Methods and compositions for genetically modifying lymphocytes in blood or enriched PBMCs

Abstract

The present disclosure provides methods and compositions for genetically modifying lymphocytes, such as T cells and / or NK cells, in shorter time periods than previously possible, and / or in whole blood or components thereof. In some embodiments, a lymphocyte-depletion filter assembly is used before or after forming a reaction mixture, and the lymphocytes are contacted with recombinant retroviral particles in a closed system to genetically modify the lymphocytes.

Description

[Technical Field]

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

[0002] Sequence Listing This application incorporates by reference the Electronic Sequence Listing material filed concurrently herewith, which was submitted as a text (.txt) file entitled "F1_001_WO_05_Sequence_Listing_September_02_2019.txt" created on September 2, 2019, has a file size of 450 KB, and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to the field of immunology, or more specifically to the genetic modification of T lymphocytes or other immune cells and methods for controlling the proliferation of such cells. [Background technology]

[0004] Lymphocytes isolated from a subject (e.g., a patient) can be activated in vitro and genetically modified to express synthetic proteins that enable redirected engagement with other cells and the environment based on an incorporated genetic program. Examples of such synthetic proteins include recombinant T cell receptors (TCRs) and chimeric antigen receptors (CARs). One currently used CAR 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] Although recombinant retroviruses have demonstrated efficacy in infecting non-dividing cells, resting CD4 and CD8 lymphocytes are resistant to gene transduction by these vectors. To overcome this difficulty, these cells are typically activated in vitro using stimulatory reagents before genetic modification with the CAR gene vector can occur. Following stimulation and transduction, the genetically modified cells are expanded in vitro and then reintroduced into lymphocyte-depleted patients. Upon antigen engagement in vivo, the intracellular signaling portion of the CAR can initiate activation-associated responses in immune cells and the release of cytolytic molecules, leading to target cell death.

[0006] Current methods require extensive engineering and manufacturing of T cells that are expanded ex vivo before reinfusion into the patient, as well as lymphodepleting chemotherapy to release cytokines and deplete competing receptors to promote T cell engraftment. Such CAR therapies cannot control their in vivo proliferation rate after introduction into the body, nor can they be safely directed to targets expressed outside the tumor. As a result, CAR therapies today typically require fewer than 1 × 10 5 ~1×10 8Ex vivo expanded cells are infused for 12–28 days at a dose of 100 cells / kg and directed to the target, e.g., tumor, with generally acceptable off-tumor on-target toxicity. These relatively long ex vivo expansion times pose challenges to scalability, as well as cell viability, sterility, and sample integrity. Therefore, safer, more effective, and scalable T cell or NK cell therapies are highly sought after.

[0007] Improved methods and compositions for studying lymphocytes are needed because understanding the processes that promote lymphocyte transduction, proliferation, and survival is central to a variety of potential commercial applications, including immunological processes. For example, it would be useful to identify methods and compositions that can be used to better characterize and understand how lymphocytes can be genetically modified and the factors that influence lymphocyte survival and proliferation. Furthermore, it would be useful to identify compositions that promote lymphocyte proliferation and survival. Such compositions could be used to study the regulation of such processes. In addition to methods and compositions for studying lymphocytes, improved virus packaging cell lines and methods for making and using them are needed. For example, such cell lines and methods would be useful in analyzing different components of recombinant viruses, such as recombinant retroviral particles, and in methods using packaging cell lines for the production of recombinant retroviral particles.

[0008] More recent methods have been developed that can be performed without prior activation and ex vivo expansion. However, it would be highly desirable to further reduce the complexity and time required for such methods, especially if such methods involve, for example, drawing the subject's blood in an infusion center and then reinjecting it into the subject on the same day. Furthermore, simpler and faster methods alone, or methods requiring less specialized equipment, could popularize these cell therapy processes, which are currently only routinely performed in highly specialized medical centers.

[0009] Some groups have attempted to simplify ex vivo cell therapy by eliminating the need for extensive ex vivo transduction by intravenously injecting viral particles to transduce cells in vivo. However, such methods require large amounts of vector and risk inactivation of retroviral particles by clotting factors and / or other enzymes present in vivo. Finally, such methods risk high levels of transduction of non-target cells / organs. Summary of the Invention

[0010] Provided herein are methods, compositions, and kits that help overcome 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. Accordingly, in some aspects, provided herein are methods, compositions, and kits for genetically modifying lymphocytes, particularly T cells and / or NK cells, and / or modulating the activity of transduced and / or genetically modified T cells and / or NK cells. Such methods, compositions, and kits offer improved efficacy and safety over current technologies, particularly for 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. In exemplary embodiments, the transduced and / or genetically modified T cells and / or NK cells produced and / or used by the methods provided herein are delivered from a retroviral (e.g., lentiviral) genome via a retroviral (e.g., lentiviral) particle and comprise a combination of functionalities that provide improved functionality to such cells and methods that utilize such cells, such as research methods, commercial production methods, and adoptive cell therapy, etc. For example, such cells can be generated ex vivo in a shorter time and have improved growth properties that can be better regulated.

[0011] In some aspects, 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 aspects can be performed much more quickly 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 expressing a TCR or CAR.

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

[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 disclosed combinations of methods, compositions, and kits or functional elements therein throughout the sections. [Brief explanation of the drawings]

[0014] [Figure 1A]

[0023] Figure 1A is a flowchart of a non-limiting exemplary cell processing workflow. Figure 1A is a flowchart of a process using a system with PBMC enrichment before T cells and NK cells in PBMCs are contacted with retroviral particles. Figure 1B is a flowchart of a process in which blood cell fractionation or enrichment is not performed before T cells and NK cells in whole blood are contacted with retroviral particles, and PBMC enrichment is performed after transduction. [Figure 1B] (same as above) [Figure 2] FIG. 2 is a diagram of a non-limiting exemplary leukocyte depletion filter assembly (200) with associated blood processing bags, tubing, valves, and a filter enclosure (210) containing a leukocyte depletion filter set. [Figure 3A] Figure 3 shows histograms of experimental results using different pseudotyping elements. Figure 3A shows a histogram of the total number of viable cells per well 6 days after transduction. Figure 3B shows a histogram of the percentage of transduced CD3+ cells, as measured by eTAG expression. [Figure 3B] (same as above) [Figure 4A]Figure 4A shows a histogram of the results of an experiment using a transduction reaction mixture containing whole blood, lentiviral particles, and the anticoagulants EDTA or heparin, without PBMC enrichment before the reaction mixture was formed. This process was performed by contacting whole blood with the indicated lentiviral particles F1-3-23G or F1-3-23GU for 4 hours, followed by a density gradient centrifugation-based PBMC enrichment procedure. Figure 4A shows a histogram of the absolute cell count per uL of the viable lymphocyte population. Figure 4B shows a histogram of the percentage (%) of CD3+eTag+ cells in the viable lymphocyte population 6 days after transduction. [Figure 4B] (same as above) [Figure 5] Histograms showing the number of CD3+FLAG+ cells per μl of culture medium on day 6 after transduction of unstimulated PBMCs with different recombinant lentiviral particles at an MOI of 1 for the indicated time periods. In addition to the same CAR, F1-3-253 encoded an anti-CD19 CAR, and F1-3-451 encoded a CLE. Lentiviral particles were pseudotyped with VSV-G [VSV-G], and optionally UCHT1ScFvFc-GPI [VSV-G+U] is shown. Samples were treated with the reverse transcription inhibitor (RT inb) dapivirine or the integration inhibitor (INT inb) dolutegravir, as indicated. [Figure 6] FIG. 1 is a schematic diagram of non-limiting exemplary transgene expression cassettes comprising polynucleotide sequences encoding the CAR and library candidate CLEs analyzed in Example 6.

[0015] definition As used herein, the term "chimeric antigen receptor" or "CAR" or "CARs" refers to an engineered receptor that transfers antigen specificity to cells, such as T cells, NK cells, macrophages, and stem cells. The CARs of the present invention comprise 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 (CSDs). 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 antibodies to redirect the specificity and reactivity of T cells toward selected targets in an MHC-unrestricted manner. MHC-unrestricted antigen recognition allows CAR-expressing T cells to recognize antigens independently of antigen processing, thereby circumventing a major mechanism of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the endogenous T cell receptor (TCR) alpha and beta chains.

[0016] As used herein, the term "microenvironment" refers to any part or region of a tissue or body that has a constant or temporary, physical, or chemical difference from other regions of the tissue or body. For example, as used herein, "tumor microenvironment" refers to the environment in which a tumor resides, which is the acellular region within the tumor and the region immediately outside the tumor tissue, but does not relate to the intracellular compartments of the cancer cells themselves. The tumor microenvironment can refer to any and all conditions in the tumor environment, including conditions that create a structural and / or functional environment for malignant processes 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, osmolality, osmolality, oxidative stress, the concentration of one or more solutes, the concentration of electrolytes, the concentration of glucose, the concentration of hyaluronan, the concentration of lactate or lactate, the concentration of albumin, the level of adenosine, the level of R-2-hydroxyglutarate, the concentration of pyruvate, the concentration of oxygen, and / or the presence of oxidizing agents, reducing agents, or cofactors, as well as other conditions understood by those skilled in the art.

[0017] As used interchangeably herein, the terms "polynucleotide" and "nucleic acid" refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.Thus, this 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 fragments of antibodies that retain specific binding to an antigen. Antibody fragments may be, 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 including 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, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFvs, and tandem tri-scFvs. Unless otherwise specified, the term "antibody" should be understood to include functional antibody fragments thereof. The term also includes intact or full-length antibodies, including antibodies of any class or subclass, 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 antibodies produces two identical antigen-binding fragments, called "Fab" fragments (each fragment containing a single antigen-binding site) and a residual "Fe" fragment (a name reflecting the ability to readily crystallize). Pepsin treatment produces an F(ab')2 fragment, which has two antigen-binding sites and is capable of cross-linking antigen.

[0020] As used interchangeably herein, the terms "single-chain Fv," "scFv," or "sFv" antibody fragment refer to the V of an antibody. H and V L In some embodiments, the Fv polypeptide comprises V domains, and these domains are present in a single polypeptide chain. H Domains and V L The sFv may further comprise a polypeptide linker or spacer between the domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg 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 that are isolated from a host without further molecular evolution to alter their affinity when produced in scFv format under specific conditions such as those disclosed in U.S. Pat. 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, expressed as the dissociation constant (Kd). The affinity may be 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 unrelated 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 after dilution. The terms "immunoreactive" and "preferentially bind" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0023] As used herein, the term "binding" refers to a direct association between two molecules by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions, including, for example, interactions such as salt bridges and water bridges. Nonspecific binding refers to binding that occurs between two molecules with a specific binding strength of about 10 -7 Binding with an affinity less than M, e.g., 10 -6 M, 10 -5 M, 10 -4 It refers to binding with an affinity such as M.

[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, a cell is generated that expresses 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, including fusions of polypeptides, regions of polypeptides, and functional mutants or fragments thereof, as well as polynucleotides, including microRNAs and shRNAs, 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 that has functional and / or structural properties.

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

[0029] As used herein, the term "isolated" means that a 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 coexisting materials in the natural system is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, and still be isolated in that such a vector or composition is not part of its natural environment.

[0030] As used herein, a "polypeptide" is a single chain of amino acid residues linked by peptide bonds. A polypeptide does not fold into a fixed structure and does not have any post-translational modifications. A "protein" is a polypeptide that folds into a fixed structure. "Polypeptide" and "protein" are used interchangeably herein.

[0031] As used herein, a polypeptide can be "purified" to remove contaminating components of the polypeptide's natural environment, e.g., materials that would interfere with diagnostic or therapeutic uses of the polypeptide, such as enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. The polypeptide can be purified (1) to greater than 90%, 95%, or 98%, e.g., greater than 99%, by weight of the antibody as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of 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 cells" generally includes white blood cells (leukocytes) 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 cells" refers to T helper cells (CD4 + cells), cytotoxic T cells (CD8 + It includes all types of immune cells that express CD3, including T-cells, T regulatory cells (Tregs), and gamma-delta T cells.

[0034] As used herein, "cytotoxic cells" include CD8 +T cells, natural killer (NK) cells, NK-T cells, γδT cells, CD4 + These include neutrophils, a subpopulation of cells that are capable of mediating cytotoxic responses, and neutrophils, which are cells that can mediate cytotoxic responses.

[0035] As used herein, the term "stem cells" 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), and committed progenitor cells (hematopoietic stem cells (HSC), bone marrow-derived cells, etc.).

[0036] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a disease and / or adverse effects resulting from the disease are partially or completely cured. As used herein, "treatment" covers any treatment of a disease in a mammal, e.g., a human, and includes: (a) preventing the disease from occurring 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 development; and (c) palliating 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, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, dogs, cats, and ungulates (e.g., horses, cattle, sheep, pigs, goats).

[0038] As used herein, the term "therapeutically effective amount" or "effective amount" refers to the amount of a drug, or the combined amount of two drugs, that, when administered to a mammal or other subject for treating a disease, is sufficient to affect such treatment for the disease. The "therapeutically effective amount" will vary depending on the drug, the disease and its severity, and the age, weight, etc., of the subject to be treated.

[0039] As used herein, the term "evolution" or "evolving" refers to the use of one or more methods of mutagenesis to generate different polynucleotides encoding different polypeptides that are themselves improved biomolecules and / or contribute to the generation of another improved biomolecule. "Physiological" or "normal" or "normal physiological" conditions include, but are not limited to, pressure, temperature, pH, ionic strength, osmolality, osmolality, oxidative stress, the concentration of one or more solutes, electrolytes, glucose, hyaluronan, lactate or lactate, albumin, adenosine, R-2-hydroxyglutarate, pyruvate, oxygen, and / or the presence of oxidizing agents, reducing agents, or cofactors, and other conditions considered to be within the normal range in the tissue or organ at the site of administration or action in a subject.

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

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

[0042] As used herein, a pseudotyping element may include a "binding polypeptide," which includes one or more polypeptides, typically glycoproteins, that identify and bind to target host cells, and one or more "fusogenic polypeptides" that mediate fusion of the retroviral and target host cell membranes, thereby allowing 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 "target-engaging element," and a "fusogenic polypeptide" may also be referred to as a "fusogenic element."

[0043] "Resting" lymphocytes, such as resting T cells, are lymphocytes in the G0 phase of the cell cycle that do not express activation markers such as Ki-67. Resting lymphocytes include naive T cells that have never encountered a specific antigen and memory T cells that have been altered by previous antigen encounters. "Resting" lymphocytes are also sometimes called "resting" lymphocytes.

[0044] As used herein, "lymphodepletion" includes methods of reducing the number of lymphocytes in a subject, for example, by administration of a lymphodepleting agent. Lymphodepletion can also be achieved by fractionated partial or whole-body radiation therapy. A lymphodepleting agent may be a compound or composition that, when administered to a mammal, can reduce the number of functional lymphocytes in 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 lymphodepleting agents include, but are not limited to, fludarabine, cyclophosphamide, cladribine, denileukin diftitox, or combinations thereof.

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

[0046] As used herein, "double-stranded RNA" or "dsRNA" or "RNA duplex" refers to an RNA molecule composed of two strands. A double-stranded molecule includes two RNA strands that hybridize to form a double-stranded RNA structure, or a molecule composed of a single RNA strand that doubles to itself to form a double-stranded structure. Most, but not all, bases in the double-stranded region are base-paired. The double-stranded region contains a sequence complementary to a target RNA. The sequence complementary to the target RNA is an antisense sequence and is often 18-29, 19-29, 19-21, or 25-28 nucleotides in length, or in some embodiments, between a lower end of 18, 19, 20, 21, 22, 23, 24, or 25 and an upper end of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, 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, connected by a loop that is not part of the duplex. In certain embodiments, the loop comprises at least 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In other embodiments, the loop comprises 2 to 40, 3 to 40, 3 to 21, or 19 to 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 nucleotides at the lower end and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 nucleotides 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 containing a microRNA processing element. A microRNA processing element is a minimal nucleic acid sequence that contributes to the generation of a mature microRNA from a precursor microRNA. Often, these elements are found within a 40-nucleotide sequence adjacent to a microRNA stem-loop structure. In some cases, microRNA processing elements are found within a stretch of nucleotide sequence 5 to 4,000 nucleotides in length adjacent to a microRNA stem-loop structure.

[0048] The term "linker" when used in reference to multiple inhibitory RNA molecules refers to the connecting means that joins two inhibitory RNA molecules.

[0049] As used herein, "recombinant retrovirus" refers to a replication-incompetent or "replication-defective" retrovirus unless explicitly described as a replication-competent retrovirus. The terms "recombinant retrovirus" and "recombinant retroviral particle" are used interchangeably herein. Such retroviruses / retroviral particles can be any type of retroviral particle, including, for example, gammaretroviruses and, in exemplary embodiments, lentiviruses. As is known, such retroviral particles, e.g., lentiviral 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, genome, or retroviral (e.g., lentiviral) expression vector (which is typically a plasmid encoding a gene of interest or other coding sequence). Thus, retroviral (e.g., lentiviral) expression vectors contain sequences that facilitate expression and packaging after transfection into cells (e.g., 5'LTR and 3'LTR flanking the psi packaging element and target heterologous coding sequence, etc.). The terms "lentivirus" and "lentiviral particle" are used interchangeably herein.

[0050] The "framework" of a miRNA consists of the "5'microRNA flanking sequence" and / or "3'microRNA flanking sequence" surrounding the miRNA and, in some cases, the loop sequence separating the stem of a stem-loop structure within the miRNA. In some instances, the "framework" is derived from a naturally occurring miRNA, such as, for example, miR-155. 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 an RNA molecule of any length 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 element. Those skilled in the art will understand whether a 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 virus particles used for infection per number of cells.The functional titration of virus particle number can be performed using FACS and reporter expression.

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

[0055] It is to be understood that the present disclosure, and the aspects and embodiments provided herein, are not limited to particular examples disclosed, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of disclosing particular examples and embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0056] Where a range of values ​​is provided, it is understood that, to the 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 that stated range, is included in the disclosure unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limits within the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. When multiple lower values ​​and multiple upper values ​​are provided in overlapping ranges, one of ordinary skill in the art will recognize that the selected range includes the lower value that is smaller than the upper value. All headings in this application are for the convenience of the reader and are not limiting.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although any methods and materials similar or equivalent to those described herein can also be used to practice or test the present invention, preferred methods and materials are described here.All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials for which the publications are cited.

[0058] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "chimeric antigen receptor" includes a plurality of such chimeric antigen receptors and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude optional elements. Accordingly, this statement is intended to serve as an antecedent basis for using exclusive language such as "solely," "only," and the like in connection with the recitation of claim elements, or for using a "negative" limitation.

[0059] It will be understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present invention are specifically embraced by the present invention and are disclosed herein as if all combinations were individually and expressly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each such subcombination was individually and expressly disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

[0060] The present disclosure overcomes the challenges 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 more rapid process for transducing lymphocytes, avoiding several steps that require specialized devices. Furthermore, the methods offer better control over post-transduction processing, as any such processing is performed ex vivo, thus allowing for the option of removing various unwanted cells. Thus, the methods provide an important step toward the generalization of cell therapy.

