Methods and compositions for delivery of retroviral particles

Abstract

The present disclosure provides methods and compositions for modifying lymphocytes, such as T cells and / or NK cells, for example, in vivo. The disclosed compositions can include replication-incompetent recombinant retroviral particles (RIPs), formulations thereof, and administration of RIP formulations to a subject. Additionally, the compositions can include chemokines, which can include chemokines delivered to and / or associated with the RIPs.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 200,329, filed March 1, 2021, U.S. Provisional Patent Application No. 63 / 197,315, filed June 4, 2021, U.S. Provisional Patent Application No. 63 / 261,099, filed September 10, 2021, International Patent Application No. PCT / US2021 / 020922, filed March 4, 2021, and International Patent Application No. PCT / US2021 / 048532, filed August 31, 2021. International patent application PCT / US2021 / 048532, filed August 31, 2021, claims priority to international patent application PCT / US2021 / 020922, filed March 4, 2021, international patent application PCT / US2020 / 048843, filed August 31, 2020, U.S. Provisional Patent Application No. 63 / 200,329, filed March 1, 2021, and U.S. Provisional Patent Application No. 63 / 136,177, filed January 11, 2021. International patent application PCT / US2021 / 020922, filed March 4, 2021, claims priority to U.S. Provisional Patent Application No. 62 / 985,741, filed March 5, 2020, International patent application PCT / US2020 / 048843, filed August 31, 2020, U.S. Provisional Patent Application No. 63 / 136,177, filed January 11, 2021, and U.S. Provisional Patent Application No. 63 / 200,329, filed March 1, 2021. International Patent Application PCT / US2020 / 048843, filed August 31, 2020, is a continuation-in-part of International Patent Application PCT / US2019 / 049259, filed September 2, 2019, as well as U.S. Provisional Patent Application No. 62 / 894,849, filed September 1, 2019, and U.S. Provisional Patent Application No. 62 / 894,852, filed September 1, 2019. This application claims the benefit of U.S. Provisional Patent Application No. 62 / 894,853, filed September 1, 2019, U.S. Provisional Patent Application No. 62 / 894,926, filed September 2, 2019, U.S. Provisional Patent Application No. 62 / 943,207, filed December 3, 2019, and U.S. Provisional Patent Application No. 62 / 985,741, filed March 5, 2020.International Patent Application PCT / US2019 / 049259 is a continuation-in-part of International Patent Application PCT / US2018 / 051392, filed September 17, 2018, and claims the benefit of 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 / 894,853, filed September 1, 2019. International Patent Application PCT / US2018 / 051392 is a continuation-in-part of International Patent Application PCT / US2018 / 020818, filed March 3, 2018, and 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 patent application PCT / US2018 / 020818 is a continuation-in-part of international patent application PCT / US2017 / 023112, filed March 19, 2017, which is a continuation-in-part of international patent application PCT / US2017 / 041277, filed July 8, 2017, which is a continuation-in-part of U.S. patent application 15 / 462,855, filed March 19, 2017, which is a continuation-in-part of U.S. patent application 15 / 6 This application is a continuation-in-part of U.S. Provisional Patent Application No. 44,778, filed on March 3, 2017, and claims the benefit of U.S. Provisional Patent Application No. 62 / 467,039, filed on September 18, 2017, U.S. Provisional Patent Application No. 62 / 560,176, filed on September 27, 2017, U.S. Provisional Patent Application No. 62 / 564,253, filed on September 27, 2017, and U.S. Provisional Patent Application No. 62 / 564,991, filed on September 28, 2017.International Patent Application 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 Patent Application PCT / US2017 / 041277 claims the benefit of International Patent Application PCT / US2017 / 023112, filed March 19, 2017, U.S. Patent Application Serial No. 15 / 462,855, filed March 19, 2017, U.S. Provisional Patent Application Serial No. 62 / 360,041, filed July 8, 2016, and U.S. Provisional Patent Application Serial 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 / 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 / 644,778 is a continuation-in-part of International Patent Application PCT / US2017 / 023112, filed March 19, 2017, and a continuation-in-part of U.S. 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. All applications cited in this paragraph are incorporated herein by reference in their entirety.

[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_003_W0_03_Sequence_Listing", created on March 1, 2022, has a file size of 506 KB, and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to the fields of immunology and immunotherapy, and more particularly to methods for genetically modifying lymphocytes and retroviruses therefor, and methods of using the same. [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 engineered genetic program. Examples of such synthetic proteins include engineered 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 shown efficacy in infecting non-dividing cells, resting CD4 and CD8 lymphocytes are resistant to genetic transduction by these vectors. To overcome this difficulty, these cells are typically activated in vitro using stimulating reagents before genetic modification with CAR gene vectors 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-related responses in immune cells and release cytolytic molecules, inducing target cell death.

[0006] Such 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 not only result in numerous unacceptable adverse events for patients, but also lack the ability to control the rate of in vivo proliferation after introduction into the body or safely target targets expressed outside the tumor. As a result, CAR therapies today typically deliver less than 1×10 5 ~1×10 8 Cells expanded ex vivo for 12–28 days using a dose of 100 cells / kg are infused and directed to the target, e.g., tumor, with generally acceptable off-tumor on-target toxicity. These relatively long ex vivo expansion times pose scalability challenges, as well as cell viability and sterility issues, as well as sample identity issues. Therefore, there is a great need for safer, more effective, and scalable T cell or NK cell therapies. Further reductions in the complexity and time required for such methods would be highly desirable, especially if such methods involve, for example, drawing a subject's blood within an infusion center and then reinjecting it into the subject on the same day. Furthermore, simpler and more rapid methods alone, or methods requiring less specialized equipment, could democratize these cell therapy processes, which are currently routinely performed only in highly specialized medical centers.

[0007] Because our understanding of the processes that drive lymphocyte transduction, proliferation, and survival is central to a variety of potential commercial applications, including immunological processes, improved methods and compositions for studying lymphocytes are needed. 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, as well as the factors that influence lymphocyte survival and proliferation. Furthermore, it would be useful to identify compositions that drive 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 for methods using packaging cell lines for the production of recombinant retroviral particles.

[0008] Furthermore, there remains a need for improved compositions and methods for inducing lymphocyte proliferation and / or survival in blood, organs, and tissues, as well as preferentially and specifically in the tumor microenvironment. Previous methods have used cells with constitutively expressing CARs that, upon binding to a target antigen, induce the expression of secreted cytokines under the control of a CAR-stimulation-inducible promoter. These secreted cytokines nonspecifically bind to and stimulate T cells and NK cells, reducing the amount of cytokines available to stimulate CAR T cells or NK cells. The cytokines may also diffuse away, further reducing the cytokines available to stimulate CAR T cells or NK cells. These previous methods typically required multiple transduction of transcription units on separate vectors and long blood cell processing times, thus requiring cancer patients to wait days, weeks, or even months after their blood was drawn to receive genetically engineered blood cells. Previous methods of one-step CAR-T cell transduction using vectors encoding two or more transcription units resulted in low viral titers and / or low expression of one or more of the transcription units, each of which represents a major obstacle to commercialization as a general therapeutic approach. Thus, there remains a need for more efficient methods for generating CAR-T cells that survive and proliferate in blood, organs, and tissues, and preferentially and specifically in the suppressive tumor microenvironment.

[0009] Furthermore, there remains a need for safer methods for performing therapies involving genetic modification of cells, particularly lymphocytes. Ex vivo cell treatment methods have inherent safety concerns regarding manipulating cells outside a subject's body and then returning the cells to the correct subject without contamination and unacceptable cytotoxicity. Summary of the Invention

[0010] Provided herein are methods, uses, compositions, and kits that simplify and expedite the process of genetically modifying lymphocytes, in exemplary embodiments, T cells and / or NK cells. Some aspects and embodiments provided herein are well-suited for point-of-care cell processing, eliminating the need to transport cells to specialized processing facilities. Furthermore, the methods, uses, compositions, and kits provided herein, in exemplary embodiments, help overcome issues related to the efficacy and safety of methods for transducing and / or modifying lymphocytes, such as T cells and / or NK cells. Certain embodiments of such methods are useful for implementing adoptive cell therapy using these cells. Thus, in some aspects, provided herein are methods, compositions, and kits for modifying lymphocytes, particularly T cells and / or NK cells, and / or for modulating the activity of transduced, genetically modified, and / or modified T cells and / or NK cells. Such methods, compositions, and kits provide improved efficacy and safety over current technologies, particularly for T cells and / or NK cells expressing engineered T cell receptors (TCRs), chimeric antigen receptors (CARs), and, in exemplary embodiments, microenvironment-restricted biological ("MRB") CARs. The transduced and / or modified, and in exemplary embodiments, genetically modified, T cells and / or NK cells produced by and / or used in the methods provided herein comprise functionalities and combinations of functionalities delivered from retroviral (e.g., lentiviral) genomes via retroviral (e.g., lentiviral) particles, in exemplary embodiments, ex vivo, or in certain exemplary aspects, in vivo, providing improved features for such cells and methods utilizing such cells, such as research methods, commercial production methods, and adoptive cell therapy. For example, such cells can be produced in vivo or generated ex vivo in shorter time periods and have improved growth characteristics that can be better regulated.In exemplary embodiments, such methods, uses, compositions, and kits comprise or are adapted for intramuscular delivery, or in further exemplary embodiments, subcutaneous delivery, to a subject.

[0011] In some aspects, methods are provided for transducing and / or modifying, and in exemplary embodiments, genetically modifying, lymphocytes such as T cells and / or NK cells, as well as in exemplary embodiments, in vivo or ex vivo methods for transducing, genetically modifying, and / or modifying resting or divided T cells and / or NK cells. Some of these aspects involve little or no ex vivo cell processing and, in exemplary embodiments, can be performed much more quickly than previous methods. This can facilitate more efficient research, more effective commercial production, safer methods for modifying lymphocytes of interest, and improved methods for treating patients. The methods, uses, compositions, and kits provided herein can be used as research tools, in commercial production, and in adoptive cell therapy using replication-incompetent retroviral particles (RIPs) and transduced and / or modified, and in exemplary embodiments, genetically modified, T cells and / or NK cells expressing a TCR or CAR.

[0012] For example, with respect to the methods, uses, and compositions provided herein relating to the transduction of lymphocytes, such as T cells and / or NK cells, methods and related uses and compositions are provided herein that involve ex vivo, or in certain exemplary embodiments, in situ or in vivo transduction reactions involving RIPs and T cells and / or NK cells. In some exemplary embodiments, such transduction reactions may be ex vivo transduction reactions, including enriched PBMCs, TNCs, or blood cells without prior cell enrichment, e.g., in the case of simplified whole blood, and may be rapid methods for performing ex vivo cell processing for CAR-T therapy. Such methods require less specialized instrumentation and training. Furthermore, such methods have a lower risk of non-target cell transduction compared to in vivo transduction methods. Further provided herein are methods, uses, and compositions, including embodiments of the methods described above, including specific target inhibitory RNAs, activating elements, polypeptide lymphoproliferative elements and the polynucleotides encoding them, pseudotyping elements, chemokines, and artificial antigen-presenting cells, which can optionally be combined with any other aspects provided herein to provide methods, uses, and compositions that potently induce lymphocyte expansion in vitro, ex vivo, and in vivo, particularly T cells and / or NK cells. In some embodiments, the modified lymphocytes are capable of engrafting in a lymphoreplete environment. In some embodiments, the patient or subject is not lymphodepleted prior to reinfusion of the modified and / or genetically modified T cells and / or NK cells.

[0013] In some aspects and embodiments, genetic constructs are provided herein that are particularly suited to providing genetically modified T cells and / or NK cells with the ability to survive and proliferate in a more controllable manner. In contrast to lymphoproliferation elements operably linked to secreted cytokines or constitutive promoters operably linked to inducible promoters, such aspects and embodiments provide inducible promoters operably linked to membrane-bound lymphoproliferation elements that, when induced by CAR binding to its target, can induce proliferation of T cells and / or NK cells, such as those present in the tumor microenvironment.

[0014] In some aspects, provided herein are methods of delivering, administering, and / or injecting a replication-incompetent recombinant retroviral particle (RIP) to a subject, the methods comprising administering to the subject a RIP formulation comprising the RIP, which may be any of the RIPs disclosed herein.

[0015] 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]

[0016] [Figure 1] FIG. 1 shows a FACS plot of peripheral blood from NSG-SGM3 CD34 humanized mice 19 days after IP injection of GCAR-19GU RIP.

[0017] [Figure 2] Figure 2 shows a graph of the number of B cells per μl of blood in NSG-SGM3 CD34 humanized mice at various days after mock IP injection with PBS or IP injection with GCAR-19GU RIP. The graph represents the average of five mice in each group.

[0018] [Figure 3] FIG. 3 shows a FACS plot of peripheral blood from NSG-MHC1 / 2 DKO PBMC-humanized mice 19 days after IP injection of GCAR-19GU RIP.

[0019] 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 CAR of the present invention comprises 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, an IAD can utilize the antigen-binding properties of antibodies to redirect the specificity and reactivity of T cells toward a selected target in an MHC-unrestricted manner. MHC-unrestricted antigen recognition confers on CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thereby bypassing 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.

[0020] As used herein, the term cell "aggregate" means a mass of cells that are adhered to one another.

[0021] As used herein, the term "constitutive T cell or NK cell promoter" refers to a promoter that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0022] As used herein, the term "inducible promoter" or "activatable promoter" refers to a promoter that, when operably linked to a polynucleotide encoding or specifying a gene product, causes a gene product to be produced in a cell only when a promoter-specific inducer is substantially present in the cell. Inducible promoters have no or low levels of basal transcriptional activity, but transcriptional activity increases, sometimes greatly, in the presence of an inducing signal.

[0023] As used herein, the term "insulator" refers to a cis-regulatory element that mediates intra- and inter-chromosomal interactions and can block interactions between enhancers and promoters. Typically, insulators are 200-2000 base pairs in length and contain clustered binding sites for sequence-specific DNA-binding proteins.

[0024] 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, concentration of one or more solutes, electrolyte concentration, glucose concentration, hyaluronan concentration, lactate or lactate concentration, albumin concentration, adenosine level, R-2-hydroxyglutarate level, pyruvate concentration, oxygen concentration, and / or the presence of oxidizing agents, reducing agents, or cofactors, as well as other conditions understood by those skilled in the art.

[0025] 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 polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0026] As used herein, an "approved biologic" is a macromolecule that meets the requirements for biologics set forth by a government regulatory authority, such as, but not limited to, the United States Food and Drug Administration (USFDA), the European Medicines Agency (EMA), the China Food and Drug Administration (China FDA), or the Japan Pharmaceutical and Food Safety Bureau (PFSB), and has been approved by such authority as a stand-alone biologic or as part of a combination product or method.

[0027] 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 camelid single-chain variable fragments (scFv), bivalent scFv, trivalent scFv, camelid VHH, 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.

[0028] As used herein, the term "antibody fragment" includes a portion of an intact antibody, e.g., 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 "Fc" fragment (a name reflecting the ability to crystallize readily). Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0029] 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).

[0030] As used herein, "naturally occurring" VH and VL domains refer to VH and VL domains isolated from a host without further molecular evolution to alter their affinity when 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.

[0031] As used herein, "antibody mimic" refers to an organic compound that specifically binds to a target sequence and has a structure different from that of naturally occurring antibodies. Antibody mimics may include proteins, nucleic acids, or small molecules, and those skilled in the art will understand when each type is relevant. The target sequence to which an antibody mimic of the present disclosure specifically binds may be an antigen. Antibody mimics can offer superior properties over antibodies, including, but not limited to, superior solubility, tissue penetration, thermal and enzymatic stability (e.g., resistance to enzymatic degradation), and lower production costs. Antibody mimetics include, but are not limited to, affibodies, afflilins, affimers, affitins, alphabodies, alphamab, anticalins, peptide aptamers, armadillo repeat proteins, atrimers, avimers (also known as avidity multimers), C-type lectin domains, cysteine-knot miniproteins, cyclic peptides, cytotoxic T-lymphocyte associated protein-4, DARPins (Designed Ankyrin Repeat Protein), fibrinogen domain, fibronectin binding domain (FN3 domain) (e.g., adnectin or monobody), fynomer, knottin, Kunitz domain peptide, nanofitin, leucine-rich repeat domain, lipocalin domain, mAb 2 or Fcab™, nanobody, nanoCLAMP, Obody, Pronectin, single-chain TCR, tetratricopeptide repeat domain, or V-like domain.

[0032] As used herein, "complementarity-determining region" or "CDR" refers to the three hypervariable regions in each variable chain of immunoglobulins and T-cell receptors, sandwiched between four "framework" regions of the chain. CDRs are primarily responsible for binding specificity. The CDRs of each immunoglobulin chain are numbered and designated CDR1, CDR2, and CDR3, starting from the N-terminus, and are typically identified by the chain in which the particular CDR is located. Thus, HCDR3 is located in the variable domain of the antibody's heavy chain, while LCDR1 is the CDR1 derived from the variable domain of the antibody's light chain. The sequences of framework regions of different light chains or different heavy chains are relatively conserved within a species. The framework region of an antibody is the combined framework region of the constituent light and heavy chains, and serves to position and align the CDRs in three-dimensional space. The amino acid sequences of the CDRs and framework regions can be determined using various definitions known in the art, such as Kabat, Chothia, the international ImMunoGeneTics database (IMGT), and AbM (see, e.g., Johnson and Wu, Nucleic Acids Res. 2000 Jan 1;28(1):214-218 and Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia & Lesk, (1987) J. Mol. Biol. 196, 901-917; Chothia et al. (1989) Nature 342, 877-883; Chothia et al. (1992) J. Mol. Biol. 227, 799-817; Al-Lazikam et al., J. Mol. Biol. 1997, 273(4)). Unless otherwise indicated, the CDRs herein are determined using "Fab Analysis" on the World Wide Web at vbase2.org (Retter et al., Nucleic Acids Res., 33:D671-D674 and Mollova et al., BMC Systems Bio., S1, p30).

[0033] As used herein, the term "idiotype" refers to the segment of an antibody or antibody mimetic that determines its specificity for an antigen, e.g., the structure of the variable region of an antibody, T cell receptor, or antibody mimetic, which is a shared characteristic among a group of antibodies, T cell receptors, or antibody mimetics based on antigen-binding specificity and is therefore also the structure of the variable region. An antibody idiotype typically includes the variable region, e.g., CDRs and framework regions. For antibodies, the idiotype is located in the Fab region. For antibodies formed from multiple chains, e.g., heavy and light chains, idiotype expression typically requires the participation of both the heavy and light chain variable regions that form the antigen-binding site. For antibodies formed from a single chain, e.g., scFv, idiotype expression typically requires the participation of the variable regions of a single polypeptide that form the antigen-binding site. For antibody mimetics, the idiotype varies depending on the type of antibody mimetic but includes the region necessary for binding to the cognate antigen.

[0034] As used herein, a "therapeutic antibody" or "therapeutic antibody mimetic" is an antibody or antibody mimetic that has been shown to have therapeutic activity using in vivo, e.g., human, assays.

[0035] As used herein, the term "recognize" refers to the ability of one molecule to bind to another molecule, for example, the ability of a receptor to bind to its ligand or the ability of an antibody to bind to its target.

[0036] 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 an antibody for an unrelated amino acid sequence. The affinity of an antibody for a 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.

[0037] As used herein, the term "binding" refers to a direct association between two molecules through 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 This refers to binding with affinity such as M.

[0038] As used herein, 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.

[0039] As used herein, the term "element" includes polynucleotides including polypeptide fusions, polypeptide regions, and functional variants or fragments thereof, as well as microRNAs and shRNAs, and functional variant or fragment polynucleotides thereof.

[0040] As used herein, the term "region" is any segment of a polypeptide or polynucleotide.

[0041] As used herein, a "domain" is a region of a polypeptide or polynucleotide that has functional and / or structural properties.

[0042] 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 may be composed of natural or synthetic polypeptides. The stalk may be derived from the hinge or hinge region of an immunoglobulin (e.g., IgG1), which is generally defined as extending from Glu216 to Pro230 of human IgG1 (Burton (1985) Molec. Immunol., 22:161-206). Hinge regions of other IgG isotypes may 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 may be naturally occurring or non-naturally occurring, including, but not limited to, altered hinge regions, as disclosed in U.S. Patent No. 5,677,425. The stalk may comprise 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 serve a similar function in providing flexibility and spacing to adjacent regions.

[0043] As used herein, the term "isolated" means that a material is removed from its original environment (e.g., the natural environment if it occurs naturally). 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.

[0044] 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.

[0045] 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.

[0046] As used herein, the term "immune cells" generally includes 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).

[0047] As used herein, "T cells" refers to T helper cells (CD4 + cells), cytotoxic T cells (CD8 +These include all types of immune cells that express CD3, including T cells (regulatory T cells), regulatory T cells (Tregs), and gamma-delta T cells. NKT cells that are CD3+, CD56+, and either CD4+ or CD8+ are considered to be a type of T cell herein. Surface expression of CD3 can be transiently reduced or eliminated in T cells, as observed in some of the methods of modifying T cells disclosed herein. Such modified CD4+ or CD8+ lymphocytes with transiently reduced / eliminated CD3 surface expression are still considered T cells in this disclosure. References herein to "CD" or markers of cluster of differentiation, e.g., CD3+, CD4+, CD8+, CD56+, etc., refer to the surface expression of such polypeptides. It is understood that surface expression is a continuum of positive and negative and can be analyzed by FACS analysis. In FACS analysis, cells are scored as positive or negative based on user cutoffs known in the art. For example, low or intermediate expression of a surface marker, such as CD3lo or CD3int, as determined by FACS analysis is considered surface marker negative (eg, CD3-) herein.

[0048] As used herein, "NK cells" include lymphocytes that express CD56 on their surface (CD56+ lymphocytes). NKT cells that are CD3+, CD56+, and either CD4+ or CD8+ are considered herein to be a type of NK cell.

[0049] 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.

[0050] As used herein, the term "stem cells" generally includes pluripotent or multipotent stem cells, including, 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.).

[0051] 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.

[0052] 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, ungulates (e.g., horses, cattle, sheep, pigs, goats), and the like.

[0053] 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 being treated.

[0054] As used herein, the term "evolution" or "evolving" refers to the use of one or more methods of mutagenesis to generate different polynucleotides that encode 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, 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, as well as other conditions considered to be within the normal range in the tissue or organ at the site of administration or action in a subject.

[0055] As used herein, a "transduced cell" or "stably transduced cell" is a cell that contains an exogenous nucleic acid integrated into the genome of the cell. 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 and regardless of the method used to introduce the exogenous nucleic acid into the cell. An exogenous nucleic acid within a cell that is not integrated into the genome of the cell may be referred to herein as "extrachromosomal." As used herein, a "modified cell" is a cell that is associated with a recombinant nucleic acid vector (also referred to herein as a "polynucleotide vector" or "gene vector") or that has been genetically modified with an exogenous nucleic acid; in an exemplary embodiment, the recombinant nucleic acid vector is a replication-incompetent recombinant retroviral particle (also referred to herein as a "RIR retroviral particle" or "RIP") containing an exogenous nucleic acid. Typically, in compositions and methods involving replication-incompetent recombinant retroviral particles, modified cells associate with the replication-incompetent recombinant retroviral particles through interactions between proteins on the surface of the cells and proteins on the surface of the replication-incompetent recombinant retroviral particles containing pseudotyping elements and / or T cell activation elements. In compositions and methods involving transfection of nucleic acids within lipid-based reagents, such as liposome reagents, the lipid-based reagent containing the nucleic acid, a type of recombinant nucleic acid vector, associates with the lipid bilayer of the modified cells before fusing with or being internalized by the modified cells. Similarly, in compositions and methods involving chemical-based transfection of nucleic acids, such as polyethyleneimine (PEI)- or calcium phosphate-based transfection, the nucleic acid typically associates with a positively charged transfection reagent to form a recombinant nucleic acid vector that associates with the negatively charged membrane of the modified cells before being internalized by the modified cells. Other means or methods for stably transfecting or genetically modifying cells include electroporation, ballistic delivery, and microinjection.As used herein, a "polypeptide" can include part or the entire protein molecule, as well as any post-translational or other modifications.

[0056] 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 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 association element," and a "fusogenic polypeptide" may also be referred to as a "fusogenic element."

[0057] "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 can include naive T cells that have never encountered a particular antigen, and memory T cells that have been altered by previous encounter with an antigen. "Resting" lymphocytes can also be referred to as "resting" lymphocytes.

[0058] As used herein, "lymphodepletion" includes methods of reducing the number of lymphocytes in a subject, for example, by administering a lymphodepleting agent. Lymphodepletion can also be achieved by fractionated radiation therapy of a portion of the body or the whole body. 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, alemtuzumab, or combinations thereof.

[0059] 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 any other RNA that is susceptible to functional inhibition by RNAi. As used herein, "inhibitory RNA molecule" refers to an RNA molecule that, when present in a cell, causes RNAi, resulting in a reduction 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, an miRNA (either endogenous or artificial) or shRNA, a precursor of an miRNA (i.e., pri-miRNA or pre-miRNA) or shRNA, or a dsRNA that is directly transcribed or introduced into a cell or subject as an isolated nucleic acid.

[0060] 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, a lower limit of 18, 19, 20, 21, 22, 23, 24, or 25 to an upper limit of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, with a given range always having a lower limit that is lower than the upper limit. Such structures typically include a 5' stem, a loop, and a 3' stem connected by a loop adjacent to each stem that is not part of the duplex. In certain embodiments, the loop includes at least 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In other embodiments, the loop includes 2 to 40, 3 to 40, 3 to 21, or 19 to 21 nucleotides, or in some embodiments, a lower limit of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 to an upper limit of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40, with a given range always having a lower limit that is lower than the upper limit.

[0061] 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 production of a mature microRNA from a precursor microRNA. Often, these elements are found within a 40-nucleotide sequence adjacent to the microRNA stem-loop structure. In some cases, microRNA processing elements are found within stretches of nucleotide sequence 5 to 4,000 nucleotides in length adjacent to the microRNA stem-loop structure.

