects provided herein. In a method of genetically modifying T cells and / or NK cells in blood or its components, the reaction mixture may comprise at least one, two, three, four, five, or all of the additional blood components disclosed herein, and in an exemplary embodiment, comprises one or more anticoagulants.

[0107] In any of the aspects provided herein, the transduction reaction mixture can be incubated at 23 - 39 °C, in some exemplary embodiments at 37 °C, in any incubation step after the initial contact of the retroviral particles and lymphocytes. In certain embodiments, the transduction reaction can be carried out at 37 - 39 °C for faster fusion / transduction. When contacted in the transduction reaction mixture, the cells and retroviral particles can be immediately processed to remove retroviral particles that remain free in the suspension and are not associated with the cells. Optionally, the cells in the suspension, and the retroviral particles that are free in the suspension or associated with the cells in the suspension, can be incubated for various lengths of time as provided herein for the contacting step of the methods provided herein. Prior to further steps, washing can be performed regardless of whether such cells are studied in vitro, studied ex vivo, or introduced into a subject.

[0108] Disclosed herein are exemplary methods that are much shorter and simpler than previous methods for genetically modifying lymphocytes, particularly NK cells, and in exemplary embodiments, T cells. Thus, in some embodiments, the contacting step in any of the methods provided herein for transducing and / or genetically modifying PBMCs or lymphocytes, typically T cells and / or NK cells, can be performed (or occur) during any of the periods provided herein including, but not limited to, the periods provided in the section on exemplary embodiments. For example, the contacting may be for less than 24 hours, such as less than 12 hours, less than 8 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes, or less than 15 minutes, but in any case, there is at least an initial contacting step where the retroviral particles and the cells contact in suspension in the transduction reaction mixture, after which the retroviral particles remaining in suspension that do not associate with the cells are separated from the cells and typically discarded. Without intending to be limited by theory, it should be noted that the contacting is typically considered to begin when a solution containing retroviral particles is added to a solution containing lymphocytes (such as T cells and / or NK cells), at which point the retroviral particles and the lymphocytes are combined together.

[0109] After such initial contact, in some embodiments, the reaction mixture containing cells and retroviral particles in suspension is incubated for a specified period without removing retroviral particles that remain free in solution and do not associate with cells. This incubation may sometimes be referred to herein as an optional incubation. Thus, in exemplary embodiments, the contact (including the initial contact and the optional incubation) can be carried out (or can occur) from a lower limit of the range of 30 seconds, or 1, 2, 5, 10, 15, 30 or 45 minutes, or 1, 2, 3, 4, 5, 6, 7 or 8 hours, to an upper limit of the range of 10 minutes, 15 minutes, 30 minutes, or 1, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, and 72 hours (as generally indicated herein, the lower limit of the selected range is less than the upper limit of the selected range). In certain exemplary embodiments, the contact step can be carried out from a lower limit of the range of 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, or 30 minutes to an upper limit of the range of 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, or 12 hours. In some embodiments, the contact step is carried out from a lower limit of the range of 30 seconds, 1 minute, and 5 minutes to an upper limit of the range of 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, or 8 hours. Thus, in some embodiments, after the reaction mixture is formed by adding retroviral particles to lymphocytes, the reaction mixture can be incubated for between 5 minutes and 12 hours, between 5 minutes and 10 hours, between 5 minutes and 8 hours, between 5 minutes and 6 hours, between 5 minutes and 4 hours, between 5 minutes and 2 hours, between 5 minutes and 1 hour, between 5 minutes and 30 minutes, or between 5 minutes and 15 minutes. In other embodiments, the reaction mixture can be incubated for between 15 minutes and 12 hours, between 15 minutes and 10 hours, between 15 minutes and 8 hours, between 15 minutes and 6 hours, between 15 minutes and 4 hours, between 15 minutes and 2 hours, between 15 minutes and 1 hour, between 15 minutes and 45 minutes, or between 15 minutes and 30 minutes. In other embodiments, the reaction mixture can be incubated for between 30 minutes and 12 hours, between 30 minutes and 10 hours, between 30 minutes and 8 hours, between 30 minutes and 6 hours, between 30 minutes and 4 hours, between 30 minutes and 2 hours, between 30 minutes and 1 hour, between 30 minutes and 45 minutes.In other embodiments, the reaction mixture can be incubated for between 1 hour and 12 hours, between 1 hour and 8 hours, between 1 hour and 4 hours, or between 1 hour and 2 hours. In another exemplary embodiment, the contact is carried out during only the first contact step without further incubation in the reaction mixture (without further incubation in the reaction mixture containing free retroviral particles in suspension and cells in suspension), or with an incubation in the reaction mixture for 5 minutes, 10 minutes, 15 minutes, 30 minutes or 1 hour.

[0110] After the indicated period for the initial contact and any optional incubation that can be part of the contact step, the blood cells or its T cell and / or NK cell-containing fraction in the reaction mixture are separated from retroviral particles that do not associate with such cells. For example, this can be carried out using a PBMC enrichment procedure (e.g., a ficoll gradient in a Sepax unit), or in a particular exemplary embodiment provided herein, by filtering the reaction mixture on a leukoreduction filter set assembly and then harvesting the leukocytes containing T cells and NK cells. In another embodiment, this can be carried out by centrifuging the reaction mixture at a relative centrifugal force of less than 500 g, such as 400 g, or between 300 and 490 g, or between 350 and 450 g. Such centrifugation to separate retroviral particles from cells can be carried out, for example, for between 5 minutes and 15 minutes, or between 5 minutes and 10 minutes. In an exemplary embodiment of separating cells from retroviral particles that do not associate with the cells using centrifugal force, such g-forces are typically lower than the g-forces successfully used in spinoculation procedures.

[0111] In some exemplary embodiments, the methods provided herein do not include performing spinoculation in any manner. In some embodiments, spinoculation is included as part of the contacting step. In exemplary embodiments, when spinoculation is performed, since the spinoculation time provides the incubation time of the above optional incubation, there is no additional incubation as part of the contact. In other embodiments, there is additional incubation after spinoculation for between 15 minutes and 4 hours, or between 15 minutes and 2 hours, or between 15 minutes and 1 hour. Spinoculation can be performed, for example, for between 30 minutes and 120 minutes, typically at least 60 minutes, for example between 60 minutes and 180 minutes, or between 60 minutes and 90 minutes. Spinoculation is typically performed at a relative centrifugal force of at least 800 g, more typically at least 1,200 g, in a centrifuge, for example between 800 g and 2,400 g, or between 800 g and 1,800 g, or between 1,200 g and 2,400 g, or between 1,200 g and 1,800 g. After spinoculation, such methods typically include an additional step of resuspending the pelleted cells and retroviral particles and then removing retroviral particles that do not associate with the cells according to the steps above when spinoculation is not performed.

[0112] In embodiments that include an optional incubation as part of the contacting step and spinoculation, the spinoculation can be performed between 4°C and 42°C, or between 20°C and 37°C. In certain exemplary embodiments, spinoculation is not performed and the contact and associated optional incubation are performed at 20 - 25°C for 4 hours or less, 2 hours or less, 1 hour or less, 30 minutes or less, 15 minutes or less, or between 15 minutes and 2 hours, 15 minutes and 1 hour, or 15 minutes and 30 minutes.

[0113] In some embodiments of the methods and compositions disclosed herein, between 5% and 85% of the total lymphocytes harvested from blood are genetically modified. In some embodiments, the percentage of genetically modified and / or transduced lymphocytes is between 1, 5, and 10% of the lower limit of the range and 15, 20, 25, 30, 40, 50, 60, 70, 80, and 85% of the upper limit of the range. In some embodiments, the percentage of genetically modified and / or transduced T cells and NK cells is at least 5%, at least 10%, at least 15%, or at least 20%. As shown in the examples herein, in the exemplary methods provided herein for transducing lymphocytes in whole blood, between 1% and 20%, or between 1% and 15%, or between 5% and 15%, or between 7% and 12%, or about 10% of the lymphocytes are genetically modified and / or transduced.

[0114] A method of genetically modifying lymphocytes provided according to any method herein typically involves inserting into a cell a polynucleotide comprising one or more transcription units encoding a CAR or a lymphoproliferative element, or in an exemplary embodiment, both a CAR and a lymphoproliferative element, according to any of the embodiments of the CARs and lymphoproliferative elements provided herein. Such CARs and lymphoproliferative elements can be provided to support shorter and more simplified methods provided herein that can support gene modification and / or expansion of transduced T cells and / or NK cells after contact and any incubation. Thus, in an exemplary embodiment of any method provided herein, the lymphoproliferative element can be delivered from the genome of retroviral particles within genetically modified and / or transduced T cells and / or NK cells, such that those cells have the increased proliferation and / or survival characteristics disclosed in the section on lymphoproliferative elements herein. In an exemplary embodiment of any method provided herein, the genetically modified T cells or NK cells can engraft in vivo in a mouse and / or can be enriched in vivo in a mouse for at least 7, 14, or 28 days. One of skill in the art will recognize that such mice can be treated or otherwise genetically modified such that any immunological differences between genetically modified T cells and / or NK cells do not result in an immune response induced in the mouse against any components of the lymphocytes transduced with replication-incompetent recombinant retroviral particles.

[0115] In the contacting step, for example, when cells and retroviral particles first come into contact, or during any subsequent optional incubation period with a reaction mixture containing retroviral particles and cells in suspension in a medium, or during cell culture and / or various washing steps in any of the embodiments provided herein, the medium can include a basal medium such as a commercially available medium for ex vivo T cell and / or NK cell culture. Non-limiting examples of such media include X-VIVO™ 15 serum-free hematopoietic cell culture medium of known composition (Lonza) (2018 catalog numbers BE02-060F, BE02-00Q, BE-02-061Q, 04-744Q, or 04-418Q), ImmunoCult™-XF T cell expansion medium (STEMCELL Technologies) (2018 catalog number 10981), PRIME-XV® T cell expansion XSFM (Irvine Scientific) (2018 catalog number 91141), AIM V® medium CTS™ (Therapeutic Grade) (Thermo Fisher Scientific (hereinafter referred to as "ThermoFisher")), or CTS™ Optimizer™ medium (Thermo Fisher) (2018 catalog numbers A10221-01 (basal medium (bottle)), and A10484-02 (additive), A10221-03 (basal medium (bag)), A1048501 (basal medium and additive kit (bottle)), and A1048503 (basal medium and additive kit (bag)). Such media may be serum-free formulations of known composition manufactured in accordance with cGMP. The medium may be xeno-free and complete. In some embodiments, the basal medium has been cleared by regulatory authorities for use in ex vivo cell processing, such as a device that has cleared FDA 510(k).In some embodiments, the medium is a basal medium, with or without the attached T cell growth additive of catalog number A1048501 (CTS™ OpTmizer™ T Cell Expansion SFM, bottle format) or A1048503 (CTS™ OpTmizer™ T Cell Expansion SFM, bag format), both available from Thermo Fisher (Waltham, MA). Additives such as human serum albumin, human AB+ serum, and / or serum from the subject may be added to the transduction reaction mixture. Auxiliary cytokines may be added to the transduction reaction mixture such as IL2, IL7, or IL15, or those found in human serum. In certain embodiments, dGTP may be added to the transduction reaction.

[0116] In some embodiments of any of the methods herein that include the step of genetically modifying lymphocytes (e.g., T cells and / or NK cells), the cells can be contacted with retroviral particles without prior activation. In some embodiments of any of the methods herein that include the step of genetically modifying T cells and / or NK cells, the T cells and / or NK cells are not incubated on a substrate that adheres to monocytes for more than 4 hours, or in another embodiment more than 6 hours, or in another embodiment more than 8 hours prior to transduction. In one exemplary embodiment, the T cells and / or NK cells are incubated overnight on an adherent substrate to remove monocytes prior to transduction. In another embodiment, the method can include incubating the T cells and / or NK cells on an adherent substrate that binds to monocytes within 30 minutes, 1 hour, or 2 hours prior to transduction. In another embodiment, the T cells and / or NK cells are not subjected to the step of removing monocytes by incubation on an adherent substrate prior to the transduction step. In another embodiment, the T cells and / or NK cells are not incubated or exposed to bovine serum, such as fetal bovine serum, during or prior to the contacting step and / or the genetic modification and / or transduction step.

[0117] Some or all steps of the methods for genetic modification provided herein, or the use of such methods, are performed in a closed system. Thus, reaction mixtures formed by such methods, as well as genetically modified and / or transduced lymphocytes (e.g., T cells and / or NK cells) produced by such methods, can be contained within such a closed system. A closed system is a cell processing system that is generally closed or completely closed to the environment, such as the environment within a room or even within a draft, or the environment outside conduits such as the tubes and chambers of a system in which cells are processed and / or transported. One of the greatest risks to safety and regulatory control in cell processing procedures is the risk of contamination due to frequent exposure to the environment, as seen in conventional open cell culture systems. In particular, several commercial processes have been developed that focus on the use of disposable (single-use) equipment to mitigate this risk in the absence of antibiotics. However, even when used in a sterile state, there is always a risk of contamination by opening the flask to take a sample or adding additional growth medium. To overcome this problem, the methods provided herein are typically ex vivo methods and are typically performed within a closed system. Such processes can be designed and operated so that the product is not exposed to the external environment. Movement of materials is performed through sterile connections such as sterile tubing and sterile welded connections. Air for gas exchange can occur through a gas permeable membrane and through a 0.2 μm filter to prevent exposure to the environment. In some exemplary embodiments, the method is performed on T cells, for example, to provide genetically modified T cells.

[0118] Such a method of a closed system can be performed using commercially available devices. Various closed system devices can be used in the various steps within the method, and in order to prevent cells or media from being exposed to the environment, the cells can be moved between these devices using tubing and connections such as welding, luer, spike, or autoclave ports. For example, blood can be collected in an IV bag or syringe, optionally containing an anticoagulant, and transferred to a Sepax 2 device (Biosafe) for PBMC enrichment and isolation. In other embodiments, whole blood can be filtered and leukocytes can be collected using a leukocyte removal filter assembly. The isolated PBMCs or isolated leukocytes can be transferred to the chamber of a G-Rex device for optional activation, transduction, and optional expansion. Alternatively, the collected blood can be converted into a blood bag, for example the bag in which it was collected. Finally, the Sepax 2 device can be used to harvest the cells and collect them in another bag. This method can be performed with any device or combination of devices compatible with closed system T cell and / or NK cell generation. Non-limiting examples of such devices include G-Rex devices (Wilson Wolf), GatheRex (Wilson Wolf), Sepax 2 (Biosafe), WAVE Bioreactors (General Electric), CultiLife Cell Culture bags (Takara), PermaLife bags (OriGen), CliniMACS Prodigy (Miltenyi Biotec), and VueLife bags (Saint-Gobain). In an exemplary embodiment, the optional activation, transduction, and optional expansion can be performed within the same chamber or container of the closed system. For example, in an exemplary embodiment, the chamber can be the chamber of a G-Rex device, and the PBMCs or leukocytes can be transferred to the chamber of the G-Rex device after they have been enriched and isolated, and can remain within the same chamber of the G-Rex device until they are harvested.

[0119] The methods provided herein can include transferring blood and cells and / or fractions thereof, as well as lymphocytes before or after they contact retroviral particles, between containers within a closed system, and thus without environmental exposure. The containers used in the closed system can be, for example, tubes, bags, syringes, or other containers. In some embodiments, the containers are those used in a research facility. In some embodiments, the containers are those used in commercial production. In other embodiments, the containers can be collection containers used in a blood collection process. The methods for genetic modification herein typically include a contacting step in which lymphocytes are contacted with replication-incompetent recombinant retroviral particles. The contacting in some embodiments can be carried out within a container, for example, within a blood bag. The blood and its various lymphocyte-containing fractions can be transferred within the closed system from one container to another (e.g., from a first container to a second container) for contacting. The second container can be a cell processing compartment of a closed device such as a G-Rex device. In some embodiments, after contacting, the genetically modified (e.g., transduced) cells can be transferred to a different container within the closed system (i.e., without exposure to the environment). At either before or after this transfer, the cells are typically washed within the closed system to remove substantially all or all of the retroviral particles. In some embodiments, the processes disclosed herein from blood collection to contacting (e.g., transduction), optional incubation and isolation after incubation, and optional washing are carried out from a lower limit of 15 minutes, 30 minutes, or 1, 2, 3, or 4 hours to an upper limit of 4, 8, 10, or 12 hours.

[0120] Although not limited to theory, in a non-limiting, exemplary method, delivery of a polynucleotide encoding a lymphoproliferative factor to ex vivo resting T cells and / or NK cells (which can be integrated into the genome of the T cells or NK cells) provides the cells with an in vivo expansion driving force without the need to lymphodeplete the host. Thus, in an exemplary embodiment, the subject is not exposed to a lymphodepleting agent within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days, or within 1 month, 2 months, 3 months, or 6 months, during contact, and / or within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days, or within 1 month, 2 months, 3 months, or 6 months after the modified T cells and / or NK cells are reintroduced into the subject. Further, in a non-limiting, exemplary embodiment, the methods provided herein can be performed without exposing the subject to a lymphodepleting agent during the step in which replication-incompetent recombinant retroviral particles are contacting the subject's resting T cells and / or resting NK cells and / or throughout the ex vivo method. Thus, methods for expanding genetically modified T cells and / or NK cells in a subject in vivo are a feature of some embodiments of the present disclosure. In an exemplary embodiment, such methods have no or substantially no ex vivo proliferation.

[0121] In non-limiting, exemplary embodiments of any aspect provided herein, the entire method / process from blood collection from a subject after ex vivo transduction of T cells and / or NK cells to reintroduction of blood into the subject can be carried out over a period of less than 48 hours, less than 36 hours, less than 24 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, 2 hours, or less than 2 hours. In other embodiments, the entire method / process from blood collection / blood draw from a subject to reintroduction of blood into the subject after ex vivo transduction of T cells and / or NK cells is, in the non-limiting, exemplary embodiments herein, between 1 hour and 12 hours, or between 2 hours and 8 hours, or between 1 hour and 3 hours, or between 2 hours and 4 hours, or between 2 hours and 6 hours, or between 4 hours and 12 hours, or between 4 hours and 24 hours, or between 8 hours and 24 hours, or between 8 hours and 36 hours, or between 8 hours and 48 hours, or between 12 hours and 24 hours, or between 12 hours and 36 hours, or between 12 hours and 48 hours, or over a period from the lower limits of the range of 15, 30, 60, 90, 120, 180, and 240 minutes to the upper limits of the range of 120, 180, and 240, 300, 360, 420, and 480 minutes. In other embodiments, the entire method / process from blood collection / blood draw from a subject to reintroduction of blood into the subject after ex vivo transduction of T cells and / or NK cells is carried out over a period from the lower limits of the range of 1, 2, 3, 4, 6, 8, 10, and 12 hours to the upper limits of the range of 8, 9, 10, 11, 12, 18, 24, 36, or 48 hours. In some embodiments, the genetically modified T cells and / or NK cells are separated from replication-incompetent recombinant retroviral particles after the period during which contact occurs.