[0061] Exemplary methods and compositions for genetically modifying lymphocytes, such as NK cells and, in exemplary embodiments, T cells, can be performed in a shorter time than previous methods. Additionally, compositions with many uses are provided, including for use in these improved methods. Some of these compositions are genetically modified lymphocytes with improved growth and survival qualities, including, for example, in vitro culture in the absence of growth factors. Such genetically modified lymphocytes have practical applications, for example, as research tools to better understand the factors that affect T cell proliferation and survival, and in commercial production for the production of specific factors, such as growth factors and immunomodulators, that can be harvested and tested or used in commercial products.

[0062] Methods for transducing and / or genetically modifying lymphocytes In certain aspects, provided herein are methods for transducing and / or genetically modifying lymphocytes, e.g., 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), the method comprising contacting 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 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 exemplary embodiments, preactivation of the T cells and / or NK cells is not required, and an activating element, which may be any of the activating elements provided herein, is present in the reaction mixture in which the contacting occurs. In further exemplary embodiments, the activating element is present on the surface of the replication-incompetent recombinant retroviral particles. In an exemplary embodiment, the activating element is anti-CD3, such as an anti-CD3 scFv or an 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 subsequent optional incubation, including the step of removing non-cell-associated retroviral particles, can be performed (or occur) within 72, 48, or 24 hours, or within any of the contact time ranges provided herein. However, in exemplary embodiments, contacting is performed for less than 2 hours, less than 1 hour, less than 30 minutes, or less than 15 minutes, although in all cases there is at least an initial contacting step in which the retroviral particles and cells are contacted in suspension in the transduction reaction mixture. This contacting typically includes an initial step in which non-cell-associated retroviral particles of the reaction mixture are separated from the cells and then further processed. Such suspension may involve settling the cells and retroviral particles, or inducing such settling by applying a force, such as centrifugal force, to the bottom of a container or chamber, as discussed in further detail herein. In exemplary embodiments, such g-forces are lower than those successfully used in spinoculation procedures. Further contact times, and discussion regarding contacting and optional incubation, are discussed further herein. In further exemplary embodiments, contacting is performed during the initial contacting step only (without further incubation in the reaction mixture containing the free retroviral particles in suspension and the cells in suspension) without further incubation in the reaction mixture, or with incubation in the reaction mixture for 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 1 hour, which may be a step to separate the free retroviral particles in the reaction mixture from the cell-associated particles.

[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. Furthermore, 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, e.g., in this section and in the exemplary embodiments section, and such methods include embodiments provided throughout the specification as further discussed herein. For example, any of the embodiments of aspects for transducing and / or genetically modifying PBMCs or lymphocytes, e.g., NK cells, or in exemplary embodiments, T cells, provided in this section and in the exemplary embodiments section, etc., can include any of the embodiments of replication-incompetent recombinant retroviral particles provided herein that include one or more lymphoproliferative elements, CARs, pseudotyping elements, riboswitches, activation elements, membrane-bound cytokines, miRNAs, Kozak 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 carried out in vitro or ex vivo. One skilled 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 embodiment involving such steps.

[0065] In certain exemplary embodiments, cells are genetically modified and / or transduced without the need for prior activation or stimulation, whether in vivo, in vitro, or ex vivo. In certain exemplary embodiments, cells are activated during contact, and are not activated 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 retroviral particle surface is not required to genetically modify and / or transduce cells. Therefore, no such activation or stimulation elements are required other than those present on the retroviral particle before, during, or after contact. Thus, as discussed in more detail herein, these exemplary embodiments, which 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. Furthermore, 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, radically simplifying the delivery of such therapy, for example, by providing a point-of-care method.

[0066] Compositions and methods for lymphocyte transduction in whole blood lymphocytes - Patent Application 20070122999 In certain aspects, provided herein are methods for 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 lymphocytes with replication-incompetent recombinant retroviral particles in a reaction mixture representing a separate aspect provided herein. In exemplary embodiments, the reaction mixture comprises lymphocytes and replication-incompetent recombinant retroviral particles, a T cell activation element, and one or more additional blood components, as described below, present such that in exemplary embodiments, the reaction mixture comprises at least 10% whole blood, and the replication-incompetent recombinant retroviral particles typically comprise pseudotyping elements on their surface. In such methods, contact (and incubation under contact conditions) promotes association of 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 aspect 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-prepared component that is not a PBMC, e.g., the reaction mixture comprises an effective amount of an anticoagulant and one or more blood-prepared components that are not PBMCs. In exemplary embodiments, such blood or blood-prepared components that are not PBMCs are one or more (e.g., at least one, two, three, four, or five) or all of the following additional components: a) red blood cells (wherein the red blood cells occupy 1-60% of the volume of the reaction mixture); b) neutrophils (wherein the neutrophils comprise 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 comprise at least 0.05% of the leukocytes in the reaction mixture); d) eosinophils (wherein the reaction mixture comprises at least 0.1% of the leukocytes in the reaction mixture); e) plasma, wherein the plasma comprises at least 1% of the volume of the reaction mixture; and f) Anticoagulants (Such blood or blood preparation components a-f above are referred to herein as ("non-PBMC blood or blood preparation components of interest")).

[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 aspects of T cells and / or NK cells provided herein) because, in these exemplary embodiments, the reaction mixture comprises at least 10% whole blood, and in certain exemplary embodiments, at least 25%, 50%, 75%, 90%, or 95% whole blood, or, for example, 25% to 95% whole blood. In these exemplary embodiments, such reaction mixtures are formed by combining whole blood with an anticoagulant (e.g., collecting the whole blood in a blood collection tube containing an anticoagulant) and adding a solution of recombinant retrovirus to the blood along with the anticoagulant. Thus, in exemplary embodiments, the reaction mixture comprises an anticoagulant, as described in more detail herein. In some embodiments, the whole blood is not or does not comprise umbilical cord blood.

[0068] The reaction mixture in these embodiments typically does not include a PBMC enrichment step prior to forming the transduction reaction mixture. Thus, such reaction mixtures typically include additional components listed in a) through f) above that are not PBMCs. Furthermore, in exemplary embodiments, the reaction mixture is substantially whole blood or includes whole blood, and thus the reaction mixture includes all of the additional components listed in a) through e) above. "Substantially whole blood" refers to blood that has been separated from an individual, not subjected to a PBMC enrichment step, and diluted by less than 50% with other solutions. For example, this dilution may be due to the addition of an anticoagulant and a volume of liquid containing retroviral particles. Additional reaction mixture embodiments for methods and compositions related to transduction of lymphocytes in whole blood are provided herein.

[0069] In another aspect, provided herein are genetically modified lymphocytes, in exemplary embodiments, genetically modified T cells and / or NK cells, produced by the above-described methods of transducing and / or genetically modifying lymphocytes in whole blood. In yet another aspect, provided herein is the use of replication-incompetent recombinant retroviral particles in the manufacture of a kit for genetically modifying a subject's lymphocytes, in exemplary embodiments, T cells and / or NK cells, wherein the use of the kit comprises the above-described methods of transducing and / or genetically modifying lymphocytes in whole blood. In another aspect, provided herein are methods for administering genetically modified lymphocytes to a subject, wherein the genetically modified lymphocytes are produced by the above-described methods of transducing and / or genetically modifying lymphocytes in whole blood. Aspects provided herein, including such methods of transducing and / or genetically modifying lymphocytes in whole blood, the use of such methods in the manufacture of kits, 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 "composition and method aspects for transducing lymphocytes in whole blood." An exemplary embodiment of such an aspect includes contacting T cells and / or NK cells with retroviral particles in whole blood, although it should be noted that such an aspect also includes 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 following a PBMC enrichment procedure.

[0070] Various elements or steps of such method aspects for transducing lymphocytes in whole blood are provided herein, e.g., in this section and in the Exemplary Embodiments section, and such methods, as further discussed herein, include embodiments provided throughout the specification. Those skilled in the art will recognize that many embodiments provided elsewhere herein can be applied to any of the composition and method aspects for transducing lymphocytes in whole blood. For example, any embodiment of the composition and method aspects for transducing lymphocytes in whole blood provided in this section and / or the Exemplary Embodiments section, etc., can include any of the replication-incompetent recombinant retroviral particle embodiments provided herein, including those that include one or more polypeptide lymphoproliferative elements, inhibitory RNAs, CARs, pseudotyping elements, riboswitches, activating elements, membrane-bound cytokines, miRNAs, Kozak 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 the present specification, as discussed in more detail herein, the pseudotyping element typically binds to lymphocytes (e.g., T cells and / or NK cells), and in exemplary embodiments, resting T cells and / or resting NK cells, and can promote membrane fusion alone or in combination with other proteins of a replication-incompetent recombinant retroviral particle. In certain exemplary embodiments, the retroviral particle is a lentiviral particle. 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 the compositions and methods for transducing lymphocytes in whole blood provided herein. In some exemplary embodiments, blood is collected with an anticoagulant present in the collection container (e.g., a tube or bag), for example, using standard blood collection protocols known in the art. Anticoagulants that can be used in the compositions and methods for transducing lymphocytes in whole blood provided herein include compounds or biological agents that block or limit the thrombin blood clotting cascade. Anticoagulants include metal chelators, preferably calcium ion chelators such as citrate (e.g., containing free citrate ions), including solutions of citrate containing one or more components such as citric acid, sodium citrate, phosphate, adenine, and mono- or monopolysaccharides, such as dextrose, oxalate, and EDTA; heparin and heparin analogs, such as unfractionated heparin, low molecular weight heparin, and other synthetic sugars; and vitamin K antagonists, such as coumarin. Exemplary citrate compositions include acid citrate dextrose (ACD) (also referred to as Anticoagulant Citrate Dextrose Solution A and Solution B (USP 26, 2002, p. 158)); and citrate phosphate dextrose (CPD) solution (which can also be prepared as CPD-A1, as known in the art). Thus, anticoagulant compositions can also include phosphate or monobasic phosphate ions, adenine, and monosaccharides or polysaccharides.

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

[0074] In some embodiments, heparin is present in the reaction mixture at a concentration of, for example, between 0.1-5x, between 0.25-2.5x, between 0.5-2x, between 0.75-1.5x, between 0.8-1.2x, between 0.9-1.1x, about 1x, or 1x the concentration of heparin in commercially available heparin blood collection tubes. Heparin is a glycosaminoglycan anticoagulant with 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 reaction mixture. In some embodiments, an effective concentration of EDTA, e.g., 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, between 1-2 mg / ml, or about 1.5 mg / ml. The reaction mixture in embodiments of the compositions and methods for transducing lymphocytes in whole blood provided herein may include two or more anticoagulants whose combined effective doses prevent clotting of the blood prior to formation of the reaction mixture and / or the reaction mixture itself.

[0075] In some embodiments, an anticoagulant may be administered to a subject before blood is drawn from the subject for ex vivo transduction, so that clotting of the drawn blood is inhibited at least partially and at least throughout the contacting step and any subsequent incubation period. In such embodiments, for example, acid citrate dextrose may be administered to a subject at between 80 mg / kg / day and 5 mg / kg / day (mg refers to mg of citrate, kg applies to the mammal being treated). Heparin may be delivered at a dose of, for example, between 5 units / kg / hour and 30 units / kg / hour.

[0076] In addition to, or instead of, an 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 0.1-75% of the volume of the reaction mixture); b) neutrophils (wherein the neutrophils comprise 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 comprise at least 0.05% of the leukocytes in the reaction mixture); d) eosinophils (wherein the reaction mixture comprises at least 0.1% of the leukocytes in the reaction mixture); e) plasma, wherein the plasma comprises at least 1% of the volume of the reaction mixture; and f) platelets (wherein the platelets are at least 1 x 10 per liter of reaction mixture) 6 containing platelets).

[0077] With respect to 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 end of the range, to 25, 50, 60, or 75% of the volume of the reaction mixture at the upper end of the range. In exemplary embodiments, the red blood cells occupy between 1-60%, between 10-60%, between 20-60%, between 30-60%, between 40-60%, between 40-50%, between 42-48%, between 44-46%, about 45% or 45%.

[0078] With respect to neutrophils, in some embodiments, neutrophils may comprise between 0.1, 0.5, 1, 5, 10, 20, 25, 35, or 40% of the leukocytes of the reaction mixture at the lower end of the range, and 25, 50, 60, 70, 75, and 80% of the leukocytes of the reaction mixture at the higher end of the range, e.g., between 25% and 70%, or between 30% and 60%, or between 40% and 60% of the leukocytes of the reaction mixture. In some embodiments, there are more neutrophils than T cells and / or NK cells in the reaction mixtures herein.

[0079] With respect to eosinophils, in some embodiments, eosinophils may comprise 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 leukocytes in the reaction mixture at the lower end 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 leukocytes in the reaction mixture at the higher end of the range. In exemplary embodiments, eosinophils comprise between 0.05-10.0%, between 0.1-9%, between 0.2-8%, between 0.2-6%, between 0.5-4%, between 0.8-4%, or between 1-4% of the leukocytes in the reaction mixture.

[0080] With respect to basophils, in some embodiments, basophils may comprise 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 end of the range, and between 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 higher end of the range. In exemplary embodiments, basophils comprise 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] With respect to plasma, in some embodiments, the plasma may comprise between 0.1, 0.5, 1, 5, 10, 25, 35, or 45% of the volume of the reaction mixture at the lower end of the range, to 25, 50, 60, 70, and 80% of the volume of the reaction mixture at the higher end of the range. In exemplary embodiments, the plasma comprises between 0.1-80%, between 1-80%, between 5-80%, between 10-80%, between 30-80%, between 40-80%, between 45-70%, between 50-60%, between 52-58%, between 54-56%, about 55%, or 55% of the reaction mixture.

[0082] With respect to platelets, in some embodiments, platelets are present at the lower end of the range, typically 1 x 10 per mL of reaction mixture. 5 , 1×10 6 , 1×10 7 , or 1 × 10 8 Platelets range from 1 x 10 to 1 x 10 per 1 mL reaction mixture at the upper end of the range 9 , 1×10 10 , 1×10 11 , 1×10 12 , 2 × 10 13 , or 2 × 10 14 In an exemplary embodiment, the platelets may comprise 1 x 10 platelets per ml of reaction mixture. 5 ~1×10 12 Platelets between 1 x 10 6 ~1×10 11 Platelets between 1 x 10 7 ~1×10 10Between 1 x 10 platelets per mL 8 ~1×10 9 platelets between 10 and 10, or 1 x 10 8 ~5×10 8 Contains platelets between the cells.

[0083] Exemplary Cell Treatment Methods for Genetically Modifying T Cells and / or NK Cells in the Presence of Blood or Components Thereof It is noteworthy that some embodiments of the methods for genetic modification provided herein do not include a step of drawing blood from a subject. However, as shown in FIG. 1, some methods provided herein include a step (110) in which blood is drawn from a subject. As discussed in more detail herein, blood can be drawn or obtained from a subject by any suitable method known in the art. For example, blood can be drawn by venipuncture or any other blood collection method in which a blood sample is drawn. In some embodiments, the amount of blood drawn is between 25 ml and 250 ml, e.g., between 25 ml and 60 ml, between 50 ml and 90 ml, between 75 ml and 125 ml, between 90 ml and 120 ml, or between 95 ml and 110 ml.

[0084] Regardless of whether blood is collected from a subject, in any of the aspects of the methods provided herein for genetically modifying lymphocytes (e.g., T cells and / or NK cells), the lymphocytes are contacted with replication-incompetent retroviral particles in a reaction mixture. In an exemplary embodiment, this contacting, and the reaction mixture in which the contacting occurs, occurs within a closed cell processing system, as discussed in more detail herein. In conventional closed cell processing methods involving ex vivo genetic modification and / or transduction of lymphocytes, particularly autologous cell therapy methods, many steps, such as PBMC enrichment, washing, cell activation, transduction, expansion, harvesting, and optionally reintroduction, are carried out over several days. In more recent methods (see FIG. 1A), some of the steps and time involved in this ex vivo cell processing have been shortened (see, e.g., WO2019 / 055946). These more recent methods (and further improved cell treatment methods provided herein) further use a rapid ex vivo transduction process that does not involve, or minimally involves, preactivation (e.g., contacting lymphocytes, e.g., T cells and / or NK cells, with an activator for less than 30, 15, 10, or 5 minutes prior to contact 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 contacting step occurs. In exemplary embodiments, the T cell and / or NK cell activation elements are associated with the surface of retroviral particles present in the reaction mixture. In exemplary embodiments, 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 in a closed system, followed by cell counting, transfer, and addition of media, which takes at least about 45 minutes before the lymphocytes contact the retroviral particles to form the transduction reaction mixture (130A).As discussed in more detail herein, following the "viral transduction" step, which is typically a contacting step involving incubation, the lymphocytes are typically washed from any remaining retroviral particles in suspension using, for example, Sepax (140A), collected (150A), and the final product is typically placed into an infusion bag for reinfusion or a cryovial for storage (160A). As discussed in more detail herein, conventional PBMC enrichment procedures typically involve Ficoll density gradients and centrifugal (e.g., centrifugation) or centripetal (e.g., Sepax) forces, or leukophoresis to enrich the PBMCs.

[0085] As shown in the examples provided herein, it has surprisingly been discovered that lymphocytes (e.g., T cells and / or NK cells) can be contacted with replication-incompetent retroviral particles in a reaction mixture of whole blood containing an anticoagulant, and a significant proportion of the lymphocytes can be genetically modified and transduced. Thus, it has been discovered that effective genetic modification of lymphocytes with recombinant retroviral particles can be carried out in the presence of blood components and blood cells in addition to PBMCs. Furthermore, based on the surprising findings discussed above regarding the effective genetic modification of T cells and, optionally, NK cells with retroviral particles, even when contacting is performed with whole blood, exemplary embodiments herein provide a further simplified method in which lymphocytes are genetically modified and / or transduced by directly adding replication-incompetent retroviral particles to whole blood to form a reaction mixture (130B), thereby contacting cells in whole blood with replication-incompetent retroviral particles, with an optional incubation period provided herein. Thus, such further improved methods in this exemplary embodiment typically do not include a lymphocyte enrichment step before contacting lymphocytes in whole blood containing an anticoagulant with retroviral particles. This further improved method, like the other cell processing methods described herein, is typically performed in a closed cell processing system and may involve minimal or no preactivation of lymphocytes prior to contact with retroviral particles. In these simplified methods, lymphocytes in whole blood can be contacted with retroviral particles directly within the blood bag. After the contacting step (130B) in such methods, the lymphocytes contacted with retroviral particles are washed and enriched using a PBMC enrichment procedure (135B), which reduces neutrophils and facilitates reintroduction into the subject. Therefore, in such embodiments, the PBMC enrichment procedure and lymphocyte enrichment filtration are typically not performed prior to contacting the cells in whole blood containing anticoagulant with recombinant retroviral particles. However, in the embodiment of Figure 1B, such a PBMC enrichment method is performed, for example, using Sepax with a Ficoll gradient (135B) after contacting (130B) with optional incubation.After PBMC enrichment, the lymphocytes are optionally further washed from residual retroviral particles, for example using Sepax (140B), collected (150B), and the final product is typically placed into an infusion bag for reinfusion or into cryovials for storage (160B).