[0062] The term "linker" when used in reference to multiple inhibitory RNA molecules refers to a connecting means that joins two inhibitory RNA molecules.

[0063] 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 or other coding sequence of interest). Thus, retroviral (e.g., lentiviral) expression vectors contain sequences (e.g., 5'LTR and 3'LTR flanking the psi packaging element and the target heterologous coding sequence) that facilitate expression and packaging after transfection into cells. The terms "lentivirus" and "lentiviral particle" are used interchangeably herein.

[0064] 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 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.

[0065] As used herein, the term "miRNA precursor" refers to an RNA molecule of any length that can be enzymatically processed into an miRNA, such as a primary RNA transcript, a pri-miRNA, or a pre-miRNA.

[0066] 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, for example, a lymphoproliferation element. Those skilled in the art will understand whether a construct refers to an isolated polynucleotide or an isolated polypeptide, depending on the context.

[0067] As used herein, "MOI" refers to the multiplicity of infection ratio, and MOI is equal to the ratio of the number of virus particles used for infection per number of cells.As a non-limiting example, FACS and reporter expression can be used to carry out functional titration of virus particle number.

[0068] "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).

[0069] As used herein, the term "syncytium-inducing polypeptide" refers to a membrane polypeptide or a portion thereof that causes cell fusion, resulting in the formation of a syncytium. Syncytium-inducing polypeptides are certain fusogenic envelope proteins as disclosed herein. As used herein, syncytium-inducing polypeptides include proteins naturally produced by viruses, particularly so-called fusogenic membrane proteins (FMPs) and fusogenic membrane glycoproteins, which mediate virus-cell fusion and cell-cell fusion of infected cells. As used herein, syncytium-inducing polypeptides also include non-viral polypeptides known to mediate cell-cell fusion in vivo. A "viral fusogenic membrane glycoprotein" is a viral-derived fusogenic membrane protein that naturally mediates membrane fusion between a virus and its target host cell. As used herein, a syncytium-inducing polypeptide (or portion thereof) or a fusogenic membrane glycoprotein (or portion thereof) when isolated from a virus has the ability to mediate or fuse fusion between a cell expressing the fusogenic membrane glycoprotein and a cell expressing a receptor for the fusogenic membrane glycoprotein. An example of a fusogenic membrane protein includes, but is not limited to, fertilin b.The viral fusogenic membrane glycoprotein subset of fusogenic membrane proteins includes, but is not limited to: G-type glycoproteins of rabies virus, Mokola virus, vesicular stomatitis virus, and Toga virus; mouse hepatitis virus JHM surface projection protein; protein); spike glycoprotein and membrane glycoprotein of porcine respiratory coronavirus; spike glycoprotein and its precursor of avian infectious bronchitis virus; spike protein of bovine enteric coronavirus; F and H, HN, or G genes of measles virus, canine distemper virus, Newcastle disease virus, human parainfluenza virus type 3, simian virus 41, Sendai virus, and human respiratory syncytial virus; gH and chaperone protein gL of human herpesvirus type 1 and simian varicella virus; gB of human, bovine, and cercopithicine herpesvirus; envelope glycoproteins of Friend murine leukemia virus and Mason-Pfizer monkey virus; influenza hemagglutinin; G protein of vesicular stomatitis virus; hemagglutinin-neuraminidase and glycoproteins F1 and F2 of mumps virus; and membrane glycoprotein from Venezuelan equine encephalomyelitis.

[0070] It is recognized herein that some syncytium-inducing polypeptides function alone, while others require multiple distinct polypeptides for fusion-promoting activity. As used herein, the singular term "syncytium-inducing polypeptide" is meant to encompass one fusion-promoting polypeptide as well as each of the polypeptides required for fusion promotion when multiple polypeptides are required.

[0071] As used herein, the term "about" refers to a value that is 10% lower or 10% higher than the disclosed value. For example, "about 1% sucrose" includes 0.9% to 1.1% sucrose.

[0072] As used herein, the term "transduction unit" refers to the number of viral particles in solution that are capable of transducing target cells, and can be calculated by transducing any target cells, such as Jurkat cells, and measuring transgene expression in the target cells, for example, as determined by serial dilution and analysis of transgene expression by qPCR of the lentiviral genome using the Lenti-X™ qRT-PCR Titration Kit (Takara, #631235).

[0073] 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.

[0074] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, and any other stated or intervening value within that stated range, to the tenth of the unit of the lower limit, is encompassed within 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 embodiments herein, subject to any specifically excluded limits in the stated ranges. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the embodiments herein. 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 less than the higher value. All headings in this application are for the convenience of the reader and are not limiting.

[0075] 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 the aspects and embodiments provided herein belong.All methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of any aspect or embodiment provided herein, but preferred methods and materials are described herein.All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials for which the publications are cited.

[0076] It must be noted that as used in this specification and 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 any optional element. Accordingly, this statement is intended to serve as a predicate for using exclusive terminology such as "solely," "only," or a "negative" limitation in connection with the recitation of claim elements.

[0077] As used herein, the term "or" is understood to be inclusive unless specifically stated otherwise or clear from the context. When used herein, for example, in the phrase "A and / or B," the term "and / or" includes each of the following: A and B; A or B; A alone; and B alone. Similarly, when used in the phrase "A, B and / or C," for example, the term "and / or" includes each of the following: A, B and C; A, B or C; A or B; A or C; B or C; A and B; A and C; B and C; A alone; B alone; and C alone. This logic extends to any number of items in a list connected with "and / or."

[0078] 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 just as if each and every combination were individually and explicitly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such subcombination were individually and explicitly disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

[0079] The present disclosure overcomes the challenges of the prior art by providing improved methods and compositions for modifying, and in exemplary embodiments, genetically modifying, lymphocytes, such as NK cells and, in exemplary embodiments, T cells. Some of the methods and compositions herein offer a simplified and more rapid process for transducing or transfecting lymphocytes, avoiding several steps that require specialized devices. Thus, the methods provide an important step toward the generalization of cell therapy. Exemplary methods and compositions for modifying lymphocytes, such as NK cells and, in exemplary embodiments, T cells, are performed ex vivo or in vivo by administering replication-incompetent recombinant retroviral particles (RIPs) directly to a subject. In some aspects and embodiments herein, the methods for modifying T cells and / or NK cells are performed in less time than conventional methods, and indeed, in some embodiments, provide a rapid, point-of-care method. Additionally, compositions are provided that have many uses, including in these improved methods, including RIPs and / or cell preparations and / or compositions adapted for perilymphatic, intranodal, or subcutaneous administration. Some of these compositions include engineered lymphocytes, and in exemplary embodiments, genetically modified lymphocytes, with improved proliferation and survival, including in vivo expansion and engraftment and / or ex vivo and / or in vitro culture, e.g., in the absence of growth factors. Such RIP formulations, as well as engineered lymphocytes, and in exemplary embodiments, genetically modified lymphocytes, have utility as research tools to more easily transduce T cells and NK cells in vivo, ex vivo, or in vitro, e.g., to understand factors that affect T cell proliferation and survival, and for commercial production purposes, e.g., for the generation of specific factors, such as growth factors and immunomodulatory agents, that can be harvested and tested or used in commercial products.Such RIP formulations, as well as modified and genetically modified lymphocytes, also have utility in the treatment of many disorders, e.g., immune cell and gene therapy, and in exemplary embodiments, in the treatment of hyperproliferative cell disorders such as cancer.

[0080] Exemplary methods and compositions for immune cell therapy herein include, are compatible with, are effective against, and / or are adapted for perilymphatic, subcutaneous, intraperitoneal, or intramuscular delivery, as well as cell preparations and / or RIP preparations for such delivery. Some of these delivery methods and cell preparations (i.e., delivery compositions) promote cell aggregation. Such cell aggregation promotes cell proliferation and survival, which in some embodiments is further enhanced by the addition of antigens, growth factors, and immunomodulators to the cell preparation or the site of administration of the cell preparation.

[0081] Also provided herein are methods and compositions that overcome challenges associated with resistance to CAR therapy by CAR cancer cells, such as loss of target antigen availability (e.g., epitope or antigen masking) through genetic modification of malignant cells.

[0082] In vivo methods for genetically modifying T cells and / or NK cells Aspects and embodiments provided herein are characterized by the direct injection of RIPs into a subject, in which T cells and / or NK cells are modified in vivo. It is further noted that while many of the aspects and embodiments provided herein are discussed in terms of recombinant retroviral particles, it is intended, and will be recognized by those skilled in the art, that many different recombinant nucleic acid vectors, including but not limited to those provided herein, can be used and / or included in such methods and compositions. Accordingly, in some aspects and embodiments, the methods provided herein involve administering (e.g., delivering, transducing, or injecting) viral particles directly to a subject. In some aspects and embodiments, provided herein are methods for administering viral particles to a subject to modify, and in exemplary embodiments, genetically modify and / or transduce, T cells and / or NK cells in vivo, i.e., in the subject. In exemplary embodiments, the viral particles are retroviral particles. In further exemplary embodiments, the retroviral particles are replication-incompetent recombinant retroviral particles (RIPs). Many of the aspects and embodiments herein refer to RIPs, and one of skill in the art will understand how other viral and / or retroviral particles may be used.

[0083] In related aspects and embodiments, provided herein are RIP formulations, modified compositions comprising RIPs, delivery solutions comprising RIPs, RIPs for use in methods, methods of making RIP formulations, in vivo compositions comprising RIPs, and in vivo reaction mixtures comprising RIPs. In exemplary embodiments, the RIPs comprise one or more polynucleotides encoding an engineered signaling polypeptide, such as a CAR, an engineered TCR, and / or a lymphoproliferative element (LE). In certain exemplary embodiments, the LEs are constitutively active LEs. In exemplary embodiments, the RIP formulations comprise an activating element either in the RIP solution or on the RIP surface to promote in vivo genetic modification of T cells and / or NK cells. In exemplary embodiments, the RIPs comprise a binding element and a fusogenic element on the RIP surface. In some embodiments, one or both of the binding element and the fusogenic element can be a pseudotyping element. In some embodiments, the activating element is a binding element, and the fusogenic element is a pseudotyping element.

[0084] In one aspect, provided herein is a method of administering a replication-incompetent recombinant retroviral particle (RIP) to a subject, the method comprising: administering to the subject a formulation comprising the RIP, wherein the RIP: a) an activation element on the surface of the RIP, and b) a polynucleotide comprising one or more transcription units, each of the one or more transcription units encoding one or more engineered signaling polypeptides.

[0085] The engineered signaling polypeptide may be any of the engineered signaling polypeptides otherwise disclosed herein, e.g., a CAR, an engineered TCR, and / or a lymphoproliferative element.

[0086] In another aspect, provided herein is a method of administering a replication-incompetent recombinant retroviral particle (RIP) to a subject, the method comprising: administering to the subject a formulation comprising the RIP, wherein the RIP: a) an activation element on the surface of the RIP, and b) A polynucleotide encoding a lymphoproliferative element (LE) and a polynucleotide encoding a chimeric antigen receptor (CAR), wherein the LE is constitutively active.

[0087] The activation element may be any of the activation elements otherwise disclosed herein. In some embodiments, the RIP further comprises a binding element and / or a fusogenic element on the RIP surface. Any of the binding elements or fusogenic elements otherwise disclosed herein may be used.

[0088] In any of the aspects and embodiments herein involving administering a RIP to a subject, including delivering or introducing a RIP to a subject and / or injecting a RIP to a subject, and in any of the aspects and embodiments herein involving a method of treating a subject comprising administering a RIP to a subject, the administering may include administering the RIP intravenously or perivascularly. In exemplary embodiments, the RIP may be administered perivascularly. Perivascular administration includes intratumoral, intralymphatic, and perilymphatic administration. Thus, in some embodiments, the RIP may be administered intratumorally, intralymphatically, or, in exemplary embodiments, perilymphatically. Perilymphatic administration includes subcutaneous, interstitial, intraperitoneal, intramuscular, and intradermal administration, and in some embodiments, the RIP may be administered subcutaneously, interstitially, intraperitoneally, intramuscularly, or intradermal. In some embodiments, the RIP may be delivered intralymphatically or subcutaneously. Any formulation herein, including a formulation that includes a RIP, may be referred to as a composition, such as a modified composition, if it includes a RIP, or as a delivery solution.

[0089] Introduction or administration of a RIP into a subject may be direct delivery to a lymph node. In exemplary embodiments, the RIP may be injected or administered to a basal lymph node, an axillary lymph node, and / or a cervical lymph node. In some embodiments, the lymph node to which the RIP is delivered contains lymph node metastasis. In some embodiments, the lymph node is a lymph node that drains a tumor. In some embodiments, the lymph node is not a lymph node that drains a tumor. In some embodiments, the lymph node contains TILs.

[0090] The RIP formulations herein may include or have components to improve efficacy upon introduction into a subject, for example; in a non-limiting example, the components may be introduced by subcutaneous delivery. In some embodiments, the RIP is formulated to include a means for retaining the RIP at or near the delivery site, or to be effective, configured, and / or adapted to retain the RIP near the delivery site. In some embodiments, the RIP is formulated to be effective, adapted, and / or configured, or having a means for retaining the RIP at or near the delivery site. In some embodiments, the RIP delivered subcutaneously comprises a membrane-bound cytokine. In some embodiments, the RIP delivered subcutaneously comprises a membrane-bound chemokine. In some embodiments, the RIP is delivered in a depot formulation. In some embodiments, the RIP is delivered in a hydrogel. Depot formulations and hydrogels are discussed in detail herein. In some embodiments, the RIP is formulated to include a means for inhibiting local absorption into the blood. In some embodiments, the RIP is formulated to be effective, adapted to inhibit, and / or configured to inhibit local absorption into the blood. In some embodiments, the RIP is formulated to include a means for entry into lymphatic vessels. In some embodiments, the RIP is effective, adapted to enter, and / or configured to enter lymphatic vessels. In some embodiments, the RIP is delivered in a soluble formulation. In some embodiments, the RIP is formulated with an effective amount of one or more excipients (lymphogenesis enhancers) that promote lymphatic absorption. In some embodiments, the lymphogenesis-promoting agent is selected from human serum albumin, hyaluronidase, non-sulfated dextran, colloids comprising dextran with a molecular weight greater than 10K, 40K, 100K, 200K, 500K, 1000K, or 2000K kDa, and other agents that reduce the local residence time of the RIP at the delivery site (e.g., subcutaneously).

[0091] In some embodiments, the RIP is formulated for delivery in a buffer solution, typically containing a salt in an amount effective for in vivo delivery. In some embodiments, the formulation contains phosphate buffered saline (PBS). In some embodiments, the formulation further contains lactose, such as, for example, 0.25% lactose to 10% lactose, e.g., 0.5% to 10%, 0.5% to 8%, 1% to 8%, 1% to 10%, 2% to 8%, 2% to 6%, 3% to 6%, 3% to 5%, 3.5% to 4.5%, 3.6% to 4.4%, 3.7% to 4.3%, 3.8% to 4.2%, 3.9% to 4.1%, about 4% lactose, or 4% lactose.

[0092] In some embodiments of any of the RIP formulations herein, or the cell formulations herein, the formulation, and / or the RIP formulation and / or cell formulation delivered to a subject has a volume of 0.5 to 25 ml, 0.5 to 20 ml, 0.5 to 10 ml, 0.5 to 7.5 ml, 0.5 to 6 ml, 1.0 to 25 ml, 1.0 to 20 ml, 1.0 to 7.5 ml, 1.0 to 5.0 ml, 1 to 5 ml, 2.5 to 25 ml, 2.5 to 20 ml, 2.5 to 7.5 ml, or 2.5 to 5.0 ml.

[0093] In some embodiments, the cell preparation may be mixed with the RIP preparation immediately prior to injection or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 minutes of injection, such that some cells, most of the cells, or all of the cells are unmodified, ungenetically modified, or untransduced.

[0094] In some embodiments, the method of administering a RIP to a subject, comprising administering a RIP formulation, further comprises administering (sometimes referred to herein as co-administering) a cell preparation (also referred to herein as a cell suspension or cell mixture) to the subject. In some embodiments, the cell preparation comprises whole blood collected from the subject. In some embodiments, the cell preparation contains neutrophils from the subject. In some embodiments, the cell preparation contains dendritic cells from the subject. In some embodiments, the cell preparation contains macrophages from the subject. In some embodiments, the whole blood is subjected to a PBMC enrichment procedure and a TNC enrichment procedure, and the cell preparation comprises the enriched cells. In some embodiments, the cell preparation contains PBMCs. In exemplary embodiments, the cell preparation administered to the subject comprises T cells and / or NK cells. In some embodiments, the cells, and in exemplary embodiments, the PBMCs, are not / are not exposed to a vector such as a RIP ex vivo, but are or have been exposed to an activating element (e.g., anti-CD3 and / or anti-CD28) prior to co-administration. Thereby, the PBMCs are in an activated state when co-administered, and / or their signaling pathways are engaged ex vivo and activated after co-administration to a subject, and may or may not be exposed to an activating element. The RIP co-administered with such co-administered cells may, in any embodiment, have an activating element on its surface and / or encode an LE. However, in certain exemplary embodiments, the RIP co-administered with activated cells does not include either an activating element and / or a polynucleotide encoding an LE on its surface, or both, and in exemplary embodiments, such activated cells have not been exposed to a vector (e.g., a RIP) ex vivo.

[0095] In some embodiments, the cells are modified, i.e., the cells are contacted with a RIP prior to administration to the subject. In exemplary embodiments, the cells are unmodified, i.e., the cells are not contacted with a RIP prior to administration to the subject.

[0096] In some embodiments, the RIP formulation and the cell formulation may be administered, delivered, introduced, or injected within 0.5, 1, 2, 3, 4, or 5 cm of each other at a target delivery site in a subject, such as on the surface of the subject's skin. In some embodiments, the administration of the cell formulation to the subject occurs simultaneously with administration of the RIP formulation to the subject, or within 1, 2, 3, 4, 5, 10, 15, 30, 45, or 60 minutes, or within 1, 2, 3, 4, 5, 6, 7, or 8 hours of administration of the RIP formulation to the subject.

[0097] In some embodiments, methods of administering a RIP and / or cells to a subject include administering to the subject at least 0.1 ml, 0.5 ml, 1 ml, 1.5 ml, 2 ml, 2.5 ml, 3 ml, 4 ml, or 5 ml, 10 ml, 15 ml, 20 ml, or 25 ml of any of the formulations disclosed herein that include a RIP. In some embodiments, methods of administering a RIP and / or cells to a subject include administering to the subject 0.1 to 20 ml of a RIP formulation. In some embodiments, methods of administering a RIP and / or cells to a subject include administering to the subject 1 to 10 ml, 1 to 20 ml, or 1 to 25 ml of a RIP formulation. In exemplary embodiments, methods of administering a RIP and / or cells to a subject include administering 2 to 3 ml of a RIP formulation. In some embodiments, methods of administering a RIP and / or cells to a subject may include any of the routes of administration disclosed herein. In exemplary embodiments, the method of administering a RIP and / or cells to a subject can include administering the RIP and / or cells perilymphatically. In some embodiments, the method of administering a RIP and / or cells to a subject can include either a single dose or multiple doses. In some embodiments, the method of administering a RIP and / or cells to a subject can include 1, 2, 3, 4, more than 5, or more doses.

[0098] In some embodiments, the method of administering a RIP to a subject comprises administering at least 1, 10, 100, 1000, 10 4 , 10 5 , 106 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 In some embodiments, the method of administering a RIP to a subject comprises administering to the subject a total of 1 x 10 total transducing units (TU). 5 ~4×10 9 In some embodiments, the method of administering a RIP to a subject comprises administering a total of 1 x 10 TU to the subject. 6 ~5×10 9 In some embodiments, the TU administered may be measured in kg of the subject's weight, i.e., in units of TU / kg. In some embodiments, the method of administering a RIP to a subject includes administering at least 1, 10, 100, 1000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 In some embodiments, the method of administering a RIP to a subject comprises administering 1 to 10 TU / kg of RIP to a subject. 14 TU / kg, 10 3 ~10 14 TU / kg, or 10 6 ~10 14 TU / kg, or 10 3 ~10 8 TU / kg, or 10 4 ~10 7 This includes administering TU / kg to the subject, where kg refers to the weight of the subject. In some embodiments, the administered TU may be measured in terms of the number of target cells present in 1 ml of the subject's blood, i.e., TU / target cell / ml of subject's blood.

[0099] In some embodiments, the method of administering RIP to a subject comprises administering 1 ng / kg / day to 500 mg / kg / day to the subject. In some embodiments, the method of administering RIP to a subject comprises administering 1 ng / kg / day to 100 mg / kg / day for 1, 2, 3, 4, 5, 6, or 7 days or consecutive days, or multiple days over 1, 2, 3, 4, 5, or 6 months, for example, one day per month for 1, 2, 3, 4, 5, or 6 months.

[0100] In some embodiments, the method of administering a RIP to a subject comprises administering at least 1.0×10 4 , 1.0×10 5 , 1.0×10 6 , or 1.0 × 10 7 In some embodiments, the method of administering a RIP to a subject comprises administering 1.0 x 10 genome copies (GC) to the subject. 4 ~1.0×10 15 In some embodiments, the method of administering a RIP to a subject comprises administering 1.0×10 9 ~1.0×10 15 In some embodiments, the administered dose may be in units of GC / kg of subject. In some embodiments, the method of administering RIP to a subject comprises administering 1.0×10 9 ~1.0×10 15 GC / kg to the subject.

[0101] In some embodiments, the method of administering a RIP to a subject comprises administering at least 1.0×10 4 , 1.0×10 5 , 1.0×10 6 , or 1.0 × 10 7 In some embodiments, the method of administering a RIP to a subject comprises administering 1.0 x 10 infectious units to the subject. 4 ~1.0×10 15 In some embodiments, the method of administering a RIP to a subject comprises administering 1.0 x 10 infectious units to the subject. 9 ~1.0×10 15The method involves administering infectious units per kg of body weight to a subject. Infectious units can be quantified by techniques available in the art, including determining the number of viral particles by plaque assay, fluorescence microscopy, and titer. For example, the number of adenoviral particles can be determined by measuring absorbance at A260. Similarly, infectious units can be determined by quantitative immunofluorescence of vector-specific proteins using monoclonal antibodies or by plaque assay. In some embodiments, the method for calculating infectious units involves plaque assay, in which a titer of virus is grown on a cell monolayer and the number of plaques is counted after several days to several weeks. For example, the infectious titer is determined by plaque assay, e.g., an assay that evaluates cytopathic effect (CPE). In some embodiments, a CPE assay is performed by serially diluting the virus on a monolayer of cells, e.g., HFF cells, overlaid with agarose. After incubation for a period until cytopathic effect appears, e.g., about 3 to 28 days, often 7 to 10 days, the cells are fixed and cell-free foci, visualized as plaques, are assessed. In some embodiments, infectious units can be determined using the endpoint dilution (TCID50) method, which determines the virus dilution that infects 50% of the cell culture, and thus generally allows for the determination of a titer within a particular range, e.g., 1 log.

[0102] In some embodiments, the method of administering a RIP to a subject comprises administering at least 1.0×10 4 , 1.0×10 5 , 1.0×10 6 , or 1.0 × 10 7 In some embodiments, the method of administering a RIP to a subject comprises administering 1.0 x 10 plaque-forming units (PFU) to the subject. 4 ~1.0×10 15 In some embodiments, the method of administering a RIP to a subject comprises administering 1.0 x 10 PFU to the subject. 9 ~1.0×10 15In some embodiments, the method of administering a RIP to a subject comprises administering 1.0 x 10 PFU to the subject. 9 ~1.0×10 15 This involves administering PFU / kg body weight to a subject.

[0103] In some embodiments, the method of administering a RIP to a subject comprises administering at least 1.0×10 RIP as otherwise disclosed herein. 3 , 1.0×10 4 , 1.0×10 5 , 1.0×10 6 , or 1.0 × 10 7 In some embodiments, the method of administering a RIP to a subject comprises administering 1.0 x 10 dimming units (DU) to the subject. 3 ~1.0×10 15 In some embodiments, the method of administering RIP to a subject comprises administering 1.0 x 10 DU to the subject. 3 ~1.0×10 6 In some embodiments, the method of administering RIP to a subject comprises administering 10 to 1.0 x 10 DU. 13 DU / kg body weight to the subject.

[0104] In some embodiments, the amount of RIP administered to a subject can be such that it does not cause too many integrations (also called integrants) in individual cells.In some embodiments, when measured as lentigenome copy number per cell genome, it produces an average of 5, 4, or 3 or less integrants.In exemplary embodiments, it produces an average of 3 or less integrants.In further exemplary embodiments, it produces an average of 2 or less integrants. Treatment method

[0105] The present disclosure provides various therapeutic methods, including administering, delivering, and / or injecting replication-incompetent recombinant retroviral particles (RIPs) directly to a subject. In exemplary embodiments, such RIPs have an activation element associated with their surface and include a polynucleotide encoding one or more of a lymphoproliferative element (LE), an engineered T cell receptor (engineered TCR), and a chimeric antigen receptor (CAR). In exemplary embodiments, the LE is a constitutively active LE. In exemplary embodiments, the RIP encodes both an LE and a CAR. Such RIPs are capable of, adapted to, and / or effective in transducing T cells and / or NK cells in vivo, whereby the transduced T cells and NK cells in vivo express the LE, the engineered TCR, and / or the CAR. The engineered TCR or CAR of the present disclosure is expressed by T lymphocytes or NK cells, and when present therein, can mediate cytotoxicity against target cells. Such methods herein typically involve administering to a subject a substantially purified or purified RIP as provided herein. The engineered TCR or CAR binds to an antigen present on a target cell, thereby mediating killing of the target cell by T lymphocytes or NK cells genetically modified to produce the engineered T cell receptor or CAR. In some embodiments, the ASTR of the engineered T cell receptor or CAR binds to an antigen present on the surface of the target cell.