[0122] The methods provided herein for genetically modifying lymphocytes and related methods for performing adoptive cell therapy can be carried out in a significantly shorter time than previous methods, enabling fundamental improvements in patient care and safety as well as product manufacturability. Thus, such processes are expected to be advantageous from the perspective of regulatory agencies responsible for approving such processes when carried out in vivo for therapeutic purposes. For example, in a non-limiting example of any of the aspects provided herein, including a subject, the subject can remain in the same building (e.g., an infusion clinic) or room as the device that processes the blood or sample for as long as the sample is being processed, until the modified T cells and / or NK cells are reintroduced into the patient. In a non-limiting, exemplary embodiment, the subject can remain within 100, 50, 25, or 12 feet or arm's length of the line of the site and / or the blood or cells being processed throughout the method / process from blood collection / sampling from the subject to reintroduction of the blood into the subject after ex vivo transduction of T cells and / or NK cells. In other non-limiting, exemplary embodiments, the subject remains awake and / or at least one person can continuously monitor the blood or cells of the subject being processed throughout the method / process from blood collection / sampling from the subject to reintroduction of the blood into the subject after ex vivo transduction of T cells and / or NK cells. Due to the improvements provided herein, adoptive cell therapy, and / or the method / process for transducing resting T cells and / or NK cells from blood collection / sampling from the subject to reintroduction of the blood into the subject after ex vivo transduction of T cells and / or NK cells can be carried out with continuous human monitoring. In other non-limiting, exemplary embodiments, blood cells are not incubated in a room without a person at any point throughout the method / process from blood collection / sampling from the subject to reintroduction of the blood into the subject after ex vivo transduction of T cells and / or NK cells.In other non-limiting exemplary embodiments, the entire method / process from blood collection / blood sampling from a subject to the reintroduction of blood into the subject after ex vivo transduction of T cells and / or NK cells is performed next to the subject, and / or in the same room as the subject, and / or next to the subject's bed or chair. Thus, misidentification of sample IDs and long and costly incubations over several days or weeks can be avoided. This is further provided by the fact that the methods provided herein are readily adaptable to closed and automated blood processing systems where the blood samples and their components to be reintroduced into the subject only contact disposable single-use components.

[0123] The methods provided herein for genetically modifying and / or transducing lymphocytes such as T cells and / or NK cells may be part of a method for performing adoptive cell therapy. Typically, a method for performing adoptive cell therapy includes the step of collecting blood from a subject and the step of returning genetically modified and / or transduced lymphocytes (e.g., T cells and / or NK cells) to the subject. The present disclosure provides various treatment methods using CARs. The CARs of the present disclosure, when present in T lymphocytes or NK cells, can mediate cytotoxicity against target cells. The CARs of the present disclosure bind to antigens present on target cells, thereby mediating the killing of target cells by T lymphocytes or NK cells genetically modified to produce the CAR. The ASTR of the CAR binds to antigens present on the surface of the target cells. The present disclosure provides a method of killing or inhibiting the growth of target cells, the method comprising contacting a cytotoxic immune effector cell (e.g., a cytotoxic T lymphocyte or NK cell) genetically modified to produce a CAR of a subject such that the T cell or NK cell recognizes an antigen present on the surface of the target cell and mediates the killing of the target cell. The target cells may be, for example, cancer cells, and the autologous cell therapy herein may be, in some exemplary embodiments, a method for treating cancer. In these embodiments, the subject may be an animal or human suspected of having cancer, or more typically, a subject known to have cancer.

[0124] In some embodiments of any of the methods provided herein for genetically modifying lymphocytes (e.g., T cells and / or NK cells), and in aspects related to the use of replication-incompetent recombinant retroviral particles in the manufacture of a kit for genetically modifying T cells and / or NK cells of a subject, the genetically modified and / or transduced lymphocytes (e.g., T cells and / or NK cells) or a population thereof are introduced or reintroduced into the subject. The introduction or reintroduction of the genetically modified lymphocytes into the subject can occur via any pathway known in the art. For example, the introduction or reintroduction can be delivery via injection into a blood vessel of the subject. In some embodiments, the genetically modified and / or transduced lymphocytes (e.g., T cells and / or NK cells) or a population thereof are subjected to four or fewer cell divisions ex vivo prior to being introduced or reintroduced into the subject. In some embodiments, the lymphocytes used in such methods are resting T cells and / or resting NK cells that have been in contact with replication-incompetent recombinant retroviral particles for between 1 hour and 12 hours. In some embodiments, between the time when blood is drawn from the subject and the time when the genetically modified T cells and / or NK cells are reintroduced into the subject, a time of within 12 hours, within 10 hours, within 8 hours, within 6 hours, within 4 hours, within 2 hours, or within 1 hour elapses. In some embodiments, all steps after the blood is drawn and before the blood is reintroduced are performed in a closed system where a person monitors the closed system throughout the process.

[0125] In some embodiments of the methods and compositions disclosed herein, genetically modified T cells and / or NK cells are returned, reintroduced, reinfused, or otherwise delivered to a subject without additional ex vivo manipulation such as stimulation and / or activation of the T cells and / or NK cells. In conventional methods, ex vivo manipulation is used for stimulation / activation of T cells and / or NK cells and for expansion of genetically modified T cells and / or NK cells prior to introducing the genetically modified T cells and / or NK cells into a subject. In prior art methods, this generally takes several days or weeks and the subject has to return to the clinic for transfusions several days or weeks after the initial blood draw. In some embodiments of the methods and compositions disclosed herein, T cells and / or NK cells are not ex vivo stimulated, for example, by exposure to anti-CD3 / anti-CD28 solid supports such as beads coated with anti-CD3 / anti-CD28, prior to contacting the T cells and / or NK cells with replication-incompetent recombinant retroviral particles. Thus, provided herein are methods without ex vivo expansion. In other embodiments, genetically modified T cells and / or NK cells are not expanded ex vivo or are expanded only for a small number of cell divisions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 rounds of cell division), but rather expand in vivo, i.e., within the subject, or preferentially expand in vivo. In some embodiments, no additional media is added to allow for further expansion of the cells. In some embodiments, cell production of primary blood lymphocytes (PBL) does not occur while the PBL are in contact with replication-incompetent recombinant retroviral particles. In an exemplary embodiment, cell production of PBL does not occur while the PBL are ex vivo. In conventional methods of adoptive cell therapy, the subject has lymphocyte depletion prior to reinfusion of the genetically modified T cells and / or NK cells. In some embodiments, the patient or subject does not have lymphocyte depletion prior to blood draw. In some embodiments, the patient or subject does not have lymphocyte depletion prior to reinfusion of the genetically modified T cells and / or NK cells.However, embodiments of the methods and compositions disclosed herein can also be used with pre-activated or pre-stimulated T cells and / or NK cells. In some embodiments, the T cells and / or NK cells can be stimulated ex vivo by exposure to an anti-CD3 / anti-CD28 solid support prior to contacting the T cells and / or NK cells with replication-incompetent recombinant retroviral particles. In some embodiments, the T cells and / or NK cells can be exposed to the anti-CD3 / anti-CD28 solid support for less than 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, or 24 hours prior to the T cells and / or NK cells contacting replication-incompetent recombinant retroviral particles (including no exposure). In an exemplary embodiment, the T cells and / or NK cells can be exposed to the anti-CD3 / anti-CD28 solid support for less than 1, 2, 3, 4, 6, or 8 hours prior to the T cells and / or NK cells contacting replication-incompetent recombinant retroviral particles.

[0126] In some exemplary embodiments, the cells are introduced or reintroduced into the subject by intravenous or arterial injection. In any of the embodiments disclosed herein, the number of T cells and / or NK cells re-infused into the subject can range from a lower limit of 1×10 3 , 2.5×10 3 , 5×10 3 , 1×10 4 , 2.5×10 4 , 5×10 4 , 1×10 5 , 2.5×10 5 , 5×10 5 , 1×10 6 , 2.5×10 6 , 5×10 6 , and 1×10 7 cells / kg to an upper limit of 5×10 4 , 1×10 5 , 2.5×10 5 , 5×10 5 , 1×10 6 , 2.5×10 6 , 5×10 6 , 1×10 7 , 2.5×107 , 5×10 7 , and 1×10 8 It may also be between cells / kg. In an exemplary embodiment, the number of T cells and / or NK cells re-infused or otherwise delivered to a subject is from a lower limit of the range of 1×10 4 , 2.5×10 4 , 5×10 4 , and 1×10 5 cells / kg to an upper limit of the range of 2.5×10 4 , 5×10 4 , 1×10 5 , 2.5×10 5 , 5×10 5 , and 1×10 6 cells / kg. In some embodiments, the number of PBLs re-infused or otherwise delivered to a subject is less than 5×10 5 , 1×10 6 , 2.5×10 6 , 5×10 6 , 1×10 7 , 2.5×10 7 , 5×10 7 , and 1×10 8 cells, may be the lower limit of the range, or may be the upper limit of the range of 2.5×10 6 , 5×10 6 , 1×10 7 , 2.5×10 7 , 5×10 7 , 1×10 8 , 2.5×10 8 , 5×10 8 , and 1×10 9 cells. In some embodiments, the number of T cells and / or NK cells available for infusion or re-infusion into a 70 kg subject or patient is between 7×10 5 ~2.5×10 8 cells. In other embodiments, the number of T cells and / or NK cells available for transduction is about 7×10 6 plus or minus 10%.

[0127] Engineered signaling polypeptide(s) In some embodiments, the replication - incompetent recombinant retroviral particles used to contact T cells and / or NK cells have a polynucleotide or nucleic acid having one or more transcription units encoding one or more engineered signaling polypeptides. In some embodiments, the engineered signaling polypeptide comprises, in combination with one or more intracellular activation domains, optionally one or more regulatory domains (e.g., co - stimulatory domains), and optionally one or more T - cell survival motifs, any combination of an extracellular domain (e.g., an antigen - specific targeting region or ASTR), a stalk, and a transmembrane domain. In exemplary embodiments, at least one, two, or all of the engineered signaling polypeptides are lymphoproliferative elements (LEs) such as chimeric antigen receptors (CARs) or chimeric lymphoproliferative elements (CLEs). In some embodiments, at least one, two, or all of the engineered signaling polypeptides are recombinant T - cell receptors (TCRs). In some embodiments, when two signaling polypeptides are utilized, one encodes a lymphoproliferative element and the other encodes a chimeric antigen receptor (CAR) comprising an antigen - specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. For any domain of the engineered signaling polypeptides disclosed herein, exemplary sequences can be found in WO2019 / 055946, which is hereby incorporated by reference in its entirety. Those skilled in the art will recognize that such engineered polypeptides can also be referred to as recombinant polypeptides. Engineered signaling polypeptides such as CARs, recombinant TCRs, LEs, and CLEs provided herein are typically transgenes for lymphocytes, particularly T cells and NK cells, especially T cells and / or NK cells engineered using the methods and compositions provided herein, for expressing such signaling polypeptides.

[0128] Extracellular domain In some embodiments, the engineered signaling polypeptide comprises an extracellular domain that is a member of a specific binding pair. For example, in some embodiments, the extracellular domain may be the extracellular domain of a cytokine receptor, or a mutant thereof, or a hormone receptor, or a mutant thereof. Such mutant extracellular domains in some embodiments have been reported to be constitutively active when expressed in at least some cell types. In an exemplary embodiment, such extracellular and transmembrane domains do not include a ligand-binding region. Such domains are present in the engineered signaling polypeptide and are thought not to bind ligands when expressed in B cells, T cells, and / or NK cells. Mutations of such receptor mutants can occur in the region near the extracellular membrane. Without being limited by theory, mutations in at least some extracellular domains (and some extracellular transmembrane domains) of the engineered signaling polypeptides provided herein are involved in signaling of the engineered signaling polypeptide in the absence of ligand by bringing together activation chains that are not normally together. Further embodiments regarding extracellular domains that include mutations in the extracellular domain can be found, for example, in the section on lymphoproliferative elements herein.

[0129] In certain exemplary embodiments, the extracellular domain includes a dimerization motif. In an exemplary embodiment, the dimerization motif includes a leucine zipper. In some embodiments, the leucine zipper is derived from a jun polypeptide, such as c-jun. Further embodiments regarding extracellular domains that include a dimerization motif can be found, for example, in the section on lymphoproliferative elements herein.

[0130] In certain embodiments, the extracellular domain is an antigen-specific targeting region (ASTR), which may also be referred to herein as an antigen-binding domain. Specific binding pairs include, but are not limited to, antigen-antibody binding pairs, ligand-receptor binding pairs, and the like. Thus, members of specific binding pairs suitable for use with the engineered signal transduction polypeptides of the present disclosure include ASTRs that are antibodies, antigens, ligands, ligand-binding domains of receptors, receptors, ligand-binding domains of receptors, and affibodies.

[0131] An ASTR suitable for use with the engineered signal transduction polypeptides of the present disclosure may be any antigen-binding polypeptide. In certain embodiments, the ASTR is an antibody such as a full-length antibody, single-chain antibody, Fab fragment, Fab’ fragment, (Fab’)2 fragment, Fv fragment, and bivalent single-chain antibody or diabody.

[0132] In some embodiments, the ASTR is a single-chain Fv (scFv). In some embodiments, the heavy chain is positioned at the N-terminus of the light chain in the engineered signal transduction polypeptide. In other embodiments, the light chain is positioned at the N-terminus of the heavy chain in the engineered signal transduction polypeptide. In any of the disclosed embodiments, the heavy and light chains may be separated by a linker, as discussed in more detail herein. In any of the disclosed embodiments, the heavy or light chain may be at the N-terminus of the engineered signal transduction polypeptide and is typically the C-terminus of another domain such as a signal sequence or peptide.

[0133] Other antibody-based recognition domains (cAb VHH (camel antibody variable domain) and humanized versions, IgNAR VH (shark antibody variable domain) and humanized versions, sdAb VH (single domain antibody variable domain) and “camelized” antibody variable domains are suitable for use with the engineered signal transduction polypeptides and methods of using the engineered signal transduction polypeptides of the present disclosure. In some cases, they are T cell receptor (TCR)-based recognition domains.

[0134] Certain embodiments of any aspect or embodiment of the specification that includes a CAR comprise CARs having an extracellular domain engineered to employ an endogenous TCR signaling complex and a CD3Z signaling pathway. In one embodiment, the chimeric antigen receptor ASTR is fused to one of the endogenous TCR complex chains (e.g., TCR alpha, CD3E, etc.) to facilitate incorporation into the TCR complex and signaling via the endogenous CD3Z chain. In other embodiments, the CAR comprises a first scFv or protein that binds to the TCR complex and a second scFv or protein that binds to a target antigen (e.g., a tumor antigen). In another embodiment, the TCR may be a single-chain TCR (scTv, a single-chain two-domain TCR including VαVβ). Finally, it is also possible to generate an scFv that recognizes a specific MHC / peptide complex and thereby functions as a surrogate TCR. Such a peptide / MHC scFv binding factor can be used in many of the same configurations as a CAR.

[0135] In some embodiments, ASTR may be a multispecificity, e.g., a bispecific antibody. A multispecific antibody has binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for one target antigen and the other is for another target antigen. In certain embodiments, the bispecific antibody may bind to two different epitopes of a target antigen. Bispecific antibodies can also be used to localize a cytotoxic agent to cells expressing the target antigen. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0136] ASTRs suitable for use in engineered signaling polypeptides of the present disclosure can have various antigen-binding specificities. In some cases, the antigen-binding domain is specific for an epitope present in an antigen expressed (synthesized) by a target cell. In one example, the target cell is a cancer cell-associated antigen. Cancer cell-associated antigens can be, for example, antigens associated with breast cancer cells, B-cell lymphoma, Hodgkin lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma, lung cancer cells (e.g., small cell lung cancer cells), non-Hodgkin B-cell lymphoma (B-NHL) cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells (e.g., small cell lung cancer cells), melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, glioma, glioblastoma, medulloblastoma, colorectal cancer cells, and the like. Cancer cell-associated antigens can also be expressed by non-cancerous cells.

[0137] Non-limiting examples of antigens to which an ASTR of an engineered signaling polypeptide can bind include, for example, CD19, CD20, CD38, CD30, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high-molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-α2, GD2, Axl, Ror2, and the like.

[0138] In some embodiments, a member of a specific binding pair suitable for use in an engineered signaling polypeptide is an ASTR that is a ligand of a receptor. Ligands include, but are not limited to, hormones (e.g., erythropoietin, growth hormone, leptin, etc.); cytokines (e.g., interferons, interleukins, certain hormones, etc.); growth factors (e.g., heregulin; vascular endothelial growth factor (VEGF), etc.); integrin-binding peptides (e.g., peptides containing the sequence Arg-Gly-Asp (SEQ ID NO: 1)), and the like.

[0139] When a member of a specific binding pair of an engineered signal transduction polypeptide is a ligand, the engineered signal transduction polypeptide can be activated in the presence of the second member of the specific binding pair, and the second member of the specific binding pair is a receptor for the ligand. For example, when the ligand is VEGF, the second member of the specific binding pair may be a VEGF receptor including a soluble VEGF receptor.

[0140] As described above, in some cases, the member of the specific binding pair included in the engineered signal transduction polypeptide is a receptor, such as a receptor for a ligand, a co-receptor, etc., which is ASTR. The receptor may be a ligand-binding fragment of the receptor. Suitable receptors include, but are not limited to, growth factor receptors (such as VEGF receptor); killer cell lectin-like receptor subfamily K, member 1 (NKG2D) polypeptide (receptor for MICA, MICB, and ULBP6); cytokine receptors (such as IL-13 receptor; IL-2 receptor, etc.); CD27; natural cytotoxicity receptor (NCR) (such as NKP30 (NCR3 / CD337) polypeptide (receptor for HLA-B-associated transcript 3 (BAT3) and B7-H6), etc.).

[0141] In certain embodiments of any of the aspects provided herein that include ASTR, the ASTR can be directed to an intermediate protein that links the ASTR to a target molecule expressed on the target cell. The intermediate protein may be endogenously expressed or exogenously introduced, and may be native, engineered, or chemically modified. In certain embodiments, the ASTR may be an anti-tag ASTR such that at least one tagged intermediate, typically an antibody-tag conjugate, is included between a tag recognized by the ASTR and a target molecule expressed on the target cell, typically a protein target. Thus, in such embodiments, the ASTR binds to the tag, and the tag is conjugated to an antibody against an antigen on a target cell such as a cancer cell. Non-limiting examples of tags include fluorescein isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase, and maltose binding protein. Therefore, the ASTR includes a molecule that binds to the tag.