[0086] Figure 2 provides a non-limiting illustrative example of a cell-processing, leukocyte-depletion filtration assembly (200) for concentrating nucleated cells that can be used as a leukocyte-depletion filter in the method of Figure 1. The illustrative leukocyte-depletion filtration assembly (200), which in the illustrative embodiment is a single-use filtration assembly, comprises 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 bag, valve, and / or channel / tubing configuration that provides the ability to enrich, concentrate, wash, and collect retained leukocytes or nucleated red blood cells using perfusion and counterperfusion (see, e.g., EP 2602315 A1, incorporated herein by reference in its entirety). In the illustrative embodiment, the leukocyte-depletion filtration assembly (200) is a commercially available HemaTrate filter (Cook Regenetec, Indianapolis, IN). Leukocyte depletion filtration assemblies can be used to enrich for total nucleated cells (TNCs), including granulocytes, which are removed in the PBMC enrichment procedure of a closed cell processing system. Because filter assemblies with leukocyte depletion medium of EP 2602315 A1, such as HemaTrate filters, and the exemplary leukocyte depletion filter assembly of Figure 2 do not remove granulocytes, they are not considered PBMC enrichment assemblies or filters herein, and methods incorporating them are not considered PBMC enrichment procedures or steps herein.

[0087] The leukocyte depletion filter assembly (200) of Figure 2 is a single-use sterile assembly containing various tubing and valves, typically needleless valves, that enable the isolation of leukocytes from blood cell preparations containing whole blood and leukocytes, as well as the rapid washing and enrichment of the 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 transduction / contacting reaction mixture containing whole blood, anticoagulant, and retroviral particles, is connected to the assembly (200) at the first assembly opening (217) of the inlet tubing (255). Lymphocytes, including some T cells and / or NK cells with associated retroviral particles, and potentially genetically modified at this point, as well as other blood cells and components in the whole blood reaction mixture, and anticoagulant, enter the inlet tubing (255) through the first assembly opening (217) by gravity when the clamp on the first inlet tubing (255) is released. The genetically modified T cells and / or NK cells pass through inlet valve (247) and collection valve (245), enter filter enclosure (210) through filter enclosure inlet (225), and contact a leukocyte depletion IV filter set (e.g., SKU J1472A Jorgensen Labs) within filter enclosure (210). Nucleated red blood cells, including white blood cells, are retained by the filter, while other blood components pass through the filter and enter outlet tubing (256) through filter enclosure outlet (226), then through outlet valve (247) and are collected in waste collection bag (216), which may be, for example, a 2 L 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 tubing (255). The buffer moves by gravity through the second assembly opening (218) and into the inlet tubing (255) when the clamp on the inlet tubing (255) is released. The buffer passes through the inlet valve (247) and collection valve (245), enters the filter housing (210) through the filter housing inlet (225), and passes through the leukocyte reduction filter set within the filter housing (210) to rinse retained lymphocytes from the filter. The buffer travels from the filter enclosure outlet (226) to the outlet tubing (256), then passes through the outlet valve (247) and is collected in the waste collection bag (216), which 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 that has been replaced in place of the first waste collection bag, before the buffer enters the second assembly opening (218). The optional wash step may optionally be performed multiple times by repeating the above process with additional buffer.

[0089] Once all or substantially all of the reaction mixture in blood bag (215) has passed through filter (210) and an optional washing step has been optionally performed, a reverse perfusion process is initiated to move fluid in the opposite direction of assembly (200) to collect lymphocytes retained on the filter set within filter enclosure (210). The exemplary embodiment of the leukocyte depletion filter assembly herein is adaptable for reperfusion. Prior to initiating the reverse perfusion process in exemplary assembly (200), outlet valve (247) is switched to the reperfusion position, and collection valve (245) is switched to the collection position. To initiate reperfusion, a buffer solution (e.g., PBS) in syringe (266) (which may be, for example, a 25 ml syringe) is passed through outlet tubing (256) by injection using syringe (266). The buffer then enters the filter enclosure (210) through the filter enclosure outlet (226), causing the lymphocytes retained in the filter set to move from the filter enclosure (210) through the filter enclosure inlet (225) and into the inlet tubing (255). The lymphocytes, including some T cells and / or NK cells with associated retroviral particles (some of which may now be genetically modified and / or transduced), then pass through the collection valve (245) and are collected in the cell sample collection bag (265), which may be a 25 ml cryopreservation bag.

[0090] In some aspects, kits for genetically modifying NK cells and / or, in exemplary embodiments, T cells are provided herein. The kits include a leukocyte depletion filter assembly and any of the replication-incompetent retroviral vector embodiments disclosed herein, typically contained in a tube or vial. The leukocyte depletion filtration assembly of such kits typically includes a leukocyte depletion filter or leukocyte depletion filter set in a filter enclosure, as well as multiple connected sterile tubing and multiple valves connected thereto, typically adapted for use in a single-use closed blood processing system, as illustrated by the exemplary assembly in FIG. 2. In exemplary embodiments, such kits include an anticoagulant, a blood processing buffer bag, a blood processing waste collection bag, a blood processing cell sample collection bag, and a sterile syringe, optionally including a blood collection bag. In exemplary embodiments, the kits include a T cell activating element, such as anti-CD3, as disclosed in detail herein. Such an activating element can be provided in solution within the tube or vial containing the retroviral particles or in a separate tube or vial. In exemplary embodiments, the activating element is anti-CD3 associated with the surface of the replication-incompetent retroviral particles. In exemplary embodiments, the replication-incompetent recombinant retroviral particles in the kit comprise a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and / or NK cells, wherein 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 a method for genetically modifying lymphocytes Some embodiments of any of the methods used in any of the aspects provided herein, typically methods for genetically modifying lymphocytes, PBMCs, and in exemplary embodiments, NK cells and / or, in further exemplary embodiments, T cells, may include a step of drawing blood from a subject. Blood includes blood components, including blood cells such as lymphocytes (e.g., T cells and NK cells), which may 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 notable that certain embodiments do not include such a step. However, in embodiments that involve drawing 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, blood can be collected by venipuncture or any other blood collection method in which a blood sample is collected. In some embodiments, the amount of blood drawn is between 50 ml and 250 ml, e.g., between 75 ml and 125 ml, or between 90 ml and 120 ml, or between 95 ml and 110 ml. In some embodiments, the amount of blood drawn is at the lower end of the range of 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 The volume can be from 900 ml to the upper end of the range of 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 1 L. In some embodiments, lymphocytes (e.g., T cells and / or NK cells) can be obtained by apheresis.In some embodiments, the volume of blood collected and processed during apheresis can be between 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.25, or 1.5 times the subject's total blood volume at the lower end of the range and 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 subject's total blood volume at the upper end of the range. Total blood volume in humans is typically in the range of 4.5 to 6 L, and therefore, much more blood is collected and processed during apheresis than when blood is collected and then the lymphocytes therein are genetically modified and / or transduced, as in the exemplary embodiments herein.

[0092] Regardless of whether blood is collected from a subject, in any of the aspects of the methods provided herein for genetically modifying lymphocytes (e.g., T cells and / or NK cells), the lymphocytes are contacted with replication-incompetent retroviral particles in a reaction mixture. The contacting in any of the embodiments provided herein can be carried out in a chamber of a closed system adapted for processing blood cells, for example, in a blood bag, 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 certain exemplary reaction mixtures provided herein, there is a multiplicity of infection (MOI) of between 0.1-50, 0.5-50, 0.5-20, 0.5-10, 1-25, 1-15, 1-10, 1-5, 2-15, 2-10, 2-7, 2-3, 3-10, 3-15, or 5-15; or an MOI of at least 1 and less than 6, 11, or 51; or in some embodiments, between 5 and 10 MOI units of replication-incompetent recombinant retroviral particles. 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, certain embodiments can use a higher MOI than methods in which PBMCs are isolated and used in the reaction mixture. For example, an exemplary embodiment of a composition and method for transducing lymphocytes in whole blood comprises 1×10 6 Assuming PBMCs / ml of blood, retroviral particles having an MOI of 1-50, 2-25, 2.5-20, 2.5-10, 4-6, or about 5, and in some embodiments, 5-20, 5-15, 10-20, or 10-15 can be used.

[0093] In exemplary embodiments, this contacting, and the reaction mixture in which the contacting occurs, occurs within a closed cell processing system, as discussed in more detail herein. Packaging cells, and in exemplary embodiments, packaging cell lines, particularly packaging cells provided in certain exemplary embodiments, can be used to generate replication-incompetent recombinant retroviral particles. The lymphocytes in the reaction mixture may be PBMCs, or, in aspects of the present specification that provide compositions and methods for transducing lymphocytes in whole blood, may be additional blood components, including anticoagulants and / or additional types of blood cells other than PBMCs, as discussed herein. Indeed, in exemplary embodiments of these composition and method aspects for transducing lymphocytes in whole blood, the reaction mixture may essentially be whole blood, and typically includes anticoagulants, retroviral particles, and a small volume of solution in which the retroviral particles are delivered to the whole blood.

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

[0095] In the reaction mixtures associated with the embodiments of the compositions and methods for genetically modifying lymphocytes in whole blood provided herein, lymphocytes, including NK cells and T cells, may be present at 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 step prior to forming the reaction mixture. For example, in some embodiments of these embodiments, 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 of embodiments for genetically modifying lymphocytes in whole blood are provided in detail in other sections of this specification.

[0096] As disclosed herein, aspects of the compositions and methods for transducing lymphocytes in whole blood typically do not include a PBMC enrichment step for the blood sample before lymphocytes from the blood sample are contacted 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), including peripheral blood lymphocytes (PBLs), such as T cells and / or NK cells, are isolated from other components of the blood sample, e.g., using a PBMC enrichment procedure, before being combined with retroviral particles in the reaction mixture.

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

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

[0099] As further non-limiting examples of PBMC enrichment procedures, in some embodiments of the transduction or genetic modification methods 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, which draws blood from a subject, passes the blood through a device that selects specific cell types (e.g., PBMCs), and returns the remainder to the subject. Density gradient centrifugation can be performed after apheresis. In some embodiments, PBMCs are isolated using a leukocyte reduction filter assembly. In some embodiments, magnetic bead-activated cell sorting is then used to purify specific cell populations, such as PBLs, or subsets thereof according to cell phenotype (i.e., positive selection) before they are used in the reaction mixtures herein.

[0100] Other purification methods, such as substrate adhesion, which utilizes a substrate that mimics the environment T cells encounter during mobilization, can also be used to purify T cells before adding them to the reaction mixture, or negative selection, in which unwanted cells are targeted for removal using antibody conjugates before the reaction mixture for the contacting step is formed. In some embodiments, red blood cell rosetting can be used to remove red blood cells before forming the reaction mixture. In other embodiments, hematopoietic stem cells can be removed before 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, an ABC transporter inhibitor and / or substrate is not present before contacting, during contacting, or both before and during contacting (i.e., not present in the reaction mixture in which contacting occurs), with or without optional incubation, or is not present in any step of the method.

[0101] In certain exemplary embodiments of any aspect provided herein, the lymphocytes are genetically modified and / or transduced without prior activation or stimulation, whether in vivo, in vitro, or ex vivo, and / or without the need for prior activation or stimulation; and / or further, in some embodiments, without ex vivo or in vitro activation or stimulation after initial contact, with or without optional incubation, or without the need for ex vivo or in vitro activation or stimulation after initial 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 retroviral viral particles in the reaction mixture. In methods for genetically modifying lymphocytes, such as T cells and / or NK cells, in blood or a component thereof, 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 are contacted in a state where they are 95-100% resting cells (KI-67 -) In some embodiments, the T cells and / or NK cells contacted by the replication-incompetent recombinant retroviral particles comprise 90, 91, 92, 93, 94, and 95% resting cells at the low end of the range to 96, 97, 98, 99, or 100% resting cells at the high end of the range. In some embodiments, the T cells and / or NK cells comprise naive cells. In some exemplary embodiments, subembodiments 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 facilitate the transduction of T cells and / or NK cells by the replication-incompetent recombinant retroviral particles. Without being limited by theory, during the contact period, the replication-incompetent recombinant retroviral particles recognize and bind to T cells and / or NK cells at the point at which the retroviral and host cell membranes begin to fuse. Then, as the next step in the transduction process, genetic material from the replication-incompetent recombinant retroviral particles enters the T cells and / or NK cells, at which point the T cells and / or NK cells become "genetically modified," as that term is used herein. It is worth noting that such a process can occur hours or days after contact is initiated, even after unrelated retroviral particles have been washed away. The genetic material then typically integrates into the genomic DNA of the T cells and / or NK cells, at which point the T cells and / or NK cells become "transduced," as that term is used herein. Therefore, in an exemplary embodiment, any method for genetically modifying lymphocytes (e.g., T cells and / or NK cells) herein is a method for transducing lymphocytes (e.g., T cells and / or NK cells). It is believed that by day 6 in vivo or ex vivo, after contact has begun, the majority of genetically modified cells are 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 this disclosure, transduced T cells and / or NK cells include 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 methods herein that recite "reintroducing" transduced cells, it is understood that such cells are typically not in a transformed state when taken from the subject's blood.

[0103] Many of the methods provided herein involve genetic modification and transduction of T cells and / or NK cells. Methods for genetic modification and transduction of 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. In exemplary embodiments, the methods provided herein do not require ex vivo stimulation or activation. Thus, this common step in previous methods can be avoided in this method, although ex vivo stimulatory molecules, such as anti-CD3 and / or anti-CD28 beads, may be present during the subsequent contact and optional incubation. However, in the exemplary methods provided herein, ex vivo stimulation is not required.

[0104] In certain exemplary embodiments of any aspect herein, blood cells such as lymphocytes, particularly T cells and / or NK cells, are activated during contact or the optional subsequent incubation, but not at all prior to contact or for periods exceeding 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, or 8 hours. In certain exemplary embodiments, activation by factors not present on the retroviral particle surface is not required to genetically modify the lymphocytes. Therefore, no such activation or stimulation factors are required other than the retroviral particles before, during, or after contact. Thus, as discussed in more detail herein, these exemplary embodiments, which do not require pre-activation or stimulation, provide the ability to rapidly conduct in vitro experiments aimed at better understanding T cells and the biological mechanisms therein. Furthermore, 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, radically simplifying the delivery of such therapy, e.g., by providing a point-of-care method.

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

[0106] The contacting step of the methods for transducing and / or genetically modifying lymphocytes in whole blood provided herein typically involves an initial stage in which retroviral particles, typically a population of retroviral particles, are contacted with a population of blood cells, typically including anticoagulants and / or additional blood components other than PBMCs that are not present after a PBMC enrichment procedure, while suspended in a liquid buffer and / or medium to form a transduction reaction mixture. This contacting may be followed by an optional incubation period in this reaction mixture, similar to other aspects provided herein, in which the reaction mixture includes blood cells, including retroviral particles and lymphocytes (e.g., T cells and / or NK cells), in suspension. In methods for genetically modifying T cells and / or NK cells in blood or a component thereof, the reaction mixture may include at least one, two, three, four, five, or all of the additional blood components disclosed herein, and in exemplary embodiments, includes one or more anticoagulants.

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

[0108] Disclosed herein are exemplary methods for genetically modifying lymphocytes, particularly NK cells, and in exemplary embodiments, T cells, that are significantly shorter and simpler than previous methods. 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 carried out (or occur) for any of the time periods provided herein, including but not limited to those provided in the exemplary embodiments section. For example, the contacting can be for less than 24 hours, e.g., 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 in which retroviral particles and cells are contacted in suspension in a transduction reaction mixture, after which any retroviral particles remaining in suspension that are not associated with cells are separated from the cells and typically discarded, as discussed in more detail herein. While not intending to be limited by theory, it is noted that contacting is believed to begin when the retroviral particles and lymphocytes are combined together, typically by adding a solution containing the retroviral particles to a solution containing lymphocytes (e.g., T cells and / or NK cells).

[0109] After this initial contact, in some embodiments, the reaction mixture containing the cells and retroviral particles in suspension is incubated for a specified period without removing the retroviral particles that remain free in the solution and are not associated with cells. This incubation may be referred to herein as optional incubation. Thus, in exemplary embodiments, contact (including initial contact and optional incubation) can be carried out (or occur) from the lower end 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 the upper end 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 end of a selected range is less than the upper end of the selected range). In certain exemplary embodiments, the contacting step can be carried out for a time period ranging from 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, or 30 minutes, on the lower end of the range, to 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, or 12 hours, on the higher end of the range. In some embodiments, the contacting step can be carried out for a time period ranging from 30 seconds, 1 minute, and 5 minutes, on the lower end of the range, to 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, or 8 hours, on the higher end of the range. Thus, in some embodiments, after a reaction mixture is formed by adding retroviral particles to lymphocytes, the reaction mixture can be incubated for 5 minutes to 12 hours, 5 minutes to 10 hours, 5 minutes to 8 hours, 5 minutes to 6 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, 5 minutes to 1 hour, 5 minutes to 30 minutes, or 5 minutes to 15 minutes. In other embodiments, the reaction mixture may be incubated for 15 minutes to 12 hours, 15 minutes to 10 hours, 15 minutes to 8 hours, 15 minutes to 6 hours, 15 minutes to 4 hours, 15 minutes to 2 hours, 15 minutes to 1 hour, 15 minutes to 45 minutes, or 15 minutes to 30 minutes. In other embodiments, the reaction mixture may be incubated for 30 minutes to 12 hours, 30 minutes to 10 hours, 30 minutes to 8 hours, 30 minutes to 6 hours, 30 minutes to 4 hours, 30 minutes to 2 hours, 30 minutes to 1 hour, or 30 minutes to 45 minutes.In other embodiments, the reaction mixture may be incubated for between 1 and 12 hours, between 1 and 8 hours, between 1 and 4 hours, or between 1 and 2 hours. In another exemplary embodiment, the contacting is carried out for only the initial contacting step without further incubation in the reaction mixture (no further incubation in the reaction mixture containing free retroviral particles in suspension and cells in suspension), or with 5, 10, 15, 30 minutes, or 1 hour of incubation in the reaction mixture.

[0110] After the initial contact and the optional incubation period that may be part of the contacting step, the blood cells in the reaction mixture, or their T cell- and / or NK cell-containing fractions, are separated from retroviral particles not associated with such cells. For example, this can be performed using a PBMC enrichment procedure (e.g., a Ficoll gradient in a Sepax unit) or, in certain exemplary embodiments provided herein, by filtering the reaction mixture over a leukocyte-reduction filter set assembly and then harvesting the leukocytes, including T cells and NK cells. In another embodiment, this can be performed by centrifuging the reaction mixture at a relative centrifugal force of less than 500 g, e.g., 400 g, or between 300 and 490 g, or between 350 and 450 g. Such centrifugation to separate retroviral particles from cells can be performed, for example, for between 5 and 15 minutes, or between 5 and 10 minutes. In exemplary embodiments in which centrifugal force is used to separate cells from non-cell-associated retroviral particles, such g-forces are typically lower than those successfully used in spinoculation procedures.