[0106] In some aspects, the present disclosure provides methods for killing or inhibiting the growth of targeted cancer cells, the methods involving contacting the cells with cytotoxic immune effector cells (e.g., cytotoxic T cells or NK cells) that have been genetically modified to produce an engineered T cell receptor or CAR, whereby the T lymphocytes or NK cells recognize antigens present on the surface of the target cells and mediate killing of the target cells.

[0107] In some aspects, the disclosure provides methods of treating a disease or disorder in an individual having the disease or disorder, the method comprising: a. introducing into a subject an expression vector comprising a polynucleotide sequence encoding a CAR and / or a LE (e.g., a constitutively active LE) to generate engineered cells, e.g., engineered cytotoxic cells, T cells, and / or NK cells. The disclosure also provides methods of treating a disease or disorder in an individual having the disease or disorder, the method comprising: introducing into a subject an expression vector comprising a polynucleotide sequence encoding a CAR to generate engineered cytotoxic cells; and b. administering the engineered cytotoxic cells to the subject. In some embodiments, the treatment method comprises administering both the expression vector (e.g., a RIP formulation) and the engineered cytotoxic cells (e.g., a cell formulation comprising modified, genetically modified, or transduced T cells and / or NK cells). In some embodiments, the method of treatment comprises administering an expression vector (e.g., a RIP formulation) and unmodified cells (e.g., a cell formulation comprising unmodified cells (e.g., T cells and / or NK cells) that have not been contacted with a RIP). In exemplary embodiments of such embodiments, the unmodified cells may be derived from the subject. In such embodiments, the unmodified cells from the cell formulation and cells present in the subject prior to administration may be modified by the RIP in the RIP formulation. In some embodiments, the RIP formulation modifies cells in the subject to generate a sustained cell population in the subject, as otherwise disclosed herein. In some embodiments, the cells modified by the RIP formulation include cells of a separately administered cell formulation.

[0108] In some embodiments, RIPs and / or cells administered to a subject for therapeutic purposes may include administration of a cytokine, as otherwise disclosed herein. In some embodiments, the cytokine is part of a formulation or is co-administered with a formulation. In some embodiments, the cytokine may be a membrane-bound cytokine on the surface of the RIP or cell, and in an exemplary embodiment, may be a membrane-bound chemokine. Cells administered to a subject containing a membrane-bound cytokine typically result from de novo fusion of the cell with a RIP expressing the membrane-bound cytokine, rather than from expression of the membrane-bound cytokine in the cell. In exemplary embodiments, the cytokine or membrane-bound cytokine may comprise one or more of IL-1, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, TNFα, IFNγ, GM-CSF, CCL1, CCL2 (MCP-1), CCL3, CCL5, CCL7 (MCP-3), CCL8 (MCP-2), CCL19, CCL20, CCL21, CCL22, CCL28, CXCL1, CXCL9, CXCL10, CXCL11, CXCL12, CXCL14 (BRAK) or CX3CL1, or a variant of any of them, or an active fragment of any of them. In exemplary embodiments, the cytokine or membrane-bound cytokine, in exemplary embodiments the chemokine or membrane-bound chemokine, may be CCL1, CCL2 (MCP-1), CCL3, CCL5, CCL7 (MCP-3), CCL8 (MCP-2), CCL19, CCL20, CCL21, CCL22 or CCL28, or a variant of any of them, or an active fragment of any of them.

[0109] The methods provided herein, including, but not limited to, adoptive cell therapy, methods for generating sustained cell populations, and methods for delivering formulations (e.g., modified T cell and / or NK cell formulations, RIP formulations, and / or unmodified T cell and / or NK cell formulations), are particularly adapted for the treatment of cancer. Such cancers may be of any type. For example, such methods may be used to treat ovarian cancer, soft tissue sarcoma, peripheral T cell carcinoma, colorectal cancer, intrahepatic cholangiocarcinoma, glioblastoma, esophageal cancer, cutaneous T cell lymphoma, non-Hodgkin's lymphoma, urothelial carcinoma, basal cell carcinoma, epithelioid sarcoma, pancreatic cancer, non-small cell lung carcinoma, Hodgkin's lymphoma, renal cell carcinoma, mesothelioma, metastatic uveal melanoma, renal cancer, hematological cancer, HER2-expressing cancer, non-melanoma skin cancer, and liposarcoma. , hepatocellular carcinoma, small lymphocytic lymphoma, prostate cancer, breast cancer, anal cancer, marginal zone lymphoma, cutaneous squamous cell carcinoma, thyroid cancer, medullary thyroid cancer, triple-negative breast cancer, neuroendocrine system prostate cancer, bladder cancer, paraganglioma, medulloblastoma, superficial basal cell carcinoma, head and neck squamous cell carcinoma, hematological malignancies, melanoma, B-cell lymphoma, relapsed / refractory acute myeloid leukemia, angiosarcoma, osteosarcoma sarcoma), refractory cervical cancer, bile duct cancer, osteosarcoma, biliary tract cancer, castration-resistant prostate cancer, gastroesophageal adenocarcinoma, rhabdomyosarcoma, carcinoma, non-muscle-invasive bladder cancer, uveal melanoma, small cell lung cancer, cervical cancer, primary open-angle glaucoma, follicular lymphoma, synovial sarcoma, liver cancer, carcinosarcoma, leptomeningeal brain tumor, T-cell lymphoma, lymphoma, small cell lung cancer, mantle cell lymphoma, B-cell malignancies, endometrial cancer, myxoid / round cell liposarcoma, metastatic Merkel cell carcinoma, neuroblastoma The present invention can be used to treat subjects with, or tumors related to, chronic lymphocytic leukemia, tenosynovial giant cell tumor, sarcoma, acute myeloid leukemia, skin cancer, nasopharyngeal carcinoma, relapsed / refractory Ewing's sarcoma, bone cancer, glioma, salivary gland carcinoma, gastric cancer, benign tumors, low-grade serous ovarian cancer, metastatic breast cancer, multiple myeloma, diffuse large B-cell lymphoma, relapsed / refractory lymphoma, metastatic colorectal cancer, advanced malignancies, acute lymphoblastic leukemia, mesothelin-expressing solid tumors.

[0110] In some embodiments, the methods herein can be used to treat tumors that express any one or more of the tumor-associated and / or tumor-specific antigens presented herein, and engineered T cell receptors and CARs can be designed to recognize targets such as, for example, any of the tumor-associated and / or tumor-specific antigens otherwise disclosed herein. By way of non-limiting example, such tumor-associated or tumor-specific antigens include the hematological tumor antigens presented elsewhere herein, and in some non-limiting embodiments, the following antigens, most or all of which are believed to be associated with solid tumors: AXL, CD44v6, CAIX, CEA, CD133, c-Met, EGFR, EGFRvIII, Epcam, EphA2, GD2, GPC3, GUCY2C, HER1, HER2, ICAM-1, IL13Rα2, IL11Rα, Kras, Kras G12D, L1CAM, MAGE, MET, mesothelin, MUC1, MUC16 ecto, NKG2D, NY-ESO-1, PSCA, ROR-2, WT-1.

[0111] In some embodiments, any of the methods provided herein involving an administration step may be combined with the administration of another cancer therapy; in certain embodiments, the other cancer therapy may be, for example, a subcutaneously delivered cancer vaccine. In other embodiments, and optionally further combined with cancer vaccine administration, such methods provided herein involving the administration of genetically modified T cells and / or NK cells to a subject, particularly those in which the subject has, is suffering from, or is suspected of having cancer, may further include delivering to the subject an effective amount of an immune checkpoint inhibitor. This delivery of the checkpoint inhibitor may occur before, after, or simultaneously with the administration of the genetically modified T cells and / or NK cells. Immune checkpoint inhibitors are known, and various compounds have been approved and are in clinical development. Checkpoint molecules, many of which are targets of checkpoint inhibitor compounds, include, but are not limited to, anti-PD1 antibodies.

[0112] In some embodiments, the administration is for treating cancer in a subject, wherein a tumor in the subject regresses within 60 days, 45 days, 30 days, or 14 days of the administration. In some embodiments, the tumor is a blood cancer, such as, for example, DLBCL, and in exemplary examples, expresses any of the blood cancer antigens provided herein. In other embodiments, the tumor is a solid tumor that expresses a solid tumor antigen, and in certain exemplary embodiments, is a HER2-positive solid tumor, such as, but not limited to, breast cancer. In some embodiments, the administration is for treating cancer in a subject, wherein the subject achieves stable disease, at least partial remission, or complete remission, in exemplary embodiments, according to RECIST 1.1 criteria, within 90 days, 75 days, 60 days, 45 days, 30 days, or 14 days of the administration. In some embodiments, the tumor shrinks by at least 10%, 20%, 25%, 30%, 50%, or more. In some embodiments, a partial remission is achieved when the total tumor lesions have decreased by 30% or more, no new lesions have appeared at least four weeks after the last scan, and / or any pathological lymph nodes have decreased to less than 10 mm in the short axis. In some embodiments, a complete remission is achieved when all target and non-target lesions have disappeared. In some embodiments, the administration is for the purpose of treating cancer in a subject, where the subject achieves at least partial remission or complete remission within 60 days, 45 days, 30 days, or 14 days of the administration. In some embodiments, the subject is a human afflicted with cancer. In some embodiments, the cell preparation is administered two, three, four, five, six, or more times, or in exemplary embodiments, administered only once to the subject prior to stable disease, or in exemplary embodiments, until partial or complete remission is achieved. In some embodiments, the second formulation is administered to the subject at a second, third, fourth, etc. time point between 1 day and 1 month, 2 months, 3 months, 6 months, or 12 months after administration of the first cell formulation, in which case the second formulation may be the same as the first formulation or may comprise any of the formulations provided herein.

[0113] In any of the aspects provided herein, including intramuscular administration, and in exemplary embodiments, subcutaneous administration of RIP and / or modified and / or unmodified lymphocytes (e.g., modified T cells and / or NK cells), in certain embodiments, administration by any route provided herein, is performed to a mammalian subject that has undergone a lymphodepletion process, as known in the art. However, in exemplary embodiments, administration of the modified T cells and / or NK cells or RIP is performed in a manner that does not require lymphodepletion of the subject for the purposes of engraftment in the subject and / or for the purposes of reducing tumor volume in the subject, or is performed in a manner that is performed to a mammalian (e.g., human) subject that has not undergone lymphodepletion in the past few days, weeks, or months, or prior to such administration (e.g., subcutaneous administration). In certain embodiments, administration is performed to a mammalian (e.g., human) subject that does not suffer from low white blood cell counts, lymphopenia, or lymphocytopenia. In certain embodiments, subcutaneous administration is performed in subjects with lymphocyte counts in the normal range (i.e., 1,000 to 4,800 lymphocytes per microliter (μL) of blood). In certain embodiments, subcutaneous administration is performed in subjects with 1,000 to 5,000, more than 300, more than 500, more than 1,000, more than 1,500, or more than 2,000 lymphocytes per μL of blood. In certain embodiments, administration, e.g., subcutaneous administration, is carried out to a mammalian subject (e.g., a human subject) that has a lymphatic system, has an effective number of lymphocytes, has at least 50%, 60%, 75%, 80%, or 90% of the normal number of lymphocytes in a healthy human, and / or has more than 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, or 95% of its own lymphocytes prior to said administration.

[0114] RIP preparations, cell preparations, and methods of use thereof For example, recombinant viral particle preparations are disclosed in the methods and compositions provided herein for modifying, by way of non-limiting example, human cells, primary cells, T cells, and / or NK cells to generate genetically modified and / or transduced human cells, primary cells, T cells, and / or NK cells. Recombinant viral particles and their preparations are themselves aspects of the present disclosure. Typically, the recombinant viral particles included in the aspects provided herein are recombinant retroviral particles and are replication-incompetent. This means that the recombinant retroviral particles are incapable of replicating once they leave the packaging cell, i.e., they are unable to replicate in a subject, for example, when administered to a subject. In practice, unless otherwise indicated herein, retroviral particles are replication-incompetent, and if such retroviral particles contain nucleic acids in their genome that are not native to the retrovirus, they are "recombinant retroviral particles." In an exemplary embodiment, the recombinant retroviral particle is a lentiviral particle. Replication-incompetent recombinant retroviral particles are referred to herein as RIPs, and preparations containing RIPs may be referred to as RIP preparations. Those skilled in the art will appreciate how the various aspects and embodiments disclosed herein may be modified for other viral and retroviral particles or for non-viral recombinant vectors. For example, provided herein are cell preparations comprising T cells and / or NK cells. Such preparations, in exemplary embodiments, are provided by the methods provided herein. Any of the cell preparations provided herein may, in a non-limiting example, comprise autologous CAR-T cells. In one aspect, provided herein are cell preparations comprising a population of autologous CAR-T cells, such as modified, genetically modified, transfected, transfected, and / or stably integrated autologous CAR-T cells in a delivery solution.

[0115] In some embodiments, the delivery solution, RIP formulation, or cell formulation is compatible with, effective against, or adapted for perilymphatic, subcutaneous, or intramuscular delivery, maintaining the cells in a localized aggregated state and allowing for controlled release of the cells into the circulation. For example, in some embodiments, the concentration of unmodified or modified cells in a delivery solution or formulation intended for perilymphatic, subcutaneous, or intramuscular delivery is higher than the concentration typically delivered intravenously. In some embodiments, the concentration of leukocytes in a delivery solution or formulation intended for perilymphatic, subcutaneous, or intramuscular delivery is about 1.5×10 8 cells / ml, approximately 5 x 10 8 cells / ml, approximately 1 x 10 9 cells / ml ~ 1.2 x 10 9 greater than cells / ml.

[0116] In exemplary embodiments, cells, e.g., a mixture of modified and unmodified lymphocytes as described herein, or unmodified cells alone, or in other embodiments, viral particles (e.g., RIPs), are formulated in a delivery solution or cell preparation such that they are capable of, effective in, and compatible with perilymphatic, intralymphatic, subcutaneous, or intramuscular administration. Indeed, certain embodiments of the commercial container and kit aspects provided herein are or include sterile perilymphatic, intralymphatic, subcutaneous, and / or intramuscular delivery solution containers, which in some embodiments are refrigerated. Such delivery solutions are capable of, effective in, and compatible with perilymphatic, subcutaneous, intralymphatic, or intramuscular administration, and in exemplary embodiments, subcutaneous administration.

[0117] To achieve this, such delivery solutions and / or formulations, e.g., RIP formulations and cell formulations, typically have a pH and ionic composition that provides an environment in which the administered RIP retains its transduction capacity (i.e., is effective, compatible, or adapted to transduce cells, in exemplary embodiments, NK cells and / or T cells, in vivo) and / or an environment in which the administered cells (e.g., NK cells and / or T cells) can survive until administration, e.g., for at least 1 hour, typically at least 4 hours. Such a pH is typically between 6.5 and 8.0, or between 7.0 and 8.0, or between 7.2 and 7.6, and may be maintained by a buffer, such as a phosphate buffer or bicarbonate, present at a concentration effective to maintain the pH within the target range. In any of the aspects and embodiments of the delivery solutions and / or formulations disclosed herein, the delivery solutions and / or formulations may include one or more of any of the buffers or salts, including concentrations, disclosed in the exemplary embodiments section, such as PBS, HBSS, saline, Ringer's lactate solution, Plasma-Lyte, etc. In any of the aspects and embodiments of the delivery solutions and / or formulations disclosed herein, the delivery solutions and / or formulations may include one or more, e.g., two or more, three or more, four or more, or five or more, of any of the other components, including concentrations, disclosed in the exemplary embodiments section, such as DMSO, human serum albumin (HSA), colloids (e.g., dextran (40 kDa to 2 MDa), hetastarch, albumin, PEG (5 kDa to 100 kDa), sugars (e.g., dextrose, lactose, sucrose, or trehalose), etc.

[0118] In some embodiments, the delivery solution and / or formulation is or includes a multi-electrolyte solution. For example, the delivery solution may be or include a sterile, non-pyrogenic, isotonic solution in a container, such as a single-dose container. In certain embodiments, such solutions are suitable for or adapted for intravenous or intraperitoneal administration, as well as perilymphatic, subcutaneous, and / or intramuscular administration. In any of the aspects and embodiments of the delivery solution and / or formulation disclosed herein, the delivery solution and / or formulation may include any of the multi-electrolyte solutions, including the concentrations disclosed in the exemplary embodiments section; for example, a multi-electrolyte injection solution may be Plasma-Lyte A Injection pH 7.4, available from various vendors.

[0119] In some embodiments, the delivery solution and / or formulation is frozen and then thawed and administered to a subject. In some embodiments, the delivery solution and / or formulation may be stored below 0, -15, or -70°C for a certain number of days. In any of the aspects and embodiments of the delivery solution and / or formulation disclosed herein, the delivery solution and / or formulation may include any of the frozen storage temperatures and frozen storage times disclosed in the exemplary embodiments section. In some embodiments, the delivery solution and / or formulation may be frozen for 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 5, 6, 9, or 12 months, or indefinitely, and then administered, or in the exemplary embodiments of cell-containing cell formulations and delivery solutions, re-administered back to the subject. During the period that the RIP or cells are frozen, or at any time prior to administration to a subject, or prior to readministration back to a subject, the RIP and / or cells may be tested for various quality control properties as disclosed elsewhere herein, e.g., viral concentration, purity, and / or potency, and / or the RIP may be tested for one or more cell and / or gene therapy quality control tests.

[0120] In other embodiments, the delivery solution and / or formulation is not frozen and is then administered to the subject. In some embodiments, the delivery solution and / or formulation may be stored at 2-8°C for a certain number of days. In any of the aspects and embodiments of the delivery solution and / or formulation disclosed herein, the delivery solution and / or formulation may include any of the non-frozen storage temperatures and non-frozen storage times disclosed in the exemplary embodiments section.

[0121] In some embodiments, the delivery solution and / or formulation includes DMSO. In an exemplary embodiment, the delivery solution and / or formulation may include about 6% DMSO (v / v). In some embodiments, the delivery solution and / or formulation does not contain DMSO. In any of the aspects and embodiments of the delivery solution and / or formulation disclosed herein, the delivery solution and / or formulation may include any of the DMSO concentrations disclosed in the exemplary embodiments section.

[0122] In some embodiments, the delivery solution and / or formulation comprises a colloid. In some embodiments, the delivery solution and / or formulation comprises dextran (40 kDa to 2,000 kDa, or 40 kDa to 2×10 6 The delivery solution and / or formulation may include one or more of the following: cellulose (cellulose acetate), hetastarch, albumin, and PEG (5 kDa-100 kDa). In some embodiments, the delivery solution and / or formulation includes human serum albumin (HSA). In exemplary embodiments, the delivery solution and / or formulation may include 2.5%-7.5% HSA (w / v) (e.g., 25-75 mg / ml). In some embodiments, the delivery solution or formulation does not include HSA. In any of the aspects and embodiments of the delivery solution and / or formulation disclosed herein, the delivery solution and / or formulation may include any of the colloids and respective concentrations disclosed in the exemplary embodiments section.

[0123] In some embodiments, the delivery solution and / or formulation comprises 1%-10% DMSO and 0.20%-5% HSA. In further exemplary embodiments, the delivery solution and / or formulation comprises 2%-8%, 3%-7%, or 4.5%-8% DMSO and 0.25%-7.5%, 0.25%-6%, or 0.25%-5% HSA. In other exemplary embodiments, the delivery solution and / or formulation comprises 5%-7.5% DMSO and 4%-6% HSA.

[0124] In some embodiments, the delivery solution and / or formulation includes a sugar. In some embodiments, the delivery solution and / or formulation may include one or more of dextrose, lactose, trehalose, and / or sucrose. In some embodiments, the delivery solution and / or formulation may include 3-7% dextrose (w / v) (e.g., 30-70 mg / ml). In some embodiments, the delivery solution and / or formulation may include 2-8% lactose. In some embodiments, the delivery solution and / or formulation may include 2-8% sucrose. In some embodiments, the delivery solution and / or formulation may include 2-8% trehalose. In any of the aspects and embodiments of the delivery solution and / or formulation disclosed herein, the delivery solution and / or formulation may include any of the sugars and respective concentrations disclosed in the Exemplary Embodiments section.

[0125] Other components that may be included in the delivery solution and / or formulation are disclosed in more detail herein and in the exemplary embodiments and may be delivered in the same delivery solution and / or formulation or in different delivery solutions and / or formulations, for example, in a delivery solution and a RIP formulation, or in a first delivery solution and a second delivery solution. Furthermore, these other components may be delivered together with the delivery solution and / or formulation, or may be delivered several days (e.g., 1, 2, 3, 4, 5, 6, or 7 days), several weeks (e.g., 1, 2, 4, or 4 weeks), or several months (e.g., 1, 2, 3, 6, 12, or 24 months) before or after the first delivery solution and / or formulation. Moreover, persistence of genetically modified CAR-T cells near the site of subcutaneous administration further illustrates the advantage of certain embodiments provided herein, where subcutaneous administration is performed near the site of neoplastic (e.g., cancerous) cells, e.g., a tumor, or near (e.g., within 1, 2, 3, 4, 5, 10, 20, or 30 cm) organs containing the tumor, including, e.g., the spleen or lymph nodes in the case of blood cancers.

[0126] In some embodiments of any of the delivery solutions and / or formulations provided herein, the delivery solution and / or formulation may be substantially free of bovine proteins, as disclosed in exemplary embodiments herein. In some embodiments of any of the delivery solutions and / or formulations provided herein, the delivery solution and / or formulation may be substantially free of non-human and non-viral proteins, as disclosed in exemplary embodiments herein.

[0127] The purity of a RIP in a delivery solution or RIP formulation may be determined using the ratio of the amount of protein from the host cells used to produce the RIP to the transducing units (amount of host cell protein / TU). In some embodiments, the ratio of host cell protein to TU may be 10, 5, 3, 2, or 1 ng or less of host cell protein / TU, or may be 750, 500, 400, 300, 200, 100, 50, 40, 30, 20, or 10 pg or less of host cell protein / TU. In any of the aspects and embodiments herein that produce a RIP, the host cell used to produce the RIP may be a human cell. In some embodiments, the host cell may be a primary cell. In some embodiments, the host cell may be an immortalized cell. In some embodiments, the host cell may be a HEK, HEK-293, HEK-293T, HEK-293E, HEK-293FT, HEK-293S, HEK-293SG, HEK-293FTM, HEK-293SGGD, HEK-293A, 293RTV, GP2-293, MDCK, C127, COS-7, A549, HeLa, CHO, mouse myeloma, PerC6, 91-1, or Vero cell. In exemplary embodiments, the host cell is a HEK, HEK293, HEK293T, HEK293A, PerC6, or 91-1.

[0128] The potency of the RIP present in the delivery solution or RIP formulation can be determined using the ratio of TU to p24 protein (in ng), which in some embodiments may be 100, 200, 300, 400, 500, 1,000, 4,000, 10,000, 12,500, or 15,000 or more TU / ng of p24 protein.

[0129] In some embodiments, the concentration of the RIP present in the delivery solution and / or RIP formulation is at least 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 2 × 108 , 5×10 8 , or 1 × 10 9 In some embodiments, the concentration of the RIP present in the delivery solution and / or in the RIP formulation may be any of the concentrations disclosed in the Exemplary Embodiments section herein.

[0130] In some embodiments, such as those in which the sample does not undergo a PBMC isolation or granulocyte depletion procedure, at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the neutrophils, basophils, and / or eosinophils present in the blood sample subjected to the modified methods herein or in the blood sample co-administered with the RIP formulation as unmodified cells (i.e., in this case, the blood has not previously been contacted with the RIP) are present in the cell preparation, including at the time of the optional delivery (i.e., administration) step. In some embodiments, such as those in which the sample does not undergo a B cell depletion procedure, at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the B cells present in the blood sample subjected to the modified methods herein or in the blood sample co-administered with the RIP formulation as unmodified cells are present in the cell preparation, including at the time of the optional delivery step. In some embodiments, such as those in which the sample does not undergo a monocyte depletion procedure, at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the monocytes present in the blood sample subjected to the modification methods herein, or in the blood sample co-administered with the RIP formulation as unmodified cells, are present in the cell formulation, including at the time of the optional delivery step.

[0131] In some embodiments, and in exemplary embodiments in which the cell preparation is administered subcutaneously or intramuscularly, the volume of the cell preparation comprising modified and / or unmodified lymphocytes is smaller than conventional CAR-T methods, which are typically infusion delivery methods, and may be less than about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 10 mL, about 15 mL, about 20 mL, or about 25 mL.

[0132] A short time between the withdrawal (collection) of blood and the reintroduction of unmodified or modified lymphocytes into the subject is beneficial, meaning that in some embodiments, some lymphocytes are associated with a recombinant nucleic acid vector, in exemplary embodiments, associated with a replication-incompetent recombinant retroviral particle, and have not yet been genetically modified. In some embodiments, at least 5% of the modified lymphocytes are not genetically modified. In some embodiments, the modified lymphocytes are genetically modified and contain a polynucleotide that is extrachromosomal or integrated into the genome. In some embodiments, the polynucleotide may be extrachromosomal in at least 5% of the modified lymphocytes. In some embodiments, at least 5% of the modified lymphocytes are not transduced. In some embodiments, including co-administration with a RIP in a RIP formulation or delivery solution, the lymphocytes are not contacted with a RIP disclosed herein, and therefore, none of the lymphocytes are modified, genetically modified, or transduced with the RIP.