[0142] Stalk In some embodiments, the engineered signaling polypeptide is extracellular and includes a stalk located in the portion of the engineered signaling polypeptide that intervenes between ASTR and the transmembrane domain. In some embodiments, the stalk has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to the wild-type CD8 stalk region (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO: 2)), at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to the wild-type CD28 stalk region (FCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 3)), or at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to the wild-type immunoglobulin heavy chain stalk region. In the engineered signaling polypeptide, the stalk used allows the antigen-specific targeting region, and typically the entire engineered signaling polypeptide, to retain increased binding to the target antigen.

[0143] The stalk region can have a length of about 4 amino acids to about 50 amino acids, such as about 4 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, or about 40 aa to about 50 aa.

[0144] In some embodiments, the stalk of the engineered signaling polypeptide includes at least one cysteine. For example, in some embodiments, the stalk can include the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 4). If present, the cysteine of the stalk of the first engineered signaling polypeptide can be available to form a disulfide bond with the stalk of the second engineered signaling polypeptide.

[0145] The stalk may contain an immunoglobulin hinge region amino acid sequence known in the art. See, for example, Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res. 14:1779. By way of non-limiting example, the immunoglobulin hinge region may contain a domain having at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to any continuous stretch of at least 10, 15, 20, or all of the amino acids of the following amino acid sequences: DKTHT (SEQ ID NO:5); CPPC (SEQ ID NO:4); CPEPKSCDTPPPCPR (SEQ ID NO:6) (see, for example, Glaser et al. (2005) J. Biol. Chem. 280:41494); ELKTPLGDTTHT (SEQ ID NO:7); KSCDKTHTCP (SEQ ID NO:8); KCCVDCP (SEQ ID NO:9); KYGPPCP (SEQ ID NO:10); EPKSCDKTHTCPPCP (SEQ ID NO:11) (human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO:12) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO:13) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO:14) (human IgG4 hinge), etc. The stalk may contain a hinge region having the amino acid sequence of the human IgG1, IgG2, IgG3, or IgG4 hinge region. The stalk may contain one or more amino acid substitutions and / or insertions and / or deletions compared to the wild-type (naturally occurring) hinge region. For example, His229 of the human IgG1 hinge can be substituted with Tyr such that the stalk contains the sequence EPKSCDKTYTCPPCP (SEQ ID NO:15) (see, for example, Yan et al. (2012) J. Biol. Chem. 287:5891). The stalk may contain an amino acid sequence derived from human CD8, for example, the stalk may contain the amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:16), or a variant thereof.

[0146] Transmembrane domain The engineered signaling polypeptides of the present disclosure can include a transmembrane domain for insertion into the eukaryotic cell membrane. The transmembrane domain can be interposed between the ASTR and the co-stimulatory domain. The transmembrane domain can be interposed between the stalk and the co-stimulatory domain such that the chimeric antigen receptor comprises, in order from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus), ASTR; stalk; transmembrane domain; and activation domain.

[0147] Any transmembrane (TM) domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell is suitable for use in the aspects and embodiments disclosed herein.

[0148] Non-limiting examples of TM domains suitable for any of the aspects or embodiments provided herein include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous series of amino acids of any of the following TM domains or combined stalk and TM domains: a) CD8 alpha TM (SEQ ID NO: 17); b) CD8 beta TM (SEQ ID NO: 18); c) CD4 stalk (SEQ ID NO: 19); d) CD3Z TM (SEQ ID NO: 20); e) CD28TM (SEQ ID NO: 21); f) CD134 (OX40) TM: (SEQ ID NO: 22); g) CD7TM (SEQ ID NO: 23); h) CD8 stalk and TM (SEQ ID NO: 24); and i) CD28 stalk and TM (SEQ ID NO: 25).

[0149] As a non-limiting example, the transmembrane domain of one aspect of the invention can have at least 80%, 90%, or 95% sequence identity to, or can have 100% sequence identity to, the transmembrane domain of SEQ ID NO: 17, or can have 100% sequence identity to any of the transmembrane domains from each of the following genes: CD8 beta transmembrane domain, CD4 transmembrane domain, CD3 zeta transmembrane domain, CD28 transmembrane domain, CD134 transmembrane domain, or CD7 transmembrane domain.

[0150] Intracellular activation domain When activated, an intracellular activation domain suitable for use in the engineered signaling polypeptides of the present disclosure typically induces the production of one or more cytokines; increases cell death; and / or CD8 + T cells, CD4 + Increases the proliferation of T cells, CD4 T cells, NKT cells, γδ T cells, and / or neutrophils. The activation domain may also be referred to herein as the activation domain. The activation domain can be used in a CAR or the lymphoproliferative elements provided herein.

[0151] In some embodiments, the intracellular activation domain comprises at least one (e.g., 1, 2, 3, 4, 5, 6, etc.) ITAM motif as described below. In some embodiments, the intracellular activation domain of one aspect of the invention can have at least 80%, 90%, or 95% sequence identity to the CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10 / CD28, or ZAP70 domain, or can have 100% sequence identity, as described below.

[0152] An intracellular activation domain suitable for use in the engineered signaling polypeptides of the present disclosure includes an intracellular signaling polypeptide that includes an immunoreceptor tyrosine-based activation motif (ITAM). The ITAM motif is YX1X2L / I, where X1 and X2 are independently any amino acid. In some embodiments, the intracellular activation domain of the engineered signaling polypeptide comprises 1, 2, 3, 4, or 5 ITAM motifs. In some embodiments, the ITAM motif is repeated twice in the intracellular activation domain, where the first and second instances of the ITAM motif are separated from each other by 6 - 8 amino acids, e.g., (YX1X2L / I)(X3) n(YX1X2L / I), where n is an integer from 6 to 8, and each of the six to eight X3s can be any amino acid. In some embodiments, the intracellular activation domain of the engineered signaling polypeptide comprises three ITAM motifs.

[0153] A suitable intracellular activation domain may be a portion containing an ITAM motif derived from a polypeptide containing an ITAM motif. For example, a suitable intracellular activation domain may be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable intracellular activation domain need not include the entire sequence of the protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, CD3Z (CD3 zeta); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD79A (antigen receptor complex-associated protein alpha chain); CD79B (antigen receptor complex-associated protein beta chain) DAP12; and FCER1G (Fc epsilon receptor I gamma chain).

[0154] In some embodiments, the intracellular activation domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). For example, a suitable intracellular activation domain may have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a domain containing a continuous stretch of at least 10, 15, 20 or all of the amino acids in the following sequence, or to a continuous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa of either of the following amino acid sequences (2 isoforms).

Chemical formula

[0155] Similarly, a suitable intracellular activation domain polypeptide may contain an ITAM motif that includes a portion of the full-length CD3 zeta amino acid sequence. Thus, a suitable intracellular activation domain may have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids in the following sequences, or to a continuous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa of any of the following amino acid sequences: RVKFSRSADAPAYQQGQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPRRKNPQEGL[YNELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR (SEQ ID NO: 28); RVKFSRSADAPAYQQGQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPQRRKNPQEGL[YNELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR (SEQ ID NO: 29); NQL[YNELNLGRREEYDVL]DKR (SEQ ID NO: 30); EGL[YNELQKDKMAEAYSEI]GMK (SEQ ID NO: 31); or DGL[YQGLSTATKDTYDAL]HMQ (SEQ ID NO: 32) (the ITAM motif is shown in parentheses).

[0156] In some embodiments, the intracellular activation domain is derived from the T cell surface glycoprotein CD3 delta chain (CD3D; CD3-delta; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; also known as T cell surface glycoprotein CD3 delta chain, etc.). Thus, a suitable intracellular activation domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids in the following sequence, or to a continuous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa of any of the following amino acid sequences: MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQV[YQPLRDRDDAQYSHL]GGNWARNK (SEQ ID NO: 33) or MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRTADTQALLRNDQV[YQPLRDRDDAQYSHL]GGNWARNK (SEQ ID NO: 34) (the ITAM motif is shown in parentheses).

[0157] Similarly, a suitable intracellular activation domain polypeptide may include the ITAM motif-containing portion of the full-length CD3 delta amino acid sequence. Thus, a suitable intracellular activation domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids in the following sequence: DQV[YQPLRDRDDAQYSHL]GGN (SEQ ID NO: 35) (the ITAM motif is shown in parentheses).

[0158] In some embodiments, the intracellular activation domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). Thus, a suitable intracellular activation domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids in the following sequence, or to a continuous stretch of approximately 100 amino acids to approximately 110 amino acids (aa), approximately 110 aa to approximately 115 aa, approximately 115 aa to approximately 120 aa, approximately 120 aa to approximately 130 aa, approximately 130 aa to approximately 140 aa, approximately 140 aa to approximately 150 aa, or approximately 150 aa to approximately 160 aa of the following amino acid sequence: MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD[YEPIRKGQRDLYSGL]NQRRI (SEQ ID NO: 36) (the ITAM motif is shown in parentheses).

[0159] Similarly, a suitable intracellular activation domain polypeptide may include the ITAM motif-containing portion of the full-length CD3 epsilon amino acid sequence. Thus, a suitable intracellular activation domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids in the following sequence:

Chemical Formula

[0160] In some embodiments, the intracellular activation domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). Thus, a suitable intracellular activation domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids in the following sequence, or to a continuous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa of the following amino acid sequence: MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQL[YQPLKDREDDQYSHL]QGNQLRRN (SEQ ID NO: 38) (The ITAM motif is shown in parentheses).

[0161] Similarly, a suitable intracellular activation domain polypeptide may include the ITAM motif-containing portion of the full-length CD3 gamma amino acid sequence. Thus, a suitable intracellular activation domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a contiguous stretch of at least 10, 15, 20 or all of the amino acids in the following sequence: [Chemical formula] (The ITAM motif is shown in parentheses).

[0162] In some embodiments, the intracellular activation domain is derived from CD79A (also known as B cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; Ig-alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein, etc.). Thus, a suitable intracellular activation domain can have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids in the following sequence, or to a continuous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa of any of the following amino acid sequences: MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENL[YEGLNLDDCSMYEDI]SRGLQGTYQDVGSLNIGDVQLEKP (SEQ ID NO: 40) or MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNEPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENL[YEGLNLDDCSMYEDI]SRGLQGTYQDVGSLNIGDVQLEKP (SEQ ID NO: 41) (the ITAM motif is shown in brackets).

[0163] Similarly, a suitable intracellular activation domain polypeptide may comprise the ITAM motif-containing portion of the full-length CD79A amino acid sequence. Thus, a suitable intracellular activation domain may comprise a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a contiguous stretch of at least 10, 15, 20 or all of the amino acids in the following sequence: ENL[YEGLNLDDCSMYEDI]SRG (SEQ ID NO: 42) (the ITAM motif is shown in parentheses).

[0164] In some embodiments, the intracellular activation domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase binding protein; killer activation receptor-associated protein; killer activation receptor-associated protein, etc.). For example, a suitable intracellular activation domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids in the following sequence, or to a continuous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa of any of the following amino acid sequences (4 isoforms): MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESP[YQELQGQRSDVYSDL]NTQRPYYK (SEQ ID NO: 43), MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESP[YQELQGQRSDVYSDL]NTQ (SEQ ID NO: 44), MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESP[YQELQGQRSDVYSDL]NTQRPYYK (SEQ ID NO: 45), or MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESP[YQELQGQRSDVYSDL]NTQRPYYK (SEQ ID NO: 46) (the ITAM motif is shown in brackets).

[0165] Similarly, a suitable intracellular activation domain polypeptide may include the ITAM motif-containing portion of the full-length DAP12 amino acid sequence. Thus, a suitable intracellular activation domain may have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a domain including a continuous stretch of at least 10, 15, 20 or all of the amino acids in the following sequences: [Chemical formula] (The ITAM motif is shown in parentheses).

[0166] In some embodiments, the intracellular activation domain is derived from FCER1G (FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc-epsilonRI-gamma; fcR gamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; also known as immunoglobulin E receptor, high affinity, gamma chain, etc.). For example, a suitable intracellular activation domain may have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a domain including a continuous stretch of at least 10, 15, 20 or all of the amino acids in the following sequences, or to a continuous stretch of about 50 amino acids to about 60 amino acids (aa), about 60 aa to about 70 aa, about 70 aa to about 80 aa, or about 80 to about 88 aa of the following amino acid sequence: MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKSDGV[YTGLSTRNQETYETL]KHEKPPQ (SEQ ID NO: 48) (The ITAM motif is shown in parentheses).

[0167] Similarly, a suitable intracellular activation domain polypeptide may include the ITAM motif-containing portion of the full-length FCER1G amino acid sequence. Thus, a suitable intracellular activation domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids in the following sequences:

Chem.

[0168] An intracellular activation domain suitable for use in the engineered signaling polypeptides of the present disclosure includes a DAP10 / CD28 type signaling chain. An example of the DAP10 signaling chain is amino acid sequence number 50. In some embodiments, a suitable intracellular activation domain includes a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids in sequence number 50.

[0169] An example of the CD28 signaling chain is the amino acid sequence of sequence number 51. In some embodiments, a suitable intracellular domain includes a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids in sequence number 51.

[0170] Intracellular activation domains suitable for use in the engineered signaling polypeptides of the present disclosure include the ZAP70 polypeptide. For example, suitable intracellular activation domains can include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids in the following sequences, or to a continuous stretch of about 300 to about 400 amino acids, about 400 to about 500 amino acids, or about 500 to 619 amino acids of SEQ ID NO: 52.

[0171] Regulatory domain The regulatory domain can alter the effect of the intracellular activation domain in the engineered signaling polypeptide, including enhancing or attenuating the downstream effect of the activation domain, or altering the nature of the response. Regulatory domains suitable for use in the engineered signaling polypeptides of the present disclosure include co-stimulatory domains. Regulatory domains suitable for inclusion in the engineered signaling polypeptide can have a length of about 30 amino acids to about 70 amino acids (aa), for example, the regulatory domain can have a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa. In other cases, the regulatory domain can have a length of about 70aa to about 100aa, about 100aa to about 200aa, or greater than 200aa.

[0172] Costimulatory domains typically enhance and / or alter the nature of the response to activation domains. Costimulatory domains suitable for use in the engineered signaling polypeptides of the present disclosure are generally polypeptides derived from receptors. In some embodiments, the costimulatory domain dimerizes homotypically. The costimulatory domain of interest may be the intracellular portion of a transmembrane protein (i.e., the costimulatory domain may be derived from a transmembrane protein). Non-limiting examples of suitable costimulatory polypeptides include, but are not limited to, 4-1BB (CD137), CD27, CD28, CD28 deletion Lck binding (ICΔ), ICOS, OX40, BTLA, CD27, CD30, GITR, and HVEM. For example, the costimulatory domain of one aspect of the invention may have at least 80%, 90%, or 95% sequence identity to the costimulatory domain of 4-1BB (CD137), CD27, CD28, CD28 deletion Lck binding (ICΔ), ICOS, OX40, BTLA, CD27, CD30, GITR, or HVEM. For example, the costimulatory domain of one aspect of the invention may have at least 80%, 90%, or 95% sequence identity to the costimulatory domain of a non-limiting example of a suitable costimulatory polypeptide including, but not limited to, 4-1BB (CD137), CD27, CD28, CD28 deletion Lck binding (ICΔ), ICOS, OX40, BTLA, CD27, CD30, GITR, and HVEM. For example, the costimulatory domain of one aspect of the invention may have at least 80%, 90%, or 95% sequence identity to the costimulatory domain of 4-1BB (CD137), CD27, CD28, CD28 deletion Lck binding (ICΔ), ICOS, OX40, BTLA, CD27, CD30, GITR, or HVEM.

[0173] Co-stimulatory domains suitable for inclusion in an engineered signaling polypeptide can have a length of from about 30 amino acids to about 70 amino acids (aa), for example, the co-stimulatory domain can have a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, or about 65 aa to about 70 aa. In other cases, the co-stimulatory domain can have a length of from about 70 aa to about 100 aa, from about 100 aa to about 200 aa, or more than 200 aa.

[0174] In some embodiments, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein CD137 (also known as TNFRSF9; CD137; 4-1BB; CDw137; ILA, etc.). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids in SEQ ID NO: 53. In some of these embodiments, the co-stimulatory domain has a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, or about 65 aa to about 70 aa.

[0175] In some embodiments, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp44). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids in SEQ ID NO: 54. In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

[0176] In some embodiments, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein CD28 deletion Lck binding (ICΔ). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids in SEQ ID NO: 55. In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

[0177] In some embodiments, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a contiguous stretch of at least 10, 15, 20 or all of the amino acids in SEQ ID NO: 56. In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

[0178] In some embodiments, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein OX40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX-40, TXGP1L). OX40 contains a p85 PI3K binding motif at residues 34-57 and a TRAF binding motif at residues 76-102, and these residues are from SEQ ID NO: 296 (in Table 1) respectively. In some embodiments, the co-stimulatory domain may include the p85 PI3K binding motif of OX40. In some embodiments, the co-stimulatory domain may include the TRAF binding motif of OX40. The lysines corresponding to amino acids 17 and 41 of SEQ ID NO: 296 are potentially negative regulatory sites that function as part of a ubiquitination targeting motif. In some embodiments, one or both of these lysines in the co-stimulatory domain of OX40 are mutated arginine or another amino acid. In some embodiments, a suitable co-stimulatory domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids at SEQ ID NO: 57. In some of these embodiments, the co-stimulatory domain has a length of about 20aa to about 25aa, about 25aa to about 30aa, 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, or about 45aa to about 50aa. In an exemplary embodiment, the co-stimulatory domain has a length of about 20aa to about 50aa, such as 20aa to 45aa, or 20aa to 42aa.

[0179] In some embodiments, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein CD27 (also known as S152, T14, TNFRSF7, and Tp55). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids in SEQ ID NO: 58. In some of these embodiments, the co-stimulatory domain has a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, or about 45 aa to about 50 aa.

[0180] In some embodiments, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein BTLA (also known as BTLAl and CD272). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20 or all of the amino acids in SEQ ID NO: 59.

[0181] In some embodiments, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of about 100 amino acids (aa) to about 110 amino acids, about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, about 150 aa to about 160 aa, or about 160 aa to about 185 aa in SEQ ID NO: 60.

[0182] In some embodiments, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a contiguous stretch of at least 10, 15, 20 or all of the amino acids in SEQ ID NO: 61. In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

[0183] In some embodiments, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a contiguous stretch of at least 10, 15, 20 or all of the amino acids in SEQ ID NO: 62. In some of these embodiments, the co-stimulatory domains of both the first and second polypeptides have a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

[0184] Linker In some embodiments, the engineered signaling polypeptide comprises a linker between any two adjacent domains. For example, the linker may be between a transmembrane domain and a first co-stimulatory domain. As another example, the ASTR may be an antibody, and the linker may be between the heavy chain and the light chain. As another example, the linker may be between the ASTR, the transmembrane domain, and the co-stimulatory domain. As another example, the linker may be between the co-stimulatory domain of a second polypeptide and the intracellular activation domain. As another example, the linker may be between the ASTR and the intracellular signaling domain.