[0111] In some exemplary embodiments, the methods provided herein in any aspect do not include spinoculation. In some embodiments, spinoculation is included as part of the contacting step. In exemplary embodiments, when spinoculation is performed, there is no additional incubation as part of the contacting, as the time of spinoculation provides the incubation time for the optional incubation described above. In other embodiments, there is an 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, for example, 30 minutes to 120 minutes, typically at least 60 minutes, e.g., 60 minutes to 180 minutes, or 60 minutes to 90 minutes. Spinoculation is typically carried out in a centrifuge at a relative centrifugal force of at least 800 g, more typically at least 1,200 g, e.g., between 800 g and 2400 g, or between 800 g and 1800 g, or between 1200 g and 2400 g, or between 1200 g and 1800 g. After spinoculation, such methods typically include the additional steps of resuspending the pelleted cells and retroviral particles, and then removing retroviral particles that are not associated with cells according to the steps described above if spinoculation is not performed.

[0112] The contacting step, including optional incubation, and spinoculation in embodiments including spinoculation, can be carried out between 4° C. and 42° C., or between 20° C. and 37° C. In certain exemplary embodiments, no spinoculation is performed, and the contacting and associated optional incubation is 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 15 minutes to 2 hours, 15 minutes to 1 hour, or 15 minutes to 30 minutes.

[0113] In some embodiments of the methods and compositions disclosed herein, between 5% and 85% of the total lymphocytes collected from blood are genetically modified. In some embodiments, the percentage of genetically modified and / or transduced lymphocytes is between 1, 5, and 10% at the lower end of the range and 15, 20, 25, 30, 40, 50, 60, 70, 80, and 85% at the upper end 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 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] The methods for genetically modifying lymphocytes provided according to any of the methods herein typically involve the insertion into cells of a polynucleotide comprising one or more transcription units encoding a CAR or lymphoproliferative element, or in exemplary embodiments, encoding both a CAR and a lymphoproliferative element, according to any of the CAR and lymphoproliferative element embodiments provided herein. Such CARs and lymphoproliferative elements may be provided to support the shorter, more simplified methods provided herein, which can support the expansion of genetically modified and / or transduced T cells and / or NK cells after contact and optional incubation. Thus, in exemplary embodiments of any of the methods provided herein, lymphoproliferative elements may be delivered from the genome of retroviral particles within genetically modified and / or transduced T cells and / or NK cells, resulting in the cells having the increased proliferation and / or survival characteristics disclosed in the lymphoproliferative element section herein. In exemplary embodiments of any of the methods provided herein, genetically modified T cells or NK cells can engraft in vivo in mice and / or be enriched in mice for at least 7, 14, or 28 days in vivo. Those skilled in the art will recognize that such mice can be treated or otherwise genetically modified so that any immunological differences between the genetically modified T cells and / or NK cells do not result in an immune response being elicited in the mice against any component of the lymphocytes transduced by the replication-incompetent recombinant retroviral particles.

[0115] Media that can be included in the contacting step, e.g., when the cells and retroviral particles are first contacted, or during any subsequent optional incubation period with a reaction mixture comprising the retroviral particles and cells in suspension in the media in any embodiment provided herein, or that can be used during cell culture and / or various washing steps in any embodiment provided herein, can comprise a basal medium, such as commercially available media for ex vivo T cell and / or NK cell culture. Non-limiting examples of such media include X-VIVO™ 15 Chemically Defined Serum-Free Hematopoietic Cell Medium (Lonza) (2018 Catalog Nos. BE02-060F, BE02-00Q, BE-02-061Q, 04-744Q, or 04-418Q), ImmunoCult™-XF T Cell Expansion Medium (STEMCELL Technologies) (2018 Catalog No. 10981), PRIME-XV® T Cell Expansion XSFM (Irvine Scientific) (2018 Catalog No. 91141), AIM V® Medium CTS™ (Therapeutic Grade) (Thermo Fisher Scientific (referred to herein as "ThermoFisher"), or CTS™ Optimizer™ Media (Thermo Fisher (2018 catalog numbers A10221-01 (basal medium (bottle)), and A10484-02 (additives), A10221-03 (basal medium (bag)), A1048501 (basal medium and additive kit (bottle)), and A1048503 (basal medium and additive kit (bag)). Such media may be chemically defined, serum-free formulations manufactured in accordance with cGMP. Media may be xeno-free and complete. In some embodiments, the basal media has been cleared by regulatory agencies for use in ex vivo cell processing, such as in an FDA 510(k) cleared device.In some embodiments, the medium is basal medium with or without the accompanying T cell expansion supplements, 2018 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 a subject may be added to the transduction reaction mixture. Supplementary 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 comprising 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 comprising genetically modifying T cells and / or NK cells, the T cells and / or NK cells are not incubated on a monocyte-adherent substrate for more than 4 hours, in one embodiment, or more than 6 hours, in another embodiment, or 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 may include incubating the T cells and / or NK cells on an adherent substrate that binds monocytes for 30 minutes, 1 hour, or 2 hours prior to transduction. In another embodiment, the T cells and / or NK cells are not subjected to a step of removing monocytes by incubation on an adherent substrate prior to said transduction step. In another embodiment, the T cells and / or NK cells are not incubated with or exposed to bovine serum, e.g., cell culture bovine serum, e.g., fetal bovine serum, prior to or during the contacting step and / or 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, may be carried out in a closed system. Thus, the reaction mixture formed by such methods, as well as the genetically modified and / or transduced lymphocytes (e.g., T cells and / or NK cells) produced by such methods, may be contained within such a closed system. A closed system is a cell processing system that is generally or completely closed off from the environment, such as the environment within a room, or even a fume hood, or the environment outside the conduits, such as the tubes and chambers, of the system through which the 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 found in traditional open cell culture systems. To mitigate this risk, particularly in the absence of antibiotics, several commercial processes have been developed that focus on the use of disposable (single-use) equipment. However, even when used under sterile conditions, there is always a risk of contamination by opening a 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 carried out in a closed system. Such processes may be designed and operated so that the product is not exposed to the external environment. Material transfer occurs through sterile connections, such as sterile tubing and sterile welded connections. Air for gas exchange can occur through a gas-permeable membrane and 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 closed-system methods can be performed using commercially available devices. Various closed-system devices can be used at various steps within the method, and cells can be transferred between these devices using tubing and connections, such as welds, luers, spikes, or Clave ports, to prevent exposure of cells or media to the environment. For example, blood can be collected into an IV bag or syringe, optionally containing an anticoagulant, and then transferred to a Sepax 2 device (Biosafe) for PBMC enrichment and isolation. In other embodiments, whole blood can be filtered to collect leukocytes using a leukocyte reduction filter assembly. Isolated PBMCs or isolated leukocytes can be transferred to a chamber in a G-Rex device for optional activation, transduction, and optional expansion. Alternatively, the collected blood can be converted into a blood bag, such as the bag from which it was collected. Finally, a Sepax 2 device can be used to harvest and collect cells in a separate 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 the G-Rex device (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 exemplary embodiments, optional activation, transduction, and optional expansion may be performed within the same chamber or vessel of a closed system. For example, in exemplary embodiments, the chamber may be a chamber of a G-Rex device, and PBMCs or leukocytes may be transferred to the chamber of the G-Rex device after they have been concentrated and isolated, and may remain in the same chamber of the G-Rex device until recovery.

[0119] The methods provided herein may include transferring blood and cells and / or fractions thereof, as well as lymphocytes before or after they have been contacted with retroviral particles, between containers within a closed system, thus avoiding environmental exposure. The containers used in the closed system may be, for example, tubes, bags, syringes, or other containers. In some embodiments, the containers are containers used in research facilities. In some embodiments, the containers are containers used in commercial production. In other embodiments, the containers may be collection containers used in blood collection processes. The genetic modification methods herein typically include a contacting step in which lymphocytes are contacted with replication-incompetent recombinant retroviral particles. In some embodiments, the contacting may be performed within a container, such as a blood bag. Blood and its various lymphocyte-containing fractions may be transferred from one container to another within the closed system (e.g., from a first container to a second container) for contacting. The second container may 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 may be transferred to a different container within the closed system (i.e., without environmental exposure). Either before or after this transfer, the cells are typically washed in a 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 post-incubation isolation, and optional washing, are carried out for periods ranging from 15 minutes, 30 minutes, or 1, 2, 3, or 4 hours on the low end of the range, to 4, 8, 10, or 12 hours on the high end of the range.

[0120] Without being bound by theory, in a non-limiting exemplary method, delivery of a polynucleotide encoding a lymphoproliferative element to resting T cells and / or NK cells ex vivo (which may be incorporated into the genome of the T cells or NK cells) provides the cells with the impetus for in vivo expansion 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, 2, 3, or 6 months of the contacting, during the contacting, and / or within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days, or within 1, 2, 3, or 6 months of the reintroduction of the modified T cells and / or NK cells into the subject. Furthermore, in non-limiting exemplary embodiments, the methods provided herein can be performed without exposing the subject to lymphodepletion agents during the step in which the replication-incompetent recombinant retroviral particles contact the subject's resting T cells and / or resting NK cells, and / or during the entire ex vivo method. Thus, methods of 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 exemplary embodiments, such methods involve no or substantially no ex vivo proliferation.

[0121] In non-limiting exemplary embodiments of any aspect provided herein, this entire method / process from drawing blood from the subject following ex vivo transduction of T cells and / or NK cells to reintroduction of the blood into the subject may occur 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 withdrawal / collection of blood from the subject to reintroduction of the blood into the subject following ex vivo transduction of T cells and / or NK cells, in non-limiting exemplary embodiments herein, is carried out over a period of 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 of from 15, 30, 60, 90, 120, 180, and 240 minutes at the lower end of the range to 120, 180, and 240, 300, 360, 420, and 480 minutes at the upper end of the range. In other embodiments, the entire method / process from withdrawal / collection of blood from the subject to reintroduction of the blood into the subject after ex vivo transduction of the T cells and / or NK cells occurs over a period of time ranging from 1, 2, 3, 4, 6, 8, 10, and 12 hours at the low end of the range to 8, 9, 10, 11, 12, 18, 24, 36, or 48 hours at the high end of the range. In some embodiments, the genetically modified T cells and / or NK cells are separated from the replication-incompetent recombinant retroviral particles after the period during which contacting occurs.

[0122] The methods provided herein for genetically modifying lymphocytes and related methods for performing adoptive cell therapy can be performed in significantly less time than previous methods, enabling fundamental improvements in patient care and safety and product manufacturability. Therefore, such processes, when performed in vivo for therapeutic purposes, are expected to be advantageous from the perspective of regulatory agencies responsible for approving such processes. For example, in non-limiting examples of any aspect provided herein that includes a subject, the subject can remain in the same building (e.g., infusion clinic) or room as the blood or sample processing equipment throughout the entire process of processing the sample before the modified T cells and / or NK cells are reintroduced into the patient. In non-limiting exemplary embodiments, the subject remains within 100, 50, 25, or 12 feet or arms' distance from the site and / or the blood or cells being processed throughout the entire method / process, from blood collection / collection from the subject to reintroduction of the blood into the subject after ex vivo transduction of the T cells and / or NK cells. In other non-limiting exemplary embodiments, the subject remains awake and / or at least one person can continue to monitor the subject's blood or cells being treated throughout the entire method / process, and / or continuously, from blood draw / collection to reintroduction of the blood into the subject after ex vivo transduction of the T cells and / or NK cells. Because of the improvements provided herein, the entire method / process for adoptive cell therapy and / or transduction of resting T cells and / or NK cells from blood draw / collection from the subject to reintroduction of the blood into the subject after ex vivo transduction of the T cells and / or NK cells can be performed with continuous human monitoring. In other non-limiting exemplary embodiments, the blood cells are not incubated in an unattended room at any point throughout the method / process, from blood draw / collection from the subject to reintroduction of the blood into the subject after ex vivo transduction of the T cells and / or NK cells.In other non-limiting exemplary embodiments, the entire method / process, from blood withdrawal / collection from the subject to reintroduction of the 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, sample ID mix-ups and lengthy and costly incubations over days or weeks can be avoided. This is further provided by the fact that the methods provided herein are easily adaptable to closed and automated blood processing systems, in which the blood sample and its components reintroduced into the subject come into contact only with disposable, single-use components.

[0123] The methods for genetically modifying and / or transducing lymphocytes, such as T cells and / or NK cells, provided herein may be part of a method for performing adoptive cell therapy. Typically, methods for performing adoptive cell therapy include withdrawing blood from a subject and infusing genetically modified and / or transduced lymphocytes (e.g., T cells and / or NK cells) back into 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 the 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 methods for killing or inhibiting the growth of target cells, comprising contacting a cytotoxic immune effector cell (e.g., a cytotoxic T lymphocyte or NK cell) genetically modified to produce a CAR of interest with the T cell or NK cell, such that the T cell or NK cell recognizes the 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, in some exemplary embodiments, be 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), as well as aspects relating to the use of replication-incompetent recombinant retroviral particles in the manufacture of kits for genetically modifying T cells and / or NK cells in a subject, 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. Introduction or reintroduction of genetically modified lymphocytes into a subject can be via any route known in the art. For example, introduction or reintroduction can be via injection into the subject's blood vessels. In some embodiments, genetically modified and / or transduced lymphocytes (e.g., T cells and / or NK cells), or a population thereof, undergo no more than four cell divisions ex vivo before 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 1 to 12 hours. In some embodiments, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 2 hours, or less than 1 hour elapses between the time blood is drawn from the subject and the time the genetically modified T cells and / or NK cells are reintroduced into the subject. In some embodiments, all steps after blood is drawn and before the blood is reintroduced are performed in a closed system with a person monitoring the closed system throughout the process.

[0125] In some embodiments of the methods and compositions disclosed herein, the genetically modified T cells and / or NK cells are returned to, 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 to stimulate / activate T cells and / or NK cells and expand the genetically modified T cells and / or NK cells before introducing them into a subject. In prior art methods, this typically takes several days or weeks, requiring the subject to return to the clinic for a transfusion several days or weeks after the initial blood draw. In some embodiments of the methods and compositions disclosed herein, the T cells and / or NK cells are not stimulated ex vivo by exposure to an anti-CD3 / anti-CD28 solid support, such as anti-CD3 / anti-CD28-coated beads, before 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, the genetically modified T cells and / or NK cells are not expanded ex vivo, or are expanded for only a few cell divisions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 rounds of cell division), but rather are expanded or predominantly expanded in vivo, i.e., within the subject. In some embodiments, no additional medium is added to allow further expansion of the cells. In some embodiments, cell production of primary blood lymphocytes (PBLs) does not occur while the PBLs are contacted with the replication-incompetent recombinant retroviral particles. In an exemplary embodiment, cell production of PBLs does not occur while the PBLs are ex vivo. In traditional methods of adoptive cell therapy, subjects are lymphodepleted before reinfusing the genetically modified T cells and / or NK cells. In some embodiments, the patient or subject is not lymphodepleted before blood is drawn. In some embodiments, the patient or subject is not lymphodepleted before reinfusing 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, 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, 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 (including no exposure) before contacting the T cells and / or NK cells with replication-incompetent recombinant retroviral particles. In exemplary embodiments, 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 before contacting the T cells and / or NK cells with replication-incompetent recombinant retroviral particles.

[0126] In some exemplary embodiments, the cells are introduced or reintroduced into a subject by intravenous or intraarterial infusion. In any of the embodiments disclosed herein, the number of T cells and / or NK cells reinfused into a subject is at the lower end of the range of 1 x 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 the upper end of the range of 5 x 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 In an exemplary embodiment, the number of T cells and / or NK cells reinfused or otherwise delivered to a subject may be between 1 x 10 cells / kg at the lower end of the range and 1 x 10 cells / kg at the lower end of the range. 4 , 2.5×10 4 , 5×10 4 , and 1 × 10 5 cells / kg to the upper end of the range of 2.5 x 10 4 , 5×10 4 , 1×10 5 , 2.5×10 5 , 5×10 5 , and 1 × 10 6 In some embodiments, the number of PBLs reinfused or otherwise delivered to a subject may be between 5 x 10 cells / kg. 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, at the lower end of the range, or 2.5 x 10 cells, at the upper end of the range. 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 In some embodiments, the number of T cells and / or NK cells available for infusion or reinfusion into a 70 kg subject or patient may be 7 x 10 5 ~2.5×10 8 In other embodiments, the number of T cells and / or NK cells available for transduction is between about 7 x 10 6 It's plus or minus 10%.

[0127] Engineered signaling polypeptide(s) In some embodiments, 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 polypeptides comprise any combination of an extracellular domain (e.g., an antigen-specific targeting region or ASTR), a stalk, and a transmembrane domain, in combination with one or more intracellular activation domains, optionally one or more regulatory domains (e.g., costimulatory domains), and optionally one or more T cell survival motifs. 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. Exemplary sequences for any domain of the engineered signaling polypeptides disclosed herein can be found in WO2019 / 055946, which is incorporated herein by reference in its entirety. Those skilled in the art will recognize that such engineered polypeptides may also be referred to as recombinant polypeptides. The engineered signaling polypeptides provided herein, such as CARs, recombinant TCRs, LEs, and CLEs, are typically transgenes into lymphocytes, particularly T cells and NK cells, especially T cells and / or NK cells engineered using the methods and compositions provided herein, to express 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 exemplary embodiments, such extracellular and transmembrane domains do not comprise a ligand-binding region. Such domains are present in engineered signaling polypeptides and are believed not to bind ligands when expressed in B cells, T cells, and / or NK cells. Mutations in such receptor mutants may occur in the extracellular juxtamembrane region. Without being limited by theory, mutations in at least some extracellular domains (and some extracellular transmembrane domains) of the engineered signaling polypeptides provided herein may contribute to signal transduction of the engineered signaling polypeptide in the absence of ligand by bringing together activation chains that are not normally present together. Further embodiments relating to extracellular domains containing mutations in the extracellular domain can be found, for example, in the lymphoproliferative element section of this specification.

[0129] In certain exemplary embodiments, the extracellular domain comprises a dimerization motif. In exemplary embodiments, the dimerization motif comprises a leucine zipper. In some embodiments, the leucine zipper is derived from a jun polypeptide, e.g., c-jun. Further embodiments regarding extracellular domains comprising a dimerization motif can be found, for example, in the lymphoproliferative element section herein.

[0130] In certain embodiments, the extracellular domain is an antigen-specific targeting region (ASTR), sometimes 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, specific binding pair members suitable for use in the engineered signaling polypeptides of the present disclosure include antibody ASTRs, antigens, ligands, receptor-binding domains of ligands, receptors, ligand-binding domains of receptors, and affibodies.