[0133] Short contact times in certain embodiments also result in many of the engineered lymphocytes in the cell preparations herein having on their surface binding polypeptides, fusogenic polypeptides, and, in some embodiments, T cell activation elements generated on the surface of the retroviral particle via association with the recombinant retroviral particle or by fusion of the retroviral envelope with the plasma membrane, including at the time of the optional delivery step. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the engineered lymphocytes in the cell preparation comprise a pseudotyping element and / or a T cell activation element, e.g., a T cell activating antibody. In some embodiments, the pseudotyping element and / or T cell activation element can be bound to the surface of the engineered lymphocyte, e.g., via a T cell receptor, CD28, OX40, 4-1BB, ICOS, CD9, CD53, CD63, CD81, or CD82, and / or the pseudotyping element and / or T cell activation element can be present in the plasma membrane of the engineered lymphocyte. In some embodiments, including RIP formulations or co-administration with RIPs in a delivery solution, the lymphocytes are not contacted with a RIP disclosed herein; therefore, none of the lymphocytes contain pseudotyping elements or T cell activation elements bound to their surface; therefore, none of the lymphocytes are engineered, genetically modified, or transduced lymphocytes. In some aspects, provided herein are cell preparations, e.g., comprising T cells and / or NK cells. Such preparations, in exemplary embodiments, are provided by the methods provided herein. Any of the cell preparations provided herein may contain autologous CAR-T cells.In one embodiment, provided herein are cell preparations comprising populations of unmodified, modified, genetically modified, transfected, transfected, and / or stably integrated T cells and / or NK cells, and in a non-limiting example, provided herein are cell preparations comprising autologous CAR-T cells and / or NK cells in a delivery solution.

[0134] For an advantageously short period of time, lymphocytes are contacted with the recombinant nucleic acid vector, and the modified lymphocytes are ex vivo after such contact in some exemplary embodiments provided herein, in which some or all of the T and NK cells have not yet expressed the recombinant nucleic acid or have not yet integrated the recombinant nucleic acid into their genomes; in these embodiments, some of the retroviral particles may be associated with, but not fused to, the target cell membrane before being used or contained in any of the methods or compositions provided herein, including, but not limited to, being introduced or reintroduced back into a subject, or before being used to prepare a cell preparation. Thus, various aspects and embodiments of cell preparations are provided herein that can be produced from these exemplary methods, such as, for example, rapid point-of-care methods involving subcutaneous administration in exemplary embodiments. Such cell preparations, including, but not limited to, those described immediately below and in the exemplary embodiments section, may be present at the time of collection of the cells after they have been contacted with the recombinant retroviral vector and optionally rinsed, and may be present up to (and including) the time of administration to a subject, in exemplary embodiments, subcutaneously.

[0135] In some embodiments, provided herein are cell preparations comprising T cells and / or NK cells, wherein less than 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 10%, or 5% of the cells in the cell preparation are T cells and / or NK cells. In some embodiments, none of the cells have been contacted with a RIP disclosed herein, and therefore none of the cells have been modified, genetically modified, or transduced. In some embodiments, cell preparations are provided that include lymphocytes, NK cells, and / or T cells, wherein at least 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the lymphocytes, NK cells, and / or in exemplary embodiments, T cells in the cell preparation are modified cells, e.g., modified with a polynucleotide comprising a nucleic acid encoding an anti-idiotype polypeptide provided herein. Such polynucleotides may optionally encode a CAR, TCR, inhibitory RNA, or LE provided herein. In some embodiments, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and 70% of the lower end of the range of lymphocytes are modified, and 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% of the upper end of the range of lymphocytes are modified cells, e.g., 5%-95%, 10%-90%, 25%-75%, and 25%-95%. In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the modified lymphocytes in the cell preparation are not genetically modified, transduced, or stably transfected.In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and 70% of the lower end of the range of modified lymphocytes are not genetically modified, transduced, or stably transfected, and 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% or all of the upper end of the range of modified lymphocytes are not genetically modified, transduced, or stably transfected, e.g., 5%-95%, 10%-90%, 25%-75%, and 25%-95%. In some embodiments, the polynucleotides of the genetically modified lymphocytes may be extrachromosomal or integrated into the genome in these cell preparations formed after contacting and incubation, and optionally at the time of administration. In some embodiments of these cell preparations, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the genetically modified lymphocytes have extrachromosomal polynucleotides. In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and 70% of the lower end of the range of engineered or genetically modified lymphocytes have extrachromosomal polynucleotides, and 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% or all of the upper end of the range of engineered or genetically modified lymphocytes have extrachromosomal polynucleotides, e.g., 5%-95%, 10%-90%, 25%-75%, and 25%-95%.In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the modified or genetically modified lymphocytes are not transduced or stably transfected in these cell preparations, e.g., as a result of the methods for genetically modifying T cells and / or NK cells provided herein. In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and 70% of the lower end of the range of engineered or genetically modified lymphocytes are not transduced, and 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% or all of the upper end of the range of engineered or genetically modified lymphocytes are not transduced or stably transfected, e.g., 5%-95%, 10%-90%, 25%-75%, and 25%-95%.

[0136] In certain embodiments disclosed herein, including subcutaneous delivery of a solution and cell formulations adapted for subcutaneous delivery, fewer modified or genetically modified lymphocytes are able to engraft when delivered intravenously compared to when delivered subcutaneously, hi some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% fewer lymphocytes engraft when delivered intravenously compared to when delivered subcutaneously.

[0137] In some embodiments, cell preparations (including such preparations present at the time of collection of the cells after the cells have been contacted with a recombinant retroviral vector and optionally rinsed, and present up to and including the time of administration to a subject) comprise at least two of unmodified lymphocytes, modified lymphocytes, and genetically modified lymphocytes. In some embodiments, such cell preparations comprise more unmodified lymphocytes than modified lymphocytes. In some embodiments of such cell preparations produced by the methods provided herein, the percentage of modified, genetically modified, transduced, and / or stably transfected T cells and NK cells is at least 5%, at least 10%, at least 15%, or at least 20%. As illustrated in the Examples herein, in exemplary methods for transducing lymphocytes in whole blood, lymphocytes in the whole blood added to or used to create the reaction mixture, in some embodiments, 1% to 20%, or 5% to 20%, or 1% to 15%, or 5% to 15%, or 7% to 12%, or about 10%, of the T cells and / or NK cells, are genetically modified and / or transduced and present in the resulting cell preparation. In some embodiments, the lymphocytes are not contacted with a recombinant nucleic acid vector, such as a replication-incompetent recombinant retroviral particle, and are unmodified. In certain exemplary embodiments, the lymphocytes are tumor-infiltrating lymphocytes. In some embodiments, the lymphocytes are tumor-infiltrating lymphocytes before or after contacting the tumor-infiltrating lymphocytes with a recombinant nucleic acid vector. In some embodiments, the lymphocytes include both tumor-infiltrating lymphocytes and T cells and / or NK cells before or after contacting the T cells and / or NK cells with a recombinant nucleic acid vector.

[0138] In some embodiments, cell preparations are provided herein wherein at least 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the engineered T and / or NK cells in the cell preparation do not express a CAR, or in certain embodiments, a transposase, and / or do not have a CAR associated with their cell membrane. In other embodiments, cell preparations are provided herein wherein at least 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the engineered T and / or NK cells in the cell preparation contain a recombinant viral reverse transcriptase or integrase. Without being limited by theory, unlike conventional CAR-T cell treatment methods in which cells are cultured ex vivo for days or weeks, and many cell divisions, in the exemplary methods provided herein in which T cells and / or NK cells are contacted with retroviral particles for modifying T cells and / or NK cells within hours of delivery, some or most of the reverse transcriptase and integrase present in the retroviral particles that transfer into the T cells and / or NK cells after fusing with the retroviral particles are still present in the modified T cells and / or NK cells at the time of delivery. In some embodiments, cell preparations are provided herein in which at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the modified T and NK cells in the cell preparation do not express recombinant mRNA (e.g., encoding CAR and / or recombinant transposase). In some embodiments, cell preparations provided herein, wherein at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the modified T and NK cells in the cell preparation do not have a recombinant nucleic acid stably integrated into their genome.In some embodiments, more than 50%, 60%, 70%, 75%, 80%, or 90% of the cells, NK cells, and / or T cells in the cell preparation are viable.

[0139] In further embodiments, cell preparations containing modified lymphocytes that can be introduced or reintroduced in the methods herein include monocytes and / or B cells. In some embodiments, a portion of the B cells are modified during the contacting step with a recombinant nucleic acid vector, e.g., a naked DNA vector, or, in exemplary embodiments, replication-incompetent recombinant retrovirus particles. In some embodiments, at least a portion of the B cells, but not more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, are modified in the cell preparations that can be optionally administered or re-administered. In exemplary embodiments, a portion of the B cells is not modified in such preparations and methods. In further exemplary embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the B cells are unmodified in such formulations and methods. Thus, in some embodiments, modified lymphocytes are present in a cell preparation along with unmodified lymphocytes, which is optionally delivered intramuscularly or subcutaneously to the subject. In some embodiments, the modified lymphocytes present in the cell preparation and optionally introduced into the subject may be allogeneic lymphocytes. In such embodiments, the lymphocytes are derived from another human, and the lymphocytes derived from the subject are unmodified. In some embodiments, blood is not drawn from the subject to collect the lymphocytes.

[0140] In exemplary embodiments, neutrophils are present in a cell preparation, such as, by way of non-limiting example, a cell preparation for subcutaneous delivery of modified T cells and / or NK cells, at a concentration that is too high for intravenous delivery when considering the safety of the subject to whom the cell preparation is administered. Without being limited by theory, and as discussed elsewhere herein, intravenous injection or delivery of neutrophils can result in lung damage, e.g., as a result of transfusion-related acute lung injury (TRALI) and / or acute respiratory distress syndrome (ARDS). For example, this situation can arise when a method for producing modified lymphocytes does not involve a PBMC enrichment step before a cell preparation containing the modified lymphocytes is prepared, and before the solution is optionally delivered subcutaneously to a subject. Thus, in some embodiments, neutrophils are present in the cell preparation, e.g., at the time of the optional delivery step. More specifically, in some embodiments, at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the neutrophils present in the blood sample subjected to the modifying methods herein are present in the cell preparation, including at the time of the optional delivery step. In some embodiments, at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 75% of the cells present in the cell preparation, including at the time of the optional delivery step, are neutrophils. In some embodiments, at the lower end of the range of cells present in the cell preparation, including at the time of the optional delivery step, 5%, 10%, 15%, 20%, 25%, 30%, or 40% are neutrophils, and at the upper end of the range of cells present in the cell preparation, 30%, 40%, 50%, 60%, 70%, or 75% are neutrophils, e.g., at the time of the optional delivery step, 5% to 50%, 20% to 50%, 30% to 75%, or 50% to 75% of the cells present in the cell preparation are neutrophils.

[0141] In some embodiments, at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the monocytes present in the blood sample subjected to the modifying methods herein are present in the cell preparation, including at the time of the optional delivery step. In some embodiments, at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the B cells present in the blood sample subjected to the modifying methods herein are present in the resulting cell preparation, including at the time of the optional delivery step. In some embodiments, the cell preparation may comprise a PBMC fraction containing modified T and NK cells. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, 85%, 90%, or 95%, or 1% to 95%, 5% to 95%, 5% to 50%, or 10% to 50% of the modified T and NK cells in the cell preparation are genetically modified.

[0142] The volume of the administered delivery solution, RIP formulation, or cell preparation, or other solution varies depending on the route of administration, as described elsewhere herein. Delivery solutions, RIP formulations, or cell preparations injected perilymphatic, subcutaneously, or intramuscularly typically have a smaller volume than those delivered via infusion. In some embodiments, the volume of the delivery solution, RIP formulation, or cell preparation is 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 15 ml, 20 ml, 25 ml, 30 ml, 35 ml, 40 ml, 45 ml, or 50 ml or less. In some embodiments, the volume of the delivery solution, RIP formulation, or cell formulation may be between a lower range of 0.20 mL, 0.25 mL, 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, 15 mL, 20 mL, or 25 mL and an upper range of 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 75 mL, 100 mL, 125 mL, 250 mL, 500 mL, or 1000 mL. Thus, by way of non-limiting example, the volume may be between 0.2 mL and 10 mL, 0.5 mL and 10 mL, 0.5 mL and 2 mL, 1 mL and 250 mL, 1 mL and 100 mL, 10 mL and 100 mL, or 1 mL and 10 mL. In certain exemplary embodiments, the delivery solution, RIP formulation, or cell formulation may be less than 10 mL, may be between 1 mL and 25 mL, and in exemplary embodiments, may be between 1 mL and 3 mL, 1 mL and 5 mL, or 1 mL and 10 mL. In exemplary embodiments, the delivery volume, RIP formulation, or cell formulation volume may be between 0.20 mL, 0.25 mL, 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL at the lower end of the range and 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, and 50 mL at the upper end of the range. In an exemplary embodiment of a cell formulation, a 70 kg subject will receive 7.0 x 10 7 1.0 x 10 cells / mL by subcutaneous administration of 1 mL of T cell delivery formulation 6The delivery solution, RIP formulation, or cell formulation is administered at a volume of at least 2, 3, 4, 5, 10, 15, 20, or 25 mL. In some embodiments, the delivery solution, RIP formulation, or cell formulation may contain hyaluronidase when the volume of the solution is at least 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, 15 mL, 20 mL, or 25 mL. In embodiments herein in which lymphocytes are specifically modified and then filtered, and / or in which transduction is specifically performed on a filter, the delivery solution can be used to resuspend and / or elute the cells from the filter in a volume that may be one of those provided above. Thus, in some embodiments, the delivery solution provided herein is an elution solution.

[0143] In some embodiments, unmodified lymphocytes, modified lymphocytes, and in exemplary embodiments, genetically modified lymphocytes are introduced or reintroduced into a subject by intradermal, intratumoral, or intramuscular administration, in exemplary embodiments, perilymphatic or subcutaneous administration using a cell preparation present in a subcutaneous delivery device, such as a sterile syringe adapted to deliver a solution subcutaneously. In some embodiments, a subcutaneous delivery device is used that holds a solution (e.g., a delivery solution, RIP formulation, or cell preparation herein), and in exemplary embodiments, has an opening or openable end that is the open end of a needle for subcutaneous administration of the solution (e.g., a delivery solution, RIP formulation, or cell preparation) from the liquid-retaining portion of the device. Such a subcutaneous delivery device is effective for subcutaneous delivery, and in exemplary embodiments, is adapted for subcutaneous delivery, or is effective for or adapted for subcutaneous injection. Non-limiting examples of subcutaneous delivery devices adapted to deliver solutions subcutaneously include indwelling subcutaneous catheters, such as, for example, Insuflon® (Becton Dickinson), and subcutaneous catheters, such as, for example, winged, needleless, closed-type indwelling subcutaneous catheter systems, such as, for example, Saf-T-Intima® (Becton Dickinson). In some embodiments, the delivery device may include a pump, such as an infusion pump or a peristaltic pump. In some embodiments, the delivery solution, RIP formulation, or cell formulation is fluidly connected to any of the needles disclosed herein, e.g., a needle compatible with, effective for, adapted for, or adapted to deliver subcutaneously, or effective for subcutaneous delivery. In exemplary embodiments, the needle may have a gauge of 26-30. In some embodiments, the subcutaneous delivery device is a subcutaneous delivery pen. Such a pen may include a syringe effective for, or adapted for, subcutaneous delivery enclosed within a housing and may include a needle guard. Examples of such pens include those used to deliver sumatriptan.In some embodiments, the delivery solution, RIP formulation, or cell formulation is present in a subcutaneous delivery device, such as a syringe, and a needle is inserted into the subject's skin, where the RIP and / or unmodified and modified T cells and / or NK cells are present in the syringe (i.e., the subject receiving the subcutaneous injection is the source of the RIP or autologous cells to be injected), and in some embodiments, the needle is placed with its open end in the subject's subcutaneous tissue. In exemplary embodiments, the subcutaneous delivery device (e.g., a syringe) may include a needle suitable for subcutaneous administration. Subcutaneous administration typically uses a needle with a smaller diameter than those used in intravenous catheters for blood transfusions, which can be, for example, a 16-gauge needle. A delivery device, such as a syringe, adapted for intramuscular, and in exemplary embodiments, subcutaneous, delivery is any delivery device (e.g., syringe) that can be successfully used for intramuscular or subcutaneous delivery, including delivery devices (e.g., syringes) effective for and adapted for intramuscular or subcutaneous delivery, as well as general-purpose syringes and, at least in some embodiments, syringes specifically designed for other purposes that can be successfully used for intramuscular or subcutaneous delivery. As is known, for subcutaneous injections, in exemplary embodiments using a syringe, the needle is inserted through the skin at a 45° to 90° angle. Thus, some embodiments include subcutaneously injecting a delivery solution, RIP formulation, or cell formulation at a 45° to 90° angle relative to the skin, as well as including a delivery solution, RIP formulation, or cell formulation contained within a syringe or other subcutaneous delivery device whose needle is at a 45° to 90° angle relative to the subject's skin. Syringes effective for intramuscular, and in exemplary embodiments, subcutaneous, delivery or effective for intramuscular or subcutaneous injection are typically syringes having parameters effective for intramuscular or subcutaneous delivery, e.g., needles having a 20-22 gauge and a length of 1 inch to 1.5 inches are typically effective for intramuscular delivery, and needles having a 26-30 gauge and a length of 0.5 inches to 0.625 inches are typically effective for subcutaneous delivery. A syringe adapted for subcutaneous delivery or adapted to inject subcutaneously is any syringe made specifically for subcutaneous delivery.One such syringe adapted for subcutaneous delivery uses a core annular flow, enabling subcutaneous delivery of highly concentrated biologics that are not normally deliverable subcutaneously (Jayaprakash V et al. Adv Healthc Mater. 2020 Aug 24; e2001022). Another syringe adapted for subcutaneous delivery uses a shorter needle than commonly used (Pager A, Expert Opin Drug Deliv. 2020 Aug 9; 1-14). Another syringe adapted for subcutaneous delivery uses a 29G / 5 bevel needle with a Thermo Plastic Elastomer (TPE) needle shield (Jaber A et al. BMC Neurol. 2008 Oct 10; 8:38). In exemplary embodiments, the outer diameter of the needle is less than 0.026". In some embodiments, the outer diameter of the needle is at most 0.01625", 0.01865", 0.01825", 0.02025", 0.02255", or 0.02525". In some embodiments, the needle is a 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 26, 27, 28, 29, or 30 gauge needle. In some embodiments, the needle is 1 inch or 0.5 inches long or less. In exemplary embodiments, the needle is a 26, 26s, 27, 28, 29, or 30 gauge needle and the needle is 0.5 inches to 0.625 inches long. In some embodiments, the needle may be a winged infusion set, also known as a butterfly needle or scalp vein needle. In some embodiments, the introduction or reintroduction may be performed using a subcutaneous catheter.

[0144] Without being limited by theory, in contrast to intravenous delivery, in which components of the delivery solution, RIP formulation, or cell formulation disperse rapidly, the subcutaneous and intramuscular delivery methods provided herein maintain the cells of the delivery solution, RIP formulation, or cell formulation in close proximity within the subject, e.g., in exemplary embodiments, for up to several days, weeks, or even months as a controlled release, creating a local environment for T cell and / or NK cell activation and expansion while maintaining an environment similar to that faced by T cells and NK cells in lymphoid organs, e.g., the spleen or lymph nodes. Large protein molecules, e.g., antibodies, greater than 20 kDa, are absorbed through the subcutaneous site and then absorbed into the bloodstream over a 24-72 hour period via lymphatic vessels. The controlled release of modified, genetically modified, and / or transduced T cells or NK cells from cell preparations injected at a local injection site using the perilymphatic, subcutaneous, or intramuscular methods provided herein has been found to involve an initial expansion phase at the injection site, after which at least a portion, typically a majority, of the modified cells migrate through the blood and lymphatic vessels to the target site of expression, e.g., a tumor, and then become detectable throughout the body. RIPs derived from RIP preparations injected at a local injection site using the perilymphatic, subcutaneous, or intramuscular methods provided herein can transduce T cells and NK cells present in a subject, or, when co-administered with PBMCs, e.g., T cells and / or NK cells, isolated from a subject, can transduce the co-administered T cells and / or NK cells. In some embodiments, controlled release local injection of modified, genetically modified, and / or transduced cells (either from a cell formulation or from a RIP formulation and then modified) allows expansion of the genetically modified cells at the site of subcutaneous administration for several days (e.g., up to 5, 7, 14, 17, 21, or 28 days) or months (e.g., up to 1, 2, 3, 6, 12, or 24 months), and migration of the genetically modified CAR-T or CAR-NK cells from the subcutaneous administration site to other sites in the body, e.g., tumors.Thus, the genetically modified CAR-T cells may migrate from the subcutaneous administration site and appear in the lymphatics or circulation several days (e.g., 1, 2, 3, 4, 5, 6, or 7 days), weeks (e.g., 1, 2, 4, or 4 weeks), or even months (e.g., 1, 2, 3, 6, 12, or 24 months) after the modified T cells and / or NK cells are subcutaneously injected into a subject.

[0145] The subcutaneous persistence of genetically modified T cells, e.g., CAR-T cells, and / or NK cells provides a beneficial local environment, allowing other components, natural or unnatural to the subject, such as molecules (ions), macromolecules (e.g., DNA, RNA, peptides, and polypeptides), and / or other cells that may affect the modified CAR-T cells, to be recruited to or near the delivery site of the modified CAR-T cells or delivered subcutaneously. Indeed, after delivery of modified T cells and / or NK cells, lymphatic tertiary structures, including lymphatic vasculature, have been observed. Without being limited by theory, it is believed that such lymphatic vasculature provides a venue for subcutaneously administered modified T cells and / or NK cells to access the local lymphatic circulation, after which the cells gain access to the systemic circulation, e.g., blood. Such tertiary lymphatic structures have been observed to contain activated lymphocytes. Accordingly, provided herein are lymphatic structures, including aggregates of actively dividing genetically modified T cells and / or NK cells, and lymphatic vasculature in close proximity to the aggregates. In some embodiments, the tertiary lymphoid structures and / or genetically modified CAR-T cells can persist near the site of subcutaneous administration for at least 1, 2, 3, 4, 5, 6, or 7 days, 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 months. In exemplary embodiments, the tertiary lymphoid structures and / or genetically modified CAR-T cells can persist near the site of subcutaneous administration for at least 1, 2, 3, 4, 5, 6, or 7 days, 1, 2, 3, or 4 weeks, or 1, 2, or 3 months. In some embodiments, the tertiary lymphoid structures and / or genetically modified CAR-T cells can persist near the site of subcutaneous administration for 1 day to 24 months, 7 days to 12 months, 2 weeks to 6 months, 3 weeks to 8 weeks, or 4 weeks to 6 weeks. In exemplary embodiments, in some embodiments, the tertiary lymphoid structures and / or genetically modified CAR-T cells can persist near the subcutaneous administration site for 1 week to 3, 4, 5, 6, 7, 8, 9, or 10 weeks, e.g., 1 week to 8 weeks, 1 week to 7 weeks, or 1 week to 6 weeks.In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the cells of the tertiary lymphoid structures and / or genetically modified cells can remain localized within 1, 2, 3, 4, or 5 cm of the administration site.

[0146] A homogeneous single-cell suspension is ideal for intravenous delivery but is not required for subcutaneous or intramuscular administration. In some embodiments, cell formulations for subcutaneous or intramuscular delivery are cell depot formulations or emulsions that promote cell aggregation; the delivery solutions used to prepare such depot cell formulations herein contain accessory components that provide the depot properties. In some embodiments, the cells can be aggregated in the formulation, e.g., before the formulation is administered to a subject, or within 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute of the cells, e.g., modified lymphocytes provided herein, being formulated in a delivery solution containing an aggregating agent to produce the formulation. In some embodiments, at least 10%, 20%, 25%, 50%, 75%, 90%, 95%, or 99% of the cells in the cell formulations provided herein are aggregated. Such aggregates can be determined, for example, using microscopic counting of individual cells versus cells associated with at least one other cell, or by counting the average number of cells associated with the cells in the formulation. In some embodiments, the cell formulations are designed for controlled or delayed release, with tissue expansion to accommodate cell expansion.

[0147] In some embodiments, the delivery solutions provided herein for subcutaneous or intramuscular delivery are depot formulations. Depot (i.e., sustained-release) formulations are typically aqueous or oily suspensions or solutions.

[0148] Thus, in some embodiments, the delivery solution or cell preparation includes components that form an artificial extracellular matrix, such as a hydrogel. In some embodiments, the depot delivery solution includes an effective amount of alginate, collagen, and / or dextran to form the depot preparation. Certain polymers that can be used to create gel-forming biomaterials and that can be included in the delivery solutions and cell preparations provided herein are composed of poly(ethylene glycol) (PEG) and its copolymers, and aliphatic polyesters such as poly(lactic acid) (PLA), poly(D,L-lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone) (PCL), and polyphosphazene. Other polymers that can be used include thermosensitive triblock copolymers based on poly(N-(2-hydroxypropylmethacrylamide lactate)) and poly(ethylene glycol) (p(HPMAm-lac)-PEG), which can spontaneously self-assemble in a physiological environment (Vermonden et al. 2006, Langmuir 22:10180-10184).