[0185] Linker peptides can have any of a variety of amino acid sequences. Proteins can generally be linked by spacer peptides of a flexible nature, although other chemical linkages are not excluded. The linker may be a peptide between about 1 and about 100 amino acids in length, or between about 1 and about 25 amino acids in length. These linkers can be generated by joining proteins using oligonucleotides encoding synthetic linkers. Peptide linkers with some degree of flexibility can be used. The linking peptide can have virtually any amino acid sequence, considering that suitable linkers generally result in flexible peptides. The use of small amino acids such as glycine and alanine serves to form flexible peptides. The formation of such sequences is routine for those skilled in the art.

[0186] Suitable linkers can be readily selected and can be any of a suitable different lengths such as from 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0187] Exemplary flexible linkers are glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , GSGGSn , GGGS n , and GGGGS n (wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are interesting because both of these amino acids are relatively unstructured and can thus function as neutral tethers between components. Glycine is of particular interest because it has much more phi-psi space accessible than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, GGGGSGGGGSGGGGS (SEQ ID NO: 63), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 64), GGGGSGGGSGGGGS (SEQ ID NO: 65), GGSG (SEQ ID NO: 66), GGSGG (SEQ ID NO: 67), GSGSG (SEQ ID NO: 68), GSGGG (SEQ ID NO: 69), GGGSG (SEQ ID NO: 70), GSSSG (SEQ ID NO: 71), etc. Those skilled in the art will recognize that the design of a peptide conjugated to any of the above elements may include all or part of a flexible linker, and as a result, the linker may include a flexible linker as well as one or more portions that provide a less flexible structure.

[0188] Combination In some embodiments, the polynucleotide provided by the replication-incompetent recombinant retroviral particle has one or more transcription units encoding a specific combination of one or more engineered signaling polypeptides. In some of the methods and compositions provided herein, the genetically modified T cells comprise a combination of one or more engineered signaling polypeptides after transduction of the T cells with the replication-incompetent recombinant retroviral particle. References to a first polypeptide, a second polypeptide, a third polypeptide, etc. are for convenience only, and elements on the "first polypeptide" and elements on the "second polypeptide" will typically be in a further element or step for that particular polypeptide, it being understood that the elements are on different polypeptides that are referred to as first or second only for reference and convention.

[0189] In some embodiments, the first engineered signaling polypeptide comprises an extracellular antigen-binding domain capable of binding to an antigen and an intracellular signaling domain. In other embodiments, the first engineered signaling polypeptide also comprises a T cell survival motif and / or a transmembrane domain. In some embodiments, the first engineered signaling polypeptide does not comprise a co-stimulatory domain, while in other embodiments, the first engineered signaling polypeptide comprises a co-stimulatory domain.

[0190] In some embodiments, the second engineered signaling polypeptide comprises a lymphoproliferative gene product and optionally an extracellular antigen-binding domain. In some embodiments, the second engineered signaling polypeptide also comprises one or more of a T cell survival motif, an intracellular signaling domain, and one or more co-stimulatory domains. In other embodiments, when two engineered signaling polypeptides are used, at least one is a CAR.

[0191] In one embodiment, one or more engineered signaling polypeptides are expressed under a T cell-specific promoter or a common promoter under the same transcript, and in the transcript, the nucleic acid encoding the engineered signaling polypeptide is separated by one or more internal ribosome entry sites (IRES) or nucleic acids encoding one or more protease cleavage peptides.

[0192] In certain embodiments, the polypeptide encodes two engineered signaling polypeptides, the first engineered signaling polypeptide comprising a first extracellular antigen-binding domain capable of binding to a first antigen and a first intracellular signaling domain rather than a co-stimulatory domain, and the second polypeptide comprising a second extracellular antigen-binding domain capable of binding to VEGF and a second intracellular signaling domain such as a signaling domain of a co-stimulatory molecule. In certain embodiments, the first antigen is PSCA, PSMA, or BCMA. In certain embodiments, the first extracellular antigen-binding domain comprises an antibody or a fragment thereof (e.g., scFv), e.g., an antibody or a fragment thereof specific for PSCA, PSMA, or BCMA. In certain embodiments, the second extracellular antigen-binding domain that binds to VEGF is a receptor for VEGF, i.e., VEGFR. In certain embodiments, the VEGFR is VEGFR1, VEGFR2, or VEGFR3. In certain embodiments, the VEGFR is VEGFR2.

[0193] In certain embodiments, the polynucleotide encodes two engineered signaling polypeptides, the first engineered signaling polypeptide comprising an extracellular tumor antigen-binding domain and a CD3ζ signaling domain, and the second engineered signaling polypeptide comprising an antigen-binding domain (the antigen being an angiogenesis or vasculogenesis factor) and one or more co-stimulatory molecule signaling domains. The angiogenesis factor may be, for example, VEGF. The one or more co-stimulatory molecule signaling motifs may include, for example, co-stimulatory signaling domains from each of CD27, CD28, OX40, ICOS, and 4-1BB.

[0194] In certain embodiments, the polynucleotide encodes two engineered signaling polypeptides, the first engineered signaling polypeptide comprising an extracellular tumor antigen binding domain and a CD3ζ signaling domain, and the second polypeptide comprising an antigen binding domain capable of binding to VEGF, as well as co-stimulatory signaling domains from each of CD27, CD28, OX40, ICOS, and 4-1BB. In further embodiments, the first signaling polypeptide or the second signaling polypeptide also has a T cell survival motif. In some embodiments, the T cell survival motif is or is derived from the intracellular signaling domain of the interleukin-7 receptor (IL-7R), the intracellular signaling domain of the interleukin-12 receptor, the intracellular signaling domain of the interleukin-15 receptor, the intracellular signaling domain of the interleukin-21 receptor, or the intracellular signaling domain of the transforming growth factor β (TGFβ) receptor or TGFβ decoy receptor (TGF-β-dominant negative receptor II (DNRII)).

[0195] In certain embodiments, the polynucleotide encodes two engineered signaling polypeptides, the first engineered signaling polypeptide comprising an extracellular tumor antigen binding domain and a CD3ζ signaling domain, and the second engineered signaling polypeptide comprising an antigen binding domain capable of binding to VEGF, an IL-7 receptor intracellular T cell survival motif, as well as co-stimulatory signaling domains from each of CD27, CD28, OX40, ICOS, and 4-1BB.

[0196] In some embodiments, three or more signaling polypeptides are encoded by a polynucleotide. In certain embodiments, only one of the engineered signaling polypeptides comprises an antigen-binding domain that binds to a tumor-associated antigen or a tumor-specific antigen, and each of the remaining engineered signaling polypeptides comprises an antigen-binding domain that binds to an antigen that is not a tumor-associated antigen or a tumor-specific antigen. In other embodiments, two or more engineered signaling polypeptides comprise an antigen-binding domain that binds to one or more tumor-associated antigens or tumor-specific antigens, and at least one of the engineered signaling polypeptides comprises an antigen-binding domain that does not bind to a tumor-associated antigen or a tumor-specific antigen.

[0197] In some embodiments, the tumor-associated antigen or tumor-specific antigen is Her2, prostate stem cell antigen (PSCA), PSMA (prostate-specific membrane antigen), B-cell maturation antigen (BCMA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein Melan-A (melanoma antigen recognized by T lymphocytes;MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, dimeric form of pyruvate kinase isoenzyme type M2 (tumor M2-PK), CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EphA2, CSPG4, CD138, FAP (fibroblast activation protein), CD171, kappa, lambda, 5T4, αvβ6 integrin, integrin ανβ3 (CD61), galectin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), Ral-B, B7-H3, B7-H6, CAIX, CD20, CD33, CD44, CD44v6, CD44v7 / 8, CD123, EGFR, EGP2, EGP40, EpCAM, fetal AchR, FRα, GD3, HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-11Rα, IL-13Rα2, Lewis-Y, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, ROR1, survivin, TAG72, TEM, VEGFR2, EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostain, TARP (T cell receptor gamma alternative reading frame protein), Trp-p8, STEAP1 (prostate transmembrane epithelial antigen of the prostate 1), an abnormal ras protein, or an abnormal p53 protein.;

[0198] In some embodiments, the first engineered signaling polypeptide comprises a first extracellular antigen-binding domain that binds to a first antigen and a first intracellular signaling domain, and the second engineered signaling polypeptide comprises a second extracellular antigen-binding domain that binds to a second antigen, or a receptor that binds to the second antigen, and a second intracellular signaling domain, and the second engineered signaling polypeptide does not comprise a co-stimulatory domain. In certain embodiments, the first antigen-binding domain and the second antigen-binding domain are independently the antigen-binding portion of a receptor or the antigen-binding portion of an antibody. In certain embodiments, either or both of the first antigen-binding domain or the second antigen-binding domain are scFv antibody fragments. In certain embodiments, the first engineered signaling polypeptide and / or the second engineered signaling polypeptide further comprises a transmembrane domain. In certain embodiments, the first engineered signaling polypeptide or the second engineered signaling polypeptide comprises a T cell survival motif, such as any of the T cell survival motifs described herein.

[0199] In another embodiment, the first engineered signaling polypeptide comprises a first extracellular antigen-binding domain that binds to HER2, and the second engineered signaling polypeptide comprises a second extracellular antigen-binding domain that binds to MUC-1.

[0200] In another embodiment, the second extracellular antigen-binding domain of the second engineered signaling polypeptide binds to an interleukin.

[0201] In another embodiment, the second extracellular antigen-binding domain of the second engineered signaling polypeptide binds to a damage-associated molecular pattern molecule (DAMP; also known as an alarmin). In other embodiments, the DAMP is a heat shock protein, a chromatin-associated protein high-mobility group box 1 (HMGB1), S100A8 (also known as MRP8, or calgranulin A), S100A9 (also known as MRP14, or calgranulin B), serum amyloid A (SAA), deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparan sulfate.

[0202] In certain embodiments, the second antigen is an antigen on an antibody that binds to an antigen presented by a tumor cell.

[0203] In some embodiments, signal transduction activation via the second engineered signaling polypeptide is non-antigenic but is associated with hypoxia. In certain embodiments, the hypoxia is induced by the activation of hypoxia-inducible factor-1α (HIF-1α), HIF-1β, HIF-2α, HIF-2β, HIF-3α, or HIF-3β.

[0204] In some embodiments, the expression of one or more engineered signaling polypeptides is regulated by regulatory elements disclosed in more detail herein.

[0205] Additional sequences Engineered signaling polypeptides such as CARs may further include one or more additional polypeptide domains, such domains including, but not limited to, signal sequences; epitope tags; affinity domains; and polypeptides whose presence or activity can be detected, for example, by an antibody assay or because they generate a detectable signal (detectable markers). Non-limiting examples of additional domains for any of the aspects or embodiments provided herein include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the following sequences described below: signal sequences, epitope tags, affinity domains, or polypeptides that generate a detectable signal.

[0206] Signal sequences suitable for use in a subject's CAR, for example, in the first polypeptide of the subject's CAR, include any eukaryotic signal sequence, including natural-occurring signal sequences, synthetic (e.g., artificial) signal sequences, etc. In some embodiments, for example, the signal sequence may be the CD8 signal sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 72).

[0207] Suitable epitope tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA; SEQ ID NO: 73); FLAG (e.g., DYKDDDDK; SEQ ID NO: 74); c-myc (e.g., EQKLISEEDL; SEQ ID NO: 75), etc.

[0208] An affinity domain can interact with a binding partner, such as one immobilized on a solid support, and contains a peptide sequence useful for identification or purification. DNA sequences encoding multiple consecutive single amino acids such as histidine, when fused to an expressed protein, can bind to a resin column such as nickel sepharose with high affinity and thus be used for one-step purification of recombinant proteins. Exemplary affinity domains include His5 (HHHHH; SEQ ID NO: 76), HisX6 (HHHHHH; SEQ ID NO: 77), c-myc (EQKLISEEDL; SEQ ID NO: 75), Flag (DYKDDDDK; SEQ ID NO: 74), Strep Tag (WSHPQFEK; SEQ ID NO: 78), hemagglutinin, such as HA Tag (YPYDVPDYA; SEQ ID NO: 73), GST, thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO: 79), Phe-His-His-Thr (SEQ ID NO: 80), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-terminal RNA tag, WEAAAREACCRECCARA (SEQ ID NO: 81), metal binding domains, such as zinc binding domains or calcium binding domains from calcium binding proteins such as calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, frequenin, caltractin, calpain large subunit, S100 proteins, parvalbumin, calbindin D9K, calbindin D28K, and calretinin, intein, biotin, streptavidin, MyoD, Id, leucine zipper sequences, and maltose binding protein.

[0209] Suitable detectable signal generating proteins include, for example, fluorescent proteins; enzymes that catalyze reactions to generate a detectable signal as a product, and the like.

[0210] Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFPl, Posilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and Kindling protein, phycobiliproteins and phycobiliprotein conjugates including B-phycoerythrin, R-phycoerythrin and allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), etc. Any of a variety of fluorescent and colored proteins from Anthozoan species are suitable for use, as described, for example, in Matz et al. (1999) Nature Biotechnol. 17:969-973.

[0211] Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, glucose oxidase (GO), etc.

[0212] Recognition and / or elimination domain Any of the replication-incompetent recombinant retroviral particles provided herein can include, as part of or separate from, a nucleic acid encoding any of the engineered signaling polypeptides provided herein, a nucleic acid encoding a recognition or elimination domain. Thus, any of the engineered signaling polypeptides provided herein can include a recognition or elimination domain. For example, any of the CARs disclosed herein can include a recognition or elimination domain. Additionally, the recognition or elimination domain can be expressed together with or even fused to any of the lymphoproliferative elements disclosed herein. The recognition or elimination domain is expressed in T cells and / or NK cells but not in replication-incompetent recombinant retroviral particles.

[0213] In some embodiments, the recognition or removal domain can be derived from the enzyme thymidine kinase from herpes simplex virus (HSV-tk) or inducible caspase-9. In some embodiments, the recognition or removal domain can include a modified endogenous cell surface molecule, as disclosed, for example, in U.S. Patent No. 8,802,374. The modified endogenous cell surface molecule can be any modified cell surface-associated receptor, ligand, glycoprotein, cell adhesion molecule, antigen, integrin, or cluster of differentiation (CD). In some embodiments, the modified endogenous cell surface molecule is a truncated tyrosine kinase receptor. In one aspect, the truncated tyrosine kinase receptor is a member of the epidermal growth factor receptor (EGFR) family (e.g., ErbB1, ErbB2, ErbB3, and ErbB4). In some embodiments, the recognition domain can be a polypeptide recognized by an antibody that recognizes the extracellular domain of an EGFR member. In some embodiments, the recognition domain can be at least 20 contiguous amino acids of an EGFR family member, or, for example, 20 to 50 contiguous amino acids of an EGFR family member. For example, SEQ ID NO: 82 is an exemplary polypeptide that is recognized by an antibody that recognizes the extracellular domain of an EGFR member and binds under appropriate conditions. Such extracellular EGFR epitopes may be referred to herein as eTags. In an exemplary embodiment, such epitopes are recognized by commercially available anti-EGFR monoclonal antibodies.

[0214] The epidermal growth factor receptor, also known as EGFR, ErbB1, and HER1, is a cell surface receptor for members of the epidermal growth factor family of extracellular ligands. Changes in EGFR activity are associated with certain cancers. In some embodiments, a gene encoding an EGFR polypeptide, including the human epidermal growth factor receptor (EGFR), encodes a polypeptide comprising a membrane-distal EGF binding domain and a cytoplasmic signaling tail, but is constructed by removal of a nucleic acid sequence that retains an extracellular membrane-proximal epitope recognized by an anti-EGFR antibody. Preferably, the antibody is a known commercially available anti-EGFR monoclonal antibody such as cetuximab, matuzumab, necitumumab, or panitumumab.

[0215] Other researchers have shown that by combining biotinylated cetuximab with anti-biotin microbeads and applying it to immunomagnetic selection, T cells transduced with a lentivirus with an EGFRt-containing construct can be concentrated from 2% to over 90% purity of the population without observable toxicity to the cell preparation. Furthermore, other researchers have shown that the constitutive expression of this inactive EGFR molecule does not affect the phenotype or effector function of T cells directed by a co-expressed chimeric antigen receptor (CAR), CD19R. Additionally, some researchers have shown through flow cytometry analysis that EGFR was successfully utilized as an in vivo tracking marker for mouse T cell engraftment. Furthermore, it has been demonstrated that EGFR has the potential as a suicide gene via the Erbitux®-mediated antibody-dependent cell cytotoxicity (ADCC) pathway. The inventors of the present disclosure have successfully expressed eTag in PBMCs using a lentiviral vector and have found that in vitro expression of eTag by PBMCs exposed to cetuximab provides an effective removal mechanism for PBMCs. Thus, EGFR can be used as a non-immunogenic selection tool, a tracking marker, and a suicide gene for transduced T cells with immunotherapeutic potential. EGFR nucleic acids can also be detected by means well known in the art.

[0216] In some embodiments provided herein, EGFR is expressed as part of a single polypeptide that also includes a CAR, or as part of a single polypeptide that includes a lymphoproliferative element. In some embodiments, the amino acid sequence encoding the EGFR recognition domain can be separated from the amino acid sequence encoding the chimeric antigen receptor by a cleavage signal and / or a ribosome skip sequence. The ribosome skip and / or cleavage signal can be any ribosome skip and / or cleavage signal known in the art. Without being limited by theory, the ribosome skip sequence can be, for example, T2A (also referred to herein as 2A-1) having the amino acid sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 83). Without being limited by theory, other examples of cleavage signals and ribosome skip sequences include FMDV 2A (F2A); equine rhinitis A virus 2A (abbreviated as E2A); porcine teschovirus-1 2A (P2A); and Thosea asigna virus 2A (T2A). In some embodiments, the polynucleotide sequence encoding the recognition domain can be on the same transcript as the CAR or lymphoproliferative element, but can be separated from the polynucleotide sequence encoding the CAR or lymphoproliferative element by an internal ribosome entry site.

[0217] In other embodiments empirically exemplified herein, the recognition domain can be expressed as part of a fusion polypeptide fused to a lymphoproliferative element. Such constructs provide the advantage of occupying less genomic space on the RNA genome compared to separate polypeptides, particularly when combined with other "space-saving" elements provided herein. In one exemplary embodiment, the eTag is expressed as a fusion polypeptide fused to an IL7Rα mutant, as experimentally demonstrated herein.