[0131] ASTRs suitable for use in the engineered signaling polypeptides of the present disclosure can be any antigen-binding polypeptide, hi certain embodiments, the ASTR is an antibody, such as a full-length antibody, a single-chain antibody, a Fab fragment, a Fab' fragment, a (Fab')2 fragment, an Fv fragment, and a 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 N-terminal to the light chain in the engineered signaling polypeptide. In other embodiments, the light chain is positioned N-terminal to the heavy chain in the engineered signaling polypeptide. In any of the disclosed embodiments, the heavy and light chains can be separated by a linker, as discussed in more detail herein. In any of the disclosed embodiments, the heavy or light chain can be at the N-terminus of the engineered signaling polypeptide, typically at the C-terminus of another domain, such as a signal sequence or peptide.

[0133] Other antibody-based recognition domains (cAb VHHs (camelized antibody variable domains) and humanized versions, IgNAR VHs (shark antibody variable domains) and humanized versions, sdAb VHs (single domain antibody variable domains) and "camelized" antibody variable domains are suitable for use with engineered signaling polypeptides and methods of using the engineered signaling polypeptides of the disclosure. In some cases, are T cell receptor (TCR)-based recognition domains.

[0134] Certain embodiments of any aspect or embodiment herein involving a CAR include CARs with an extracellular domain engineered to recruit the endogenous TCR signaling complex and CD3Z signaling pathway. In one embodiment, a chimeric antigen receptor ASTR is fused to one of the endogenous TCR complex chains (e.g., TCR alpha, CD3E, etc.) to promote incorporation into the TCR complex and signaling through 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 comprising VαVβ). Finally, scFvs can also be generated to recognize specific MHC / peptide complexes, thereby functioning as surrogate TCRs. Such peptide / MHC scFv binders can be used in many configurations similar to CARs.

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

[0136] ASTRs suitable for use in the engineered signaling polypeptides of the present disclosure can have a variety of antigen-binding specificities. In some cases, the antigen-binding domain is specific for an epitope present in an antigen expressed (synthesized) by the target cell. In one example, the target cell is a cancer cell-associated antigen. The cancer cell-associated antigen may be, for example, an antigen associated with breast cancer cells, B-cell lymphoma cells, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells (e.g., small cell lung cancer cells), non-Hodgkin's 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, gliomas, glioblastomas, medulloblastomas, colorectal cancer cells, etc. The cancer cell-associated antigen may also be expressed by non-cancerous cells.

[0137] Non-limiting examples of antigens to which the engineered signaling polypeptide ASTR 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-a2, 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, which is a ligand for 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] Where the member of the specific binding pair of the engineered signaling polypeptide is a ligand, the engineered signaling polypeptide can be activated in the presence of a second member of the specific binding pair, which is a receptor for the ligand. For example, where the ligand is VEGF, the second member of the specific binding pair can be a VEGF receptor, including a soluble VEGF receptor.

[0140] As noted above, in some cases, the member of a specific binding pair included in the engineered signaling polypeptide is a receptor, e.g., an ASTR, which is a receptor for a ligand, a coreceptor, etc. The receptor may also be a ligand-binding fragment of a receptor. Suitable receptors include, but are not limited to, growth factor receptors (e.g., VEGF receptors); killer cell lectin-like receptor subfamily K, member 1 (NKG2D) polypeptides (receptors for MICA, MICB, and ULB6); cytokine receptors (e.g., IL-13 receptor; IL-2 receptor, etc.); CD27; natural cytotoxicity receptors (NCRs) (e.g., NKP30 (NCR3 / CD337) polypeptides (receptors for HLA-B-associated transcript 3 (BAT3) and B7-H6), etc.); and the like.

[0141] In certain embodiments of any of the aspects provided herein that include an ASTR, the ASTR can be directed to an intermediate protein that links the ASTR to a target molecule expressed on a target cell. The intermediate protein may be endogenously expressed or exogenously introduced, and may be natural, 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 the tag recognized by the ASTR and the target molecule, typically a protein target, expressed on the target cell. 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. Thus, an ASTR includes a molecule that binds to a tag.

[0142] Stoke In some embodiments, the engineered signaling polypeptide comprises a stalk located on the outside of the cell, in a portion of the engineered signaling polypeptide intervening between the 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 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG AVHTRGLDFA (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 engineered signaling polypeptides, the stalk used enables 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, for example, 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 an engineered signaling polypeptide comprises at least one cysteine. For example, in some embodiments, the stalk may comprise the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 4). If present, the cysteine ​​of the stalk of a first engineered signaling polypeptide may be available to form a disulfide bond with the stalk of a second engineered signaling polypeptide.

[0145] The stalk can comprise an immunoglobulin hinge region amino acid sequence known in the art. See, e.g., 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 can 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 amino acids of any of the following amino acid sequences: DKTHT (SEQ ID NO: 5); CPPC (SEQ ID NO: 4); CPEPKSCDTPPPCPR (SEQ ID NO: 6) (see, e.g., 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 comprise a hinge region having the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 hinge region. The stalk may comprise one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally occurring) hinge region. For example, His229 of the human IgG1 hinge can be substituted with Tyr such that the stalk comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 15) (see, e.g., Yan et al. (2012) J. Biol. Chem. 287:5891). The stalk can comprise an amino acid sequence derived from human CD8, for example, the stalk can comprise the amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16), or a variant thereof.

[0146] Transmembrane domain The engineered signaling polypeptides of the present disclosure may contain a transmembrane domain for insertion into a eukaryotic cell membrane. The transmembrane domain may be interposed between the ASTR and the costimulatory domain. The transmembrane domain may be interposed between the stalk and the costimulatory domain so that the chimeric antigen receptor comprises, in order from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus), an ASTR; a stalk; a transmembrane domain; and an 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 stretches 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) CD28 TM (SEQ ID NO: 21); f) CD134 (OX40) TM: (SEQ ID NO: 22); g) CD7 TM (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, a transmembrane domain of one embodiment of the present invention may have at least 80%, 90%, or 95% sequence identity, or 100% sequence identity to the transmembrane domain of SEQ ID NO: 17, or may 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 Upon activation, intracellular activation domains suitable for use in engineered signaling polypeptides of the present disclosure typically induce the production of one or more cytokines; increase cell death; and / or induce CD8 + T cells, CD4 + Increase the proliferation of T cells, NKT cells, gamma delta T cells, and / or neutrophils. The activation domain may be referred to herein as activation domain. The activation domain can be used in CAR or lymphoproliferative element provided herein.

[0151] In some embodiments, the intracellular activation domain comprises at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif, as described below. In some embodiments, the intracellular activation domain of one aspect of the invention may have at least 80%, 90%, or 95% sequence identity, or may have 100% sequence identity, to a CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10 / CD28, or ZAP70 domain, as described below.

[0152] Intracellular activation domains suitable for use in engineered signaling polypeptides of the present disclosure include intracellular signaling polypeptides that contain an immunoreceptor tyrosine-based activation motif (ITAM). An ITAM motif is YX1X2L / I, where X1 and X2 are independently any amino acid. In some embodiments, the intracellular activation domain of an engineered signaling polypeptide contains one, two, three, four, or five 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 between 6 and 8, and each of the 6 to 8 X3 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 an ITAM motif-containing portion 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 contain 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 (CD3Z, also known as T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). For example, 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 stretch of at least 10, 15, 20 or all amino acids in the following sequences, or to a stretch of about 100 to about 110 amino acids (aa), about 110 to about 115 aa, about 115 to about 120 aa, about 120 to about 130 aa, about 130 to about 140 aa, about 140 to about 150 aa, or about 150 to about 160 aa of either of the following amino acid sequences (2 isoforms): [ka] or MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPQRRKNPQEGL[YNELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR (SEQ ID NO: 27) (ITAM motifs are listed in parentheses).

[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 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 amino acids in the following sequence, or to a contiguous stretch of about 100 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]DKRRGRDPEM GGKPRRKNPQEGL[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) (ITAM motifs are shown in parentheses).

[0156] In some embodiments, the intracellular activation domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as 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; T cell surface glycoprotein CD3 delta chain, etc.). 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 stretch of at least 10, 15, 20 or all amino acids in the following sequence, or to a contiguous stretch of about 100 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: MEHSTFLSGLVLATLLSQVSPFKIPIEELE DRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQV[YQPLRDRDDAQYSHL]GGNWARNK (SEQ ID NO: 33) or MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRTADTQALLRNDQV[YQPLRDRDDAQYSHL]GGNWARNK (SEQ ID NO: 34) (ITAM motifs are shown in brackets).

[0157] Similarly, a suitable intracellular activation domain polypeptide can comprise an ITAM motif-containing portion of the full-length CD3 delta amino acid sequence. Thus, a suitable 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 a stretch of at least 10, 15, 20, or all 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 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 be at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of a stretch of at least 10, 15, 20, or all of the amino acids in the following sequence, or of a contiguous stretch of about 100 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:

[0049] The invention may comprise a domain having sequence identity to: MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD[YEPIRKGQRDLYSGL]NQRRI (SEQ ID NO: 36) (the ITAM motif is shown in brackets).

[0159] Similarly, a suitable intracellular activation domain polypeptide may comprise an ITAM motif-containing portion of the full-length CD3 epsilon amino acid sequence. Accordingly, 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 stretch of at least 10, 15, 20 or all amino acids in the following sequence: [ka] (ITAM motifs are shown in brackets).

[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.). Accordingly, a suitable intracellular activation domain may be at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1100%, 1110%, 112%, 113%, 114%, 115%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, %, 99% or 100% sequence identity: MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQL[YQPLKDREDDQYSHL]QGNQLRRN (SEQ ID NO: 38) (ITAM motifs are shown in brackets).

[0161] Similarly, a suitable intracellular activation domain polypeptide can comprise an ITAM motif-containing portion of the full-length CD3 gamma amino acid sequence. Thus, a suitable 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 a stretch of at least 10, 15, 20 or all amino acids in the following sequence: [ka] (ITAM motifs are shown in brackets).

[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.). Accordingly, a suitable intracellular activation domain may be derived from at least 10, 15, 20, or all of the amino acids in the following sequence, or from a contiguous stretch of about 100 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 from any of the following amino acid sequences: It may comprise a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to about 150 aa to about 160 aa: MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPN GTLIIQNVNKSHGGIYVCRVQEGNESYQQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENL[YEGLNLDDCSMYEDI]SRGLQGTYQDVGSLNIGDVQLEKP (SEQ ID NO: 40) or MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNEPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENL[YEGLNLDDCSMYEDI]SRGLQGTYQDVGSLNIGDVQLEKP (SEQ ID NO: 41) (ITAM motifs are shown in brackets).

[0163] Similarly, a suitable intracellular activation domain polypeptide can comprise an ITAM motif-containing portion of the full-length CD79A amino acid sequence. Thus, a suitable 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 a stretch of at least 10, 15, 20, or all 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 activating protein 12; KAR-associated protein; TYRO protein tyrosine kinase binding protein; killer activating receptor-associated protein; killer activating receptor-associated protein, etc.). For example, a suitable intracellular activation domain may extend over at least 10, 15, 20, or all of the amino acids in the following sequence, or over a contiguous stretch of about 100 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 over a contiguous stretch of any of the following amino acid sequences (four isoforms): The sequence of about 150 aa to about 160 aa may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity: MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESP[YQELQGQR SDVYSDL]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) (ITAM motifs are shown in parentheses).

[0165] Similarly, a suitable intracellular activation domain polypeptide can comprise an ITAM motif-containing portion of the full-length DAP12 amino acid sequence. Thus, a suitable 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 a stretch of at least 10, 15, 20 or all amino acids in the following sequence: [ka] (ITAM motifs are shown in brackets).

[0166] In some embodiments, the intracellular activation domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc-epsilon RI-gamma; fcR gamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain, etc.). For example, a suitable 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 a stretch of at least 10, 15, 20 or all amino acids in the following sequence, or to a contiguous stretch of about 50 to about 60 amino acids (aa), about 60 to about 70 aa, about 70 to about 80 aa, or about 80 to about 88 aa of the following amino acid sequence: MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKSDGV[YTGLSTRNQETYETL]KHEKPPQ (SEQ ID NO: 48) (ITAM motifs are shown in parentheses).

[0167] Similarly, a suitable intracellular activation domain polypeptide can comprise an ITAM motif-containing portion of the full-length FCER1G amino acid sequence. Accordingly, a suitable 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 a stretch of at least 10, 15, 20 or all amino acids in the following sequence: [ka] (ITAM motifs are shown in brackets).

[0168] Intracellular activation domains suitable for use in engineered signaling polypeptides of the present disclosure include DAP10 / CD28 type signaling chains. An example of a DAP10 signaling chain is amino acid SEQ ID NO: 50. In some embodiments, suitable intracellular activation domains include domains 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 amino acids in SEQ ID NO: 50.

[0169] An example of a CD28 signaling chain is the amino acid sequence of SEQ ID NO: 51. In some embodiments, a suitable intracellular domain comprises 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 amino acids in SEQ ID NO: 51.

[0170] Intracellular activation domains suitable for use in engineered signaling polypeptides of the present disclosure include ZAP70 polypeptides. 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 stretch of at least 10, 15, 20, or all of the amino acids in the following sequence:

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

[0172] The costimulatory domain typically enhances and / or alters the nature of the response to the activation domain. Costimulatory domains suitable for use in the engineered signaling polypeptides of the present disclosure are generally receptor-derived polypeptides. In some embodiments, the costimulatory domain homodimerizes. A costimulatory domain of interest may also 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-lBB (CD137), CD27, CD28, CD28-deleted Lck-binding (ICΔ), ICOS, OX40, BTLA, CD27, CD30, GITR, and HVEM. For example, the costimulatory domain of one embodiment of the present invention may have at least 80%, 90%, or 95% sequence identity to the costimulatory domain of 4-1BB (CD137), CD27, CD28, CD28-deleted Lck-binding (ICΔ), ICOS, OX40, BTLA, CD27, CD30, GITR, or HVEM. For example, the costimulatory domain of one embodiment of the present invention may have at least 80%, 90%, or 95% sequence identity to the costimulatory domain of non-limiting examples of suitable costimulatory polypeptides, including, but not limited to, 4-1BB (CD137), CD27, CD28, CD28-deleted Lck-binding (ICΔ), ICOS, OX40, BTLA, CD27, CD30, GITR, and HVEM. For example, a costimulatory domain of one embodiment of the present invention can have at least 80%, 90%, or 95% sequence identity to the costimulatory domain of 4-1BB (CD137), CD27, CD28, CD28-deleted Lck-binding (ICΔ), ICOS, OX40, BTLA, CD27, CD30, GITR, or HVEM.

[0173] Costimulatory domains suitable for inclusion in engineered signaling polypeptides can have a length of about 30 to about 70 amino acids (aa), e.g., costimulatory domains can have a length of about 30 to about 35 aa, about 35 to about 40 aa, about 40 to about 45 aa, about 45 to about 50 aa, about 50 to about 55 aa, about 55 to about 60 aa, about 60 to about 65 aa, or about 65 to about 70 aa. In other cases, costimulatory domains can have a length of about 70 to about 100 aa, about 100 to about 200 aa, or greater than 200 aa.

[0174] In some embodiments, the costimulatory 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 costimulatory 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 amino acids in SEQ ID NO:53. In some of these embodiments, the costimulatory 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 costimulatory domain is derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp44). For example, a suitable costimulatory 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 at least 10, 15, 20, or all stretches of amino acids in SEQ ID NO: 54. In some of these embodiments, the costimulatory 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.

[0176] In some embodiments, the costimulatory domain is derived from the intracellular portion of the transmembrane protein CD28-deleted Lck-binding (ICΔ). For example, a suitable costimulatory 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 at least 10, 15, 20, or all stretches of amino acids in SEQ ID NO: 55. In some of these embodiments, the costimulatory 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.

[0177] In some embodiments, the costimulatory domain is derived from the intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). For example, a suitable costimulatory 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 at least 10, 15, 20, or all stretches of amino acids in SEQ ID NO: 56. In some of these embodiments, the costimulatory 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.

[0178] In some embodiments, the costimulatory domain is derived from the intracellular portion of the transmembrane protein OX40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX-40, and TXGP1L). OX40 contains a p85 PI3K-binding motif at residues 34-57 and a TRAF-binding motif at residues 76-102, each of which is from SEQ ID NO:296 (Table 1). In some embodiments, the costimulatory domain may contain the p85 PI3K-binding motif of OX40. In some embodiments, the costimulatory domain may contain 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 ubiquitin-targeting motif. In some embodiments, one or both of these lysines in the costimulatory domain of OX40 are mutated to arginine or another amino acid. In some embodiments, a suitable costimulatory 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 at least 10, 15, 20, or all stretches of amino acids in SEQ ID NO: 57. In some of these embodiments, the costimulatory domain has a length of about 20 aa to about 25 aa, about 25 aa to about 30 aa, 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. In exemplary embodiments, the costimulatory domain has a length of about 20 aa to about 50 aa, e.g., 20 aa to 45 aa, or 20 aa to 42 aa.

[0179] In some embodiments, the costimulatory domain is derived from the intracellular portion of the transmembrane protein CD27 (also known as S152, T14, TNFRSF7, and Tp55). For example, a suitable costimulatory 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 at least 10, 15, 20, or all stretches of amino acids in SEQ ID NO: 58. In some of these embodiments, the costimulatory 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 costimulatory domain is derived from the intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). For example, a suitable costimulatory 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 in SEQ ID NO:59.

[0181] In some embodiments, the costimulatory domain is derived from the intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). For example, a suitable costimulatory 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 stretch of about 100 to about 110 amino acids (aa), about 110 to about 115 aa, about 115 to about 120 aa, about 120 to about 130 aa, about 130 to about 140 aa, about 140 to about 150 aa, about 150 to about 160 aa, or about 160 to about 185 aa of SEQ ID NO: 60.

[0182] In some embodiments, the costimulatory 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 costimulatory 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 in SEQ ID NO:61. In some of these embodiments, the costimulatory 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.

[0183] In some embodiments, the costimulatory 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 costimulatory 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 amino acids in SEQ ID NO:62. In some of these embodiments, the costimulatory domains of both the first and second polypeptides 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.

[0184] Linker In some embodiments, the engineered signaling polypeptide includes a linker between any two adjacent domains. For example, the linker may be between the transmembrane domain and the first costimulatory 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 and the transmembrane domain and the costimulatory domain. As another example, the linker may be between the costimulatory domain and the intracellular activation domain of a second polypeptide. As another example, the linker may be between the ASTR and the intracellular signaling domain.

[0185] The linker peptide can have any of a variety of amino acid sequences. Proteins can be linked by a spacer peptide, which is generally flexible in nature, although other chemical bonds are not excluded. The linker can 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 linking 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, provided that a suitable linker has a sequence that generally results in a flexible peptide. The use of small amino acids such as glycine and alanine facilitates the formation of flexible peptides. The formation of such sequences is routine for those skilled in the art.