[0149] In some embodiments, the hydrogels used in the delivery solutions or cell formulations herein contain hyaluronic acid (HA). Such HA may be modified with 1-ethyl-3-(3-dimethylaminopropyl)-1-carbodiimide hydrochloride to have carboxylic acid groups that can react with amine groups on proteins, peptides, polymers, and linkers, such as those found on the modified lymphocytes provided herein, preferentially in the presence of N-hydroxysuccinimide. Antibodies, cytokines, and peptides can be chemically conjugated to HA using such methods to produce hydrogels for co-injection as cell emulsions in some cell formulation embodiments provided herein. Furthermore, in some embodiments, the HA in the delivery solutions and cell formulations is polymeric (e.g., Healon) and / or crosslinked via its -OH groups with an agent such as glutaraldehyde (e.g., Restylane (Abbive / Allergan)), e.g., lightly crosslinked, to reduce local catabolic effects of the material after subcutaneous injection. The HA used in the delivery solutions and cell formulations herein may have variable length and viscosity. The HA used in the delivery solutions and cell formulations herein can be further crosslinked with other glycosaminoglycans, such as chondroitin sulfate (e.g., Viscoat) or polymers or surfactants. Those skilled in the art will recognize that the porosity and degree of crosslinking of the substrate can be adjusted to ensure that cells, such as the modified lymphocytes herein, can migrate through the hydrogel. Thus, substrates, such as the hydrogel substrates used in the cell formulations herein, may be configured or adapted to allow cell migration through the substrate. The degree of substitution of the hydrogel and the concentration at crosslinking affect the porosity swelling ratio and Young's modulus (or stiffness). For example, an initial 1% substitution of HA with 1 mg / mL tyramine, when subsequently crosslinked in the presence of peroxide, results in a hydrogel with higher porosity and lower stiffness than a solution with 3% substitution and 5 mg / mL.Reducing the shear modulus is desirable in some circumstances to reduce shear forces during injection and ensure sufficient porosity and half-life for cells to grow subcutaneously into the matrix over a period of 1-2 weeks, hi some embodiments, the shear modulus is about 2.5 kPa, about 3 kPa, about 3.5 kPa, or about 4 kPa.

[0150] In some embodiments, the delivery solution, composition in the kit, or cell preparation comprises one or more cytokines, such as, for example, IL-2, IL-7, IL-15, IL-21, or variants thereof, or active fragments of any of the foregoing, and / or a cytokine receptor agonist, such as, for example, an IL-15 agonist. In some embodiments, the delivery solution, the composition in the kit, and / or the cell preparation comprises one or more of IL-1, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, TNFα, IFNγ, GM-CSF, CCL1, CCL2 (MCP-1), CCL3, CCL5, CCL7 (MCP-3), CCL8 (MCP-2), CCL19, CCL20, CCL21, CCL22, CCL28, CXCL1, CXCL9, CXCL10, CXCL11, CXCL12, CXCL14 (BRAK), CX3CL1, and variants thereof, and active fragments of any thereof. In some embodiments, the delivery solution, the composition in the kit, and / or the cell preparation does not comprise IL-2, IL-7, IL-15, or IL-21. In some embodiments, the delivery solution, the composition in the kit, and / or the cell preparation comprises one or more of IL-1, IL-12, IL-18, TNFα, IFNγ, GM-CSF, and variants thereof, and active fragments of any of them. In some embodiments, the delivery solution, the composition in the kit, and / or the cell preparation comprises one or more of CCL1, CCL2 (MCP-1), CCL3, CCL5, CCL7 (MCP-3), CCL8 (MCP-2), CCL19, CCL20, CCL21, CCL22, CCL28, and variants thereof, and active fragments of any of them. In some embodiments, the delivery solution, the composition in the kit, and / or the cell preparation comprises one or more of CCL19, CCL21, and variants thereof, and active fragments of any of the foregoing capable of binding to CCR7 and / or CXCR3. In some embodiments, the delivery solution, composition in the kit, or cell preparation comprises one or more of CXCL1, CXCL9, CXCL10, CXCL11, CXCL12, CXCL14 (BRAK), and variants thereof, and active fragments of any of them.In some embodiments, the delivery solution, the composition in the kit, and / or the cell preparation comprises one or more of CX3CL1, variants thereof, and active fragments of any of them. In some embodiments, the delivery solution, the composition in the kit, and / or the cell preparation comprises one or more polypeptides capable of binding to CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CXCR3, CXCR4, CXCR5, CXCR6, and / or Cx3cr1. In some embodiments, the delivery solution, the composition in the kit, and / or the cell preparation comprises one or more polypeptides capable of binding to CCR7, CXCR3, CXCR4, and / or CXCR6. In some embodiments, the delivery solution, the composition in the kit, and / or the cell preparation comprises one or more polypeptides capable of binding to CCR1, CC42, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CXCR3, CXCR4, CXCR5, and / or CXCR6.

[0151] In some embodiments, the cytokine does not bind to a cytokine receptor included in the delivery solution, kit, or cell preparation and / or does not bind to a cytokine receptor encoded by a polynucleotide in the delivery solution, cell preparation, or kit. In some embodiments, the cytokine may be, without being limited by theory, a modified cytokine that selectively activates a proliferation-inducing complex. In an exemplary embodiment, the modified cytokine is a modified IL-2, e.g., a fusion protein containing a circularly substituted IL-2 containing the extracellular domain of IL-2Rα (see, e.g., Lopes et al., J Immunother Cancer 2020 Apr;8(1):e000673). In some embodiments, the cytokine, modified cytokine, or cytokine receptor agonist may be administered from the cell preparation in a single or separate administration, before, simultaneously with, or after administration with the delivery solution or cell preparation. In some embodiments, two or more separate administrations may be dose-escalating. In some embodiments, two or more administrations may be the same dose. In some embodiments, two or more administrations may contain the same or different cytokines, modified cytokines, and / or cytokine receptor agonists. In some embodiments, the separate administrations may be a series of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 administrations. In some embodiments, the separate administrations are administered on consecutive days.

[0152] In some embodiments, the cell preparation contains antibodies or polypeptides capable of binding to CD2, CD3, CD28, OX40, 4-1BB, ICOS, CD9, CD53, CD63, CD81, and / or CD82. EDC-NHS reaction may be used to link such proteins to HA or via other intermediates mentioned above. In some embodiments, these cytokines, antibodies, or polypeptides are crosslinked to components of a hydrogel. The hydrogel may be mixed with the cell suspension using a syringe connector and two syringes prior to injection. In other embodiments, these cytokines, antibodies, or polypeptides are in solution. In some embodiments, the delivery solution or cell preparation contains RNA encoding these cytokines, antibodies, or polypeptides.

[0153] The proliferation and survival of genetically engineered T cells and / or NK cells expressing a CAR are promoted by signaling through the CAR when bound to their cognate antigen under appropriate conditions. In some embodiments, an antigen can be added to or coadministered with engineered and / or genetically engineered T cells and / or NK cells. In some embodiments, the antigen is a protein, glycoprotein, carbohydrate, or fragment thereof, e.g., a peptide, glycopeptide, or functional group. In some embodiments, the antigen may be soluble. In some embodiments, the antigen is derived from a non-human source. In some embodiments, the antigen may be immobilized on the surface of an artificial substrate, e.g., a hydrogel. In some embodiments, the antigen is a nucleic acid, such as DNA or RNA. In some embodiments, the nucleic acid encodes a protein or peptide antigen that is recognized by the CAR. In exemplary embodiments, the antigen may be expressed on the surface of cells containing a nucleic acid encoding the protein or peptide antigen, thereby making the cells target cells, referred to herein as feeder cells. In some embodiments, such target cells are present in large numbers in whole blood and naturally occur in cell preparations without the need for addition. For example, B cells present in whole blood, isolated TNCs, and isolated PBMCs, naturally present in cell preparations, can serve as target cells for T cells and / or NK cells expressing CARs directed against CD19 or CD22 (both expressed on B cells, as non-limiting examples). In other embodiments, such target cells are not present in whole blood or are not present in large numbers in whole blood and are therefore added externally, e.g., as feeder cells. In some embodiments, target cells can be isolated or enriched from a subject, such as from a tumor sample, using methods known in the art. In other embodiments, cells derived from a subject or from a source other than the subject, including cell lines, are modified to express an appropriate antigen. In some embodiments, target cells are treated to reduce their proliferative capacity, e.g., with radiation or a chemotherapeutic agent, and then administered to a subject.In exemplary embodiments, the antigen expressed on the target cell may comprise all or a portion of a protein containing the antigen. In further exemplary embodiments, the antigen expressed on the target cell may comprise all or a portion of the extracellular domain of the protein comprising the antigen. In some embodiments, the antigen is an antibody that recognizes the ASTR of the CAR, such as an anti-idiotypic antibody directed against the scFv domain of the CAR. In some embodiments, the antigen expressed on the target cell may be fused with a transmembrane domain that anchors it to the cell surface. Any of the transmembrane domains disclosed elsewhere herein may be used. In some embodiments, the antigen expressed on the target cell may be fused with a stalk domain. Any of the stalk domains disclosed elsewhere herein may be used. In an exemplary embodiment, the antigen may be fused with the CD8 stalk and transmembrane domain (SEQ ID NO: 24).

[0154] In exemplary embodiments, cells in a first cell mixture, e.g., cells obtained from a subject, are modified with a recombinant nucleic acid vector encoding a target antigen, which may be referred to herein as "artificial antigen-presenting cells" or "aAPCs," and cells in a separate second cell mixture from the same subject are modified to express a CAR that binds to the antigen. In some embodiments, when the modified cells modified with the vector encoding the target antigen are T cells, the cells may be referred to herein as "T-APCs." Such modified T-APCs include, by way of non-limiting example, B cells, dendritic cells, and macrophages, and in exemplary embodiments, dendritic cells and macrophages, such as when the corresponding CAR-T target is a B-cell cancer target. Modified T-APCs can also be produced using the methods provided herein, where the reaction mixture for modification (e.g., transduction) includes a T cell-binding polypeptide, such as a polypeptide directed against CD3. In further exemplary embodiments, the cell mixture is whole blood, isolated TNCs, or isolated PBMCs. For example, a first cell mixture can be modified with a recombinant nucleic acid vector encoding a fusion protein of the extracellular domain of Her2 and the transmembrane domain of PDGF, and a second cell mixture can be modified with a recombinant nucleic acid vector encoding a CAR directed against HER2. The cells can then be formulated into a delivery solution or otherwise administered to a subject at different CAR effector cell to target cell ratios. In some embodiments, the effector to target ratio when formulated or administered is (about) 10:1, (about) 9:1, (about) 8:1, (about) 7:1, (about) 6:1, (about) 5:1, (about) 4:1, (about) 3:1, (about) 2:1, (about) 1:1, (about) 1:2, (about) 1:3, (about) 1:5, (about) 1:6, (about) 1:7, (about) 1:8, (about) 1:9, or (about) 1:10. In an exemplary embodiment, the target cells are co-administered subcutaneously or intramuscularly with the modified T and / or NK cells.

[0155] The proliferation and survival of genetically modified T cells and / or NK cells expressing a CAR can also be promoted by CAR signaling initiated by cross-linking the CAR through an interaction other than through the CAR's ASTR, which binds to the CAR's cognate antigen. In some embodiments, a small molecule or protein can cross-link and activate the CAR on the surface of a cell. In an exemplary embodiment, an antibody can cross-link and activate the CAR on the cell surface. In a further exemplary embodiment, the antibody recognizes an epitope in the extracellular domain of the CAR, such as the stalk or spacer domain. In some embodiments, the epitope may be an epitope tag such as 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), HA tag (YPYDVPDYA, SEQ ID NO: 73), RYIRS (SEQ ID NO: 79), Phe-His-His-Thr (SEQ ID NO: 80), or WEAAAREACCRECCARA (SEQ ID NO: 81). In exemplary embodiments, the epitope is common to intracellular antigens that are not reactive to extracellular receptors. In some embodiments, the epitope tag is a HisX6 tag (SEQ ID NO: 77). In some embodiments, the CAR can be crosslinked and activated by adding a soluble antibody that binds to the epitope tag. In exemplary embodiments, the CAR can be crosslinked and activated by adding cells expressing an antibody or antibody mimic that binds to the epitope tag, also referred to herein as universal feeder cells. In some embodiments, the antibody or antibody mimic is associated with the cell membrane via a GPI anchor. In exemplary embodiments, the antibody or antibody mimic is associated with the cell membrane via a transmembrane domain. In further exemplary embodiments, a stalk or spacer separates the antibody or antibody mimic from the transmembrane domain.In some embodiments, the same universal feeder cells, such as those expressing an anti-HisX6 scFv attached to the CD8a stalk and transmembrane domain, can be used with cells expressing CARs containing ASTRs, which bind to different antigens but contain a HisX6 epitope tag in their stalks. These universal feeder cells can be used with cells expressing different CARs that contain a common epitope tag. Regarding universal feeder cells, it is not necessary to generate different feeder cells expressing the cognate antigen for the CAR containing a different ASTR, provided that the CAR contains an epitope tag. The epitope tag on the CAR-expressing cells is crosslinked by the universal feeder cells, contributing to the clustering and proliferation of the CAR. For example, anti-HisX6 universal feeder cells can be used with cells expressing a CAR that binds to Her2 and contains a HisX6 epitope tag, and with cells expressing a CAR that binds to Axl and contains a HisX6 epitope tag. The combination of universal feeder cells and CARs allows for the growth of CAR-T cells, which then engage their cognate antigen. Furthermore, if the ASTR of the CAR is in a restricted microenvironment, the use of antigen-binding universal feeder cells allows for expansion outside of that restricted environment.

[0156] Recombinant retroviral particles In the methods and compositions provided herein, recombinant retroviral particles are disclosed for modifying, by way of example and not limitation, human cells, primary cells, T cells, and / or NK cells to generate genetically modified and / or transduced human cells, primary cells, T cells, and / or NK cells. Such modification can be performed in vivo, ex vivo, or in vitro. Recombinant retroviral particles are themselves an aspect of the present invention. Typically, recombinant retroviral particles included in the aspects provided herein are replication-incompetent, meaning that they are unable to replicate once they leave the packaging cell. In practice, unless otherwise indicated herein, retroviral particles are replication-incompetent, and if such retroviral particles contain nucleic acids in their genome that are not native to the retrovirus, they are "recombinant retroviral particles." In an exemplary embodiment, the recombinant retroviral particles are lentiviral particles.

[0157] In some aspects, provided herein are replication-incompetent recombinant retroviral particles for use in transducing cells, typically lymphocytes, and in exemplary embodiments, T cells and / or NK cells. The replication-incompetent recombinant retroviral particles may comprise an envelope protein. In some embodiments, the envelope protein may be a pseudotyping element. In some embodiments, the envelope protein may be an activation element. In some embodiments, the replication-incompetent recombinant retroviral particles comprise both a pseudotyping element and an activation element. The replication-incompetent recombinant retroviral particles may comprise any of the pseudotyping elements discussed elsewhere herein. In some embodiments, the replication-incompetent recombinant retroviral particles may comprise any of the activation elements discussed elsewhere herein. In one aspect, provided herein is a replication-incompetent recombinant retroviral particle (RIP) comprising a polynucleotide having a nucleic acid encoding at least one of a CAR, a TCR, a LE, an anti-idiotype polypeptide, and a cytokine, and an inhibitory RNA provided in any of the aspects and embodiments herein. Such polypeptides typically comprise nucleic acids further encoding at least one of a CAR, a TCR, an LE, a cytokine, and an inhibitory RNA. In some embodiments, a RIP comprises a polynucleotide comprising: A) one or more transcription units operably linked to a promoter active in T cells and / or NK cells, the one or more transcription units encoding at least one of an anti-idiotype polypeptide, an engineered T cell receptor, or a chimeric antigen receptor (CAR), and B) a pseudotyping element and a T cell activation element on its surface, wherein the T cell activation element is not encoded by a polynucleotide in the replication-incompetent recombinant retroviral particle. In some embodiments, the T cell activation element may be any of the activation elements discussed elsewhere herein.In an exemplary embodiment, the T cell activation element may be an anti-CD3 scFvFc. In any of the embodiments disclosed herein, a pseudotyping element may be absent. In another aspect, provided herein is a replication-incompetent recombinant retroviral particle comprising a polypeptide comprising one or more transcription units operably linked to a promoter active in T cells and / or NK cells, the one or more transcription units encoding a first polypeptide comprising an engineered T cell receptor or a chimeric antigen receptor (CAR), and a second polypeptide comprising a lymphoproliferation element. In some embodiments, the lymphoproliferation element may be a chimeric lymphoproliferation element. In an exemplary embodiment, the lymphoproliferation element does not comprise IL-7 tethered to the IL-7 receptor alpha chain or a fragment thereof. In some embodiments, the lymphoproliferation element does not comprise IL-15 tethered to the IL-2 / IL-15 receptor beta chain. In some embodiments of any of the aspects or embodiments of retroviral particles provided herein, or any other aspect comprising a retroviral particle, the engineered T cell receptor, CAR, or other transgene is expressed, displayed, and / or otherwise incorporated at a reduced level on the surface of the replication-incompetent retroviral particle, with less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% surface expression compared to when the transgene is expressed from an EF1-a or PGK promoter, and in exemplary embodiments, compared to when the transgene is expressed from an EF1-a or PGK promoter in the absence of additional elements to reduce such surface expression (e.g., degrons or inhibitory RNA). In exemplary embodiments of any of the aspects of polynucleotide vectors (e.g., RIPs) provided herein, or any other aspects comprising a genetic vector, the genetic vector is substantially free of protein transcripts encoded by the nucleic acid of the genetic vector, and / or the RIP does not express or comprise a detectable amount of an engineered T cell receptor or CAR on its surface, or expresses or comprises a low amount of an engineered T cell receptor or CAR on its surface.

[0158] In some aspects, provided herein are replication-incompetent recombinant retroviral particles comprising a polypeptide comprising one or more transcription units operably linked to a promoter active in T cells and / or NK cells, the one or more transcription units encoding a first polypeptide comprising a chimeric antigen receptor (CAR) and a lymphoproliferation element (LE), in exemplary embodiments, a chimeric lymphoproliferation element (CLE), e.g., a constitutively active CLE. In exemplary embodiments, the chimeric lymphoproliferation element does not comprise a cytokine tethered to its cognate receptor or to a fragment of its cognate receptor. As further disclosed herein, in some embodiments, the LE, e.g., a CLE, comprises a first lymphoproliferation element polypeptide (LE polypeptide) and a second LE polypeptide, wherein the first LE polypeptide has a different amino acid sequence from the second LE polypeptide, and the first LE polypeptide and the second LE polypeptide are capable of, adapted to, and / or configured to dimerize with each other. Such embodiments are referred to herein as heterodimeric LEs. In exemplary embodiments, such LEs are heterodimeric LEs, wherein a first LE polypeptide and a second LE polypeptide comprise a first extracellular dimerization motif and a second extracellular dimerization motif, respectively, which are capable of, adapted to, and / or configured to dimerize with each other. In exemplary embodiments, such LEs are heterodimeric LEs, wherein a first LE polypeptide and a second LE polypeptide comprise a first intracellular dimerization motif and a second intracellular dimerization motif, respectively, which are capable of, adapted to, and / or configured to dimerize with each other.

[0159] In some aspects, provided herein is a recombinant retroviral particle comprising: (i) a pseudotyping element capable of binding to and promoting membrane fusion of the recombinant retroviral particle to T cells and / or NK cells; (ii) a polynucleotide having 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 engineered signaling polypeptide having a chimeric antigen receptor comprising an antigen-specific targeting region, a transmembrane domain, and an intracellular activation domain, and a second engineered signaling polypeptide comprising at least one lymphoproliferation element, wherein expression of the first engineered signaling polypeptide and / or the second engineered signaling polypeptide is regulated by an in vivo control element; and (iii) an activation element on its surface, wherein the activation element is capable of binding to T cells and / or NK cells, and the activation element is not encoded by a polynucleotide in the recombinant retroviral particle. In some embodiments, the promoter active in T cells and / or NK cells is not active in a packaging cell line or is active in a packaging cell line only in an inducible manner. In any of the embodiments disclosed herein, either the first or second engineered signaling polypeptide can comprise a chimeric antigen receptor, and the other engineered signaling polypeptide can comprise at least one lymphoproliferative element.

[0160] In some aspects, the present invention provides a replication-incompetent recombinant retroviral particle that comprises a polynucleotide encoding a self-driving CAR.Details regarding aspects of the composition and method that comprise such replication-incompetent recombinant retroviral particles and self-driving CARs are disclosed in more detail herein, for example, in the self-driving CAR method and composition section and in the exemplary embodiment section.

[0161] Various elements and combinations of elements contained in replication-incompetent recombinant retroviral particles are provided throughout the present disclosure, such as pseudotyping elements, activation elements, and membrane-bound cytokines, as well as nucleic acid sequences contained in the genome of replication-incompetent recombinant retroviral particles, such as, but not limited to, nucleic acid sequences encoding anti-idiotype polypeptides, nucleic acid sequences encoding CARs, nucleic acid sequences encoding lymphoproliferative elements, nucleic acids encoding cytokines, nucleic acid sequences encoding control elements such as riboswitches, promoters, particularly promoters that are constitutively active or inducible in T cells, and nucleic acids encoding inhibitory RNA molecules. Furthermore, various embodiments provided herein, such as methods for producing recombinant retroviral particles, methods for performing adoptive cell therapy, and methods for transducing T cells, produce and / or include replication-incompetent recombinant retroviral particles. The replication-incompetent recombinant retrovirus itself produced and / or contained in such methods forms a separate aspect of the present disclosure, as a replication-incompetent recombinant retroviral particle composition, which may be in isolated form. Such compositions may be in a dried (eg, lyophilized) form or may be in a suitable solution or medium known in the art for the storage and use of retroviral particles.

[0162] Thus, by way of non-limiting example, in another aspect, provided herein is a replication-incompetent recombinant retroviral particle having within its genome a polynucleotide having one or more nucleic acid sequences operably linked to a promoter active in T cells and / or NK cells, which in some cases includes a first nucleic acid sequence encoding one or more (e.g., two or more) inhibitory RNA molecules directed to one or more RNA targets, as described herein, and a second nucleic acid sequence encoding a chimeric antigen receptor, or CAR. In other embodiments, a third nucleic acid sequence is present that encodes at least one (e.g., one, two, three, or four) lymphoproliferative element described herein above that is not an inhibitory RNA molecule. In certain embodiments, the polynucleotide comprises one or more riboswitches, as provided herein, operably linked to the first nucleic acid sequence, the second nucleic acid sequence, and / or the third nucleic acid sequence, if present. In such constructs, expression of the one or more inhibitory RNAs, the CAR, and / or one or more lymphoproliferative elements that are not inhibitory RNAs is controlled by the riboswitch. In some embodiments, 2 to 10 inhibitory RNA molecules are encoded by the first nucleic acid sequence. In further embodiments, two to six inhibitory RNA molecules are encoded by the first nucleic acid sequence. In exemplary embodiments, four inhibitory RNA molecules are encoded by the first nucleic acid sequence. In some embodiments, the first nucleic acid sequence encodes one or more inhibitory RNA molecules and is located within an intron. In specific embodiments, the intron includes all or a portion of a promoter. The promoter may be a Pol I, Pol II, or Pol III promoter. In some exemplary embodiments, the promoter is a Pol II promoter. In some embodiments, the intron is adjacent to and downstream of a promoter active in T cells and / or NK cells. In some embodiments, the intron is EF1-α intron A.

[0163] Embodiments of recombinant retroviral particles herein include those in which the retroviral particle comprises a genome comprising one or more nucleic acids encoding one or more inhibitory RNA molecules. Various alternative embodiments of such nucleic acids encoding inhibitory RNA molecules (including combinations of such nucleic acids with other nucleic acids encoding lymphoproliferative elements other than CAR or inhibitory RNA molecules) that can be included in the genome of a retroviral particle are included, for example, in the section on inhibitory RNA provided herein, as well as in various other paragraphs combining these embodiments. Furthermore, various alternatives to such replication-incompetent recombinant retroviruses can be identified by the exemplary nucleic acids disclosed within the packaging cell line aspects disclosed herein. Those skilled in the art will recognize that the disclosure in this section of recombinant retroviral particles comprising a genome encoding one or more (e.g., two or more) inhibitory RNA molecules can be combined with various alternatives for such nucleic acids encoding inhibitory RNA molecules provided in other sections herein. Furthermore, those skilled in the art will recognize that such nucleic acids encoding one or more inhibitory RNA molecules can be combined with various other functional nucleic acid elements provided herein, for example, as disclosed in sections herein focusing on inhibitory RNA molecules and the nucleic acids encoding these molecules. Additionally, various embodiments of specific inhibitory RNA molecules provided elsewhere herein may be used in the recombinant retroviral particle aspects of the present disclosure.

[0164] The necessary elements of recombinant retroviral vectors, such as lentiviral vectors, are known in the art. These elements are provided in the packaging cell line section and the Examples section, and are included in details for generating replication-incompetent recombinant retroviral particles as shown in WO 2019 / 055946. For example, lentiviral particles typically contain packaging elements REV, GAG, and POL, which can be delivered to a packaging cell line via one or more packaging plasmids, a pseudotyping element, which can be delivered to a packaging cell line via a pseudotyping plasmid (various examples of which are provided herein), and a genome produced by a polynucleotide delivered to a host cell via a transfer plasmid. This polynucleotide typically contains the viral LTR and a psi packaging signal. The 5' LTR may be a chimeric 5' LTR fused to a heterologous promoter, including a 5' LTR that is independent of Tat transactivation. The transfer plasmid may be self-inactivating, for example, by deleting the U3 region of the 3' LTR. In some non-limiting embodiments, Vpu, such as a polypeptide comprising Vpu (sometimes referred to herein as a "Vpu polypeptide"), including but not limited to Src-FLAG-Vpu, is packaged within a retroviral particle, with respect to aspects and embodiments of any compositions or methods provided herein that include retroviral particles. In some non-limiting embodiments, Vpx, such as Src-FLAG-Vpx, is packaged within a retroviral particle. Without being limited by theory, upon transduction of T cells, Vpx enters the cytosol of the cell and promotes degradation of SAMHD1, increasing the pool of cytoplasmic dNTPs available for reverse transcription. In some non-limiting embodiments, Vpu and Vpx are packaged within a retroviral particle, with respect to aspects and embodiments of any compositions or methods provided herein that include retroviral particles.

[0165] Retroviral particles (e.g., lentiviral particles) included in various aspects of the present disclosure are, in exemplary embodiments, replication-incompetent for safety reasons, particularly for embodiments involving the introduction of cells transduced with such retroviral particles into a subject. When replication-incompetent retroviral particles are used to transduce cells, no retroviral particles are produced from the transduced cells. Modifications to retroviral genomes to ensure that retroviral particles containing the genome are replication-incompetent are known in the art. However, it will be understood that replication-competent recombinant retroviral particles may be used in some embodiments of any of the aspects provided herein. In some non-limiting embodiments, the retroviral particle lacks a functional integrase. In these and other embodiments, the retroviral RNA is reverse transcribed but does not integrate into the host cell genome.