[0218] Chimeric antigen receptor In some aspects of the present invention, the engineered signaling polypeptide is a chimeric antigen receptor (CAR) or a polynucleotide encoding a CAR, which, for simplicity, is referred to herein as a "CAR." The CARs of the present disclosure comprise a) at least one antigen-specific targeting region (ASTR); b) a transmembrane domain; and c) an intracellular activation domain. In an exemplary embodiment, the antigen-specific targeting region of the CAR is the scFv portion of an antibody against the target antigen. In an exemplary embodiment, the intracellular activation domain is derived from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10 / CD28, or ZAP70, and in some further exemplary embodiments, is derived from CD3z. In an exemplary embodiment, the CAR further comprises a co-stimulatory domain, such as any of the co-stimulatory domains provided in the section of regulatory domains above, and in a further exemplary embodiment, the co-stimulatory domain is the intracellular co-stimulatory domain of 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM. In some embodiments, the CAR comprises any of the transmembrane domains listed in the section of transmembrane domains above.

[0219] The CARs of the present disclosure can be present on the plasma membrane of eukaryotic cells, such as mammalian cells. Suitable mammalian cells include, but are not limited to, cytotoxic cells, T lymphocytes, stem cells, progeny of stem cells, progenitor cells, progeny of progenitor cells, and NK cells, NK-T cells, and macrophages. When present on the plasma membrane of a eukaryotic cell, the CARs of the present disclosure are active in the presence of one or more target antigens that bind to the ASTR under specific conditions. The target antigen is the second member of a specific binding pair. The target antigen of the specific binding pair may be a soluble (e.g., not bound to a cell) factor; a factor present on the surface of a cell such as a target cell; a factor presented on a solid surface; a factor present in a lipid bilayer, etc. When the ASTR is an antibody and the second member of the specific binding pair is an antigen, the antigen may be a soluble (e.g., not bound to a cell) antigen; an antigen present on the surface of a cell such as a target cell; an antigen presented on a solid surface; an antigen present in a lipid bilayer, etc.

[0220] In some cases, when the CARs of the present disclosure are present on the plasma membrane of a eukaryotic cell and are activated by one or more target antigens, they increase the expression of at least one nucleic acid in the cell. For example, in some cases, when the CARs of the present disclosure are present on the plasma membrane of a eukaryotic cell and are activated by one or more target antigens, the expression of at least one nucleic acid in the cell is increased by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold compared to the transcription level of the nucleic acid in the absence of the one or more target antigens.

[0221] As an example, the CARs of the present disclosure may include an intracellular signaling polypeptide that includes an immunoreceptor tyrosine-based activation motif (ITAM).

[0222] The CARs of the present disclosure, when present in the plasma membrane of a eukaryotic cell and when activated by one or more target antigens, can, in some cases, result in an increase in the production of one or more cytokines by the cell. For example, the CARs of the present disclosure, when present in the plasma membrane of a eukaryotic cell and when activated by one or more target antigens, can increase the production of cytokines by the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold compared to the amount of cytokines produced by the cell in the absence of one or more target antigens. Cytokines whose production can be increased include, but are not limited to, interferon gamma (IFN-γ), tumor necrosis factor-α (TNF-α), IL-2, IL-15, IL-12, IL-4, IL-5, IL-10; chemokines; growth factors, and the like.

[0223] In some embodiments, the CARs of the present disclosure, when present in the plasma membrane of a eukaryotic cell and when activated by one or more target antigens, can result in both an increase in the transcription of nucleic acids within the cell and an increase in the production of cytokines by the cell.

[0224] In some cases, when the CARs of the present disclosure are present in the plasma membrane of eukaryotic cells and are activated by one or more target antigens, they confer cytotoxic activity of the cells against target cells expressing an antigen to which the antigen-binding domain of the first polypeptide of the CAR binds on their cell surface. For example, when the eukaryotic cell is a cytotoxic cell (e.g., an NK cell or a cytotoxic T lymphocyte), the CARs of the present disclosure, when present in the plasma membrane of the cell and activated by one or more target antigens, increase the cytotoxic activity of the cell against target cells expressing one or more target antigens on their cell surface. For example, when the eukaryotic cell is an NK cell or a T lymphocyte, the CARs of the present disclosure, when present in the plasma membrane of the cell and activated by one or more target antigens, increase the cytotoxic activity of the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold as compared to the cytotoxic activity of the cell in the absence of one or more target antigens.

[0225] In some embodiments, the CARs of the present disclosure, when present in the plasma membrane of eukaryotic cells and activated by one or more target antigens, can result in other CAR activation-related events such as proliferation and expansion (due to increased cell division or anti-apoptotic responses).

[0226] In some embodiments, the CARs of the present disclosure, when present in the plasma membrane of eukaryotic cells and activated by one or more target antigens, can result in other CAR activation-related events such as intracellular signal transduction regulation, cell differentiation, or cell death.

[0227] In some embodiments, the microenvironment of the CARs of the present disclosure is restricted. This property is typically the result of the restricted nature of the microenvironment of the ASTR domain of the CAR. Thus, the CARs of the present disclosure can have a lower binding affinity, or in exemplary embodiments, can have a higher binding affinity for one or more target antigens under microenvironmental conditions than under conditions of a normal physiological environment.

[0228] In certain exemplary embodiments, the CARs provided herein include a co-stimulatory domain in addition to an intracellular activation domain, and the co-stimulatory domain is, for example, any of the intracellular signaling domains provided herein for lymphoproliferative element (LE), such as the intracellular domain of CLE. In certain exemplary embodiments, the co-stimulatory domain of the CARs herein is CLE or the first intracellular domain (P3 domain) specified herein for the P4 domain, which is shown as the effective intracellular signaling domain of CLE herein in the absence of the P3 domain. Further, in certain exemplary embodiments, the co-stimulatory domain of the CARs can include both the P3 and P4 intracellular signaling domains specified herein for CLE. Certain exemplary sub-embodiments include particularly effective P3 and P4 partner intracellular signaling domains, as specified herein for CLE. In an exemplary embodiment, the co-stimulatory domain is other than the ITAM-containing intracellular domain of the CAR, either as part of the co-stimulatory domain or, in a further exemplary embodiment, as the only co-stimulatory domain.

[0229] In these embodiments comprising a CAR having a co-stimulatory domain identified herein as the effective intracellular domain of LE, the co-stimulatory domain of the CAR may be any of the intracellular signaling domains in Table 1 provided herein. An active fragment of any of the intracellular domains in Table 1 may be the co-stimulatory domain of the CAR. In an exemplary embodiment, the ASTR of the CAR comprises an scFV. In an exemplary embodiment, in addition to the c-stimulatory intracellular domain of CLE, these CARs comprise an intracellular activation domain that is, in an exemplary embodiment, CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C. DAP10 / CD28, or the ZAP70 intracellular activation domain, or, in a further exemplary embodiment, the CD3z intracellular activation domain.

[0230] In these exemplary embodiments, the co-stimulatory domain of the CAR may comprise an intracellular domain or a functional signaling fragment thereof that includes a signaling domain from CSF2RB, CRLF2, CSF2RA, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL5RA, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL17RC, IL17RD, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL22RA1, IL23R, IL27RA, IL31RA, LEPR, LIFR, LMP1, MPL, MyD88, OSMR, or PRLR. In some embodiments, the co-stimulatory domain of the CAR may comprise an intracellular domain or a functional signaling fragment thereof that includes a signaling domain from CSF2RB, CRLF2, CSF2RA, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL5RA, IL6R, IL6ST, IL9R, IL10RA, IL10RB, IL11RA, IL13RA1, IL13RA2, IL17RB, IL17RC, IL17RD, IL18R1, IL18RAP, IL20RA, IL20RB, IL22RA1, IL31RA, LEPR, LIFR, LMP1, MPL, MyD88, OSMR, or PRLR.In some embodiments, the co-stimulatory domain of the CAR can include an intracellular domain or a functional fragment thereof that includes a signaling domain from CSF2RB, CSF2RA, CSF3R, EPOR, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL1RL1, IL2RA, IL2RG, IL5RA, IL6R, IL9R, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA2, IL15RA, IL17RD, IL21R, IL23R, IL27RA, IL31RA, LEPR, MPL, MyD88, or OSMR. In some embodiments, the co-stimulatory domain of the CAR can include an intracellular domain or a fragment thereof that includes a signaling domain from CSF2RB, CSF2RA, CSF3R, EPOR, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL1RL1, IL2RA, IL2RG, IL5RA, IL6R, IL9R, IL10RB, IL11RA, IL13RA2, IL17RD, IL31RA, LEPR, MPL, MyD88, or OSMR. In some embodiments, the co-stimulatory domain of the CAR can include an intracellular domain or a functional signaling fragment thereof that includes a signaling domain from CSF2RB, CSF3R, IFNAR1, IFNGR1, IL2RB, IL2RG, IL6ST, IL10RA, IL12RB2, IL17RC, IL17RE, IL18R1, IL27RA, IL31RA, MPL, MyD88, OSMR, or PRLR. In some embodiments, the co-stimulatory domain of the CAR can include an intracellular domain or a functional signaling fragment thereof that includes a signaling domain from CSF2RB, CSF3R, IFNGR1, IL2RB, IL2RG, IL6ST, IL10RA, IL17RE, IL31RA, MPL, or MyD88.

[0231] In some embodiments, the co-stimulatory domain of the CAR can include an intracellular domain or a fragment thereof that includes a signaling domain from CSF3R, IL6ST, IL27RA, MPL, and MyD88. In certain exemplary sub - embodiments, the intracellular activation domain of the CAR is derived from CD3z.

[0232] Recombinant T cell receptor (TCR) T cell receptor (TCR) recognizes specific protein fragments derived from intracellular and extracellular proteins. When proteins are degraded into peptide fragments, they are presented on the cell surface together with another protein called the major histocompatibility complex, or MHC, which is called the HLA (human leukocyte antigen) complex in humans. Three different combinations of T cell antigen receptors in vertebrates are αβ TCR, γδ TCR, and pre-TCR. Such combinations are formed by dimerization between members of dimerization subtypes such as the α TCR subunit and the β TCR subunit, the γ TCR subunit and the δ TCR subunit, and in the case of pre-TCR, the pTα subunit and the β TCR subunit. A set of TCR subunits dimerizes and recognizes target peptide fragments presented in the context of MHC. Pre-TCR is expressed only on the surface of immature αβ T cells, αβ TCR is expressed on the surface of mature αβ T cells and NK T cells, and γδ TCR is expressed on the surface of γδ T cells. The αβ TCR on the surface of T cells recognizes peptides presented by MHC I or MHC II, and the αβ TCR on the surface of NK T cells recognizes lipid antigens presented by CD1. γδ TCR can recognize MHC and MHC-like molecules and can also recognize non-MHC molecules such as viral glycoproteins. When a ligand is recognized, αβ TCR and γδ TCR transmit activation signals via the CD3 zeta chain, which stimulates T cell proliferation and cytokine secretion.

[0233] TCR molecules belong to the immunoglobulin superfamily, are antigen-specifically present in the V region, CDR3 is more variable than CDR1 and CDR2, and directly determines the antigen-binding specificity of TCR. When the MHC-antigen peptide complex is recognized by TCR, CDR1 and CDR2 recognize and bind to the side walls of the MHC molecule antigen-binding channel, and CDR3 binds directly to the antigen peptide. Therefore, recombinant TCRs that recognize tumor-specific protein fragments presented by MHC can be engineered.

[0234] Thus, recombinant TCRs, such as those derived from human TCRα and TCRβ pairs that recognize specific peptides with common HLAs, can be generated with specificity for tumor-specific proteins (Schmitt, TM et al., 2009). The target of the recombinant TCR can be a peptide derived from any of the antigen targets of the CAR ASTR provided herein, but more generally is derived from an intracellular tumor-specific protein such as a carcinoembryonic antigen, or a mutant of a normal intracellular protein or other cancer-specific neoepitope. Libraries of TCR subunits can be screened for their selectivity for the target antigen. Screening of native and / or recombinant TCR subunits can identify a set of TCR subunits that have high avidity and / or reactivity for the target antigen. Members of such a set of TCR subunits can be selected and cloned to produce one or more polynucleotides encoding the TCR subunits.

[0235] Polynucleotides encoding such a set of TCR subunits can be included in replication-incompetent recombinant retroviral particles for gene modification of lymphocytes, or in T cells or NK cells in exemplary embodiments, such that the lymphocytes express the recombinant TCR. Thus, in any aspect or embodiment provided herein that includes an engineered signaling polypeptide, such as an embodiment that includes one or more CARs and / or lymphoproliferative elements, the engineered signaling polypeptide can include one or more sets of recombinant γδ TCR chains, or in exemplary embodiments, αβ TCR chains, or can be such chains. The TCR chains that form the set can be co-expressed using several different techniques for co-expressing two TCR chains as disclosed herein, for expressing two or more other engineered signaling polypeptides such as CARs and lymphoproliferative elements. For example, protease cleavage epitopes such as 2A protease, internal ribosome entry site (IRES), and separate promoters can be used.

[0236] Several strategies have been employed to reduce the potential for mixed TCR dimer formation. In general, this involves modifying the constant (C) domains of the TCRα and TCRβ chains to promote preferential pairing of the introduced TCR chains with each other while reducing the likelihood of successful pairing with endogenous TCR chains. One approach that has shown some promise in vitro involves replacing the C domains of the human TCRα and TCRβ chains with their murine counterparts. Another approach involves mutations in the human TCRα common domain and TCRβ chain common regions to promote self-pairing, or the expression of endogenous TCR alpha and TCR beta miRNAs within viral gene constructs. Thus, in some embodiments provided herein that include one or more sets of TCR chains as engineered signaling polypeptides, each member of the set of TCR chains, in an exemplary embodiment the set of αβ TCR chains, includes a modified constant domain that promotes preferential pairing with each other. In some sub-embodiments, each member of the set of TCR chains, in an exemplary embodiment the set of αβ TCR chains, includes a sufficient sequence derived from a murine constant domain from the same TCR chain type, or a murine constant domain from the same TCR chain subtype, such that dimerization of the set of TCR chains with each other is preferred over or excludes dimerization with human TCR chains. In other sub-embodiments, each member of the set of TCR chains, in an exemplary embodiment the set of αβ TCR chains, includes a mutation corresponding to its constant domain, such that dimerization of the set of TCR chains with each other is preferred over or excludes dimerization with TCR chains having human constant domains. Such preferred or exclusive dimerization in exemplary embodiments is under physiological conditions.

[0237] Lymphoproliferative factor The number of peripheral T lymphocytes is maintained at a remarkably stable level throughout adulthood due to movement from the thymus, proliferation in response to antigen encounter, and cell loss due to the removal of antigen-specific effectors after antigen clearance, despite the continuous addition of cells (Marrak, P. et al. 2000. Nat Immunol 1:107-111; Freitas, A.A. et al. 2000. Annu Rev Immunol 18:83-111). The size of the peripheral T cell compartment is regulated by multiple factors that affect both proliferation and survival. However, in a lymphopenic environment, T lymphocytes divide independently of cognate antigen by an "acute homeostatic proliferation" mechanism that maintains the size of the peripheral T cell compartment. The state of lymphopenia is established in a subject or patient by growing T cells in vitro and introducing them into a lymphodepleted subject during adoptive cell therapy, resulting in enhanced engraftment and antitumor function of the transferred T cells. However, lymphodepletion of the subject is undesirable because it can cause serious side effects including immune dysfunction and death.

[0238] Studies have shown that lymphodepletion functions as an endogenous lymphocyte sink for homeostatic cytokines, thereby releasing cytokines to induce the survival and proliferation of adoptively transferred cells. Some cytokines, such as IL-7 and IL-15, are known to mediate antigen-independent proliferation of T cells and can thus induce homeostatic proliferation in a non-lymphopenic environment. However, these cytokines and their receptors have intrinsic regulatory mechanisms that prevent lymphoproliferative diseases in homeostasis.

[0239] Many of the embodiments provided herein include a lymphoproliferative element, or a nucleic acid encoding the same, typically as part of an engineered signaling polypeptide. Thus, in some aspects of the invention, the engineered signaling polypeptide is a lymphoproliferative element (LE), such as a chimeric lymphoproliferative element (CLE). Typically, an LE includes an extracellular domain, a transmembrane domain, and at least one intracellular signaling domain that drives proliferation, and in exemplary embodiments a second intracellular signaling domain.

[0240] In some embodiments, the lymphoproliferative element may include a first and / or second intracellular signaling domain. In some embodiments, the first and / or second intracellular signaling domain is CD2, CD3D, CD3E, CD3G, CD4, CD8A, CD8B, CD27, mutant delta Lck CD28, CD28, CD40, CD79A, CD79B, CRLF2, CSF2RB, CSF2RA, CSF3R, EPOR, FCER1G, FCGR2C, FCGRA2, GHR, ICOS, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL22RA1, IL23R, IL27RA, IL31RA, LEPR, LIFR, LMP1, MPL, MYD88, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18, or a functional mutant and / or fragment thereof. In an exemplary embodiment, the first intracellular signaling domain may include MyD88, or a functional mutant and / or fragment thereof. In a further exemplary embodiment, the first intracellular signaling domain may include MyD88, or a functional mutant and / or fragment thereof, and the second intracellular signaling domain may include ICOS, TNFRSF4, or TNSFR18, or a functional mutant and / or fragment thereof. In some embodiments, the first intracellular domain is MyD88 and the second intracellular domain is an intracellular domain containing an ITAM, such as the intracellular domain from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10 / CD28, or ZAP70.In some embodiments, the second intracellular signaling domain can include TNFRSF18, or a functional mutant and / or fragment thereof.

[0241] In some embodiments, the lymphoproliferative element can include a fusion of an extracellular domain and a transmembrane domain. In some embodiments, the fusion of the extracellular domain and the transmembrane domain can include eTAG IL7RA Ins PPCL (interleukin 7 receptor), Myc LMP1, LMP1, eTAG CRLF2, eTAG CSF2RB, eTAG CSF3R, eTAG EPOR, eTAG GHR, an eTAG with a cleavage after Fn F523C IL27RA, or an eTAG with a cleavage after Fn S505N MPL, or a functional mutant and / or fragment thereof. In some embodiments, the lymphoproliferative element can include an extracellular domain. In some embodiments, the extracellular domain can include an eTag having 0, 1, 2, 3, or 4 additional alanines at the carboxy terminus. In some embodiments, the extracellular domain can include Myc having 0, 1, 2, 3, or 4 additional alanines at the carboxy terminus, or a functional mutant and / or fragment thereof.

[0242] In some embodiments, the lymphoproliferative element may include a transmembrane domain. In some embodiments, the transmembrane domain is CD2, CD3D, CD3E, CD3G, CD3Z CD247, CD4, CD8A, CD8B, CD27, CD28, CD40, CD79A, CD79B, CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, FCER1G, FCGR2C, FCGRA2, GHR, ICOS, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL7RA, IL7RA Ins PPCL, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL22RA1, IL23R, IL27RA, IL31RA, LEPR, LIFR, MPL, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18, or functional mutants and / or fragments thereof.