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

[0187] An exemplary flexible linker is a glycine polymer (G) n , glycine-serine polymers (e.g., (GS) n , G.S.G.S.G.S.n ,GGGS n , and GGGGS n (where 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 of interest because both of these amino acids are relatively unstructured and can therefore function as neutral tethers between components. Glycine polymers are of particular interest because glycine has access to much more phi-psi space 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, GGGGSGGGGSGGGGGS (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 peptides conjugated to any of the above elements may include a fully or partially flexible linker, such that the linker may include a flexible linker as well as one or more moieties that confer 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 particular combination of one or more engineered signaling polypeptides. In some methods and compositions provided herein, the genetically modified T cells contain 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 it will be understood that elements on a "first polypeptide" and elements on a "second polypeptide" mean that, typically in further elements or steps relative to that particular polypeptide, the elements are on different polypeptides, referred to as first or second only by 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 costimulatory domain, while in other embodiments, the first engineered signaling polypeptide comprises a costimulatory 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 costimulatory 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 general promoter under the same transcript, where the nucleic acids encoding the engineered signaling polypeptides are separated by nucleic acids encoding one or more internal ribosome entry sites (IREs) or one or more protease cleavage peptides.

[0192] In certain embodiments, the polypeptide encodes two engineered signaling polypeptides, where the first engineered signaling polypeptide comprises a first extracellular antigen-binding domain capable of binding to a first antigen and a first intracellular signaling domain that is not a costimulatory domain, and the second polypeptide comprises a second extracellular antigen-binding domain capable of binding to VEGF and a second intracellular signaling domain, such as the signaling domain of a costimulatory 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 fragment thereof (e.g., an scFv), e.g., an antibody or 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, where the first engineered signaling polypeptide comprises an extracellular tumor antigen-binding domain and a CD3ζ signaling domain, and the second engineered signaling polypeptide comprises an antigen-binding domain (wherein the antigen is an angiogenic or vasculogenic factor) and one or more costimulatory molecule signaling domains. The angiogenic factor may be, for example, VEGF. The one or more costimulatory molecule signaling motifs may comprise, for example, a costimulatory signaling domain from each of CD27, CD28, OX40, ICOS, and 4-1BB.

[0194] In certain embodiments, the polynucleotide encodes two engineered signaling polypeptides, wherein the first engineered signaling polypeptide comprises an extracellular tumor antigen-binding domain and a CD3ζ signaling domain, and the second polypeptide comprises an antigen-binding domain capable of binding to VEGF and a costimulatory signaling domain from each of CD27, CD28, OX40, ICOS, and 4-1BB. In further embodiments, the first signaling polypeptide or the second signaling polypeptide also comprises a T cell survival motif. In some embodiments, the T cell survival motif is or is derived from the intracellular signaling domain of the IL-7 receptor (IL-7R), the intracellular signaling domain of the IL-12 receptor, the intracellular signaling domain of the IL-15 receptor, the intracellular signaling domain of the IL-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, wherein a first engineered signaling polypeptide comprises an extracellular tumor antigen binding domain and a CD3ζ signaling domain, and a second engineered signaling polypeptide comprises an antigen binding domain capable of binding to VEGF, an IL-7 receptor intracellular T-cell survival motif, and costimulatory signaling domains from each of CD27, CD28, OX40, ICOS, and 4-1BB.

[0196] In some embodiments, three or more signaling polypeptides are encoded by polynucleotides. In certain embodiments, only one of the engineered signaling polypeptides comprises an antigen-binding domain that binds to a tumor-associated or 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 or tumor-specific antigen. In other embodiments, two or more engineered signaling polypeptides comprise antigen-binding domains that bind to one or more tumor-associated 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 or tumor-specific antigen.

[0197] In some embodiments, the tumor-associated 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), macroscopic 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 αvβ3 (CD61), galactin, 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), prostein, TARP (T-cell receptor gamma alternative reading frame protein), Trp-p8, STEAP1 (prostate six-transmembrane epithelial antigen 1), abnormal ras protein, or abnormal p53 protein;

[0198] In some embodiments, the first engineered signaling polypeptide comprises a first extracellular antigen-binding domain that binds a first antigen and a first intracellular signaling domain, and the second engineered signaling polypeptide comprises a second extracellular antigen-binding domain that binds a second antigen, or a receptor that binds the second antigen and a second intracellular signaling domain, and the second engineered signaling polypeptide does not comprise a costimulatory domain. In certain embodiments, the first antigen-binding domain and the second antigen-binding domain are independently antigen-binding portions of a receptor or antigen-binding portions of an antibody. In certain embodiments, either the first antigen-binding domain or the second antigen-binding domain, or both, are scFv antibody fragments. In certain embodiments, the first engineered signaling polypeptide and / or the second engineered signaling polypeptide further comprise a transmembrane domain. In certain embodiments, the first engineered signaling polypeptide or the second engineered signaling polypeptide comprises a T cell survival motif, e.g., 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 HER2, and the second engineered signaling polypeptide comprises a second extracellular antigen binding domain that binds 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 alarmin). In other embodiments, the DAMP is a heat shock protein, 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, said second antigen is an antigen on an antibody that binds to an antigen presented by a tumor cell.

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

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

[0205] Additional arrays An engineered signaling polypeptide, such as a CAR, may further comprise one or more additional polypeptide domains, including, but not limited to, a signal sequence; an epitope tag; an affinity domain; and a polypeptide whose presence or activity can be detected, for example, by antibody assay or because it is a polypeptide that generates a detectable signal (a detectable marker). Non-limiting examples of additional domains of 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: a signal sequence, an epitope tag, an affinity domain, or a polypeptide that generates a detectable signal.

[0206] Suitable signal sequences for use in a subject CAR, e.g., in the first polypeptide of a subject CAR, include any eukaryotic signal sequence, including naturally 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); and the like.

[0208] Affinity domains include peptide sequences that can interact with a binding partner, e.g., one immobilized on a solid support, and are useful for identification or purification. DNA sequences encoding multiple consecutive single amino acids, such as histidine, when fused to an expressed protein, can be used for one-step purification of recombinant proteins by binding with high affinity to a resin column, such as nickel sepharose. 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, e.g., 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 domain, e.g., zinc-binding domain or calcium-binding domain such as those from calcium-binding proteins, e.g., calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, flekenin, caltractin, calpain large subunit, S100 protein, parvalbumin, calbindin D9K, calbindin D28K, and calretinin, intein, biotin, streptavidin, MyoD, Id, leucine zipper sequence, and maltose-binding protein.

[0209] Suitable detectable signal-generating proteins include, for example, fluorescent proteins; enzymes that catalyze a reaction that generates 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 variants of GFP (BFP), cyan fluorescent variants of GFP (CFP), yellow fluorescent variants 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, pocilloporin, Renilla GFP, and Monster. Phycobiliproteins and phycobiliprotein conjugates, including GFP, paGFP, Kaede protein and kindling protein, B-phycoerythrin, R-phycoerythrin, and allophycocyanin, are also suitable. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, and mPlum (Shaner et al. (2005) Nat. Methods 2:905-909). 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), and the like.

[0212] Recognition and / or Elimination Domains Any of the replication-incompetent recombinant retroviral particles provided herein may contain a nucleic acid encoding a recognition or elimination domain, either as part of or separately from the nucleic acid encoding any of the engineered signaling polypeptides provided herein. Thus, any of the engineered signaling polypeptides provided herein may contain a recognition or elimination domain. For example, any of the CARs disclosed herein may contain a recognition or elimination domain. Furthermore, the recognition or elimination domain may 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 the replication-incompetent recombinant retroviral particle.

[0213] In some embodiments, the recognition or elimination domain may be derived from the enzyme thymidine kinase from herpes simplex virus (HSV-tk) or inducible caspase-9. In some embodiments, the recognition or elimination domain may comprise a modified endogenous cell surface molecule, for example, as disclosed in U.S. Pat. No. 8,802,374. The modified endogenous cell surface molecule may be a 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 may be a polypeptide recognized by an antibody that recognizes the extracellular domain of an EGFR member. In some embodiments, the recognition domain may 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 and bound under appropriate conditions by antibodies that recognize the extracellular domain of an EGFR member. Such extracellular EGFR epitopes are sometimes 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. Altered EGFR activity is associated with certain cancers. In some embodiments, a gene encoding an EGFR polypeptide, including human epidermal growth factor receptor (EGFR), is constructed by removing a nucleic acid sequence that encodes a polypeptide comprising a membrane-distal EGF-binding domain and a cytoplasmic signaling tail, but 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 applying immunomagnetic selection with biotinylated cetuximab in combination with anti-biotin microbeads successfully enriched T cells transduced with lentiviral EGFRt-containing constructs from 2% of the population to over 90% purity without observable toxicity to the cell preparation. Furthermore, other researchers have shown that constitutive expression of this inactive EGFR molecule does not affect the phenotype or effector function of T cells directed by the coordinately expressed chimeric antigen receptor (CAR), CD19R. Furthermore, other researchers have demonstrated through flow cytometry analysis that EGFR has been successfully used as an in vivo tracking marker for T cell engraftment in mice. Furthermore, EGFR has been demonstrated to be a potential suicide gene via the Erbitux®-mediated antibody-dependent cellular cytotoxicity (ADCC) pathway. The inventors of the present disclosure successfully expressed eTag in PBMCs using lentiviral vectors and found that in vitro expression of eTag by PBMCs exposed to cetuximab provided an effective mechanism for PBMC elimination. 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 acid 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 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 ribosomal skipping sequence. The ribosomal skipping and / or cleavage signal can be any ribosomal skipping and / or cleavage signal known in the art. Without being limited by theory, the ribosomal skipping sequence can be, for example, T2A (also referred to herein as 2A-1), which has the amino acid sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 83). Without being limited by theory, other examples of cleavage signals and ribosomal skipping sequences include FMDV 2A (F2A); equine rhinitis A virus 2A (abbreviated as E2A); porcine teschovirus-1 2A (P2A); and Thoseaasigna virus 2A (T2A). In some embodiments, the polynucleotide sequence encoding the recognition domain may be on the same transcript as the CAR or lymphoproliferative element, but may 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, particularly in combination with other "space-saving" elements provided herein, offer the advantage of occupying less genomic space on the RNA genome compared to separate polypeptides. 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." 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 exemplary embodiments, the antigen-specific targeting region of the CAR is the scFv portion of an antibody directed against the target antigen. In exemplary embodiments, 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 exemplary embodiments, the CAR further comprises a costimulatory domain, such as any of the costimulatory domains provided in the regulatory domain section above, and in further exemplary embodiments, the costimulatory domain is the intracellular costimulatory 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 transmembrane domain section above.

[0219] The CAR of the present disclosure can reside in the plasma membrane of a eukaryotic cell, such as a mammalian cell. 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, NK cells, NK-T cells, and macrophages. When present in the plasma membrane of a eukaryotic cell, the CAR of the present disclosure is active in the presence of one or more target antigens that bind to the ASTR under certain 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 displayed on a solid surface; a factor present in a lipid bilayer; or the like. 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 displayed on a solid surface; an antigen present in a lipid bilayer; or the like.

[0220] In some cases, when the CAR of the present disclosure is present in the plasma membrane of a eukaryotic cell and is activated by one or more target antigens, it increases the expression of at least one nucleic acid in the cell. For example, in some cases, when the CAR of the present disclosure is present in the plasma membrane of a eukaryotic cell and is activated by one or more target antigens, it increases the expression of at least one nucleic acid in 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, 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 one or more target antigens.

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

[0222] When the CAR of the present disclosure is present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, in some cases, it can result in increased production of one or more cytokines by the cell. For example, when the CAR of the present disclosure is present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, it can increase cytokine production 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 cytokine 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, etc.

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

[0224] In some cases, when the CAR of the present disclosure is present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, it causes the cell to exhibit cytotoxic activity against a target cell that expresses on its cell surface an antigen to which the antigen-binding domain of the first polypeptide of the CAR binds. For example, when the eukaryotic cell is a cytotoxic cell (e.g., an NK cell or a cytotoxic T lymphocyte), the CAR of the present disclosure, when present in the plasma membrane of the cell and activated by one or more target antigens, increases the cytotoxic activity of the cell against a target cell that expresses one or more target antigens on its cell surface. For example, when the eukaryotic cell is an NK cell or a T lymphocyte, a CAR of the present disclosure, when present at the plasma membrane of the cell and when activated by one or more target antigens, increases 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 compared to the cytotoxic activity of the cell in the absence of the one or more target antigens.

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

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

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

[0228] In certain exemplary embodiments, the CARs provided herein comprise a costimulatory domain in addition to an intracellular activation domain, and the costimulatory domain is, for example, any of the intracellular signaling domains provided herein for a lymphoproliferative element (LE), such as the intracellular domain of a CLE. In certain exemplary embodiments, the costimulatory domain of the CARs herein is the first intracellular domain (P3 domain) identified herein for a CLE or the P4 domain indicated as an effective intracellular signaling domain of a CLE herein in the absence of the P3 domain. Furthermore, in certain exemplary embodiments, the costimulatory domain of a CAR may comprise both the P3 and P4 intracellular signaling domains identified herein for a CLE. Certain exemplary subembodiments include particularly effective P3 and P4 partner intracellular signaling domains as identified herein for a CLE. In exemplary embodiments, the costimulatory domain is other than the ITAM-containing intracellular domain of the CAR, either as part of the costimulatory domain, or in further exemplary embodiments, as the only costimulatory domain.

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

[0230] In these exemplary embodiments, the costimulatory domain of the CAR is selected from the group consisting of 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, IL12R, IL13R, IL14R, IL15R, IL16R, IL17R, IL18R, IL19R, IL20R, IL21R, IL22R, IL23R, IL24R, IL25R, IL26R, IL27R, IL28R, IL29R, IL30R, IL31R, IL32R, IL33R, IL34R, IL35R, IL36R, IL37R, IL38R, IL39R, IL40R, IL41R, IL42R, IL43R, IL44R, IL45R, IL46R, IL47R, IL48R, IL49R, IL50R, IL51R, IL52R, IL53R, IL54R, IL55R, IL56R, IL57R, IL58R, IL59R, IL60R, IL61R, IL62R, IL63R, IL64R, IL65R, IL66R, IL67R, IL68R, IL6 The intracellular domain may comprise a functional signaling fragment thereof, including a signaling domain from RB1, 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 costimulatory domain of a CAR may comprise an intracellular domain or a functional signaling fragment thereof, including 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 costimulatory domain of the CAR may comprise an intracellular domain or a functional fragment thereof, including 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 costimulatory domain of a CAR may comprise an intracellular domain or a fragment thereof comprising 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 costimulatory domain of a CAR may comprise an intracellular domain or a functional signaling fragment thereof comprising 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 costimulatory domain of the CAR may comprise an intracellular domain or a functional signaling fragment thereof, including a signaling domain from CSF2RB, CSF3R, IFNGR1, IL2RB, IL2RG, IL6ST, IL10RA, IL17RE, IL31RA, MPL, or MyD88.

[0231] In some embodiments, the costimulatory domain of the CAR may comprise an intracellular domain or fragment thereof, including signaling domains from CSF3R, IL6ST, IL27RA, MPL, and MyD88. In certain exemplary subembodiments, the intracellular activation domain of the CAR is derived from CD3z.

[0232] Recombinant T cell receptor (TCR) T cell receptors (TCRs) recognize specific protein fragments derived from intracellular and extracellular proteins. Once proteins are broken down into peptide fragments, they are presented on the cell surface along with another protein called the MHC, the major histocompatibility complex, or in humans, the HLA (human leukocyte antigen) complex. The three distinct T cell antigen receptor combinations in vertebrates are αβTCR, γδTCR, and pre-TCR. These combinations are formed by dimerization between members of dimerizing subtypes, such as the αTCR and βTCR subunits, the γTCR and δTCR subunits, and, in the case of pre-TCRs, the pTα and βTCR subunits. The sets of TCR subunits dimerize and recognize target peptide fragments presented in the context of the MHC. Pre-TCRs are expressed only on the surface of immature αβT cells, whereas αβTCRs are expressed on the surface of mature αβT cells and NK T cells, and γδTCRs are expressed on the surface of γδT cells. The αβ TCR on the surface of T cells recognizes peptides presented by MHC1 or MHCII, while the αβ TCR on the surface of NK T cells recognizes lipid antigens presented by CD1. The γδ TCR can recognize MHC and MHC-like molecules, as well as non-MHC molecules such as viral glycoproteins. Upon ligand recognition, the αβ and γδ TCRs transmit activation signals via the CD3 zeta chain, which stimulates T cell proliferation and cytokine secretion.

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

[0234] Thus, recombinant TCRs, such as those derived from human TCRα and TCRβ pairs that recognize specific peptides with common HLA, 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 ASTRs provided herein, but more commonly is derived from an intracellular tumor-specific protein such as 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 natural and / or recombinant TCR subunits can identify sets of TCR subunits with high avidity and / or reactivity to 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 a replication-incompetent recombinant retroviral particle to genetically modify lymphocytes, or in exemplary embodiments, T cells or NK cells, so 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 or be one or more sets of recombinant γδ TCR chains, or in exemplary embodiments, αβ TCR chains. The TCR chains that form a set can be coexpressed using several different techniques for coexpressing two TCR chains, as disclosed herein, to express 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 sites (IRES), and separate promoters, can be used.

[0236] Several strategies have been employed to reduce the likelihood of mixed TCR dimer formation. Generally, this involves modifying the constant (C) domains of the TCR α and TCR β 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 TCR α and TCR β chains with their mouse counterparts. Another approach involves mutating the human TCR α common domain and TCR β chain common region to promote self-pairing, or expressing 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 TCR chains, in exemplary embodiments the αβ TCR chains, contains a modified constant domain that promotes preferential pairing with each other. In some subembodiments, each member of the set of TCR chains, in exemplary embodiments, αβ TCR chains, comprises a murine constant domain from the same TCR chain type, or a constant domain from the same TCR chain subtype with sufficient sequence derived from 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 to the exclusion of dimerization with human TCR chains. In other subembodiments, each member of the set of TCR chains, in exemplary embodiments, αβ TCR chains, comprises a corresponding mutation in its constant domain, such that dimerization of the set of TCR chains with each other is preferred over or to the exclusion of dimerization with TCR chains having human constant domains. Such preferred or exclusive dimerization in exemplary embodiments is under physiological conditions.

[0237] Lymphoproliferative elements Despite the continuous addition of cells, peripheral T lymphocyte numbers remain at a remarkably stable level throughout adulthood due to migration from the thymus, proliferation in response to antigen encounter, and cell loss due to elimination of antigen-specific effectors after antigen clearance (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 lymphopenic environments, T lymphocytes divide independently of their cognate antigen through an "acute homeostatic proliferation" mechanism that maintains the size of the peripheral T cell compartment. A lymphopenic state is established in subjects or patients during adoptive cell therapy by expanding T cells in vitro and introducing them into lymphodepleted subjects, resulting in enhanced engraftment and antitumor function of the transferred T cells. However, lymphodepletion of a subject is undesirable because it can cause serious side effects, including immune dysfunction and death.

[0238] Studies have shown that lymphodepletion removes endogenous lymphocytes, which function as a cellular sink for homeostatic cytokines, thereby liberating cytokines to promote 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 therefore induce homeostatic proliferation in a non-lymphopenic environment. However, these cytokines and their receptors possess inherent control mechanisms that prevent lymphoproliferative disorders during homeostasis.