[0166] Those skilled in the art will recognize that the functional elements discussed herein can be delivered to packaging cells and / or T cells using different types of vectors, such as expression vectors. Exemplary aspects of the present disclosure utilize retroviral vectors, and in some particularly exemplary embodiments, lentiviral vectors. Other suitable expression vectors can be used to achieve certain embodiments of the present disclosure. Such expression vectors include viral vectors (e.g., vaccinia virus-based viral vectors, poliovirus, adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94 / 12649; WO93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984; and WO95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86,1998, Flannery et al.,PNAS 94:6916 6921,1997,Bennett et al.,Invest Opthalmol Vis Sci 38:2857 2863,1997,Jomary et al.,Gene Ther 4:683 690,1997,Rolling et al.,Hum Gene Ther 10:641 648,1999, Ali et al., Hum Mol Genet 5:591 594,1996, WO 93 / 09239 by Srivastava, Samulski et al., J. Vir. (1989) 63:3822-3828, Mendelson et al., Virol. (1988) 166:154-165, and Flotte et al. al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; or retroviral vectors (e.g., vectors derived from murine leukemia virus, spleen necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), such as gammaretroviruses; or human immunodeficiency virus (e.g., see Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999).

[0167] As disclosed herein, replication-incompetent recombinant retroviral particles are a common tool for gene delivery (Miller, Nature (1992) 357:455-460). The ability of replication-incompetent recombinant retroviral particles to deliver unrearranged nucleic acid sequences to a wide range of rodent, primate, and human somatic cells makes them well suited for introducing genes into cells. In some embodiments, the replication-incompetent recombinant retroviral particles can be derived from the alpharetrovirus, betaretrovirus, gammaretrovirus, deltaretrovirus, epsilonretrovirus, lentivirus, or spumavirus genera. Many retroviruses are suitable for use in the methods disclosed herein. For example, murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29), and avian erythroblastosis virus (AEV) can be used. A detailed list of retroviruses can be found in Coffin et al. ("Retroviruses" 1997 Cold Spring Harbor Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763). Details of the genome structure of some retroviruses can be found in the art. As an example, details of HIV can be found in NCBI Genbank (ie, genome accession number AF033819).

[0168] In exemplary embodiments, the replication-incompetent recombinant retroviral particle may be derived from the lentivirus genus. In some embodiments, the replication-incompetent recombinant retroviral particle may be derived from HIV, SIV, or FIV. In further exemplary embodiments, the replication-incompetent recombinant retroviral particle may be derived from human immunodeficiency virus (HIV), also of the lentivirus genus. Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. This increased complexity allows lentiviruses to adjust their life cycle, such as during latent infection. A typical lentivirus is the human immunodeficiency virus (HIV), the etiological agent of AIDS. In vivo, HIV can infect terminally differentiated cells that rarely divide, such as lymphocytes and macrophages.

[0169] In an exemplary embodiment, a replication-incompetent recombinant retroviral particle provided herein contains a Vpx polypeptide.

[0170] In some embodiments, the replication-incompetent recombinant retroviral particles provided herein comprise and / or contain a Vpu polypeptide.

[0171] In an exemplary embodiment, the retroviral particle is a lentiviral particle. Such retroviral particles typically contain a retroviral genome within a capsid located within the viral envelope.

[0172] In some embodiments, DNA-containing viral particles are utilized instead of recombinant retroviral particles. Such viral particles may be adenovirus, adeno-associated virus, herpesvirus, cytomegalovirus, poxvirus, avipoxvirus, influenza virus, vesicular stomatitis virus (VSV), or Sindbis virus. Those skilled in the art will understand how to modify the methods disclosed herein for use with different viruses and retroviruses or retroviral particles. When viral particles containing a DNA genome are used, those skilled in the art will understand that such genomes can contain functional units to induce integration of all or part of the DNA genome of the viral particle into the genome of T cells transduced with such viruses.

[0173] In some embodiments, the polynucleotide regions encoding HIV RRE and HIV Rev can be replaced with a polynucleotide region encoding an N-terminal RGG box RNA-binding motif and ICP27. In some embodiments, the polynucleotide region encoding HIV Rev can be replaced with one or more polynucleotide regions encoding adenovirus E1B 55-kDa and E4 Orf6.

[0174] In certain aspects, the replication-incompetent recombinant retroviral particle can include a nucleic acid encoding a self-propelled CAR, as disclosed elsewhere herein. As a non-limiting example, such an embodiment is a retroviral particle, the genome of which includes one or more first transcription units operably linked to an inducible promoter inducible in at least one of T cells or NK cells, and one or more second transcription units operably linked to a constitutive T cell or NK cell promoter, wherein the number of nucleotides between the 5' end of the one or more first transcription units and the 5' end of the one or more second transcription units is less than the number of nucleotides between the 3' end of the one or more first transcription units and the 3' end of the one or more second transcription units; a) at least one of the one or more first transcription units encodes a lymphoproliferation element; b) At least one of the one or more second transcription units encodes a first chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain.

[0175] In some embodiments, the nucleic acid in the first transcription unit or the nucleic acid in the second transcription unit may further encode an anti-idiotype polypeptide according to any of the embodiments provided herein. In some embodiments, the replication-incompetent recombinant retroviral particle may further display a T cell activation element.

[0176] Without being limited by theory, T cells that have been contacted and transduced with these replication-incompetent recombinant retroviral particles containing a nucleic acid encoding a self-driven CAR may receive an initial boost in transcription from the CAR-stimulated inducible promoter because the T cell activation element can stimulate an induction signal for the CAR-stimulated inducible promoter. Binding of the T cell activation element can induce calcium ion influx, which leads to the dephosphorylation of NFAT and its subsequent nuclear translocation and binding to the NFAT-responsive promoter. Lymphoproliferative elements transcribed and translated from these CAR-stimulated inducible promoters can result in an initial increase in proliferation in these cells. In an exemplary embodiment, the T cell activation element may be a membrane-bound anti-CD3 antibody and may be GPI-linked or otherwise displayed on the virus. In some embodiments, the membrane-bound anti-CD3 antibody may be fused to a viral envelope protein, such as MuLV, VSV-G, Henipavirus-G, or Niv-G, or a fragment thereof.

[0177] In some embodiments, the isolated replication-incompetent retroviral particles are a large batch contained in a large vessel. Such a large batch can be, for example, 10 6 ~10 8Titer in TU / mL, and 1 x 10 10 TU~1×10 13 TU, 1×10 11 TU~1×10 13 TU, 1×10 12 TU~1×10 13 TU, 1×10 10 TU~5×10 12 TU, or 1×10 11 TU~5×10 12 The retroviral particles of any aspect or embodiment provided herein can be substantially pure, as discussed in more detail herein.

[0178] Retroviral genome size In the methods and compositions provided herein, including RIPs and the genomes of RIPs (i.e., recombinant retroviral genomes), in non-limiting exemplary embodiments, the lentiviral genome has a limited number of polynucleotides that can be packaged into viral particles. In some embodiments provided herein, the polypeptide encoded by the polynucleotide coding region may be truncated or otherwise deleted to retain functional activity, such that the polynucleotide coding region is encoded by fewer nucleotides than the polynucleotide coding region of the wild-type polypeptide. In some embodiments, the polypeptide encoded by the polynucleotide coding region may be a fusion polypeptide that can be expressed from a single promoter. In some embodiments, the fusion polypeptide may have a cleavage signal to generate two or more functional polypeptides from a single fusion polypeptide and a single promoter. Furthermore, some functions that are not required after the initial ex vivo transduction are not included in the retroviral genome, but rather are present on the surface of the replication-incompetent recombinant retroviral particle via the packaging cell membrane. These various strategies are used herein to maximize the functional elements packaged into replication-incompetent recombinant retroviral particles. In some embodiments, the packaged recombinant retroviral genome may be from a lower range of 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, and 8,000 nucleotides to an upper range of 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, and 11,000 nucleotides. The packaged retroviral genome includes one or more polynucleotide regions encoding first and second manipulating signaling polypeptides, as disclosed in detail herein. In some embodiments, the packaged recombinant retroviral genome may be less than 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, or 11,000 nucleotides. Functions discussed elsewhere herein that may be packaged include retroviral sequences necessary for retroviral assembly and packaging, such as the retroviral rev, gag, and pol coding regions, and the 5'LTR and 3'LTR, or active cleaved fragments thereof, nucleic acid sequences encoding retroviral cis-acting RNA packaging elements, and cPPT / CTS elements. Additionally, in exemplary embodiments, the replication-incompetent recombinant retroviral particles herein can include any one or more or all of the following, in some embodiments in a reverse orientation relative to the 5' to 3' orientation established by the retroviral 5' LTR and 3' LTR (as shown, by way of non-limiting example, in WO 2019 / 055946): one or more polynucleotide regions encoding first and second engineered signaling polypeptides, at least one of which comprises at least one lymphoproliferation element (e.g., each comprising one or two lymphoproliferation element polypeptides); a second engineered signaling polypeptide that may comprise a chimeric antigen receptor; a control element, such as an miRNA or riboswitch, which typically regulates expression of the first and / or second engineered signaling polypeptide; a safety switch polypeptide, an intron, a promoter active in a target cell, such as a T cell, a 2A cleavage signal, and / or an IRES.

[0179] Kits and Commercial Products In another aspect, provided herein are compositions or suspensions for delivery to treat or prevent a disease, e.g., cancer or tumor growth, which comprise as an active ingredient a polynucleotide, e.g., a polynucleotide vector, illustratively a replication-incompetent recombinant retroviral particle (RIP), or modified cells, e.g., modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells, and / or modified NK cells. In another aspect, provided herein are compositions or other RIP or cell preparations for infusion to treat or prevent cancer or tumor growth, which comprise a polynucleotide, e.g., a polynucleotide vector, illustratively a RIP, or modified cells, e.g., modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells, and / or modified NK cells. The polynucleotide, e.g., a polynucleotide vector, in exemplary embodiments a RIP, or modified cells, e.g., modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells, and / or modified NK cells, of the delivery or infusion composition may include any of the aspects, embodiments, or subembodiments described above or otherwise discussed herein, including, for example, any embodiments that include nucleic acids encoding anti-idiotype polypeptides, CARs, LEs, cytokines, and / or TCRs.

[0180] In one aspect, provided herein are containers, e.g., commercially available containers or packages, or kits containing the same, according to any of the aspects and embodiments provided herein, comprising a polynucleotide, e.g., a polynucleotide vector, e.g., a RIP, or comprising an engineered cell, e.g., an engineered lymphocyte, an engineered TIL, an engineered lymphocyte other than a B cell, or an engineered T cell, and / or an engineered NK cell. By way of non-limiting example, a polynucleotide, e.g., a polynucleotide vector, e.g., a RIP, or an engineered cell, e.g., an engineered lymphocyte, an engineered TIL, an engineered lymphocyte other than a B cell, or an engineered T cell, and / or an engineered NK cell, may comprise in its genome a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and / or NK cells. In some embodiments, the nucleic acid sequence of the one or more nucleic acid sequences may encode an anti-idiotype polypeptide, an inhibitory RNA, a cytokine, a lymphoproliferative element, a TCR, and / or a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. In some embodiments, the nucleic acid sequences of the one or more nucleic acid sequences may encode one, two, or more inhibitory RNA molecules directed against one or more RNA targets.

[0181] In any aspect or embodiment, including commercially available containers and kits, the container containing the polynucleotide, e.g., a polynucleotide vector, e.g., a RIP, or modified cells, e.g., modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells, and / or modified NK cells, may be an IV bag, tube, vial, well of a plate, or other vessel for storage of the polynucleotide, e.g., a polynucleotide vector, e.g., a RIP, or modified cells, e.g., modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells, and / or modified NK cells. Indeed, some aspects provided herein include a container containing a polynucleotide, e.g., a polynucleotide vector, e.g., a RIP, or modified cells, e.g., modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells, and / or modified NK cells, wherein the biologic comprises any nucleic acid or other component disclosed herein. In exemplary embodiments, such a container contains a substantially pure polynucleotide, e.g., a polynucleotide vector, e.g., a RIP, or modified cells, e.g., modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells, and / or modified NK cells, which may be referred to herein for short as a substantially biologic. Typically, the substantially pure retroviral particle preparation and / or container is sterilized and negative for mycoplasma, replication-competent retroviruses of the same type, and adventitious viruses according to standard protocols (see, e.g., "Viral Vector Characterization: A Look at Analytical Tools"; October 10, 2018 (available at https: / / cellculturedish.com / viral-vector-characterization-analytical-tools / )). Exemplary methods for producing substantially pure biologics and cells intended for cell therapy are known. For example, for such a method involving a RIP, the viral supernatant can be purified by a combination of depth filtration, TFF, benzonase treatment, diafiltration, and formulation.In certain exemplary embodiments, a substantially pure biologic meets all of the following characteristics, based on quality control test results: a) Negative for mycoplasma. b) Less than 25 EU / mL, and in certain further exemplary embodiments, less than 10 EU / mL endotoxin. c) There are no detected replication-competent retroviruses of the same type as those intentionally detected in the detected container (e.g., lentiviruses); and d) Absence of detected adventitious viruses.

[0182] Additionally, when the biologic is a viral particle, e.g., a retroviral particle, in an exemplary embodiment, the following quality control test results are met: a) less than 1 pg of host cell DNA / transducing unit (TU), and in certain further exemplary embodiments, less than 0.3 pg / TU. b) a residual plasmid copy number / TU of less than 100, and in certain further exemplary embodiments, less than 10 copies / TU (where TU is the TU of any plasmid used to generate the recombinant retroviral particle). c) less than 1 ng of host cell protein / TU, and in certain further exemplary embodiments, less than 50 pg of host cell protein / TU; and / or d) greater than 100 TU / ng of P24 protein, and in certain further exemplary embodiments, greater than 10,000 TU / ng of P24 protein.

[0183] Retroviral particles are typically tested against release specifications, including some or all of those provided above, before being shipped to customers. Mycoplasma testing can be performed according to United States Pharmacopeia (USP) Chapter 63 or by rapid methods such as RT-PCR of samples for the absence of mycoplasma. Endotoxin testing can be performed according to USP Chapter 85 or by rapid methods such as Charles River's Endosafe® nexgen-PTS™. Replication-competent retroviruses can be tested by analyzing reverse transcriptase activity using the qPCR-based Product Enhanced Reverse Transcriptase (PERT) assay or rapid RCL assay. Detection of adventitious viruses can be performed in vitro by inoculating indicator cell lines (MRC-5, Vero, HEK 293T) with the test substance and observing the cultures for cytopathic effects for 14 days, as well as testing for red blood cell hemagglutination and hemadsorption. Host cell DNA can be quantified by qPCR of host cell genomic nucleic acids. The number of copies of the remaining viral RNA genome can be measured by qRT-PCR for nucleic acid residues of viral envelope proteins, such as VSVG. Host cell proteins can be measured by ELISA using polyclonal antibodies against host cell lysates. P24 assays for determining the physical titer of HIV-1-based lentiviral supernatants can be determined by standard ELISA.

[0184] The titer of each particle may be defined based on the p24 viral capsid protein or viral RNA genome copy number and can be converted to infectious titer by measuring transducing units (TU) of functional gene transfer in a bioassay.

[0185] Determining the infectious titer of purified bulk retroviral material and final product by bioassay and qPCR is an exemplary analytical test method for determining the infectious titer of a retrovirus. An indicator cell bank (e.g., Jurkat or F1XT (HEK293T cell variant)) can be seeded at 150,000 cells per well and subsequently exposed to serial dilutions of the retroviral product. Dilutions of purified retroviral particles are performed on the indicator cells, e.g., 1:200 to 1:1,600. A reference standard virus can be added for system suitability. After incubation with the retrovirus for 4 days (or 2-4 days), the cells are harvested, and DNA is extracted and purified. For example, a standard curve of 100 to 10,000,000 copies / well of a human genome and a unique retroviral genome sequence plasmid pDNA amplicon is used, followed by the addition of genomic DNA from the cell sample exposed to the retroviral particles. For each PCR reaction, the Cq values ​​of both the retroviral amplicon and an endogenous control, such as human RNAse P, are extrapolated back to the copy number per reaction. From these values, the integrated genome copy number is calculated. In some cases, indicator cells, such as HEK 293T, are characterized as triploid, so three copies of the single-copy gene per cell should be used for the calculation. Using the initial viable cell count per well, the volume of retrovirus added to the cells, and the genome copy number ratio, the transducing units (TU) per mL of retroviral particles can be determined. One TU is the amount of functional viral particles in solution that will result in one transgene integration when the viral solution is added to 100 recipient cells.

[0186] The potency test can include testing the potency against the release standard using purity and specific activity. For example, the release test of the potency of the final product can include measuring the number of transducing units (TU) compared to the amount of viral particles (for example, by performing p24 capsid protein ELISA, for example, by performing ELISA on lentivirus viral proteins, with a cutoff of p24 of at least 100, 1,000, 2,000, or 2,500 TU / ng), and CAR functionality, for example, by measuring the interferon gamma release by a reporter cell line exposed to genetically modified cells. Thus, the RIP formulations herein can have potency or potency ranges equivalent to any of the cutoffs and ranges of the potency tests herein.

[0187] The purity of replication-incompetent recombinant retroviral particles (RIPs) may be determined using the ratio of the amount of protein from the host cells used to produce the RIPs to the transducing units (amount of host cell protein / TU). In some embodiments, the host cell protein to TU ratio may be 10, 5, 3, 2, or 1 ng or less of host cell protein / TU, or 750, 500, 400, 300, 200, 100, 50, 40, 30, 20, or 10 pg or less of host cell protein / TU. In some embodiments, the host cell protein to TU ratio may be 1 ng or less of host cell protein / TU. In some embodiments, the host cell protein to TU ratio may be 50 pg or less of host cell protein / TU. In some embodiments, the host cell is a HEK cell line or a variant thereof. In some embodiments, the ratio of HEK protein to Tus may be 10, 5, 3, 2, or 1 ng or less of HEK protein / TU, or 750, 500, 400, 300, 200, 100, 50, 40, 30, 20, or 10 pg or less of HEK protein / TU. In some embodiments, the ratio of HEK protein to Tus may be 1 ng or less of protein / TU. In some embodiments, the ratio of HEK protein to Tus may be 50 pg or less of HEK protein / TU.

[0188] The potency of the RIP present in the delivery solution or RIP formulation can be determined using the ratio of TU to p24 protein (in ng), which in some embodiments may be 100, 200, 300, 400, 500, 1,000, 4,000, 10,000, 12,500, or 15,000 or more TU / ng of p24 protein.

[0189] In some embodiments, the purity and potency of the RIP in the substantially pure biologic may be any of the combinations provided in the Exemplary Embodiments section herein.

[0190] In some embodiments, quality control tests may include ratios of host cell protein / TU and TU / ng p24 protein, which may be the following, respectively: ≦1 ng host cell protein / TU and ≧100 TU / ng p24 protein; ≦1 ng host cell protein / TU and ≧500 TU / ng p24 protein; ≦1 ng host cell protein / TU and ≧1,000 TU / ng p24 protein; ≦1 ng host cell protein / TU and ≧5,000 TU / ng p24 protein; ≦1 ng host cell protein / TU and ≧10,000 TU / ng p24 protein; ≦1 ng host cell protein / TU and ≧12,500 TU / ng p24 protein; ≦1 ng host cell protein / TU and ≧15,000 TU / ng p24 protein; ≦50 pg host cell protein / TU and ≧100 TU / ng p24 protein; ≦50 pg host cell protein / TU and ≧500 TU / ng or greater than p24 protein; ≤50pg host cell protein / TU and ≥1,000TU / ng p24 protein; ≤50pg host cell protein / TU and ≥5,000TU / ng p24 protein; ≤50pg host cell protein / TU and ≥10,000TU / ng p24 protein; ≤50pg host cell protein / TU and ≥12,500TU / ng p24 protein; or ≤50pg host cell protein / TU and ≥15,000TU / ng p24 protein.

[0191] In some embodiments, the host cell may be a HEK 293 cell line or a variant thereof, including a HEK 293T cell line. In such embodiments, quality control tests may include the ratios of HEK protein / TU and TU / ng p24 protein, which may be, respectively, the following: less than or equal to 1 ng HEK protein / TU and more than or equal to 100 TU / ng p24 protein; less than or equal to 1 ng HEK protein / TU and more than or equal to 500 TU / ng p24 protein; less than or equal to 1 ng HEK protein / TU and more than or equal to 1,000 TU / ng p24 protein; less than or equal to 1 ng HEK protein / TU and more than or equal to 5,000 TU / ng p24 protein; less than or equal to 1 ng HEK protein / TU and more than or equal to 10,000 TU / ng p24 protein; less than or equal to 1 ng HEK protein / TU and more than or equal to 12,500 TU / ng p24 protein; less than or equal to 1 ng HEK protein / TU and more than or equal to 15,000 TU / ng p24 protein; less than or equal to 50 pg HEK protein / TU and more than or equal to 100 TU / ng p24 protein; less than or equal to 50 pg HEK protein / TU and more than or equal to 500 TU / ng p24 protein; ≤ HEK protein / TU and ≥ 1,000 TU / ng p24 protein; ≤ 50 pg HEK protein / TU and ≥ 5,000 TU / ng p24 protein; ≤ 50 pg HEK protein / TU and ≥ 10,000 TU / ng p24 protein; ≤ 50 pg HEK protein / TU and ≥ 12,500 TU / ng p24 protein; or ≤ 50 pg HEK protein / TU and ≥ 15,000 TU / ng p24 protein.

[0192]

[0193] In any of the kit or isolated replication-incompetent recombinant retroviral particle embodiments herein (including containers of such retroviral particles), sufficient recombinant retroviral particles are present in the container to achieve an MOI (number of transducing units, or TU applied per cell) in reaction mixtures made with the retroviral particles of 0.1 to 50, 0.5 to 50, 0.5 to 20, 0.5 to 10, 1 to 25, 1 to 15, 1 to 10, 1 to 5, 2 to 15, 2 to 10, 2 to 7, 2 to 3, 3 to 10, 3 to 15, or 5 to 15, or at least 0.1, 0.5, 1, 2, 2.5, 3, 5, 10, or 15, or to achieve an MOI of at least 0.1, 0.5, 1, 2, 2.5, 3, 5, 10, or 15. The transducing unit of the viral particles provided in the kit should be such that it prevents the production of too many integrants in an individual cell, allowing for an average MOI of less than 5, 4, or 3 lentigen copies per cell genome, and more preferably 1 copy per cell. For kit and isolated retroviral particle embodiments, such an MOI is 1 x 10 6 Assuming target cells / mL, for example, 1 x 10 for whole blood. 6 These may be based on 1, 2.5, 5, 10, 20, 25, 50, 100, 250, 500, or 1,000 mL of reaction mixture, assuming 1 x 10 PBMCs / mL of blood. Thus, a container of retroviral particles may contain 1 x 10 5 ~1×10 9 , 1×10 5 ~1×10 8 , 1×10 5 ~5×10 7 , 1×10 5 ~1×10 7 , 1×10 5 ~1×10 6 , 5×10 5 ~1×10 9 , 5×10 5 ~1×10 8 , 5×10 5 ~5×10 7 , 5×10 5 ~1×10 7 , 5×105 ~1×10 6 , or 1 × 10 7 ~1×10 9 , 1×10 7 ~5×10 7 , 1×10 6 ~1×10 7 , and 1 × 10 6 ~5×10 6 In certain exemplary embodiments, the container contains 1 x 10 7 ~1×10 9 , 5×10 6 ~1×10 8 , 1×10 6 ~5×10 7 , 1×10 6 ~5×10 6 , or 5 × 10 7 ~1×10 8 A retroviral particle transduction unit may contain 10 ...

[0194] Each container containing retroviral particles may contain a volume of, for example, 0.05 mL to 5 mL, 0.05 mL to 1 mL, 0.05 mL to 0.5 mL, 0.1 mL to 5 mL, 0.1 mL to 1 mL, 0.1 mL to 0.5 mL, 0.1 to 10 mL, 0.5 to 10 mL, 0.5 mL to 5 mL, 0.5 mL to 1 mL, 1.0 mL to 10.0 mL, 1.0 mL to 5.0 mL, 10 mL to 100 mL, 1 mL to 20 mL, 1 mL to 10 mL, 1 mL to 5 mL, 1 mL to 2 mL, 2 mL to 20 mL, 2 mL to 10 mL, 2 mL to 5 mL, 0.25 mL to 10 mL, 0.25 to 5 mL, or 0.25 to 2 mL.

[0195] In certain embodiments, the retroviral particles in the container are GMP-grade or cGMP-grade retroviral particles (i.e., produced under GMP or current GMP requirements in accordance with a regulatory agency), or are the product of a retroviral manufacturing step performed using a GMP system. Such retroviral particles are typically produced using the Good Manufacturing Practices (GMP) of the US FDA (i.e., US GMP or US cGMP), the EMA (i.e., EMA GMP or EMA cGMP), or the National Medical Products Administration (NMPA) of China (i.e., China FDA) (i.e., NMPA GMP or NMPA cGMP), e.g., using a GMP quality system and GMP procedural controls. These products are typically produced in facilities that meet GMP or cGMP requirements. Such products are typically manufactured under strict quality control systems based on GMP or cGMP regulations. GMP-grade retroviral particles are typically sterilized. This can be achieved, for example, by filtering the retroviral particles, e.g., substantially pure retroviral particles, through a 0.45 μm or 0.22 μm filter. GMP grade retroviral particles are typically substantially pure and prepared using good manufacturing practice testing specifications for potency, quality, and safety.