[0243] The CLE for use in any aspect or embodiment herein may include any CLE disclosed in WO2019 / 055946 (incorporated herein by reference in its entirety), most of which are designed to be constitutively active and are considered to be constitutively active. As shown therein, when the first and second intracellular signaling domains of the CLE are present, the first intracellular signaling domain is disposed between the membrane-binding motif and the second intracellular domain.

[0244] In another embodiment, the LE can provide a property that drives T cell proliferation in vivo and / or possess such property (or cells genetically modified and / or transduced with the LE can provide the drive for T cell expansion in vivo, adapt thereto, possess such property, and / or be modified therefor). Methods for performing such in vivo tests are provided in Example 6. For example, as shown in Example 6, the in vivo test utilizes a mouse model where T cells are contacted with a lentiviral vector encoding the LE and, after introduction into the mouse, T cell expansion can be measured at 15-25 days in vivo, or 19-21 days in vivo, or at about 21 days in vivo.

[0245] In some embodiments, the lymphoproliferative element may comprise any of the sequences (SEQ ID NOs: 84-302) listed in Table 1. Table 1 shows the portions of the domains tested in CLE, the names (including gene names), and the amino acid sequences. Typically, CLE includes an extracellular domain (designated P1), a transmembrane domain (designated P2), a first intracellular domain (designated P3), and a second intracellular domain (designated P4). Typically, the lymphoproliferative element includes the first intracellular domain. In an exemplary embodiment, the first intracellular domain may include S036-S0216, or any of the portions listed in Table 1, or a functional mutant and / or fragment thereof. In some embodiments, the lymphoproliferative element may include the second intracellular domain. In an exemplary embodiment, the second intracellular domain may include S036-S0216, or any of the portions listed in Table 1, or a functional mutant and / or fragment thereof. In some embodiments, the lymphoproliferative element may include the extracellular domain. In an exemplary embodiment, the extracellular domain may include any of the sequences of the portions listed as M001-M049 or E006-E015 in Table 1, or a functional mutant and / or fragment thereof. In some embodiments, the lymphoproliferative element may include the transmembrane domain. In an exemplary embodiment, the transmembrane domain may include any of the portions listed as M001-M049 or T001-T082 in Table 1, or a functional mutant and / or fragment thereof. In some embodiments, the lymphoproliferative element may be a fusion of the extracellular / transmembrane domain (M001-M049 in Table 1), the first intracellular domain (S036-S0216 in Table 1), and the second intracellular domain (S036-S216 in Table 1). In some embodiments, the lymphoproliferative element may be a fusion of the extracellular domain (E006-E015 in Table 1), the transmembrane domain (T001-T082 in Table 1), the first intracellular domain (S036-S0216 in Table 1), and the second intracellular domain (S036-S0216 in Table 1).For example, the lymphoproliferative element may be a fusion of E006, T001, S036, and S216, which may also be described as E006-T001-S036-S216. In an exemplary embodiment, the lymphoproliferative element may be the fusion E010-T072-S192-S212, E007-T054-S197-S212, E006-T006-S194-S211, E009-T073-S062-S053, E008-T001-S121-S212, E006-T044-S186-S053, or E006-T016-S186-S050.

[0246] In an exemplary embodiment, the intracellular domain of the LE, or the first intracellular domain of the LE having two or more intracellular domains, is a functional intracellular activation domain from an ITAM-containing intracellular domain, for example, CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10 / CD28, or ZAP70, and in a further exemplary sub-embodiment is other than the intracellular domain from CD3z. In an exemplary embodiment, the second intracellular domain of the LE is other than the co-stimulatory domains of 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM. In an exemplary embodiment, the extracellular domain of the LE does not include a single-chain variable fragment (scFv). In a further exemplary embodiment, the extracellular domain of the LE that activates the LE upon binding to a binding partner does not include a single-chain variable fragment (scFv).

[0247] CLE does not include both the ASTR and the activation domains from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10 / CD28, or ZAP70. Without being limited to theory, the extracellular domain and transmembrane domain are thought to ensure that the intracellular signaling domain is in an effective conformation / orientation / localization for promoting proliferation and play a supporting role in the LE. Thus, the ability of the LE to drive proliferation is thought to be provided by the intracellular domain of the LE, and the extracellular domain and transmembrane domain are thought to play a secondary role compared to the intracellular domain. The lymphoproliferative element includes an intracellular domain that is a signaling polypeptide capable of promoting the proliferation of T cells or NK cells, and this domain associates with the membrane via a membrane-associating motif (such as a transmembrane domain) and is oriented or can be oriented in an active conformation. The ASTR of the LE in an exemplary embodiment does not include an scFv. Strategies are provided herein for associating the intracellular domain with the membrane, such as by including a transmembrane domain, GPI anchor, myristoylation region, palmitoylation region, and / or prenylation region. In some embodiments, the lymphoproliferative element does not include an extracellular domain.

[0248] The extracellular domain, transmembrane domain, and intracellular domain of LE may have different lengths of respective amino acids. For example, in embodiments including replication-incompetent retroviral particles, since there is a limit to the length of polynucleotides that can be packaged into retroviral particles, in certain exemplary embodiments, an LE having a shorter amino acid sequence may be advantageous. In some embodiments, the full length of LE may be between 3 and 4000 amino acids, such as between 10 and 3000, between 10 and 2000, between 50 and 2000, or between 250 and 2000 amino acids, and in exemplary embodiments, may be between 50 and 1000, between 100 and 1000, or between 250 and 1000 amino acids. When present to form the extracellular and transmembrane domains, the extracellular domain may be between 1 and 1000 amino acids, typically between 4 and 400, between 4 and 200, between 4 and 100, between 4 and 50, between 4 and 25, or between 4 and 20 amino acids. In one embodiment, the extracellular region is GGGS of the extracellular and transmembrane domains of this aspect of the present invention. The transmembrane domain, or the transmembrane region of the extracellular domain and transmembrane domain, may be between 10 and 250 amino acids, more typically may have a length of at least 15 amino acids, for example, may have a length between 15 and 100, between 15 and 75, between 15 and 50, between 15 and 40, or between 15 and 30 amino acids. The intracellular signaling domain may be, for example, between 10 and 1000, between 10 and 750, between 10 and 500, between 10 and 250, or between 10 and 100 amino acids. In exemplary embodiments, the intracellular signaling domain may be at least 30, or between 30 and 500, between 30 and 250, between 30 and 150, between 30 and 100, between 50 and 500, between 50 and 250, between 50 and 150, or between 50 and 100 amino acids.In some embodiments, the intracellular signaling domain of a particular gene is at least 90%, 95%, 98%, 99% or 100% identical to at least 10, 25, 30, 40, or 50 amino acids from the sequence of that intracellular signaling domain, e.g., the sequences provided herein with respect to that intracellular domain, up to the size of the entire intracellular domain sequence, and may include up to an additional 1, 2, 3, 4, 5, 10, 20 or 25 amino acids, provided that such sequences can still provide any of the LE properties disclosed herein.

[0249] In some embodiments, the lymphoproliferative element is, but not limited to, a chimeric cytokine receptor such as a cytokine linked to its receptor, and typically constitutively activates the same STAT pathway as the corresponding activated wild-type cytokine receptor such as STAT3, STAT4, and in exemplary embodiments STAT5. In some embodiments, the chimeric cytokine receptor is an interleukin or fragment thereof linked or covalently attached to its cognate receptor or fragment thereof via a linker. In some embodiments, the chimeric cytokine receptor is IL7 linked to IL7Rα (also known as IL7RA). In other embodiments, the chimeric cytokine receptor is IL-7 linked to a domain of IL7Rα, such as the extracellular domain of IL-7Rα and / or the transmembrane domain of IL-7Rα. In some embodiments, the lymphoproliferative element is a cytokine receptor not linked to a cytokine, and in fact, in exemplary embodiments, the lymphoproliferative element is a constitutively active cytokine receptor not linked to a cytokine. These chimeric IL-7 receptors typically constitutively activate STAT5 upon expression.

[0250] In any exemplary embodiment of the methods and compositions provided herein that include a lymphoproliferative element, where the lymphoproliferative element is a cytokine or cytokine receptor polypeptide, or a fragment thereof that includes a signaling domain, the lymphoproliferative element can include an interleukin polypeptide covalently attached via a linker to a portion of a cognate interleukin receptor polypeptide. Typically, this portion of the cognate interleukin receptor includes the functional portion of the extracellular domain that can bind the interleukin cytokine and the transmembrane domain. In some embodiments, the intracellular domain is the intracellular portion of the cognate interleukin receptor. In some embodiments, the intracellular domain is the intracellular portion of a different cytokine receptor that can promote lymphocyte proliferation. In some embodiments, the lymphoproliferative element is an interleukin polypeptide covalently attached via a linker to its full-length cognate interleukin receptor polypeptide.

[0251] In some exemplary embodiments of the methods and compositions provided herein that include lymphoproliferative elements, the intracellular domain can be derived from a portion of the protein IL7RA. Domains, motifs, and point mutations in IL7RA that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the IL7RA polypeptide, some of which are discussed in this paragraph. The IL7RA protein has an S region rich in serine residues (corresponding to residues 359-394 of full-length IL7RA, residues 96-133 of SEQ ID NO: 248), a T region with three tyrosine residues (residues Y401, Y449, and Y456 of full-length IL7RA, residues Y138, Y18, and Y193 of SEQ ID NO: 248), and a Box1 motif (residues 272-280 of full-length IL7RA, corresponding to residues 9-17 of SEQ ID NO: 248 and 249) that can bind to the signaling kinase Jak1 (Jiang, Qiong et al. Mol. and Cell. Biol. Vol. 24(14):6501-13(2004)). In some embodiments, the lymphoproliferative elements herein can include one or more, such as all, of the domains and motifs of IL7RA disclosed herein or known to induce the proliferation and / or survival of T cells and / or NK cells. In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous stretch of amino acids of SEQ ID NO: 248 or 249. In some embodiments, the intracellular domain derived from IL7RA has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, about 65aa to about 70aa, about 70aa to about 100aa, about 100aa to about 125aa, about 125aa to 150aa, about 150 to about 175aa, or about 175aa to about 200aa.In an exemplary embodiment, the intracellular domain derived from IL7RA has a length of about 30 aa to about 200 aa. In an exemplary embodiment of a lymphoproliferative element comprising a first intracellular domain derived from IL7RA, the second intracellular domain may be derived from TNFRSF8.

[0252] In any exemplary embodiments of the methods and compositions provided herein that include lymphoproliferative elements, the intracellular domain can be derived from a portion of the protein IL12RB. Domains, motifs, and point mutations of IL12RB that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the IL12RB polypeptide, some of which are discussed in this paragraph. Full-length IL12RB contains at least one Box1 motif PXXP (SEQ ID NO: 306), where each X can be any amino acid (residues 10-12 of SEQ ID NOs: 254 and 255, and residues 107-110 and 139-142 of SEQ ID NO: 256) (Presky DH et al. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24)). In some embodiments, the lymphoproliferative element comprising the IL12RB intracellular domain can include one or more of the above Box1 motifs or other motifs, domains, or mutations of IL12RB that are known to induce the proliferation and / or survival of T cells and / or NK cells. The Box1 motif of IL12RB is known in the art, and one of ordinary skill in the art can identify the corresponding motif of the IL12RB polypeptide. In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a contiguous series of the amino acids of SEQ ID NOs: 254-256. In some embodiments, the intracellular domain derived from IL12RB has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, about 65aa to about 70aa, about 70aa to about 100aa, about 100aa to about 125aa, about 125aa to 150aa, about 150 to about 175aa, about 175aa to about 200aa, or about 200aa to about 219aa.In an exemplary embodiment, the intracellular domain derived from IL12RB has a length of about 30 aa to about 219 aa, for example, 30 aa to 92 aa, or 30 aa to 90 aa.

[0253] In any exemplary embodiment of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain can be derived from a portion of the protein IL31RA. Domains, motifs, and point mutations of IL31RA that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the IL31RA polypeptide, some of which are discussed in this paragraph. Full-length IL31RA contains the Box1 motif PXXP (SEQ ID NO: 306), where each X can be any amino acid (corresponding to residues 12-15 of SEQ ID NO: 275 and 276) (Cornelissen C et al. Eur J Cell Biol. 2012 Jun-Jul;91(6-7):552-66). In some embodiments, the lymphoproliferative element comprising the IL31RA intracellular domain can contain the Box1 motif. Full-length IL31RA also contains three phosphorylatable tyrosine residues, Y652, Y683, and Y721 (corresponding to residues Y96, Y237, and Y165 of SEQ ID NO: 275; these tyrosine residues are not present in SEQ ID NO: 276), which are important for downstream signaling (Cornelissen C et al. Eur J Cell Biol. 2012 Jun-Jul;91(6-7):552-66). All three tyrosine residues contribute to the activation of STAT1, Y652 is required for the activation of STAT5, and Y721 recruits STAT3. In some embodiments, the lymphoproliferative element having the IL31RA intracellular domain contains the Box1 motif and / or known phosphorylation sites disclosed herein. The Box1 motif and phosphorylatable tyrosine of IL31RA are known in the art, and one of ordinary skill in the art will be able to identify the corresponding motifs and phosphorylatable tyrosine in similar IL31RA polypeptides. In other embodiments, the lymphoproliferative element having the IL31RA intracellular domain does not contain known phosphorylation sites disclosed herein.In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous stretch of the amino acids of SEQ ID NO: 275 or 276. In some embodiments, the intracellular domain derived from IL31RA has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, about 65aa to about 70aa, about 70aa to about 100aa, about 100aa to about 125aa, about 125aa to 150aa, about 150 to about 175aa, or about 175aa to about 189aa. In an exemplary embodiment, the intracellular domain derived from IL31RA has a length of about 30aa to about 200aa, such as 30aa to 189aa, 30aa to 106aa.

[0254] In any exemplary embodiment of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain can be derived from the intracellular portion of the transmembrane protein CD40. Domains, motifs, and point mutations of CD40 that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the CD40 polypeptide, some of which are discussed in this paragraph. The CD40 protein contains several binding sites for TRAF proteins. Without being limited by theory, the binding sites for TRAF1, TRAF2, and TRAF3 are located in the membrane-distal domain of the intracellular portion of CD40 and contain the amino acid sequence PXQXT (SEQ ID NO: 303), where each X can be any amino acid (corresponding to amino acids 35-39 of SEQ ID NO: 208) (Elgueta et al. Immunol Rev. 2009 May;229(1):152-72). TRAF2 has also been shown to bind to the consensus sequence SXXE (SEQ ID NO: 304), where each X can be any amino acid (corresponding to amino acids 57-60 of SEQ ID NO: 208) (Elgueta et al. Immunol Rev. 2009 May;229(1):152-72). A distinct binding site for TRAF6 is located in the membrane-proximal domain of the intracellular portion of CD40 and contains the consensus sequence QXPXEX (SEQ ID NO: 305), where each X can be any amino acid (corresponding to amino acids 16-21 of SEQ ID NO: 208) (Lu et al. J Biol Chem. 2003 Nov 14;278(46):45414-8). In an exemplary embodiment, the intracellular portion of the transmembrane protein CD40 can include all of the binding sites for TRAF proteins. TRAF binding sites are known in the art, and one of ordinary skill in the art will be able to identify the corresponding TRAF binding sites in similar CD40 polypeptides. In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 10, 15, 20, or all of a contiguous series of amino acids of SEQ ID NO: 208 or SEQ ID NO: 209.In some embodiments, the intracellular domain derived from CD40 has a length of about 30 amino acids (aa) to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, or about 60 aa to about 65 aa. In an exemplary embodiment, the intracellular domain derived from CD40 has a length of about 30 aa to about 66 aa, for example, 30 aa to 65 aa, or 50 aa to 66 aa. In an exemplary embodiment of a lymphoproliferative element comprising a first intracellular domain derived from CD40, the second intracellular domain may be other than MyD88, a CD28 family member (e.g., CD28, ICOS), a pattern recognition receptor, a C-reactive protein receptor (i.e., Nodi, Nod2, PtX3-R), a TNF receptor, CD40, RANK / TRANCE-R, OX40, 4-1BB), an HSP receptor (Lox-1 and CD91), or an intracellular domain derived from CD28. Pattern recognition receptors include, but are not limited to, pattern recognition receptors for endocytosis (i.e., mannose receptor, scavenger receptor (i.e., Mac-1, LRP, peptidoglycan, teichoic acid, toxin, CD11c / CR4)), external signal pattern recognition receptors (Toll-like receptors (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10), peptidoglycan recognition protein (PGRP which binds to bacterial peptidoglycan and CD14)); internal signal pattern recognition receptors (i.e., NOD receptors 1 and 2), and RIG1.

[0255] In any exemplary embodiment of the methods and compositions provided herein that include lymphoproliferative elements, the intracellular domain can be derived from the intracellular portion of CD27. Domains, motifs, and point mutations of CD27 that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the CD27 polypeptide, some of which are discussed in this paragraph. Serine at amino acid 219 of full-length CD27 (corresponding to serine at amino acid 6 of SEQ ID NO: 205) has been shown to be phosphorylated. In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 10, 15, 20, or all of a contiguous series of amino acids of SEQ ID NO: 205. In some embodiments, the intracellular domain derived from CD27 has a length of about 30 amino acids (aa) to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, or about 45 aa to about 50 aa.

[0256] In any exemplary embodiment of the methods and compositions provided herein that include lymphoproliferative elements, the intracellular domain can be derived from the intracellular portion of CSF2RB. Domains, motifs, and point mutations of CSF2RB that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the CSF2RB polypeptide, some of which are discussed in this paragraph. Full-length CSF2RB contains a Box1 motif at amino acids 474 - 482 (corresponding to amino acids 14 - 22 of SEQ ID NO: 213). The tyrosine at amino acid 766 of full-length CSF2RB (corresponding to the tyrosine at amino acid 306 of SEQ ID NO: 213) has been shown to be phosphorylated. In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20, or all of the amino acids of SEQ ID NO: 213. In some embodiments, the intracellular domain derived from CSF2RB has a length of about 30aa - about 35aa, about 35aa - about 40aa, about 40aa - about 45aa, about 45aa - about 50aa, about 50aa - about 55aa, about 55aa - about 60aa, about 60aa - about 65aa, about 65aa - about 70aa, about 70aa - about 100aa, about 100aa - about 125aa, about 125aa - 150aa, about 150 - about 175aa, about 175aa - about 200aa, about 200aa - about 250aa, about 250aa - 300aa, about 300aa - 350aa, about 350aa - about 400aa, or about 400aa - about 450aa.