[0239] Many of the embodiments provided herein include a lymphoproliferative element, or a nucleic acid encoding 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 can comprise a first and / or second intracellular signaling domain, such as CD2, CD3D, CD3E, CD3G, CD4, CD8A, CD8B, CD27, mutant delta Lck, 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, IL In exemplary embodiments, the first intracellular signaling domain may comprise MyD88, or a functional mutant and / or fragment thereof. In further exemplary embodiments, the first intracellular signaling domain may comprise MyD88, or a functional mutant and / or fragment thereof, and the second intracellular signaling domain may comprise 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 ITAM-containing intracellular domain, e.g., an 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 may comprise TNFRSF18, or a functional mutant and / or fragment thereof.

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

[0242] In some embodiments, the lymphoproliferative element may comprise a transmembrane domain. In some embodiments, the transmembrane domain is selected from the group consisting of 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 The polypeptide may comprise 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] A CLE for use in any aspect or embodiment herein can include any CLE disclosed in WO2019 / 055946 (incorporated herein by reference in its entirety), most of which are designed to be and are considered to be constitutively active. As shown therein, when a first and second intracellular signaling domain of a 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 and / or possess properties that drive T cell proliferation in vivo (or cells genetically modified and / or transduced with the LE can provide, are adapted to, possess properties, and / or are modified to provide a drive for T cell expansion in vivo). Methods for performing such in vivo studies are provided in Example 6. For example, as shown in Example 6, in vivo studies can utilize a mouse model to measure T cell expansion at 15-25 days in vivo, or at 19-21 days in vivo, or at about 21 days in vivo after T cells are contacted with a lentiviral vector encoding the LE and introduced into mice.

[0245] In some embodiments, the lymphoproliferative element may comprise any of the sequences listed in Table 1 (SEQ ID NOS: 84-302). Table 1 shows the portions, names (including gene names), and amino acid sequences of the domains tested in the CLE. Typically, the CLE comprises 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 comprises a first intracellular domain. In exemplary embodiments, the first intracellular domain may comprise any of the portions listed as S036-S0216 or in Table 1, or functional mutants and / or fragments thereof. In some embodiments, the lymphoproliferative element may comprise a second intracellular domain. In exemplary embodiments, the second intracellular domain may comprise any of the portions listed as S036-S0216 or in Table 1, or functional mutants and / or fragments thereof. In some embodiments, the lymphoproliferative element may comprise an extracellular domain. In exemplary embodiments, the extracellular domain may comprise any of the sequences of the portions listed in Table 1 as M001-M049 or E006-E015, or functional mutants and / or fragments thereof. In some embodiments, the lymphoproliferative element may comprise a transmembrane domain. In exemplary embodiments, the transmembrane domain may comprise any of the portions listed in Table 1 as M001-M049 or T001-T082, or functional mutants and / or fragments thereof. In some embodiments, the lymphoproliferative element may be a fusion of the extracellular / transmembrane domain (M001-M049 in Table 1), a first intracellular domain (S036-S0216 in Table 1), and a second intracellular domain (S036-S216 in Table 1). In some embodiments, the lymphoproliferative element may be a fusion of an extracellular domain (E006-E015 in Table 1), a transmembrane domain (T001-T082 in Table 1), a first intracellular domain (S036-S0216 in Table 1), and a second intracellular domain (S036-S0216 in Table 1).For example, the lymphoproliferative element may be a fusion of E006, T001, S036, and S216, which is also described as E006-T001-S036-S216. In exemplary embodiments, 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 exemplary embodiments, the intracellular domain of an LE, or the first intracellular domain of an LE having two or more intracellular domains, is other than a functional intracellular activation domain from an ITAM-containing intracellular domain, e.g., CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10 / CD28, or ZAP70, and in further exemplary subembodiments, an intracellular domain from CD3z. In exemplary embodiments, the second intracellular domain of an LE is other than a costimulatory domain of 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM. In exemplary embodiments, the extracellular domain of an LE does not comprise a single-chain variable fragment (scFv). In further exemplary embodiments, the extracellular domain of an LE that activates the LE upon binding to a binding partner does not comprise a single-chain variable fragment (scFv).

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

[0248] The extracellular domain, transmembrane domain, and intracellular domain of an LE can vary in amino acid length. For example, in embodiments involving replication-incompetent retroviral particles, due to limitations on the length of polynucleotide that can be packaged into retroviral particles, an LE with a shorter amino acid sequence may be advantageous in certain exemplary embodiments. In some embodiments, the total length of the LE may be between 3 and 4,000 amino acids, e.g., between 10 and 3,000, 10 and 2,000, 50 and 2,000, or 250 and 2,000 amino acids, and in exemplary embodiments, between 50 and 1,000, 100 and 1,000, or 250 and 1,000 amino acids. The extracellular domain, if present to form the extracellular and transmembrane domains, may be between 1 and 1,000 amino acids, typically between 4 and 400, 4 and 200, 4 and 100, 4 and 50, 4 and 25, or 4 and 20 amino acids. In one embodiment, the extracellular region is GGGS for the extracellular and transmembrane domains of this aspect of the invention. The transmembrane domain, or the transmembrane region of the extracellular and transmembrane domains, can be between 10 and 250 amino acids, more typically at least 15 amino acids in length, e.g., between 15 and 100, 15 and 75, 15 and 50, 15 and 40, or 15 and 30 amino acids in length. The intracellular signaling domain can be between 10 and 1000, 10 and 750, 10 and 500, 10 and 250, or 10 and 100 amino acids in length. In exemplary embodiments, the intracellular signaling domain can be at least 30, or between 30 and 500, 30 and 250, 30 and 150, 30 and 100, 50 and 500, 50 and 250, 50 and 150, or 50 and 100 amino acids in length.In some embodiments, the intracellular signaling domain of a particular gene is at least 90%, 95%, 98%, 99%, or 100% identical to the sequence of that intracellular signaling domain, e.g., at least 10, 25, 30, 40, or 50 amino acids from the sequence provided herein for that intracellular domain, up to the size of the entire intracellular domain sequence, and may include, e.g., up to an additional 1, 2, 3, 4, 5, 10, 20, or 25 amino acids, provided that such a sequence can still provide any of the properties of LE disclosed herein.

[0249] In some embodiments, the lymphoproliferative element is a chimeric cytokine receptor, such as, but not limited to, a cytokine tethered to its receptor, which 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 a fragment thereof tethered or covalently linked via a linker to its cognate receptor or a fragment thereof. In some embodiments, the chimeric cytokine receptor is IL7 tethered to IL7Rα (also known as IL7RA). In other embodiments, the chimeric cytokine receptor is IL-7 tethered 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 that is not tethered to a cytokine; indeed, in exemplary embodiments, the lymphoproliferative element is a constitutively active cytokine receptor that is not tethered to a cytokine. These chimeric IL-7 receptors typically constitutively activate STAT5 upon expression.

[0250] In exemplary embodiments of any of the methods and compositions provided herein comprising a lymphoproliferative element, wherein the lymphoproliferative element is a cytokine or cytokine receptor polypeptide, or a fragment thereof comprising a signaling domain, the lymphoproliferative element may comprise an interleukin polypeptide covalently linked via a linker to a portion of a cognate interleukin receptor polypeptide. Typically, this portion of the cognate interleukin receptor comprises a functional portion of the extracellular domain capable of binding to an interleukin cytokine and a 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 capable of promoting lymphocyte proliferation. In some embodiments, the lymphoproliferative element is an interleukin polypeptide covalently linked via a linker to its full-length cognate interleukin receptor polypeptide.

[0251] In exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may be derived from a portion of the protein IL7RA. Domains, motifs, and point mutations of IL7RA that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and one of skill in the art can identify corresponding domains, motifs, and point mutations in IL7RA polypeptides, some of which are discussed in this paragraph. The IL7RA protein has a serine-rich S region (359-394 of full-length IL7RA, corresponding to residues 96-133 of SEQ ID NO: 248), a T region with three tyrosine residues (residues Y401, Y449, and Y456 of full-length IL7RA, corresponding to 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 NOs: 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, e.g., all, of the domains and motifs of IL7RA disclosed herein or known to induce proliferation and / or survival of T cells and / or NK cells. In some embodiments, a suitable intracellular 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 at least 10, 15, 20, or all of a 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 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, or about 175 aa to about 200 aa.In exemplary embodiments, the intracellular domain derived from IL7RA has a length of about 30 aa to about 200 aa. In exemplary embodiments of lymphoproliferative elements comprising a first intracellular domain derived from IL7RA, the second intracellular domain may be derived from TNFRSF8.

[0252] In exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may be derived from a portion of the protein IL12RB. Domains, motifs, and point mutations of IL12RB that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and those skilled in the art can identify corresponding domains, motifs, and point mutations in IL12RB polypeptides, some of which are discussed in this paragraph. Full-length IL12RB contains at least one Box 1 motif, PXXP (SEQ ID NO: 306), where each X may 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, a lymphoproliferative element comprising an IL12RB intracellular domain may comprise one or more of the above-described Box 1 motifs or other motifs, domains, or mutations of IL12RB known to induce proliferation and / or survival of T cells and / or NK cells. The Box 1 motif of IL12RB is known in the art, and one of skill in the art can identify the corresponding motif in an IL12RB polypeptide. 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 amino acids set forth in SEQ ID NOs:254-256. In some embodiments, the intracellular domain derived from IL12RB 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 100 aa, about 125 aa, about 125 aa to 150 aa, about 150 to about 175 aa, about 175 aa to about 200 aa, or about 200 aa to about 219 aa.In exemplary embodiments, 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 exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may be derived from a portion of the protein IL31RA. Domains, motifs, and point mutations of IL31RA that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and those skilled in the art can identify corresponding domains, motifs, and point mutations in IL31RA polypeptides, some of which are discussed in this paragraph. Full-length IL31RA contains the Box 1 motif PXXP (SEQ ID NO: 306), where each X may be any amino acid (corresponding to residues 12-15 of SEQ ID NOs: 275 and 276) (Cornelissen C et al. Eur J Cell Biol. 2012 Jun-Jul;91(6-7):552-66). In some embodiments, a lymphoproliferative element that includes an IL31RA intracellular domain may contain a Box 1 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 absent in SEQ ID NO: 276) that 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, while Y652 is required for the activation of STAT5, and Y721 recruits STAT3. In some embodiments, lymphoproliferative elements having an IL31RA intracellular domain contain a Box 1 motif and / or known phosphorylation sites disclosed herein. The Box 1 motif and phosphorylatable tyrosines of IL31RA are known in the art, and one of skill in the art would be able to identify corresponding motifs and phosphorylatable tyrosines in similar IL31RA polypeptides. In other embodiments, the lymphoproliferative element having an IL31RA intracellular domain does not contain any of the known phosphorylation sites disclosed herein.In some embodiments, a suitable intracellular 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 at least 10, 15, 20, or all of a stretch of amino acids of SEQ ID NO: 275 or 276. In some embodiments, the intracellular domain derived from IL31RA 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, or about 175 aa to about 189 aa. In exemplary embodiments, the intracellular domain derived from IL31RA has a length of about 30 aa to about 200 aa, e.g., 30 aa to 189 aa, 30 aa to 106 aa.

[0254] In exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may be derived from the intracellular portion of the transmembrane protein CD40. Domains, motifs, and point mutations of CD40 that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and those skilled in the art can identify corresponding domains, motifs, and point mutations in CD40 polypeptides, 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 comprise the amino acid sequence PXQXT (SEQ ID NO: 303), where each X may 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 separate 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 contain all of the binding sites for the TRAF protein. TRAF binding sites are known in the art, and one of skill in the art would be able to identify corresponding TRAF binding sites in similar CD40 polypeptides. In some embodiments, a suitable intracellular 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 at least 10, 15, 20, or all of a stretch 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 exemplary embodiments, the intracellular domain derived from CD40 has a length of about 30 aa to about 66 aa, e.g., 30 aa to 65 aa, or 50 aa to 66 aa. In exemplary embodiments of a lymphoproliferative element comprising a first intracellular domain derived from CD40, the second intracellular domain may be other than an intracellular domain derived from 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 CD28. Pattern recognition receptors include, but are not limited to, endocytic pattern recognition receptors (i.e., mannose receptor, scavenger receptors (i.e., Mac-1, LRP, peptidoglycan, techoic acid, toxins, CD11 c / CR4)), external signal pattern recognition receptors (Toll-like receptors (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10)), peptidoglycan recognition proteins (PGRP binds to bacterial peptidoglycan and CD14); internal signal pattern recognition receptors (i.e., NOD receptors 1 and 2), and RIG1.

[0255] In exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may be derived from the intracellular portion of CD27. Domains, motifs, and point mutations of CD27 that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and those skilled in the art can identify corresponding domains, motifs, and point mutations in CD27 polypeptides, some of which are discussed in this paragraph. The serine at amino acid 219 of full-length CD27 (corresponding to the serine at amino acid 6 of SEQ ID NO:205) has been shown to be phosphorylated. In some embodiments, a suitable intracellular 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 at least 10, 15, 20, or all of a stretch 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 exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may be derived from the intracellular portion of CSF2RB. Domains, motifs, and point mutations in CSF2RB that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and those skilled in the art can identify corresponding domains, motifs, and point mutations in CSF2RB polypeptides, some of which are discussed in this paragraph. Full-length CSF2RB contains a Box 1 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 may 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 stretch of amino acids of SEQ ID NO:213. In some embodiments, the intracellular domain derived from CSF2RB 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, or about 400 aa to about 450 aa.

[0257] In exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may be derived from the intracellular portion of IL2RB. Domains, motifs, and point mutations in IL2RB that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and one of skill in the art can identify corresponding domains, motifs, and point mutations in IL2RB polypeptides, some of which are discussed in this paragraph. Full-length IL2RB contains a Box 1 motif at amino acids 278-286 (corresponding to amino acids 13-21 of SEQ ID NO:240). In some embodiments, a suitable intracellular 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 at least 10, 15, 20, or all of a stretch of amino acids of SEQ ID NO:240. In some embodiments, the intracellular domain derived from IL2RB 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, or about 250 aa to 300 aa.

[0258] In exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may be derived from the intracellular portion of IL6ST. Domains, motifs, and point mutations in IL6ST that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and those skilled in the art can identify corresponding domains, motifs, and point mutations in IL6ST polypeptides, some of which are discussed in this paragraph. Full-length IL6ST contains a Box 1 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 may 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 stretch of 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 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, or about 250 aa to 300 aa.

[0259] In exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain can be derived from the intracellular portion of IL17RE. Domains, motifs, and point mutations of IL17RE that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and one of skill in the art can identify corresponding domains, motifs, and point mutations in IL17RE polypeptides, 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 at least 10, 15, 20, or all of a stretch of amino acids of SEQ ID NO:265. In some embodiments, the intracellular domain derived from IL17RE 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, or about 175 aa to about 200 aa.

[0260] In exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may be derived from the intracellular portion of IL2RG. Domains, motifs, and point mutations of IL2RG that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and one of skill in the art can identify corresponding domains, motifs, and point mutations in IL2RG polypeptides, some of which are discussed in this paragraph. Full-length IL2RG contains a Box 1 motif at amino acids 286-294 (corresponding to amino acids 3-11 of SEQ ID NO:241). In some embodiments, a suitable intracellular 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 at least 10, 15, 20, or all of a stretch of amino acids of SEQ ID NO:241. In some embodiments, the intracellular domain derived from IL2RG 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, or about 70 aa to about 100 aa.

[0261] In exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may be derived from the intracellular portion of IL18R1. Domains, motifs, and point mutations of IL18R1 that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and one of skill in the art can identify corresponding domains, motifs, and point mutations in IL18R1 polypeptides, 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 may 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 stretch of amino acids of SEQ ID NO:266. In some embodiments, the intracellular domain derived from IL18R1 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, or about 70 aa to about 100 aa.

[0262] In exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain can be derived from the intracellular portion of IL27RA. Domains, motifs, and point mutations of IL27RA that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and one of skill in the art can identify corresponding domains, motifs, and point mutations in IL27RA polypeptides, some of which are discussed in this paragraph. Full-length IL27RA contains a Box 1 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 at least 10, 15, 20, or all of a stretch of 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 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, or about 70 aa to about 100 aa.

[0263] In exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may be derived from the intracellular portion of IFNGR2. Domains, motifs, and point mutations of IFNGR2 that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and those skilled in the art can identify corresponding domains, motifs, and point mutations in IFNGR2 polypeptides, 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 may 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 stretch of amino acids of SEQ ID NO:230. In some embodiments, the intracellular domain derived from IFNGR2 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.