[0196] In some embodiments, the solution containing retroviral particles in the container does not contain detectable bovine proteins, which may be referred to as "bovine component-free." For example, bovine proteins, such as bovine serum proteins, are not used in the cultivation of packaging cells during retrovirus production, and such a solution of retroviral particles may be bovine component-free. In some embodiments, the solution of retroviral particles is GMP grade and bovine component-free. A substantially pure nucleic acid solution is typically produced in a bovine component-free, immortal component-free broth.

[0197] Retroviral particles in certain exemplary reaction mixtures provided herein, and in exemplary embodiments, lentiviral particles, are present at a multiplicity of infection (MOI) of 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 at least 1-6, 11, or less than 51 MOIs, or in some embodiments, 5-10 units of replication-incompetent recombinant retroviral particles. In some embodiments, the MOI may 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 may use a higher MOI than methods in which PBMCs are isolated and used in the reaction mixture. For example, exemplary embodiments of compositions and methods for transducing lymphocytes in whole blood use a multiplicity of infection (MOI) of 1 x 10. 6 Assuming 10 PBMC / mL of blood, retroviral particles with 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.

[0198] Surface expression of TCR complexes, including TCRα, TCRβ, and CD3, on CD4-positive (CD4+) and CD8-positive (CD8+) cells is reduced, or "dimmed," when such cells are contacted with a TCR complex, in certain exemplary embodiments herein, a polynucleotide vector (e.g., a replication-incompetent recombinant (RIR) retroviral particle) displaying a binding polypeptide that binds to a T cell activation element. This dimming is primarily the result of internalization of the TCR complex upon activation. Furthermore, the degree of dimming increases with increasing concentration of a given gene vector in the reaction mixture and correlates with the gene vector's ability to activate and enter cells. Similarly, internalization of other surface polypeptides following binding to polypeptides on the surface of the gene vector can also result in dimming of surface polypeptides on cells contacted with the gene vector, and this internalization may also occur universally in transduction using other binding polypeptides. Thus, in some embodiments, the percentage reduction in expression of a surface polypeptide on cells contacted with a gene vector comprising a binding polypeptide, compared to the expression of the surface polypeptide on cells not contacted with the gene vector, is used to quantify the potency of the gene vector and determine an appropriate dose of the gene vector to use for modifying a cell population. In an exemplary embodiment, the percentage reduction in expression of a surface TCR complex on cells contacted with the gene vector, compared to the expression of the surface TCR complex on cells not contacted with the gene vector, is used to quantify the potency of the gene vector and determine an appropriate dose of the gene vector to use for modifying a cell population. As used herein, a "Dimming Unit" (DU) is the amount of a gene vector (e.g., an RIR retroviral particle) that reduces the surface expression of a surface polypeptide in 1 ml of a cell mixture after contact with the gene vector for 4 hours at 37°C and 5% CO by 50%, compared to the surface expression of the surface polypeptide in a cell mixture not contacted with the gene vector but under similar conditions. The surface polypeptide is typically a binding partner for a binding polypeptide present on the surface of the gene vector.In some embodiments, the surface polypeptide is a TCR complex polypeptide. In some embodiments, the TCR complex polypeptide is CD3D, CD3E, CD3G, CD3Z, TCRα, or TCRβ. In an exemplary embodiment, the binding partner is CD3 and the binding polypeptide is anti-CD3.

[0199] Because the expression level of a binding polypeptide on the surface of a polynucleotide vector (sometimes referred to herein as a "gene vector") varies with different binding polypeptides and also varies with different polynucleotide vector preparations, the ability of a polynucleotide vector to reduce surface expression of a surface polypeptide must be determined for each preparation of polynucleotide vector. In some embodiments, the ability of a polynucleotide vector to reduce surface expression of a surface polypeptide is determined based on target cell number. In some embodiments, the ability of a polynucleotide vector to reduce surface expression of a surface polypeptide is determined based on cell quantity. In any of the aspects and embodiments herein, reducing surface expression of a surface polypeptide may also be referred to as dimming of the surface polypeptide. For example, if the surface expression of CD3 on a cell is reduced, CD3 on the cell will dimmed and the cell may be referred to as CD3-, even though the cell may still contain CD3 that is not yet expressed on its surface. Without being limited by theory, T cells transiently internalize and dimmed CD3 and eventually re-express CD3 on their cell surface, thereby causing the T cell to become CD3+ again.

[0200] Thus, in one aspect, there is provided herein a method for determining the amount of a polynucleotide vector preparation that extinguishes surface expression of a surface polypeptide by percent extinction on cells in an extinction volume, the method comprising: a) creating a plurality of reaction mixtures comprising a plurality of volumes of a polynucleotide vector preparation and a plurality of volumes of a cell mixture, wherein at least two of the reaction mixtures in the plurality of reaction mixtures comprise different amounts of the polynucleotide vector preparation and / or the cell mixture, wherein the cell mixture comprises a plurality of cells comprising a surface polypeptide on their surface, and wherein the polynucleotide vector preparation comprises a plurality of polynucleotide vectors comprising a binding polypeptide on their surface capable of binding to the surface polypeptide; b) incubating the reaction mixture; c) measuring the surface expression of surface polypeptides in the reaction mixture and in a non-contacting volume of the cell mixture, the non-contacting volume of the cell mixture not being contacted with the polynucleotide vector preparation; and d) determining the amount of polynucleotide vector preparation to be dimmed, the percentage dimming rate of cells in the dimming volume, the surface expression of surface polypeptides measured in the reaction mixture, the surface expression of surface polypeptides measured in a non-contacting volume of the cell mixture, and the amount of polynucleotide vector preparation and cell mixture in the reaction mixture.

[0201] In some embodiments, the amount of the cell mixture in the reaction mixture is based on volume. In some embodiments, the amount of the cell mixture in the reaction mixture is based on the number of target cells. In some embodiments, the polynucleotide vector preparation is a virus preparation. In exemplary embodiments, the virus preparation is a replication-incompetent recombinant retroviral particle preparation. In some embodiments, the percent extinction (percentage of extinct cells) is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, or 97%. In exemplary embodiments, the percent extinction is at least or about 80%, 85%, 90%, or 95%. In some embodiments, the extinction volume is 0.25 ml, 0.5 ml, 0.75 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 15 ml, 20 ml, or 25 ml. In some embodiments, the surface polypeptide may be CD3D, CD3E, CD3G, CD3Z, TCRα, TCRβ, CD16A, NKp46, 2B4, CD2, DNAM, or NKG2C, NKG2D, NKG2E, NKG2F, and / or NKG2H. In some embodiments, the surface polypeptide is a TCR complex polypeptide. In some embodiments, the TCR complex polypeptide is CD3D, CD3E, CD3G, CD3Z, TCRα, or TCRβ. In an exemplary embodiment, the surface polypeptide is CD3E. In some embodiments, the binding polypeptide may be any of the activating elements disclosed in the activating elements section herein. In such embodiments, the surface polypeptide may be a binding partner of an activating element.

[0202] In an exemplary embodiment, the cell mixture is whole blood. In a further exemplary embodiment, the cell mixture is subjected to a red blood cell depletion procedure. In some embodiments, the whole blood is collected from a healthy subject, e.g., a subject not having, known to have, or suspected to have, a disease, disorder, or condition associated with elevated expression of an antigen. In some embodiments, the whole blood is collected from a subject having a disease, disorder, or condition associated with elevated expression of an antigen, in which case the polynucleotide vector is administered to the subject or to another subject having the disease, disorder, or condition. In some embodiments, whole blood is collected from each subject, and the extinction units are calculated separately for each subject.

[0203] In some embodiments, the reaction mixture may be incubated for about 24 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, or 2 hours, or 60 minutes, 45 minutes, 30 minutes, 15 minutes, 10 minutes, or 5 minutes, or less, or just during initial contact. In some embodiments, the reaction mixture may be incubated for 10 minutes to 24 hours, or 10 minutes to 8 hours, or 1 hour to 8 hours, or 1 hour to 6 hours, or in exemplary embodiments, 3.5 to 4.5 hours, or 4 hours. In some embodiments, the reaction mixture may be incubated at about 10°C, 15°C, 20°C, 25°C, 30°C, 37°C, or 42°C. In some embodiments, the reaction mixture is incubated without CO2. In exemplary embodiments, the reaction mixture is incubated with 5% CO2.

[0204] In some embodiments, the surface expression of surface polypeptides is measured by fluorescence-activated cell sorting (FACS). In some embodiments, the antibody used in the FACS is GMP. In some embodiments, the surface expression of surface polypeptides is determined using a CD3 antibody. In some embodiments, the CD3 antibody is UCHT1, OKT-3, HIT3A, TRX4, X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111409, CLB-T3.4.2, TR-66, TR66.opt, HuM291, WT31, WT32, SPv-T3b, 11D8, XIII-141, XIII46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW24B6, OKT3D, M-T301, SMC2, F101.01, and / or SK7. In an exemplary embodiment, the CD3 antibody is PerCP mouse anti-human CD3-clone SK7 (BD, 347344). In some embodiments, cells present in the cell mixture are separated from unbound polynucleotide vectors in the incubated reaction mixture before measuring surface expression of the surface polypeptide.

[0205] In an exemplary embodiment of the above method, the polynucleotide vector preparation is a RIP preparation, the percent extinction is 50%, the extinction volume is 1 ml, the surface polypeptide is CD3, the cell mixture is whole blood drawn from a healthy subject, the reaction mixture is incubated at 37°C and 5% CO2 for 4 hours, and extinction units are calculated using the method.

[0206] Such methods can be used to determine the amount of retroviral particles in a polynucleotide vector preparation that reduces surface polypeptide expression on cells to a specific percentage. This amount can then be used to determine the amount of retroviral particles that can be used to subsequently transduce whole blood, isolated PBMCs, or isolated TNCs. In any of the aspects and embodiments provided herein that involve genetically modified and / or transduced lymphocytes, the amount of polynucleotide vector preparation, e.g., replication-incompetent recombinant retroviral particle preparation, to be added to the lymphocytes can be determined using the methods described above.

[0207] In any of the aspects or embodiments herein that include a contacting step to determine the amount of polynucleotide vector to add, extinction units (DU) can be used. 1 DU of polynucleotide vector reduces surface expression of a surface polypeptide by 50% in a 1 ml volume of cells. 10 DU of polynucleotide vector reduces surface expression of a surface polypeptide by 50% in a 10 ml cell mixture. In some embodiments, sufficient DU are added to a cell volume such that, after contact with the polynucleotide vector, surface expression of a surface polypeptide, e.g., CD3, is reduced by more than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, or 97% compared to surface expression of the surface polypeptide in a cell mixture under similar conditions but not contacted with the polynucleotide vector. In exemplary embodiments, sufficient DU are added to a cell volume such that surface expression of a surface polypeptide is reduced by 80%, 85%, 90%, or greater than 95% after contact with the polynucleotide vector compared to surface expression of the surface polypeptide in a cell mixture under similar conditions but not contacted with the polynucleotide vector. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 DU are added per ml of cell mixture. In exemplary embodiments, 5-20 DU, 5-15 DU, 10-20 DU, or 13-18 DU are added per ml of cell mixture. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 DU are added per 1,000,000 target cells. In some embodiments, the target cells are lymphocytes, such as T cells or NK cells. In exemplary embodiments, the cells are in whole blood, isolated PBMCs, or isolated TNCs. In further exemplary embodiments, the cells are the remainder of whole blood after lysing red blood cells. In some embodiments, sufficient DU is added to dim a specific percentage of a cell population. For example, CD3 on a T cell population is dimmed by more than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, or 97%.In some embodiments, sufficient dimming units of the polynucleotide vector, in exemplary embodiments, a RIP, are present to increase the proportion of surface dimmed surface polypeptides. In exemplary embodiments, dimmed surface CD3- in a cell population, in exemplary embodiments, a T cell population, is increased by at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, or 97%. In any of the aspects and embodiments herein comprising cells contacted with a polynucleotide vector, the composition comprising the cells may comprise, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 DU per ml of cells, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 DU per ml of blood, cell preparation, or cell population.

[0208] In some aspects, provided herein are kits for modifying NK cells and / or, in exemplary embodiments, T cells. In certain embodiments, such kits include one or more containers containing a polynucleotide, typically a substantially pure polynucleotide, comprising one or more first transcription units operably linked to a promoter active in T cells and / or NK cells (the one or more first transcription units encode a first chimeric antigen receptor (CAR) (sometimes referred to as a first CAR)), as well as one or more containers of accessory components (also referred to herein as accessory kit components). Polynucleotides (e.g., retroviral particles) can be stored frozen, for example, at -70°C or below (e.g., -80°C).

[0209] In some embodiments where the kit includes modified cells, e.g., modified lymphocytes, modified TILs, modified lymphocytes that are not B cells, e.g., modified T cells and / or modified NK cells, in a cell suspension in a commercially available container, e.g., a cryopreserved infusion bag, the container, e.g., a cryopreserved infusion bag, may hold up to 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, or 500 ml of blood. In some embodiments, the container, e.g., a cryopreserved infusion bag, may hold at least 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, or 500 ml of blood. In some embodiments, a container such as, for example, a cryopreserved infusion bag may hold 1, 2, 3, 4, 5, 10, 15, 20, 25, and 50 mL of blood at the low end of the range, and 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, and 500 mL of blood at the high end of the range. In some embodiments, a container such as, for example, a cryopreserved infusion bag may hold 1, 2, 3, 4, 5, 10, 15, 20, 25, and 50 mL of blood at the low end of the range, and 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, and 500 mL of blood at the high end of the range. For example, a container such as a cryopreserved infusion bag may hold 1-10 mL, 5-25 mL, 10-50 mL, 25-100 mL, 50-200 mL, or 100-500 mL of blood. In some embodiments, a container such as a cryopreserved infusion bag may contain heparin. In other embodiments, a container such as a cryopreserved infusion bag does not contain heparin.

[0210] In some embodiments, where the kit includes modified cells, e.g., modified lymphocytes, modified TILs, modified lymphocytes other than B cells, or modified T cells and / or modified NK cells, in a cell suspension in a container, e.g., a cell cryopreservation infusion bag, the number of cells delivered is 1 x 10 per kg of body weight of the subject to whom the cells are delivered. 5 cells ~1×10 9 cells, 1 x 10 6 cells ~1×10 9 cells, or 1 x 106 cells ~5×10 8 The number of cells may be sufficient to provide viable CAR-positive T cells and / or NK cells. Thus, in some embodiments, a commercial container may contain 50 to 150 kg, or 50 to 100 kg, of the aforementioned ranges. In some embodiments, a commercial container may contain 1 x 10 7 ~1×10 11 Cells, 1×10 8 ~1×10 11 cells, or 1 x 10 8 ~5×10 10 The cells include, for example, viable CAR-positive T cells and / or viable NK cells, or in an exemplary embodiment, cells that are positive for an anti-idiotype extracellular recognition domain.

[0211] In exemplary embodiments, a polynucleotide encoding any of the various polypeptides disclosed herein, such as a CAR, is located in the genome of a retroviral particle, typically a substantially pure retroviral particle, according to any of the aspects and embodiments of replication-incompetent recombinant retroviral particles provided herein. 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 first transcription units encode a first polypeptide comprising an anti-idiotype polypeptide, a CAR, a TCR, and / or an LE, and optionally a second polypeptide comprising an anti-idiotype polypeptide, a CAR, a TCR, and / or an LE, according to any of the embodiments provided herein.

[0212] The kits provided herein may include a container containing a polynucleotide, e.g., a polynucleotide vector, e.g., a RIP, or containing modified cells, e.g., modified lymphocytes, modified TILs, modified lymphocytes other than B cells, e.g., modified T cells and / or modified NK cells, and an accessory kit. The accessory kit components may include one or more of the following: a. one or more containers containing a delivery solution adapted for, and in exemplary embodiments effective for, and in further exemplary embodiments adapted for, subcutaneous and / or intramuscular administration as provided herein; b. one or more containers of a hyaluronidase provided herein; c. In an exemplary embodiment, one or more blood bags, such as a blood collection bag containing anticoagulant in the bag or a separate container, a blood processing buffer bag, a blood processing waste collection bag, and a blood processing cell sample collection bag; one or more sterile syringes suitable for, and in exemplary embodiments effective for, and in further exemplary embodiments adapted for, subcutaneous or intramuscular delivery of dT cells and / or NK cells; e. A T cell activation element as disclosed in detail herein, e.g., anti-CD3, provided in solution within the vessel containing the retroviral particles, or in a separate vessel, or in an exemplary embodiment, is associated with the surface of the replication-incompetent retroviral particles; f. one or more leukocyte depletion filtration assemblies; one or more containers containing a solution or medium compatible with, in an exemplary embodiment effective for, and in a further exemplary embodiment adapted for, the transduction of gT cells and / or NK cells; one or more containers containing a solution or medium suitable for, in an exemplary embodiment effective for, and / or in a further exemplary embodiment adapted for, rinsing hT cells and / or NK cells; i. one or more containers containing a pH adjusting agent; one or more containers containing a polynucleotide, typically a substantially pure polynucleotide (e.g., found within a recombinant retroviral particle according to any embodiment herein), comprising one or more second transcription units operably linked to a promoter active in jT cells and / or NK cells, wherein the one or more second transcription units encode polypeptides comprising second CARs directed to different target epitopes, and in particular embodiments, different antigens, found on the same target cancer cell (e.g., B cell); k. One or more containers containing the cognate antigen of the first CAR and / or the second CAR encoded by nucleic acid (e.g., a retroviral particle), and l. Instructions, either physically or digitally associated with the other kit components, for their use, e.g., to modify T cells and / or NK cells, to deliver the modified T cells and / or NK cells subcutaneously or intramuscularly to a subject, and / or to treat tumor growth or cancer in a subject.

[0213] In some embodiments, a blood bag can hold up to 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, or 500 mL of blood. In some embodiments, a blood bag can hold at least 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, or 500 mL of blood. In some embodiments, multiple blood bags can hold from 1, 2, 3, 4, 5, 10, 15, 20, 25, and 50 mL of blood at the lower end of the range to 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, and 500 mL of blood at the upper end of the range. In some embodiments, the blood bag can hold volumes of blood ranging from 1, 2, 3, 4, 5, 10, 15, 20, 25, and 50 mL at the lower end of the range to 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, and 500 mL at the upper end of the range. For example, the blood bag can hold volumes of blood ranging from 1 to 10 mL, 5 to 25 mL, 10 to 50 mL, 25 to 100 mL, 50 to 200 mL, or 100 to 500 mL. In some embodiments, the blood bag can contain heparin. In other embodiments, the blood bag does not contain heparin.

[0214] In any of the aspects and embodiments of the kits herein comprising an antigen or cognate antigen, less than 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the polypeptides in the kit are non-human, i.e., produced from a non-human source.

[0215] In some embodiments, the kit may be a single-pack / use kit, while in other embodiments, the kit is a multi-pack or multi-use kit for processing two or more blood samples from contact with a nucleic acid encoding a CAR, optionally via subcutaneous administration. Typically, the container of nucleic acid encoding a CAR (and optionally, in certain embodiments, the paired container of nucleic acid encoding a second CAR) in the kit is used for a single performance of the method for modifying T cells and / or NK cells and optionally for subcutaneous administration. The containers containing the nucleic acid encoding the CAR and optionally the second CAR are typically stored and shipped in a frozen state. Thus, a kit can include sufficient containers (e.g., vials) of nucleic acid encoding a CAR (and optionally, in certain embodiments, a paired container encoding a second CAR) for 1, 2, 3, 4, 5, 6, 10, 12, 20, 24, 50, and 100 performances of the methods for modifying T cells and / or NK cells provided herein, and thus can include 1, 2, 3, 4, 5, 6, 10, 12, 20, 24, 50, and 100 containers (e.g., vials) of nucleic acid encoding a CAR (e.g., retroviral particles), and similarly be considered 1, 2, 3, 4, 5, 6, 10, 12, 20, 24, 50, and 100 packs, performances, administrations, or X kits, respectively. Similarly, accessory components within the kit are provided for a like number of performances of the methods for modifying T cells and / or NK cells, and optionally subcutaneous administration, using the kit.

[0216] When present in such a kit, the one or more leukocyte depletion filtration assemblies typically include one or more leukocyte depletion filters or leukocyte depletion filter sets (each typically within a filter enclosure) adapted for use in a single-use closed blood processing system, as well as a plurality of connecting sterile tubings connected thereto or adapted to be connected thereto, and a plurality of connecting valves connected thereto or adapted to be connected thereto. Typically, there is one leukocyte depletion filtration assembly for each container of CAR-encoding nucleic acid in the kit. Thus, in an exemplary embodiment, a 20-pack kit includes 20 vials of CAR-encoding nucleic acid and 20 leukocyte depletion filtration assemblies. In some embodiments, the kits herein include one or more containers containing nucleic acid and one or more leukocyte depletion filtration assemblies. Such kits may optionally be intended for use in administration to a subject via any route, including, for example, injection, or in an exemplary embodiment, intramuscular delivery, and / or in a further exemplary embodiment, subcutaneous delivery. Thus, such kits optionally include other accessory components intended for use in such administration routes. The one or more containers of subcutaneous or intramuscular delivery solution are discussed in more detail herein, and are typically sterilized and may contain a combined total volume or individual volumes per container of 100 mL to 5 L, 1 mL to 1 L, 1 mL to 500 mL, 1 mL to 250 mL, 1 mL to 200 mL, 1 mL to 100 mL, 1 mL to 10 mL, or 1 mL to 5 mL, 5 mL to 1 L, 5 mL to 500 mL, 5 mL to 250 mL, 5 mL to 100 mL, 5 mL to 10 mL, or about 5 mL. In some exemplary embodiments, the kit comprises multiple containers of subcutaneous delivery solution, each container having a volume of 10 mL to 200 mL, 10 mL to 100 mL, 1 mL to 20 mL, 1 mL to 10 mL, 1 mL to 5 mL, 1 mL to 2 mL, 2 mL to 20 mL, 2 mL to 10 mL, 2 mL to 5 mL, 0.25 mL to 10 mL, 0.25 mL to 5 mL, or 0.25 mL to 2 mL. In exemplary embodiments, there is one container of delivery solution for each container of nucleic acid encoding a CAR in the kit.Thus, a 20-pack kit in an exemplary embodiment includes 20 vials of nucleic acid encoding a CAR and 20 containers of sterile delivery solution.

[0217] In certain kit aspects, provided herein are embodiments in which either or both of the containers containing the nucleic acid encoding the first CAR and, optionally, the nucleic acid encoding the second CAR are nucleic acids according to any of the self-propelled CAR embodiments provided herein. In such embodiments, the accessory components of the kit may further include one or more of the following: a. one or more containers containing a delivery solution adapted for, compatible with, and / or effective for intravenous or intraperitoneal administration as provided herein; and b. Instructions for their use, either physically or digitally associated with the other kit components, e.g., for intravenous or intraperitoneal delivery of the modified T cells and / or NK cells to a subject.

[0218] In certain aspects, provided herein is the use of a RIP in the manufacture of a kit for modifying T cells or NK cells, the use of the kit comprising contacting T cells or NK cells ex vivo with replication-incompetent recombinant retroviral particles, the replication-incompetent recombinant retroviral particles comprising a pseudotyping element on their surface and a T cell activation element on their surface, wherein said contacting promotes transduction of the T cell or NK cell with the replication-incompetent recombinant retroviral particles, thereby genetically modifying, and in exemplary embodiments, producing genetically modified T cells or NK cells.

[0219] In some aspects, provided herein are aspects including the use of replication-incompetent recombinant retroviral particles in the manufacture of kits for modifying T cells or NK cells. Details regarding polynucleotides and replication-incompetent recombinant retroviral particles containing such polynucleotides are disclosed in greater detail herein and in the Exemplary Embodiments section. In some embodiments, the T cells or NK cells may be derived from a subject. In some embodiments, the T cell activation element may be membrane-bound. In some embodiments, the contacting may be performed for a period ranging from 1, 2, 3, 4, 5, 6, 7, or 8 hours at the lower end of the range to 4, 5, 6, 7, 8, 10, 12, 15, 18, 21, and 24 hours at the higher end of the range, e.g., 1 to 12 hours. Replication-incompetent recombinant retroviral particles for use in the manufacture of kits may include any of the aspects, embodiments, or subembodiments discussed elsewhere herein.

[0220] Further, in another aspect, provided herein is a container, e.g., a commercial container or package, containing isolated packaging cells, in exemplary embodiments, isolated packaging cells from a packaging cell line, according to any of the packaging cell and / or packaging cell line aspects provided herein, or a kit comprising the same. In some embodiments, the kit includes additional containers containing additional reagents, such as buffers or reagents, for use in the methods provided herein. Further, in certain aspects, provided herein is the use of any replication-incompetent recombinant retroviral particle provided herein in any aspect in the manufacture of a kit for modifying, and in exemplary embodiments, genetically modifying, T cells or NK cells according to any aspect provided herein. Further, in certain aspects, provided herein is the use of any packaging cell or packaging cell line provided herein in any aspect in the manufacture of a kit for producing replication-incompetent recombinant retroviral particles according to any aspect provided herein.

[0221] In another aspect, provided herein is a pharmaceutical composition for treating or preventing cancer or tumor growth, comprising a replication-incompetent recombinant retroviral particle as an active ingredient. In another aspect, provided herein is an injection composition or other cellular preparation for treating or preventing cancer or tumor growth, comprising a replication-incompetent recombinant retroviral particle. The replication-incompetent recombinant retroviral particle of the pharmaceutical composition or injection composition may comprise any of the aspects, embodiments, or subembodiments discussed above or elsewhere herein.