[0257] In any exemplary embodiments of the methods and compositions provided herein that include lymphoproliferative elements, the intracellular domain can be derived from the intracellular portion of IL2RB. Domains, motifs, and point mutations of IL2RB that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the IL2RB polypeptide, some of which are discussed in this paragraph. Full-length IL2RB contains a Box1 motif at amino acids 278-286 (corresponding to amino acids 13-21 of SEQ ID NO: 240). In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a contiguous stretch of at least 10, 15, 20, or all of the amino acids of SEQ ID NO: 240. In some embodiments, the intracellular domain derived from IL2RB has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, about 65aa to about 70aa, about 70aa to about 100aa, about 100aa to about 125aa, about 125aa to 150aa, about 150 to about 175aa, about 175aa to about 200aa, about 200aa to about 250aa, or about 250aa to 300aa.

[0258] In any exemplary embodiments of the methods and compositions provided herein that include lymphoproliferative elements, the intracellular domain can be derived from the intracellular portion of IL6ST. Domains, motifs, and point mutations of IL6ST that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the IL6ST polypeptide, some of which are discussed in this paragraph. Full-length IL6ST contains a Box1 motif at amino acids 651-659 (corresponding to amino acids 10-18 of SEQ ID NO: 247). Serines at amino acids 661, 667, 782, 789, 829, and 839 of full-length IL6ST (corresponding to serines at amino acids 20, 26, 141, 148, 188, and 198 of SEQ ID NO: 247, respectively) have been shown to be phosphorylated. In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a continuous stretch of at least 10, 15, 20, or all of the amino acids of SEQ ID NO: 246 or SEQ ID NO: 247. In some embodiments, the intracellular domain derived from IL6ST has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, about 65aa to about 70aa, about 70aa to about 100aa, about 100aa to about 125aa, about 125aa to 150aa, about 150 to about 175aa, about 175aa to about 200aa, about 200aa to about 250aa, or about 250aa to 300aa.

[0259] In any exemplary embodiment of the methods and compositions provided herein that include lymphoproliferative elements, the intracellular domain can be derived from the intracellular portion of IL17RE. Domains, motifs, and point mutations of IL17RE that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the IL17RE polypeptide, some of which are discussed in this paragraph. Full-length IL17RE contains a TIR domain at amino acids 372-495 (corresponding to amino acids 13-136 of SEQ ID NO: 265). In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a contiguous stretch of at least 10, 15, 20, or all of the amino acids of SEQ ID NO: 265. In some embodiments, the intracellular domain derived from IL17RE has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, about 65aa to about 70aa, about 70aa to about 100aa, about 100aa to about 125aa, about 125aa to 150aa, about 150 to about 175aa, or about 175aa to about 200aa.

[0260] In any exemplary embodiment of the methods and compositions provided herein that include lymphoproliferative elements, the intracellular domain can be derived from the intracellular portion of IL2RG. Domains, motifs, and point mutations of IL2RG that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the IL2RG polypeptide, some of which are discussed in this paragraph. Full-length IL2RG contains the Box1 motif at amino acids 286-294 (corresponding to amino acids 3-11 of SEQ ID NO: 241). In some embodiments, the appropriate intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a contiguous stretch of amino acids of SEQ ID NO: 241. In some embodiments, the intracellular domain derived from IL2RG has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, about 65aa to about 70aa, or about 70aa to about 100aa.

[0261] In any exemplary embodiment of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain can be derived from the intracellular portion of IL18R1. Domains, motifs, and point mutations of IL18R1 that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the IL18R1 polypeptide, some of which are discussed in this paragraph. Full-length IL18R1 contains a TIR domain at amino acids 222-364 (corresponding to amino acids 28-170 of SEQ ID NO: 266). In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20, or all of the amino acids of SEQ ID NO: 266. In some embodiments, the intracellular domain derived from IL18R1 has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, about 65aa to about 70aa, or about 70aa to about 100aa.

[0262] In any exemplary embodiment of the methods and compositions provided herein that include lymphoproliferative elements, the intracellular domain can be derived from the intracellular portion of IL27RA. Domains, motifs, and point mutations of IL27RA that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the IL27RA polypeptide, some of which are discussed in this paragraph. Full-length IL27RA contains a Box1 motif at amino acids 554 - 562 (corresponding to amino acids 17 - 25 of SEQ ID NO: 273). In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20, or all of the amino acids of SEQ ID NO: 273 or SEQ ID NO: 274. In some embodiments, the intracellular domain derived from IL27RA has a length of about 30aa - about 35aa, about 35aa - about 40aa, about 40aa - about 45aa, about 45aa - about 50aa, about 50aa - about 55aa, about 55aa - about 60aa, about 60aa - about 65aa, about 65aa - about 70aa, or about 70aa - about 100aa.

[0263] In some exemplary embodiments of any of the methods and compositions provided herein that include lymphoproliferative elements, the intracellular domain can be derived from the intracellular portion of IFNGR2. Domains, motifs, and point mutations of IFNGR2 that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the IFNGR2 polypeptide, some of which are discussed in this paragraph. Full-length IFNGR2 contains a dileucine internalization motif at amino acids 276-277 (corresponding to amino acids 8-9 of SEQ ID NO: 230). In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a contiguous stretch of amino acids of SEQ ID NO: 230. In some embodiments, the intracellular domain derived from IFNGR2 has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

[0264] In any exemplary embodiments of the methods and compositions provided herein that include lymphoproliferative elements, the intracellular domain can be derived from a portion of the protein MyD88. Domains, motifs, and point mutations of MyD88 that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the MyD88 polypeptide, some of which are discussed in this paragraph. The MyD88 protein has an N-terminal death domain (corresponding to amino acids 29-106 of SEQ ID NO: 284) that mediates interaction with other death domain-containing proteins, an intermediate domain (corresponding to amino acids 107-156 of SEQ ID NO: 284) that interacts with IL-1R-associated kinases, and a C-terminal TIR domain (corresponding to amino acids 160-304 of SEQ ID NO: 284) associated with the TLR-TIR domain (Biol Res. 2007;40(2):97-112). MyD88 also has standard nuclear localization and export motifs. Point mutations have been identified in MyD88, including loss-of-function mutations L93P and R193C (corresponding to L93P and R196C of SEQ ID NO: 284), and a gain-of-function mutation L265P (corresponding to L260P of SEQ ID NO: 284) (Deguine and Barton. F1000Prime Rep. 2014 Nov 4;6:97). In some embodiments, the lymphoproliferative elements herein can include one or more, e.g., all of the domains and motifs of MyD88 disclosed herein. In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a contiguous sequence of amino acids of SEQ ID NOs: 284-293, and in exemplary embodiments can include one or more, in exemplary embodiments all of the following MyD88 domains / motifs: death domain, intermediate domain, TIR domain, nuclear localization and export motifs, and amino acids corresponding to positions L93, R193, and L265 or P265.In some embodiments, the intracellular domain derived from MyD88 has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, about 65aa to about 70aa, about 70aa to about 100aa, about 100aa to about 125aa, about 125aa to 150aa, about 150 to about 175aa, about 175aa to about 200aa, about 200aa to about 250aa, about 250aa to 300aa, or about 300aa to 350aa. In an exemplary embodiment, the intracellular domain derived from MyD88 has a length of about 30aa to about 350aa, such as 50aa to 350aa, or 100aa to 350aa, 100aa to 304aa, 100aa to 296aa, 100aa to 251aa, 100aa to 191aa, 100aa to 172aa, 100aa to 146aa, or 100aa to 127aa. In an exemplary embodiment of a lymphoproliferative element comprising a first intracellular domain derived from MyD88, the second intracellular domain may be derived from TNFRSF4 or TNFRSF8. In other exemplary embodiments of a lymphoproliferative element comprising a first intracellular domain derived from MyD88, the second intracellular domain may be other than an intracellular domain derived from a CD28 family member (e.g., CD28, ICOS), a pattern recognition receptor, a C-reactive protein receptor (i.e., Nodi, Nod2, PtX3-R), a TNF receptor (i.e., CD40, RANK / TRANCE-R, OX40, 4-1BB), an HSP receptor (Lox-1 and CD91), or CD28.

[0265] In any exemplary embodiment of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain can be derived from a portion of the transmembrane protein MPL. Domains, motifs, and point mutations of MPL that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the MPL polypeptide, some of which are discussed in this paragraph. The transmembrane MPL protein includes a Box1 motif PXXP (SEQ ID NO: 306) (each X can be any amino acid (corresponding to amino acids 17-20 of SEQ ID NO: 283)) and a Box2 motif (a region with increased serine and glutamic acid content) (corresponding to amino acids 46-64 of SEQ ID NO: 283) (Drachman and Kaushansky. Proc Natl Acad Sci USA. 1997 Mar 18;94(6):2350-5). The Box1 and Box2 motifs are involved in binding to and signaling by JAK, although the presence of the Box2 motif is not necessarily required for proliferative signaling (Murakami et al. Proc Natl Acad Sci USA. 1991 Dec 15;88(24):11349-53, Fukunaga et al. EMBO J. 1991 Oct;10(10):2855-65, and O’Neal and Lee. Lymphokine Cytokine Res. 1993 Oct;12(5):309-12). Many cytokine receptors have hydrophobic residues at positions 1, 2, and 6 relative to the Box1 motif (corresponding to amino acids 16, 15, and 11 of SEQ ID NO: 283, respectively), which form a “switch motif” that is required for cytokine-induced JAK2 activation but not for JAK2 binding (Constantinescu et al. Mol Cell. 2001 Feb;7(2):377-85; and Huang et al. Mol Cell. 2001 Dec;8(6):1327-38).Deletion of the region encompassing amino acids 70 - 95 of SEQ ID NO: 283 has been shown to support viral transformation in association with v - mpl (Benit et al. J Virol. 1994 Aug;68(8):5270 - 4), thus indicating that this region is not required for the function of mpl associated with this. Morello et al. Blood 1995 July;86(8):557 - 71 used the same deletion to show that this region is not required to stimulate transcription of a hematopoietin receptor - responsive CAT reporter gene construct, and further found that, as suggested by Drachman and Kaushansky, this deletion results in a slightly enhanced transcription expected from the removal of non - essential negative elements in this region. Thus, in some embodiments, the MPL intracellular signaling domain does not include the region encompassing amino acids 70 - 95 of SEQ ID NO: 283. In full - length MPL, lysines K553 (corresponding to K40 of SEQ ID NO: 283) and K573 (corresponding to K60 of SEQ ID NO: 283) have been shown to be negative regulatory sites that function as part of a ubiquitination targeting motif (Saur et al. Blood 2010 Feb 11;115(6):1254 - 63). Thus, in some embodiments herein, the MPL intracellular signaling domain does not include these ubiquitination targeting motif residues. In full - length MPL, tyrosine Y521 (corresponding to Y8 of SEQ ID NO: 283), Y542 (corresponding to Y29 of SEQ ID NO: 283), Y591 (corresponding to Y78 of SEQ ID NO: 283), Y626 (corresponding to Y113 of SEQ ID NO: 283), and Y631 (corresponding to Y118 of SEQ ID NO: 283) have been shown to be phosphorylated (Varghese et al. Front Endocrinol (Lausanne). 2017 Mar 31;8:59).Y521 and Y591 of full-length MPL are negative regulatory sites that function either as part of the lysosomal targeting motif (Y521) or through interaction with the adaptor protein AP2 (Y591) (Drachman and Kaushansky. Proc Natl Acad Sci USA. 1997 Mar 18;94(6):2350-5, and Hitchcock et al. Blood. 2008 Sep 15;112(6):2222-31). Y626 and Y631 of full-length MPL are positive regulatory sites (Drachman and Kaushansky. Proc Natl Acad Sci USA. 1997 Mar 18;94(6):2350-5), and the mouse homolog of Y626 is required for cell differentiation and phosphorylation of Shc (Alexander et al. EMBO J. 1996 Dec 2;15(23):6531-40), and Y626 is also required for constitutive signaling of MPL with the W515A mutation described below (Pecquet et al. Blood. 2010 Feb 4;115(5):1037-48). MPL contains the Shc phosphotyrosine-binding binding motif NXXY (SEQ ID NO: 307), where each X may be any amino acid (corresponding to amino acids 110-113 of SEQ ID NO: 283), and this tyrosine is phosphorylated and is important for TPO-dependent phosphorylation of Shc, SHIP, and STAT3 (Laminet et al. J Biol Chem. 1996 Jan 5;271(1):264-9; and van der Geer et al. Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):963-8). MPL also contains the STAT3 consensus binding sequence YXXQ (SEQ ID NO: 308), where each X may be any amino acid (corresponding to amino acids 118-121 of SEQ ID NO: 283) (Stahl et al. Science. 1995 Mar 3;267(5202):1349-53). The tyrosine of this sequence can be phosphorylated, and MPL is capable of partial STAT3 recruitment (Drachman and Kaushansky. Proc Natl Acad Sci USA. 1997 Mar 18;94(6):2350-5).MPL also includes the array YLPL (array number 309) (corresponding to amino acids 113-116 of array number 283), which is similar to the consensus binding site of STAT5 mobilizing pYLXL (array number 310), where pY is phosphotyrosine and X can be any amino acid (May et al. FEBS Lett. 1996 Sep 30;394(2):221-6). Lee et al. used computer simulations to discover that clinically relevant mutations in the transmembrane domain of MPL should activate MPL in the following order: W515K (corresponding to amino acid substitution W2K of array number 283) > S505A (corresponding to amino acid substitution S14A of array number 187) > W515I (corresponding to amino acid substitution W2I of array number 283) > S505N (corresponding to amino acid substitution S14N of array number 187, which was tested in Example 12 as part T075 (array number 188)) (PLoS One. 2011;6(8):e23396). The simulations predicted that these mutations could cause constitutive activation of JAK2, the kinase partner of MPL. In some embodiments, the intracellular portion of MPL can include one or more, or all, of the domains and motifs described herein that are present in array number 283. In some embodiments, the transmembrane portion of MPL can include one or more, or all, of the domains and motifs described herein that are present in array number 187. The domains, motifs, and point mutations of MPL provided herein are known in the art, and those skilled in the art will recognize that the MPL intracellular signaling domain in the exemplary embodiments includes one or more corresponding domains, motifs, and point mutations that have been shown to promote proliferative activity and do not include those that have been shown to inhibit MPL proliferative activity. In some embodiments, suitable intracellular domains can include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a contiguous stretch of amino acids of array number 283.In some embodiments, the intracellular domain derived from MPL has a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, about 65 aa to about 70 aa, about 70 aa to about 100 aa, about 100 aa to about 125 aa, about 125 aa to 150 aa, about 150 to about 175 aa, about 175 aa to about 200 aa, about 200 aa to about 250 aa, about 250 aa to 300 aa, about 300 aa to 350 aa, about 350 aa to about 400 aa, about 400 aa to about 450 aa, about 450 aa to about 500 aa, about 500 aa to about 550 aa, about 550 aa to about 600 aa, or about 600 aa to about 635 aa. In an exemplary embodiment, the intracellular domain derived from MPL has a length of about 30 aa to about 200 aa, such as 30 aa to 150 aa, 30 aa to 119 aa, 30 aa to 121 aa, 30 aa to 122 aa, or 50 aa to 125 aa. In an exemplary embodiment of a lymphoproliferative element comprising a first intracellular domain derived from MPL, the second intracellular domain can be derived from CD79B.

[0266] In any exemplary embodiment of the methods and compositions provided herein that include lymphoproliferative elements, the intracellular domain can be derived from a portion of the transmembrane protein CD79B, also known as B29; IGB; AGM6. Domains, motifs, and point mutations of CD79B that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the CD79B polypeptide, some of which are discussed in this paragraph. CD79B contains an ITAM motif at residues 193-212 (corresponding to amino acids 16-30 of SEQ ID NO: 211). CD79B has two tyrosines, Y196 and Y207 (corresponding to Y16 and Y27 of SEQ ID NO: 211), that are known to be phosphorylated. In some embodiments, the intracellular portion of the transmembrane protein CD79B includes an ITAM motif and / or a known phosphorylation site disclosed herein. Motifs and phosphorylatable tyrosines of CD79B are known in the art, and one of ordinary skill in the art will be able to identify the corresponding motifs and phosphorylatable tyrosines in similar CD79B polypeptides. In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a contiguous stretch of at least 10, 15, 20, or all of the amino acids of SEQ ID NO: 211. In some embodiments, the intracellular domain derived from CD79B has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, or about 45aa to about 50aa. In an exemplary embodiment, the intracellular domain derived from CD79B has a length of about 30aa to about 50aa.For example, a suitable CD79B intracellular activation domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous stretch of amino acids of the following sequence: LDKDDSKAGMEEDHT[YEGLDIDQTATYEDI]VTLRTGEVKWSVGEHPGQE (SEQ ID NO: 211) (the ITAM motif is shown in parentheses). In an exemplary embodiment of a lymphoproliferative element comprising a second intracellular domain derived from CD79B, the first intracellular domain can be derived from CSF3R.

[0267] In some exemplary embodiments of the methods and compositions provided herein that include lymphoproliferative elements, the intracellular domain can be derived from a portion of the transmembrane protein OSMR. Domains, motifs, and point mutations of OSMR that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the OSMR polypeptide, some of which are discussed in this paragraph. OSMR contains a Box1 motif at amino acids 771-779 of isoform 3 (corresponding to amino acids 16-30 of SEQ ID NO: 294). OSMR has two serines (serines at amino acids 65 and 128 of SEQ ID NO: 294) at amino acids 829 and 890 of isoform 3 that are known to be phosphorylated. In some embodiments, the intracellular portion of the protein OSMR can include the Box1 motif and the known phosphorylation sites disclosed herein. The motifs and phosphorylatable serines of OSMR are known in the art, and one of ordinary skill in the art will be able to identify the corresponding motifs and phosphorylatable serines in similar OSMR polypeptides. In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20, or all of the amino acids of SEQ ID NO: 294. In some embodiments, the intracellular domain derived from OSMR has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, about 65aa to about 70aa, about 70aa to about 100aa, about 100aa to about 125aa, about 125aa to 150aa, about 150 to about 175aa, about 175aa to about 200aa, or about 200aa to about 250aa.

[0268] In some exemplary embodiments of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain can be derived from a portion of the transmembrane protein PRLR. Domains, motifs, and point mutations in PRLR that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations in the PRLR polypeptide, some of which are discussed in this paragraph. PRLR contains a growth hormone receptor binding domain at amino acids 185-261 of isoform 6 (corresponding to amino acids 28-104 of SEQ ID NO: 295). The growth hormone receptor binding domain of PRLR is known in the art, and one of ordinary skill in the art will be able to identify the corresponding domain in a similar PRLR polypeptide. In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids of SEQ ID NO: 295. In some embodiments, the intracellular domain derived from PRLR has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, about 65aa to about 70aa, about 70aa to about 100aa, about 100aa to about 125aa, about 125aa to 150aa, about 150 to about 175aa, about 175aa to about 200aa, about 200aa to about 250aa, about 250aa to 300aa, about 300aa to 350aa, or about 350aa to about 400aa.