[0264] In exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may be derived from a portion of the protein MyD88. Domains, motifs, and point mutations of MyD88 that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and those skilled in the art can identify corresponding domains, motifs, and point mutations in MyD88 polypeptides, some of which are discussed in this section. The MyD88 protein has an N-terminal death domain (corresponding to amino acids 29-106 of SEQ ID NO:284) that mediates interactions 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 kinase, and a C-terminal TIR domain (corresponding to amino acids 160-304 of SEQ ID NO:284) that associates with the TLR-TIR domain (Biol Res. 2007;40(2):97-112). MyD88 also has canonical nuclear localization and export motifs. Point mutations have been identified in MyD88, including the loss-of-function mutations L93P and R193C (corresponding to L93P and R196C in SEQ ID NO: 284), and the gain-of-function mutation L265P (corresponding to L260P in SEQ ID NO: 284) (Deguine and Barton. F1000 Prime Rep. 2014 Nov 4;6:97). In some embodiments, the lymphoproliferative elements herein may comprise one or more, for example, all of the domains and motifs of MyD88 disclosed herein. In some embodiments, a suitable intracellular 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 at least 10, 15, 20, or all of a stretch of amino acids in SEQ ID NOs: 284-293, which in exemplary embodiments includes one or more, and 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 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, or about 300 aa to 350 aa. In exemplary embodiments, the intracellular domain derived from MyD88 has a length of about 30 aa to about 350 aa, e.g., 50 aa to 350 aa, or 100 aa to 350 aa, 100 aa to 304 aa, 100 aa to 296 aa, 100 aa to 251 aa, 100 aa to 191 aa, 100 aa to 172 aa, 100 aa to 146 aa, or 100 aa to 127 aa. In exemplary embodiments of lymphoproliferative elements comprising a first intracellular domain derived from MyD88, the second intracellular domain may be derived from TNFRSF4 or TNFRSF8. In other exemplary embodiments of lymphoproliferative elements 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 exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may be derived from a portion of the transmembrane protein MPL. Domains, motifs, and point mutations of MPL that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and those skilled in the art can identify corresponding domains, motifs, and point mutations in MPL polypeptides, some of which are discussed in this paragraph. Transmembrane MPL proteins contain a Box 1 motif PXXP (SEQ ID NO: 306), where each X can be any amino acid (corresponding to amino acids 17-20 of SEQ ID NO: 283), and a Box 2 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 JAKs and signal transduction, but the presence of the Box2 motif is not necessarily required for growth signals (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, respectively, of SEQ ID NO: 283), 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 containing 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 mpl function in this context. 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 resulted in slightly enhanced transcription, as would be expected from removal of a non-essential negative element in this region. Thus, in some embodiments, the MPL intracellular signaling domain does not include the region containing amino acids 70-95 of SEQ ID NO:283. In full-length MPL, lysines K553 (corresponding to K40 in SEQ ID NO: 283) and K573 (corresponding to K60 in 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). Therefore, in some embodiments herein, the MPL intracellular signaling domain does not contain these ubiquitination targeting motif residues. In full-length MPL, tyrosines Y521 (corresponding to Y8 in SEQ ID NO: 283), Y542 (corresponding to Y29 in SEQ ID NO: 283), Y591 (corresponding to Y78 in SEQ ID NO: 283), Y626 (corresponding to Y113 in SEQ ID NO: 283), and Y631 (corresponding to Y118 in 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 a 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 in full-length MPL are positive regulatory sites (Drachman and Kaushansky. Proc Natl Acad Sci USA. 1997 Mar 18;94(6):2350-5), 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 in MPL with the W515A mutation described below (Pecquet et al. Blood. 2010 Feb 4;115(5):1037-48). MPL contains the Shc phosphotyrosine binding motif NXXY (SEQ ID NO:307), where each X can 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 can 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 tyrosines in 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 contains the sequence YLPL (SEQ ID NO: 309) (corresponding to amino acids 113-116 of SEQ ID NO: 283), which resembles the consensus binding site for STAT5 recruitment pYLXL (SEQ ID NO: 310), where pY is phosphotyrosine and X can be any amino acid (May et al. FEBS Lett. 1996 Sep 30;394(2):221-6). Using computer simulations, Lee et al. discovered that clinically relevant mutations in the transmembrane domain of MPL should activate MPL in the following order: W515K (corresponding to the amino acid substitution W2K in SEQ ID NO: 283) > S505A (corresponding to the amino acid substitution S14A in SEQ ID NO: 187) > W515I (corresponding to the amino acid substitution W2I in SEQ ID NO: 283) > S505N (corresponding to the amino acid substitution S14N in SEQ ID NO: 187, which was tested in Example 12 as moiety T075 (SEQ ID NO: 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 may comprise one or more, or all, of the domains and motifs present in SEQ ID NO: 283 and described herein. In some embodiments, the transmembrane portion of MPL may comprise one or more, or all, of the domains and motifs present in SEQ ID NO: 187 and described herein. The domains, motifs, and point mutations of MPL provided herein are known in the art, and one of skill in the art will recognize that the MPL intracellular signaling domain herein in exemplary embodiments includes one or more corresponding domains, motifs, and point mutations shown to promote, but not inhibit, MPL proliferative activity. In some embodiments, a suitable intracellular 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 at least 10, 15, 20, or all of a stretch of amino acids of SEQ ID NO:283.In some embodiments, the intracellular domain derived from MPL 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 150 aa, 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 length. In an exemplary embodiment, the intracellular domain derived from MPL has a length of about 30 aa to about 200 aa, e.g., 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 may be derived from CD79B.

[0266] In exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may be derived from a portion of the transmembrane protein CD79B, also known as B29; IGB; and AGM6. Domains, motifs, and point mutations of CD79B that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and those skilled in the art can identify corresponding domains, motifs, and point mutations in CD79B polypeptides, 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 contains an ITAM motif and / or known phosphorylation sites disclosed herein. CD79B motifs and phosphorylatable tyrosines are known in the art, and one of skill in the art would be able to identify corresponding motifs and phosphorylatable tyrosines in similar CD79B polypeptides. 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 amino acids in SEQ ID NO: 211. In some embodiments, the intracellular domain derived from CD79B 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. In exemplary embodiments, the intracellular domain derived from CD79B has a length of about 30 aa to about 50 aa.For example, a suitable CD79B 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 at least 10, 15, 20, or all of a stretch of amino acids in the following sequence: LDKDDSKAGMEEDHT[YEGLDIDQTATYEDI]VTLRTGEVKWSVGEHPGQE (SEQ ID NO: 211) (ITAM motifs are shown in parentheses). In an exemplary embodiment of a lymphoproliferative element comprising a second intracellular domain derived from CD79B, the first intracellular domain may be derived from CSF3R.

[0267] In exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may be derived from a portion of the transmembrane protein OSMR. Domains, motifs, and point mutations of OSMR that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and one of skill in the art would be able to identify corresponding domains, motifs, and point mutations in OSMR polypeptides, some of which are discussed in this paragraph. OSMR contains a Box 1 motif at amino acids 771-779 of isoform 3 (corresponding to amino acids 16-30 of SEQ ID NO:294). OSMR has two serines at amino acids 829 and 890 of isoform 3 that are known to be phosphorylated (serines at amino acids 65 and 128 of SEQ ID NO:294). In some embodiments, the intracellular portion of the protein OSMR contains a Box 1 motif and the known phosphorylation sites disclosed herein. The motifs and phosphorylatable serines of OSMR are known in the art, and one of skill in the art would be able to identify corresponding motifs and phosphorylatable serines in analogous OSMR polypeptides. In some embodiments, a suitable intracellular 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 at least 10, 15, 20, or all of a stretch of amino acids of SEQ ID NO:294. In some embodiments, the intracellular domain derived from OSMR 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, or about 200 aa to about 250 aa.

[0268] In exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may be derived from a portion of the transmembrane protein PRLR. Domains, motifs, and point mutations in PRLR that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and one skilled in the art would be able to identify corresponding domains, motifs, and point mutations in PRLR polypeptides, 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 skilled in the art would be able to identify corresponding domains in similar PRLR polypeptides. In some embodiments, a suitable intracellular 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 at least 10, 15, 20, or all of a stretch of amino acids of SEQ ID NO:295. In some embodiments, the intracellular domain derived from PRLR 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, or about 350 aa to about 400 aa.

[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 exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may 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 those skilled in the art can identify corresponding domains, motifs, and point mutations in CD28 polypeptides, 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, a lymphoproliferative element that includes a CD28 intracellular domain may contain PI3-K and Grb2-binding 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 amino acids in SEQ ID NO: 206 or 207. In some embodiments, the intracellular domain derived from CD28 has a length of about 5 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 35 aa, or about 35 aa to about 42 aa.

[0271] In exemplary embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the intracellular domain may be derived from a portion of the protein ICOS. Domains, motifs, and point mutations in ICOS that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and those skilled in the art can identify corresponding domains, motifs, and point mutations in ICOS polypeptides, some of which are discussed in this section. 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. Those skilled in the art will understand how to mutate this residue in SEQ ID NO:225 to generate an ICOS intracellular domain that binds to Grb2 in addition to PI3-K. In some embodiments, lymphoproliferative elements comprising an ICOS intracellular domain can contain a PI3-K binding motif. In some embodiments, lymphoproliferative elements comprising an ICOS intracellular domain can contain a PI3-K binding motif mutated to further bind to Grb2. ICOS also contains a membrane-proximal motif in its cytoplasmic tail that is essential for ICOS-assisted 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, lymphoproliferative elements comprising an ICOS intracellular domain can contain a calcium signaling motif. Two other conserved motifs have been identified in full-length ICOS. The first conserved motif is at residues 170-179 (corresponding to residues 9-18 of SEQ ID NO: 225) and the second conserved motif is at 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 important functions 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 both 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 amino acids of SEQ ID NO: 225. In some embodiments, the intracellular domain derived from ICOS has a length of about 5 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 35 aa, or about 35 aa to about 38 aa.

[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). Domains, motifs, and point mutations of OX40 that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and one of skill in the art can identify corresponding domains, motifs, and point mutations in OX40 polypeptides, 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) that is important for binding 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 p85 PI3K-binding motif at residues 34-57. In some embodiments, when OX40 is present as the intracellular domain of a lymphoproliferative element, it contains the p85 PI3K-binding motif of OX40. In some embodiments, the intracellular domain of OX40 can 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 ubiquitin 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, a suitable intracellular domain of a lymphoproliferative element may 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 stretch of amino acids of SEQ ID NO:57.In some of these embodiments, the intracellular domain of OX40 has a length of about 20 aa to about 25 aa, about 25 aa to about 30 aa, 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. In exemplary embodiments, the intracellular domain of OX40 has a length of about 20 aa to about 50 aa, e.g., 20 aa to 45 aa, or 20 aa to 42 aa.

[0273] In some embodiments, the intracellular domain of a chimeric lymphoproliferative element is derived from the intracellular portion of the transmembrane protein IFNAR2. Domains, motifs, and point mutations of IFNAR2 that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and those skilled in the art can identify corresponding domains, motifs, and point mutations in IFNAR2 polypeptides, some of which are discussed in this paragraph. Full-length IFNAR2 contains a Box 1 motif and two Box 2 motifs (known as Box 2A and Box 2B). (Usacheva A et al. J Biol Chem. 2002 Dec 13;277(50):48220-6). In some embodiments, a lymphoproliferative element comprising an IFNAR2 intracellular domain may contain one or more Box 1 or Box 2 motifs. In exemplary embodiments, the IFNAR2 intracellular domain may contain one or more Box 1, Box 2A, or Box 2B motifs. IFNAR2 contains a JAK1 binding site (Gauzzi MC et al. Proc Natl Acad Sci US A. 1997 Oct 28;94(22):11839-44; Schindler et al. J Biol Chem. 2007 Jul 13;282(28):20059-63). In some embodiments, a lymphoproliferative element comprising an IFNAR2 intracellular domain may contain a JAK1 binding site. In some embodiments, a suitable intracellular domain of a 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 stretch of 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 exemplary embodiments, the intracellular domain of OX40 has a length of about 30 aa to about 251 aa, e.g., 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 proliferation and / or survival of T cells and / or NK cells are known in the art, and those skilled in the art can identify corresponding domains, motifs, and point mutations of CSF3R polypeptides, some of which are discussed in this paragraph. Full-length CSF3R contains Box 1 and Box 2 motifs, as well as a Box 3 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, a lymphoproliferative element comprising a CSF3R intracellular domain may contain one or more of Box 1, Box 2, or Box 3 motifs. CSF3R contains four tyrosine residues, Y704, Y729, Y744, and Y764, in the full-length CSF3R, which are important for binding to STAT3 (Y704 and Y744), SOCS3 (Y729), and Grb2 and p21Ras (Y764). In some embodiments, a lymphoproliferative element containing the CSF3R intracellular domain may contain one, two, three, or all of the tyrosine residues corresponding to Y704, Y729, Y744, and Y764 in the full-length CSF3R. CSF3R contains two threonine residues, T615 and T618, in the full-length CSF3R, which, when mutated 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, lymphoproliferative elements containing the CSF3R intracellular domain can contain one or more of the mutations corresponding to T615A and T618I.In some embodiments, a suitable intracellular domain of a 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 stretch of amino acids of SEQ ID NO: 216, 217, or 218. In some of these embodiments, the intracellular domain of CSF3R 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 213 aa. In exemplary embodiments, the intracellular domain of CSF3R has a length of about 30 aa to about 213 aa, for example, about 30 aa to about 186 or 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 proliferation and / or survival of T cells and / or NK cells are known in the art, and those skilled in the art can identify corresponding domains, motifs, and point mutations in EPOR polypeptides, some of which are discussed in this section. EPOR contains Box 1 (residues 257-264 of full-length EPOR) and Box 2 (residues 303-313 of full-length EPOR) motifs (Constantinescu SN. Trends Endocrinol Metab. 1999 Dec;10(1):18-23). ​​EPOR also contains an extended Box 2 motif (residues 329-372) that is important for binding to the tyrosine kinase receptor KIT (Constantinescu SN. Trends Endocrinol Metab. 1999 Dec;10(1):18-23). In some embodiments, lymphoproliferative elements comprising the EPOR intracellular domain may contain one or more of the Box1, Box2, or extended Box2 motifs. The EPOR also contains a short segment important for EPOR internalization (residues 267-276 of the full-length EPOR). In some embodiments, lymphoproliferative elements comprising the EPOR intracellular domain do not contain an internalization segment. In some embodiments, a suitable intracellular domain of a 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 stretch of amino acids of SEQ ID NO: 219 or 220. In some of these embodiments, the intracellular domain of EPOR 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 235 aa.In exemplary embodiments, the intracellular domain of the EPOR has a length of about 30 aa to about 235 aa.

[0276] In some embodiments, the intracellular domain of a 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 those skilled in the art can identify corresponding domains, motifs, and point mutations of CD3G polypeptides, some of which are discussed in this paragraph. Two serine residues, S123 and S126, of full-length CD3G 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, a lymphoproliferative element comprising a CD3G intracellular domain may contain one or more serine residues corresponding to full-length S123 and S126. Furthermore, it has been shown that phosphorylation at S126, but not at S123, is required for PKC-mediated downregulation (Dietrich J et al. EMBO J. 1994 May 1; 13(9):2156-66). In some embodiments, a lymphoproliferative element comprising a CD3G intracellular domain may contain a serine residue corresponding to full-length S123, but not to full-length S126. In some embodiments, a lymphoproliferative element comprising a CD3G intracellular domain may contain 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 serine-to-alanine mutation. Furthermore, it has been shown that a leucine-to-alanine mutation of either of the dileucine motifs, L131 and L132, of full-length CD3G prevents PKC-mediated downregulation (Dietrich J et al. EMBO J. 1994 May 1; 13(9):2156-66). In some embodiments, a lymphoproliferative element comprising the CD3G intracellular domain may comprise at least one amino acid substitution at a leucine residue corresponding to L131 or L132 of full-length CD3G. In an exemplary embodiment, the amino acid substitution may be a leucine to alanine mutation.In some embodiments, a suitable intracellular domain of a lymphoproliferative element may 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 amino acid stretch of SEQ ID NO: 199. In some of these embodiments, the intracellular domain of CD3G has a length of about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 35 aa, about 35 aa to about 40 aa, or about 40 aa to about 45 aa. In exemplary embodiments, the intracellular domain of CD3D has a length of about 30 aa to about 45 aa.

[0277] The cytoplasmic domain of TNF receptors (TNFRs), which in exemplary embodiments may be TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18, can recruit signaling molecules containing TRAFs (TNF receptor-associated factors) and / or "death domain" (DD) molecules. TNFR domains, motifs, and point mutations that induce proliferation and / or survival of T cells and / or NK cells are known in the art, and those skilled in the art can identify corresponding domains, motifs, and point mutations in TNFR polypeptides, some of which are discussed in this paragraph. Mammals have at least six TRAF molecules and several non-receptor DD molecules. Receptors and adaptor proteins that bind to TRAFs 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, which is also known in the art (Locksley RM et al. Cell. 2001 Feb 23;104(4):487-501). One skilled in the art would be able to identify TRAF and / or DD-binding motifs in different TNFR families, for example, using sequence alignment to known binding motifs. TNFRs 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, lymphoproliferative elements comprising a TNFR intracellular domain may comprise one or more TRAF-binding motifs. In some embodiments, lymphoproliferative elements comprising a TNFR intracellular domain do not comprise a DD-binding motif, or one or more DD-binding motifs are deleted or mutated within the intracellular domain. In some embodiments, lymphoproliferative elements comprising the TNFR intracellular domain can recruit TRADD and / or TRAF2.TNFRs also contain a cysteine-rich domain (CRD) that is important for ligand binding (Locksley RM et al. Cell. 2001 Feb 23;104(4):487-501). In some embodiments, lymphoproliferative elements that contain a TNFR intracellular domain do not contain a TNFR CRD.

[0278] The lymphoproliferative element and CLE that can be included in any of the embodiments disclosed herein may be any of the LEs or CLEs disclosed in WO2019 / 055946, which disclosed a CLE that promotes proliferation in cell culture of PBMCs transduced with lentiviral particles encoding a CLE between days 7 and 21, 28, 35, and / or 42 post-transduction. Furthermore, a CLE that promotes in vivo proliferation in mice in the presence or absence of an antigen recognized by a CAR was identified, and T cells expressing a CLE and one of the CARs were introduced into the mice. As exemplified herein, tests and / or criteria can be used to identify any test polypeptides that include an LE, or a test domain of an LE, such as a first intracellular domain, or a second intracellular domain, or both the first and second intracellular domains, that are indeed LEs or effective intracellular domains of LEs, or particularly effective LEs or intracellular domains of LEs. Thus, in certain embodiments, any aspect or other embodiment provided herein comprising an LE or a polynucleotide or nucleic acid encoding an LE may describe that the LE satisfies, provides the characteristics of, or provides and / or possesses the characteristics of, any one or more of the identified tests or criteria for identifying an LE provided herein, or that a cell genetically modified and / or transduced with a retroviral particle, such as a lentiviral particle, encoding an LE may provide, be adapted to achieve, possess the characteristics of, and / or be modified to achieve, one or more of the results of the described tests.In one embodiment, the LE provides, is capable of providing, and / or possesses the property of (or cells genetically modified and / or transduced with a retroviral particle encoding the LE are capable of providing, are adapted to, possess the property of, and / or are modified for) improved expansion to pre-activated PBMCs transduced with a lentivirus comprising a nucleic acid encoding an anti-CD19 CAR comprising a CD3 zeta intracellular activation domain but not a costimulatory domain, between day 7 and day 21, day 28, day 35, and / or day 42 post-transduction in the absence of exogenously added cytokines, compared to a control retroviral particle, e.g....

Claims

1. 1. A method for genetically modifying T cells, comprising: ex vivo contacting whole blood containing T cells taken from the subject with replication-incompetent recombinant retroviral particles in a reaction mixture. A method comprising: wherein the reaction mixture comprises at least 50% whole blood; wherein the reaction mixture comprises an anticoagulant, and wherein said replication-incompetent recombinant retroviral particle comprises: (a) on their surface a T cell activation element; and (b) a polynucleotide comprising one or more transcription units, wherein each of said one or more transcription units is operably linked to a promoter active in the T cell, and wherein said 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. method.

2. 10. The method of claim 1, further comprising the step of expanding the cells ex vivo, wherein the cells are expanded ex vivo by no more than four cell divisions per cell.

3. The method according to claim 1 , wherein the method does not require prior ex vivo stimulation.

4. 4. The method of claim 1, wherein the reaction mixture comprises at least 90% whole blood.

5. 5. The method of any one of claims 1 to 4, wherein after the contacting step, at least 5% of the T cells in the reaction mixture are genetically modified T cells.

6. 6. The method of claim 5, wherein when the T cells are combined with the 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 performed for 24 hours or less.

7. The method of claim 1 , wherein the T cell activation element comprises a GPI anchor.

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.

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

12. The lymphoproliferative elements are selected from the group consisting of CD2, CD3D, CD3E, CD3G, CD4, CD8A, CD8B, CD27, 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, I 12. The method of claim 11, comprising a signaling domain from L12RB2, 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 proliferation and / or survival of T cells.

13. 11. The method of 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. 13. The method of any one of claims 11 to 12, wherein the one or more transcription units further encode a second polypeptide, the second polypeptide comprising a chimeric antigen receptor and / or a recombinant TCR.

15. 15. The method of 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, TNFSF6 (FASL), CASP3, SOCS2, TIEG1, JunB, Cbx3, Tet2, or HK2.

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

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

19. 20. The method of claim 18, wherein the reaction mixture is contacted with a leukoreduction filter assembly in a closed cell processing system.

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

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

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