[0269] In some embodiments, the intracellular domain of the lymphoproliferative element is derived from the intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1).

[0270] In any exemplary embodiment of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain can be derived from a portion of the protein CD28. Domains, motifs, and point mutations of CD28 that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the CD28 polypeptide, some of which are discussed in this paragraph. Full-length CD28 contains PI3-K and Grb2 binding motifs corresponding to residues 12-15 of SEQ ID NOs: 206 and 207 (Harada et al. J Exp Med. 2003 Jan 20;197(2):257-62). In some embodiments, the lymphoproliferative element comprising the CD28 intracellular domain can include PI3-K and Grb2 binding motifs. In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a continuous stretch of at least 10, 15, 20, or all of the amino acids of SEQ ID NO: 206 or 207. In some embodiments, the intracellular domain derived from CD28 has a length of about 5aa to about 10aa, about 10aa to about 15aa, about 15aa to about 20aa, about 20aa to about 25aa, about 25aa to about 30aa, about 30aa to about 35aa, or about 35aa to about 42aa.

[0271] In some exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain can be derived from a portion of the protein ICOS. Domains, motifs, and point mutations of ICOS that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the ICOS polypeptide, some of which are discussed in this paragraph. Unlike CD28, ICOS binds to PI3-K rather than Grb2. The PI3-K binding motif of full-length ICOS corresponds to residues 19-22 of SEQ ID NO: 225. A single amino acid substitution in this motif can result in increased Grb2 binding and IL-2 production by ICOS (Harada et al. J Exp Med. 2003 Jan 20;197(2):257-62). This mutation corresponds to mutating phenylalanine 21 of SEQ ID NO: 225 to asparagine. One of ordinary skill in the art will understand how to mutate this residue of SEQ ID NO: 225 to generate an ICOS intracellular domain that binds to Grb2 in addition to PI3-K. In some embodiments, the lymphoproliferative element comprising the ICOS intracellular domain can include a PI3-K binding motif. In some embodiments, the lymphoproliferative element comprising the ICOS intracellular domain can include a PI3-K binding motif mutated to further bind to Grb2. ICOS also includes a membrane-proximal motif in the cytoplasmic tail that is essential for ICOS-mediated calcium signaling (Leconte et al. Mol Immunol. 2016 Nov;79:38-46). This calcium signaling motif corresponds to residues 5-8 of SEQ ID NO: 225. In some embodiments, the lymphoproliferative element comprising the ICOS intracellular domain can include a calcium signaling motif. Two other conserved motifs have been identified in full-length ICOS. A first conserved motif of residues 170-179 (corresponding to residues 9-18 of SEQ ID NO: 225) and a second conserved motif of residues 185-191 (corresponding to residues 24-30 of SEQ ID NO: 225) (Pedros et al. Nat Immunol. 2016 Jul;17(7):825-33).These two conserved motifs may have an important function in mediating downstream ICOS signaling. In some embodiments, a lymphoproliferative element comprising an ICOS intracellular domain may comprise at least one of the first or second conserved motifs. In some embodiments, a lymphoproliferative element comprising an ICOS intracellular domain does not comprise the first conserved motif, does not comprise the second conserved motif, or does not comprise the first and second conserved motifs. In some embodiments, a suitable intracellular domain may comprise a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a stretch of the amino acids of SEQ ID NO: 225. In some embodiments, an intracellular domain derived from ICOS has a length of about 5aa to about 10aa, about 10aa to about 15aa, about 15aa to about 20aa, about 20aa to about 25aa, about 25aa to about 30aa, about 30aa to about 35aa, or about 35aa to about 38aa.

[0272] In some embodiments, the intracellular domain of the chimeric lymphoproliferative element is derived from the intracellular portion of the transmembrane protein OX40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX-40, TXGP1L). The domains, motifs, and point mutations of OX40 that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the OX40 polypeptide, some of which are discussed in this paragraph. OX40 contains a TRAF binding motif at residues 256-263 of full-length OX40 (corresponding to residues 20-27 of SEQ ID NO: 296), which is important for the binding of TRAF1, TRAF2, TRAF3, and TRAF5 (Kawamata, S, et al. J Biol Chem. 1998 Mar 6;273(10):5808-14; Hori, T. Int J Hematol. 2006 Jan;83(1):17-22). Full-length OX40 also contains a p85PI3K binding motif at residues 34-57. In some embodiments, when OX40 is present as the intracellular domain of the lymphoproliferative element, it contains the p85 PI3K binding motif of OX40. In some embodiments, the intracellular domain of OX40 may contain the TRAF binding motif of OX40. In some embodiments, the intracellular domain of OX40 can bind to TRAF1, TRAF2, TRAF3, and TRAF5. The lysines corresponding to amino acids 17 and 41 of SEQ ID NO: 296 are potentially negative regulatory sites that function as part of a ubiquitination targeting motif. In some embodiments, one or both of these lysines in the intracellular domain of OX40 are mutated arginine or another amino acid. In some embodiments, the appropriate intracellular domain of the lymphoproliferative element can contain a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20, or all of the amino acids of SEQ ID NO: 57.In some of these embodiments, the intracellular domain of OX40 has a length of about 20aa to about 25aa, about 25aa to about 30aa, 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, or about 45aa to about 50aa. In an exemplary embodiment, the intracellular domain of OX40 has a length of about 20aa to about 50aa, such as 20aa to 45aa, or 20aa to 42aa.

[0273] In some embodiments, the intracellular domain of the chimeric lymphoproliferative element is derived from the intracellular portion of the transmembrane protein IFNAR2. Domains, motifs, and point mutations of IFNAR2 that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the IFNAR2 polypeptide, some of which are discussed in this paragraph. Full-length IFNAR2 contains a Box1 motif as well as two Box2 motifs (known as Box2A and Box2B). (Usacheva A et al. J Biol Chem. 2002 Dec 13;277(50):48220-6). In some embodiments, the lymphoproliferative element comprising the IFNAR2 intracellular domain may comprise one or more of the Box1 or Box2 motifs. In an exemplary embodiment, the IFNAR2 intracellular domain may comprise one or more of the Box1, Box2A, or Box2B motifs. IFNAR2 contains a JAK1 binding site (Gauzzi MC et al. Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):11839-44; Schindler et al. J Biol Chem. 2007 Jul 13;282(28):20059-63). In some embodiments, the lymphoproliferative element comprising the IFNAR2 intracellular domain may comprise the JAK1 binding site. In some embodiments, the appropriate intracellular domain of the lymphoproliferative element may comprise a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous stretch of the amino acids of SEQ ID NO: 227 or 228.In some of these embodiments, the intracellular domain of IFNAR2 has a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, about 65 aa to about 70 aa, about 70 aa to about 100 aa, about 100 aa to about 125 aa, about 125 aa to about 150 aa, about 150 aa to about 175 aa, about 175 aa to about 200 aa, or about 200 aa to about 251 aa. In an exemplary embodiment, the intracellular domain of OX40 has a length of about 30 aa to about 251 aa, such as 30 aa to 67 aa.

[0274] In some embodiments, the intracellular domain of the chimeric lymphoproliferative element is derived from the intracellular portion of the transmembrane protein CSF3R. Domains, motifs, and point mutations of CSF3R that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and those skilled in the art can identify the corresponding domains, motifs, and point mutations of the CSF3R polypeptide, some of which are discussed in this paragraph. Full-length CSF3R contains Box1 and Box2 motifs, as well as a Box3 motif (Nguyen-Jackson HT et al. G-CSF Receptor Structure, Function, and Intracellular Signal Transduction. Twenty Years of G-CSF, (2011) 83-105). In some embodiments, the lymphoproliferative element comprising the CSF3R intracellular domain may comprise one or more of the Box1, Box2, or Box3 motifs. CSF3R contains four tyrosine residues Y704, Y729, Y744, and Y764 in the full-length CSF3R, which are important for the binding of STAT3 (Y704 and Y744), SOCS3 (Y729), and Grb2 and p21Ras (Y764). In some embodiments, the lymphoproliferative element comprising the CSF3R intracellular domain may comprise one, two, three, or all of the tyrosine residues corresponding to Y704, Y729, Y744, and Y764 of the full-length CSF3R. CSF3R contains two threonine residues T615 and T618 in the full-length CSF3R, and mutation to alanine and isoleucine (T615A and T618I), respectively, can increase receptor dimerization and activity (Maxson et al. J Biol Chem. 2014 Feb 28;289(9):5820-7). In some embodiments, the lymphoproliferative element comprising the CSF3R intracellular domain may comprise one or more of the mutations corresponding to T615A and T618I.In some embodiments, a suitable intracellular domain of a lymphoproliferative element can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20, or all of the amino acids of SEQ ID NO: 216, 217, or 218. In some of these embodiments, the intracellular domain of CSF3R is from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100 aa to about 125 aa, from about 125 aa to about 150 aa, from about 150 aa to about 175 aa, from about 175 aa to about 200 aa, or from about 200 aa to about 213 aa in length. In an exemplary embodiment, the intracellular domain of CSF3R is from about 30 aa to about 213 aa in length, such as from about 30 aa to about 186 or from about 30 aa to about 133 aa.

[0275] In some embodiments, the intracellular domain of the chimeric lymphoproliferative element is derived from the intracellular portion of the transmembrane protein EPOR. Domains, motifs, and point mutations of EPOR that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the EPOR polypeptide, some of which are discussed in this paragraph. EPOR contains Box1 (residues 257-264 of full-length EPOR) and Box2 (residues 303-313 of full-length EPOR) motifs (Constantinescu SN. Trends Endocrinol Metab. 1999 Dec;10(1):18-23). EPOR also contains an extended Box2 motif (residues 329-372) that is important for the binding of the tyrosine kinase receptor KIT (Constantinescu SN. Trends Endocrinol Metab. 1999 Dec;10(1):18-23). In some embodiments, the lymphoproliferative element comprising the EPOR intracellular domain may comprise one or more of Box1, Box2, or the extended Box2 motif. EPOR also contains a short segment (residues 267-276 of full-length EPOR) that is important for the internalization of EPOR. In some embodiments, the lymphoproliferative element comprising the EPOR intracellular domain does not contain the internalization segment. In some embodiments, the appropriate intracellular domain of the lymphoproliferative element may comprise a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous stretch of the amino acids of SEQ ID NO: 219 or 220. In some of these embodiments, the intracellular domain of EPOR is about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, about 65aa to about 70aa, about 70aa to about 100aa, about 100aa to about 125aa, about 125aa to about 150aa, about 150aa to about 175aa, about 175aa to about 200aa, or about 200aa to about 235aa in length.In an exemplary embodiment, the intracellular domain of EPOR has a length of from about 30 aa to about 235 aa.

[0276] In some embodiments, the intracellular domain of the chimeric lymphoproliferative element is derived from the intracellular portion of the transmembrane protein CD3G. Domains, motifs, and point mutations of CD3G that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and one of ordinary skill in the art can identify the corresponding domains, motifs, and point mutations of the CD3G polypeptide, some of which are discussed in this paragraph. Two serine residues of full-length CD3G, S123 and S126, have been shown to be phosphorylated in T cells in response to ionomycin (Davies et al. J Biol Chem. 1987 Aug 15;262(23):10918-21). In some embodiments, the lymphoproliferative element comprising the CD3G intracellular domain may comprise one or more serine residues corresponding to full-length S123 and S126. Furthermore, phosphorylation at S126 rather than S123 has been shown to be required for PKC-mediated downregulation (Dietrich J et al. EMBO J. 1994 May 1;13(9):2156-66). In some embodiments, the lymphoproliferative element comprising the CD3G intracellular domain may comprise a serine residue corresponding to full-length S123 and may not comprise a serine residue corresponding to full-length S126. In some embodiments, the lymphoproliferative element comprising the CD3G intracellular domain may comprise a non-phosphorylatable amino acid substitution at the serine residue corresponding to full-length S126. In an exemplary embodiment, the amino acid substitution may be a mutation from serine to alanine. Furthermore, mutation of either leucine of the di-leucine motif of full-length CD3G, L131 and L132, from leucine to alanine has been shown to prevent PKC-mediated downregulation (Dietrich J et al. EMBO J. 1994 May 1;13(9):2156-66). In some embodiments, the lymphoproliferative element comprising the CD3G intracellular domain may comprise at least one amino acid substitution at the leucine residue corresponding to L131 or L132 of full-length CD3G. In an exemplary embodiment, the amino acid substitution may be a mutation from leucine to alanine.In some embodiments, a suitable intracellular domain of a lymphoproliferative element can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of the contiguous amino acids of SEQ ID NO: 199. In some of these embodiments, the intracellular domain of CD3G has a length of about 20aa to about 25aa, about 25aa to about 30aa, about 30aa to about 35aa, about 35aa to about 40aa, or about 40aa to about 45aa. In an exemplary embodiment, the intracellular domain of CD3D has a length of about 30aa to about 45aa.

[0277] In an exemplary embodiment, the cytoplasmic domain of a TNF receptor (TNFR) that may be TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18 can recruit signaling molecules including TRAF (TNF receptor-associated factor) and / or "death domain" (DD) molecules. Domains, motifs, and point mutations of TNFR that induce the proliferation and / or survival of T cells and / or NK cells are known in the art, and those skilled in the art can identify the corresponding domains, motifs, and point mutations of the TNFR polypeptide, some of which are discussed in this paragraph. Mammals have at least six TRAF molecules and several non-receptor DD molecules. Receptors and adapter proteins that bind to TRAF share a short consensus TRAF-binding motif known in the art (Meads et al. J Immunol. 2010 Aug 1;185(3):1606-15). The DD-binding motif is a globular bundle of approximately 60 amino acids of six conserved α-helices also known in the art (Locksley RM et al. Cell. 2001 Feb 23;104(4):487-501). Those skilled in the art will be able to identify TRAF and / or DD-binding motifs in different TNFR families, for example, using sequence alignment to known binding motifs. TNFR can recruit TRADD and TRAF2 and activate NF-κB, MAPK, and JNK (Sedger and McDermott. Cytokine Growth Factor Rev. 2014 Aug;25(4):453-72). In some embodiments, the lymphoproliferative element comprising the TNFR intracellular domain ...

Claims

**Claim 1** A method for genetically modifying T cells, comprising: ex vivo, contacting whole blood containing T cells collected from a subject with replication-incompetent recombinant retroviral particles in a reaction mixture wherein the reaction mixture comprises at least 50% whole blood, wherein the reaction mixture comprises an anticoagulant, and wherein the replication-incompetent recombinant retroviral particles comprise: (a) a T cell activation element on their surface; and (b) a polynucleotide comprising one or more transcription units, each of the one or more transcription units being operably linked to a promoter active in T cells, wherein the one or more transcription units encode a first polypeptide and / or an inhibitory RNA molecule, wherein the first polypeptide comprises a chimeric antigen receptor (CAR), a recombinant T cell receptor (TCR), and / or a lymphoproliferative element. A method. **Claim 2** The method according to claim 1, further comprising the step of expanding cells ex vivo, wherein the cells are expanded with no more than 4 cell divisions per cell ex vivo. **Claim 3** The method according to any one of claims 1 to 2, which does not require prior ex vivo stimulation. **Claim 4** The method according to any one of claims 1 to 3, wherein the reaction mixture comprises at least 90% whole blood. **Claim 5** The method according to any one of claims 1 to 4, wherein at least 5% of the T cells in the reaction mixture are genetically modified T cells after the contacting step. **Claim 6** The method according to claim 5, wherein when the T cells are combined with replication-incompetent recombinant retroviral particles to form the reaction mixture, at least 25% of the T cells are resting T cells and the contacting step is carried out in 24 hours or less. **Claim 7** The method according to any one of claims 1 to 6, wherein the T cell activation element comprises a GPI anchor. **Claim 8** The method according to any one of claims 1 to 6, wherein the T cell activation element is a fusion protein with a viral envelope protein. **Claim 9** ​ The method according to any one of claims 1 to 8, wherein the T cell activation element comprises one or more polypeptides capable of binding to CD3, CD28, OX40, 4-1BB, ICOS, CD9, CD53, CD63, CD81, or CD82.

10. The method according to claim 9, wherein the T cell activation element comprises one or more polypeptides capable of binding to CD3, and at least one of the one or more polypeptides capable of binding to CD3 is an antibody.

11. The method according to any one of claims 1 to 10, wherein the one or more transcription units encode a first polypeptide, and the first polypeptide comprises the lymphoproliferative element.

12. The lymphoproliferative element is a signal transduction domain from CD2, CD3D, CD3E, CD3G, CD4, CD8A, CD8B, CD27, CD28, CD40, CD79A, CD79B, CRLF2, CSF2RB, CSF2RA, CSF3R, EPOR, FCER1G, FCG2RC, FCGRA2, GHR, ICOS, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL22RA1, IL23R, IL27RA, IL31RA, LEPR, LIFR, LMP1, MPL, MYD88, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, TNFRSF18, or a functional variant and / or fragment thereof capable of promoting the proliferation and / or survival of T cells. The method according to claim 11.

13. The method according to any one of claims 1 to 10, wherein the one or more transcription units encode the first polypeptide, and the first polypeptide comprises the CAR and / or the recombinant TCR.

14. The method according to any one of claims 11 to 12, wherein the one or more transcription units further encode a second polypeptide, and the second polypeptide comprises a chimeric antigen receptor and / or a recombinant TCR.

15. The method according to any one of claims 1 to 14, wherein the one or more transcription units encode an inhibitory RNA molecule, and the inhibitory RNA molecule targets TCRa, TCRb, SOCS1, miR155 target, IFN gamma, cCBL, TRAIL2, I2A, ABCG1, CD3z, PD1, CTLA4, TIM3, LAG3, SMAD2, TNFRSF10B, PPP2CA, TNFRSF6 (FAS), BTLA, TIGIT, A2AR, AHR, EOMES, SMAD3, SMAD4, TGFBR2, PPP2R2D, TNFRSF6 (FASL), CASP3, SOCS2, TIEG1, JunB, Cbx3, Tet2 or HK2.

16. The method according to claim 15, wherein the inhibitory RNA molecule comprises at least one of the sequences of SEQ ID NOs: 394-401, 406-409, 438-441, or 446-449.

17. The method according to any one of claims 1 to 16, wherein the replication-incompetent recombinant retroviral particle further comprises a membrane-bound cytokine on its surface.

18. The method according to any one of claims 1 to 17, wherein the reaction mixture is in a closed cell processing system.

19. The method according to claim 18, wherein the reaction mixture is in contact with a leukocyte removal filter assembly in a closed cell processing system.

20. The method according to any one of claims 1 to 19, wherein the contacting step is performed for less than 8 hours.

21. The method according to any one of claims 1 to 20, further comprising performing a PBMC enrichment step after the contacting step.

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