[0222] Collection of lymphocytes from the subject Some embodiments of any method used in any aspect provided herein, which may include a step for modifying, and in exemplary embodiments, genetically modifying lymphocytes, PBMCs, and in exemplary embodiments, NK cells, and / or in further exemplary embodiments, T cells, by direct administration of RIP ex vivo or in vivo, may include a step for collecting blood from a subject. The blood contains 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). Of note, certain embodiments do not include such a step. However, in embodiments that include collecting blood from a subject, the blood may be collected or obtained from the subject by any suitable method known in the art, as discussed in more detail herein. Thus, the collected blood or blood-derived component is referred to as a "blood-derived product," and is typically a "peripheral blood-derived product" because it is typically isolated from peripheral blood. For example, blood-derived products may be collected by venipuncture or any other blood collection method known in the art, whereby a sample of unfractionated whole blood is collected in a tube, e.g., in a blood bag, or whereby leukocytes and lymphocytes are isolated from the blood, such as by apheresis (e.g., leukapheresis or lymphocytoplasty). In some embodiments, the amount of blood (e.g., unfractionated whole blood) collected is 1-5 mL, 5-10 mL, 10-15 mL, 15-20 mL, 20-25 mL, 5-25 mL, 25-250 mL, 25 mL-125 mL, 50 mL-100 mL, or 50 mL-250 mL, 75 mL-125 mL, 90 mL-120 mL, or 95-110 mL.In some embodiments, the amount of blood drawn is at the lower end of the range of 1, 5, 10, 15, 20, 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, 80 The volume of blood collected may be from 0, or 900 mL, to the upper 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 900 mL, or 1 L. In some embodiments, the volume of blood collected is less than 250 mL, 100 mL, 75 mL, 20 mL, 15 mL, 10 mL, or 5 mL. In some embodiments, lymphocytes (e.g., T cells and / or NK cells) may be obtained by apheresis. In some embodiments, the volume of blood collected and processed during apheresis (e.g., leukapheresis or lymphocytoplasty) ranges from 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.25, or 1.5 L of the subject's total blood volume at the lower end of the range to 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, or 2.5 L of the subject's total blood volume at the upper end of the range, e.g., 0.5-2.5, 0.5-2, 0.5-1.5, or 1-2 L of total blood volume. A human's total blood volume is typically in the range of 4.5-6 L; therefore, much more blood is typically collected and processed during apheresis than when unfractionated whole blood is collected. Whether the target blood cells (e.g., T cells) are obtained by apheresis or unfractionated whole blood is collected, for example, in a blood bag, it is contemplated that the target blood cells (e.g., T cells) therein will be treated according to the methods provided herein, which, in certain exemplary embodiments, result in target blood cells that become modified, genetically modified, and / or transduced.When apheresis (e.g., leukapheresis or lymphoplasmapheresis) is used to collect a cell fraction containing T cells and / or NK cells (e.g., to provide a leukopak or lymphoplasmaphatase), such cells are resuspended in a solution, either directly or after one or more washes, to which a recombinant vector encoding a CAR is added to form a reaction mixture provided herein. Such a reaction mixture can be used in any of the methods provided herein. In some exemplary methods in which a subject or a blood sample from a subject has a low CD3+ blood cell count, apheresis (e.g., leukapheresis or lymphoplasmapheresis) is used to collect blood cells (e.g., leukocytes or lymphocytes) for inclusion in the methods provided herein.

[0223] In aspects and embodiments in which a RIP is administered directly to a subject, T cells and / or NK cells in the subject are contacted with the RIP and modified, and in exemplary embodiments, genetically modified and transduced, in the in vivo reaction mixture formed. Details regarding such in vivo reaction mixtures are found elsewhere herein, for example, in the exemplary embodiments section herein.

[0224] In reaction mixtures related to aspects of compositions and methods intended to modify lymphocytes in whole blood provided herein, and in aspects and embodiments herein involving the co-administration of lymphocytes, such as unmodified T cells and / or NK cells, lymphocytes, including NK cells and / or T cells, may be present in the co-administered lymphocytes or in the reaction mixture at a lower percentage of blood cells and at a lower percentage of leukocytes than in methods involving a PBMC enrichment procedure prior to co-administering or forming the reaction mixture. For example, in some embodiments of these aspects, there are more granulocytes or neutrophils in the co-administered cell population or cell population in the reaction mixture than there are in NK cells, and even more than there are in T cells. Details regarding the composition of anticoagulants and one or more additional blood components present in the co-administered cell population or reaction mixture of aspects intended to modify lymphocytes in whole blood are provided in detail elsewhere herein. In some reaction mixtures provided herein, T cells may comprise, for example, 10, 20, 30, or 40% of the lymphocytes in the co-administered cell population or reaction mixture at the lower end of the range, and 40, 50, 60, 70, 80, or 90% of the lymphocytes in the co-administered cell population or reaction mixture at the higher end of the range. In exemplary embodiments, T cells comprise 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% at the lower end of the range of lymphocytes in the reaction mixture to 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14% at the higher end of the range of lymphocytes in the reaction mixture. In exemplary embodiments, T cells comprise 1-14%, 2-14%, 3-14%, 4-14%, 5-14%, 5-13%, 5-12%, 5-11%, or 5-10% of the lymphocytes in the reaction mixture. In some embodiments, T cells may comprise at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the reaction mixture.As disclosed herein, aspects of compositions and methods for transducing lymphocytes in whole blood typically do not involve blood fractionation, such as a PBMC enrichment step, before lymphocytes from the blood sample are contacted with a recombinant nucleic acid vector, e.g., retroviral particles, in the reaction mixture disclosed herein for those aspects. Thus, in some embodiments, lymphocytes in unfractionated whole blood are contacted with recombinant retroviral particles. However, in some embodiments, particularly in some aspects of the self-powered CAR methods and compositions section herein, and in some embodiments of aspects involving co-administration of a cell population comprising T cells and / or NK cells with direct administration of a RIP, neutrophils / granulocytes are separated from other blood cells, and then the cells are co-administered to a subject or contacted with a RIP in an ex vivo reaction mixture. 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 a blood sample, e.g., using a PBMC enrichment procedure, and then typically formulated into a cell preparation and co-administered to a subject, or subsequently mixed with an ex vivo reaction mixture containing retroviral particles. Those skilled in the art will appreciate that various methods known in the art can be used to enrich different blood fractions containing T cells and / or NK cells.

[0225] 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 many 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, e.g., Panda and Ravindran (2013) Isolation of Human PBMCs. BioProtoc. Vol. 3(3)). Additionally, centripetal force can be used to separate PBMCs from other blood components with Ficoll using the rotational force of the Sepax cell processing system.

[0226] In some embodiments, cells such as PBMCs can be isolated using apheresis, e.g., leukapheresis. For example, AMICUS RBCX (Fresenius-Kabi) and Trima Accel (Terumo BCT) apheresis devices and kits can be used. Cells isolated by apheresis typically contain T cells, B cells, NK cells, monocytes, granulocytes, other nucleated leukocytes, red blood cells, and / or platelets. Cells collected by apheresis can be washed to remove the plasma fraction, and placed in an appropriate buffer or medium, such as phosphate-buffered saline (PBS) or a wash solution that lacks calcium and may lack magnesium, or may lack many, but not all, divalent cations, for subsequent processing steps. In some embodiments, cells collected by apheresis can be genetically modified by any of the methods provided herein. In some embodiments, cells collected by apheresis can be used to prepare any of the cell preparations provided herein. In some embodiments, cells collected by apheresis can be resuspended in various biocompatible buffers, such as Ca-free, Mg-free PBS. Alternatively, unwanted components of a sample containing cells collected by apheresis can be removed, and the cells can be resuspended in culture medium. In some embodiments, leukapheresis can be used to isolate cells such as lymphocytes. In any of the embodiments provided herein involving PBMCs, leukopaques can be used. In any of the embodiments involving TNCs, buffy coats can be used. Another PBMC enrichment method uses an automated leukapheresis collection system (such as the SPECTRA OPTIA® APHERESIS SYSTEM manufactured by Terumo BCT, Inc., Lakewood, CO 80215, USA) to typically return effluent materials such as plasma, red blood cells, and granulocytes to the donor, while high-speed centrifugation is used to separate the inflow of whole blood from the target PBMC fraction, although this 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 requires the patient's presence and participation during the PBMC enrichment step.

[0227] Further non-limiting examples of PBMC enrichment procedures include, in some embodiments involving co-administration of cell populations comprising T cells and / or NK cells, or in some embodiments relating to the ex vivo methods of transduction, genetic modification, and / or modification described herein, PBMCs are isolated using a Sepax or Sepax2 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 may 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 (i.e., positively select) specific cell populations from PBMCs, e.g., PBLs, or subsets thereof, according to cell phenotype before they are used in the reaction mixtures described herein.

[0228] 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, erythrocyte rosetting can be used to remove erythrocytes 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, ABC transporter inhibitors and / or substrates are not present before, during, or both before and during contacting (i.e., not present in the reaction mixture in which the contacting occurs), with or without optional incubation, or are not present in any step of the method.

[0229] In exemplary embodiments of aspects herein involving replication-incompetent recombinant retroviral particles, contact between T cells and / or NK cells and replication-incompetent recombinant retroviral particles can facilitate transduction of the 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 (RIPs) identify and bind to the T cells and / or NK cells, and the T cells and NK cells become "modified," as that term is used herein. At this point, the retrovirus and host cell membranes begin to fuse, and any retroviral pseudotyping elements and / or T cell activation elements, including anti-CD3 antibodies, become incorporated into the surface of the modified T cells and / or NK cells. 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 noteworthy that such a process can occur several hours or even days after contact is initiated, and even after unassociated retroviral particles have been washed away. The genetic material is then typically incorporated into the genomic DNA of the T cells and / or NK cells, at which point the T cells and / or NK cells are "transduced," as that term is used herein. Similarly, cells can be modified, genetically modified, and / or transduced by recombinant vectors other than replication-incompetent recombinant retroviral particles. Cells also internalize and incorporate the genetic material into the genomic DNA of the T cells and / or NK cells after transfection, at which point the T cells and / or NK cells are "stably transfected," as that term is used herein. Thus, in exemplary embodiments, any method for modifying and / or 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 is initiated, the majority of the modified and genetically altered 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, or in some embodiments, stably transduced T cells and / or NK cells, include progeny of ex vivo transduced cells that retain at least a portion of the nucleic acid or polynucleotide integrated into the genome of the cells during ex vivo transduction. In methods herein that recite "reintroducing" transduced cells, it will be understood that such cells are typically not in a transduced state when taken from the subject's blood.

[0230] Introduction or reintroduction, also referred to herein as administration and re-administration to a subject in the methods provided herein, is also referred to as delivery of modified lymphocytes, and in exemplary embodiments, delivery of genetically modified lymphocytes, or in some embodiments, delivery of replication-incompetent recombinant retroviral particles (RIPs), and may be via any route known in the art. Introduction or reintroduction of such genetically modified lymphocytes typically involves suspending i) modified and / or ii) genetically modified and / or iiia) transduced or iiib) transfected cells, or iv) RIPs, in a delivery solution to create a cell or RIP formulation, which can be introduced or reintroduced into a subject as discussed in detail herein. Some embodiments relating to the introduction of RIPs involve suspending RIPs in a delivery solution to create a transduction formulation, which can be introduced into a subject. For example, RIPs, introduction of lymphocytes or modified lymphocytes, or reintroduction of lymphocytes or modified lymphocytes may be delivered into the subject's blood vessels via infusion. In some embodiments, RIPs or modified lymphocytes (e.g., T cells and / or NK cells) are administered or otherwise introduced into a subject, or in the case of lymphocytes or modified lymphocytes, reintroduced back, by intraperitoneal, intratumoral, intramuscular, intralymphatic administration, or in exemplary embodiments, by subcutaneous administration.

[0231] Some administered cells are modified with a nucleic acid encoding a lymphoproliferation element. Without being limited by theory, in a non-limiting exemplary method, delivery of a polynucleotide encoding a lymphoproliferation 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 an engine 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 direct administration of the RIP or 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, after the RIP is directly administered or the modified T cells and / or NK cells are reintroduced back into the subject. In further non-limiting embodiments, the methods provided herein may be performed without exposing the subject to a lymphodepleting agent during the in vivo or ex vivo contacting of the RIP with the subject's T cells and / or NK cells (e.g., resting T cells and / or resting NK cells) and / or during the entire ex vivo procedure or during the entire in vivo administration. Thus, methods of expanding modified, and in exemplary embodiments, 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 are free of or substantially free of ex vivo propagation.

[0232] The present disclosure provides various treatment methods using CARs. When present in T lymphocytes or NK cells, the CARs of the present disclosure can mediate cytotoxicity against target cells. The CARs of the present disclosure bind to antigens present on target cells, thereby mediating target cell killing by T lymphocytes or NK cells genetically modified to produce the CAR. The ASTR of the CAR binds to an antigen present on the surface of the target cell. 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 cell or NK cell) genetically modified to produce the target CAR with the T lymphocyte or NK cell, such that the T lymphocyte or NK cell recognizes the antigen present on the surface of the target cell and mediates killing of the target cell. The target cell may be, for example, a cancer cell, 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. In some embodiments for treating PDL-1-positive cancers, and in exemplary embodiments for treating PDL-1-positive diffuse large B-cell lymphoma (DLBCL), the genetically modified cells may be administered in combination with an anti-PDL-1 antibody or antibody mimetic.

[0233] In some exemplary embodiments, the cells are introduced or reintroduced into a subject by injection into a vein or artery, or by subcutaneous, intratumoral, or intramuscular administration, particularly if neutrophils are not present in the preparation of lymphocytes that have been contacted with retroviral particles and are ready to be reintroduced, or for embodiments in which at least 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, or 25% of the cells, or 1%-90%, 1%-75%, 1%-50%, 1%-25%, 1%-20%, 1%-10%, 5%-90%, 5%-75%, 5%-50%, 5%-25%, 5%-20%, 5%-10%, 10%-90%, 10%-75%, 10%-50%, 10%-25%, or 10%-20% of the cells in the administered cell preparation are neutrophils. Such embodiments may include co-administration or sequential administration with hyaluronidase, as discussed in further detail herein. In any of the embodiments disclosed herein, the number of lymphocytes, and in exemplary embodiments, the number of modified T cells and / or NK cells, present in the cell formulations provided herein and optionally re-infused into the subject, or in exemplary embodiments delivered subcutaneously to the subject, is greater than or equal to 1 x 10 at the lower end of the range. 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, 5 × 10 at the upper end of the range 4 , 1×10 5 , 2.5×10 5 , 5×10 5 , 1×10 6 , 2.5×10 6 , 5×10 6 , 1×10 7 , 2.5×10 7 , 5×10 7 , 1×10 8 , 1×109 , and 1 × 10 10 In certain embodiments, the number of lymphocytes, and in exemplary embodiments, the number of modified T cells and / or NK cells, present in the cell formulations herein and optionally reinfused or otherwise delivered to a subject may range from 1 x 10 at the lower end of the range to 1 x 10 at the lower end of the range. 4 , 2.5×10 4 , 5×10 4 , and 1 × 10 5 cells / kg, 2.5 × 10 at the upper end of the range 4 , 5×10 4 , 1×10 5 , 2.5×10 5 , 5×10 5 , 1×10 6 , 1×10 7 , 2.5×10 7 , 5×10 7 , and 1 × 10 8 cells / kg, or at the lower end of the range 1 x 10 4 cells / kg, 2.5 × 10 at the upper end of the range 4 , 5×10 4 , 1×10 5 , 2.5×10 5 , 5×10 5 , 1×10 6 , 1×10 7 , 2.5×10 7 , 5×10 7 , and 1 × 10 8 In some embodiments, the number of lymphocytes, and in exemplary embodiments, the number of T cells and / or NK cells, present in the cell formulations herein and optionally re-infused or delivered intratumorally, intramuscularly, subcutaneously, or otherwise to a subject may range from 5 x 10 at the lower end of the range to 5 x 10 at the lower end of the range. 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, 2.5 × 10 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 lymphocytes, and in exemplary embodiments, T cells and / or NK cells, present in the cell formulations herein and available for infusion, reinfusion, or other means of delivery (e.g., subcutaneous delivery) to a 70 kg subject or patient may range from 7 x 10 5 ~2.5×10 8 In other embodiments, the number of lymphocytes, and in exemplary embodiments, T cells and / or NK cells, present in the cell preparations herein and available for transduction is about 7 x 10 6 It's plus or minus 10%.

[0234] In any of the embodiments and aspects provided herein involving T cells, NK cells, B cells, or stem cells, the cells may be autologous or allogeneic cells. In some embodiments, the allogeneic cells may be genetically engineered allogeneic cells. Allogeneic cells, such as allogeneic T cells, and methods for genetically engineering allogeneic cells are known in the art. In some embodiments where the allogeneic cells are T cells, the T cells are genetically engineered to functionally impair and / or at least partially delete at least one component of the TCR complex. In some embodiments, the T cells are genetically engineered to reduce or eliminate expression of at least one component of the TCR complex. In some embodiments, the allogeneic cells may be modified to lack all or part of the B2 microglobulin gene. In some embodiments, the allogeneic cells may include any of the lymphoproliferation elements and / or CLEs disclosed herein. The use of lymphoproliferation elements and CLEs may reduce the number of cells required and may enhance cell production of T cells, NK cells, B cells, or stem cells. In some embodiments, the allogeneic cells may be immortalized cells. In any of the aspects or embodiments herein involving allogeneic cells, steps involving drawing blood or contacting cells with replication-incompetent recombinant retroviral particles may be excluded. For example, to treat a subject with allogeneic CAR-T cells, the T cells may be pre-genetically modified, and the genetically modified allogeneic CAR-T cells are administered to the subject without drawing blood from the subject. In some embodiments, the allogeneic cells are administered subcutaneously. In some embodiments, the allogeneic cells are administered intravenously. In some embodiments, the allogeneic cells are administered intraperitoneally.

[0235] In some embodiments of any of the methods provided herein for modifying lymphocytes (e.g., T cells and / or NK cells), as well as in some aspects for using replication-incompetent recombinant retroviral particles (RIPs) in the manufacture of kits for modifying T cells and / or NK cells in a subject, modified, genetically modified, and / or transduced lymphocytes (e.g., T cells and / or NK cells), or populations thereof, or RIPs in cell-free compositions provided herein, e.g., GMP RIP compositions, are introduced or reintroduced into a subject. Delivery, introduction, or reintroduction of modified lymphocytes, in exemplary embodiments, genetically modified lymphocytes, and direct delivery or reintroduction of RIPs into a subject may be via any route known in the art. For example, introduction or reintroduction may be via injection into a subject's blood vessels, intratumorally, intraperitoneally, intramuscularly, intralymph node, and, in certain exemplary embodiments, subcutaneously. In some embodiments, modified, genetically modified, and / or transduced lymphocytes (e.g., T cells and / or NK cells) or populations thereof undergo no more than four cell divisions ex vivo before being introduced or reintroduced into a subject. In some embodiments, the lymphocytes used in such methods are resting T cells and / or NK cells that have been in contact with replication-incompetent recombinant retroviral particles for 1 to 12 hours. In some embodiments, 12, 10, 8, 6, 4, 2, or 1 hour or less elapses between the time blood is drawn from the subject and the time the unmodified, modified, and / or genetically modified T cells and / or NK cells are formulated for delivery and / or reintroduction 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 personnel monitoring the closed system throughout the process.

[0236] Enrichment of T cells and / or NK cells by positive selection In some embodiments, any cells in a cell mixture, cell preparation, or reaction mixture useful in adoptive cell therapy, referred to herein as desired cells, such as one or more cell populations of T cells and / or NK cells, may be enriched prior to formulation for delivery. In some embodiments, the desired cells may be enriched by positive selection prior to contact with a recombinant nucleic acid vector, such as a replication-incompetent retroviral particle. In other embodiments, the desired cells may be enriched by positive selection after contacting the cell mixture, cell preparation, or reaction mixture with a recombinant nucleic acid vector, such as a replication-incompetent recombinant retroviral particle. In some embodiments, enriching one or more cell populations may be performed simultaneously with any of the genetic modification methods disclosed herein, and in exemplary embodiments, genetic modification using a replication-incompetent retroviral particle.

[0237] Mononuclear cells (such as PBMCs) or TNCs can be isolated from more complex cell mixtures, such as whole blood, by density gradient centrifugation or reverse perfusion through a leukocyte-reduction filter assembly, respectively, as described in more detail herein. In some embodiments, desired cells may possess a particular cell lineage, e.g., NK cells, T cells, and / or T cell subsets, including naive, effector, memory, suppressor, and / or regulatory T cells, and may be enriched through selection of cells expressing one or more surface molecules. In exemplary embodiments, the one or more surface molecules may include TCR components such as CD4, CD8, CD16, CD25, CD27, CD28, CD44, CD45RA, CD45RO, CD56, CD62L, CCR7, KIR, FoxP3, and / or CD3. Methods using beads conjugated to antibodies directed against one or more surface molecules can be used to enrich for desired cells using magnetic, density, and size-based separation.

[0238] In the process of such antibody-based positive selection methods, binding of one or more cell surface molecules can result in signal transduction and changes in the biology of the bound cells. For example, selection of T cells using beads with antibodies attached to CD3 can result in CD3 signal transduction and T cell activation. In other examples, binding and signal transduction can result in further cell differentiation of cells such as naive or memory T cells. In some embodiments, positive selection is not used to enrich for desired cells, such as when it is preferable for the desired cells to be left untouched rather than contacted.

[0239] In some embodiments, the desired cells may be enriched, whereby the desired cells comprise at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the cells in a cell mixture, cell preparation, or reaction mixture. In some embodiments, the desired cells may be concentrated, whereby the desired cells comprise a range of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, or 40% at the lower end of the range of cells in the cell mixture, cell preparation, or reaction mixture, and 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% at the upper end of the range of cells in the cell mixture, cell preparation, or reaction mixture. In some embodiments, the desired cells may be enriched, such that the desired cells comprise 10% to 90%, 20% to 90%, 30% to 90%, 40% to 90%, 40% to 80%, 45% to 75%, 1% to 14%, 2% to 14%, 3% to 14%, 4% to 14%, 5% to 14%, 5% to 13%, 5% to 12%, 5% to 11%, or 5% to 10% of the cells in the cell mixture, cell preparation, or reaction mixture.

[0240] Enrichment of desirable cells by depletion of unwanted cells In some embodiments, any cells in a cell population, cell mixture, cell preparation, or reaction mixture derived from whole blood, isolated TNC, or isolated PBMCs may contain one or more unwanted cell populations, referred to herein as unwanted cells, and can be depleted of unwanted cells, thereby enriching the cell mixture, cell preparation, or reaction mixture for desirable cells. In some embodiments, unwanted cells can be depleted by negative selection, followed by contact with a recombinant nucleic acid vector, such as a replication-incompetent retroviral particle, as provided in the methods for genetically modifying T cells or NK cells provided herein. In other embodiments, unwanted cells can be depleted by negative selection after contacting the cell mixture with a recombinant nucleic acid vector, such as a replication-incompetent retroviral particle, as provided in the methods for genetically modifying T cells or NK cells provided herein. In some embodiments, depletion of unwanted cells can be performed simultaneously with any of the genetic modification methods disclosed herein, and in exemplary embodiments, can be performed simultaneously with genetic modification using a replication-incompetent retroviral particle.

[0241] In some embodiments, unwanted cells may include any non-T cell or non-NK cell. In some embodiments, unwanted cells may include T cell or NK cell subsets, such as regulatory T cells or suppressor T cells. In some embodiments, unwanted cells include cancer cells. In some embodiments, unwanted cells include monocytes. In some embodiments, unwanted cells include granulocytes. In exemplary embodiments, unwanted cells include cells that express a cognate antigen for a CAR that is or will be expressed on the population of cells formulated for delivery.

[0242] In further exemplary embodiments, unwanted cells include cancer cells. Cancer cells from many types of cancer can enter the bloodstream and can be unintentionally genetically modified at low frequencies along with lymphocytes using the methods provided herein. In some embodiments, cancer cells can be derived from any cancer, including, but not limited to, renal cell carcinoma, gastric cancer, sarcoma, breast cancer, lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Hodgkin's lymphoma, non-Hodgkin's lymphoma (B-NHL), neuroblastoma, glioma, glioblastoma, medulloblastoma, colorectal cancer, ovarian cancer, prostate cancer, mesothelioma, lung cancer (e.g., small cell lung cancer), melanoma, leukemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML). In exemplary embodiments, CAR cancer cells can be derived from B-cell lymphoma. Without being limited by theory, cancer cells expressing a CAR with an ASTR that binds to an antigen expressed on their own cell surface, i.e., the CAR-expressing cancer cells, are themselves target cells (CAR cancer cells) and can block CAR-T cell binding to the antigen, also known as epitope masking, thereby preventing CAR cancer cell killing. CAR-cancer cells can lead to cancer recurrence due to immunity to CAR-T, even after initial successful treatment with CAR-T (see, e.g., Ruella et al. Nat Med. 2018 Oct;24(10):1499-1503). The methods and compositions provided herein for depleting unwanted cancer cells overcome this risk posed by genetically modified cells, such as blood cells or PBMCs, isolated from cancer patients.

[0243] Monocytes can be depleted by incubating the cell mixture with an immobilized monocyte-binding substrate, such as a standard plastic tissue culture plate, nylon or glass wool, or Sephadex resin. Without being limited by theory, monocytes preferentially adhere to the immobilized monocyte-binding substrate compared to other cells in the cell mixture, which adhere less frequently or strongly, or not at all. In some embodiments, incubation can be carried out at 37°C for at least 1 hour, or by passing the cell mixture through the resin. After incubation, the desired non-adherent cells in suspension are collected for further processing. In exemplary embodiments of the rapid ex vivo processing of lymphocytes provided herein, whole blood, TNC, or PBMCs are not incubated with the immobilized monocyte-binding substrate for at least 8, 7, 6, 5, 4, 3, 2, or 1 hour, and monocytes are not depleted by such incubation.

[0244] In exemplary embodiments, the methods herein involve depleting unwanted cells by negative selection of cells expressing one or more surface molecules, using methods known in the art for removing such cells. In exemplary embodiments, the surface molecules are tumor-associated antigens, tumor-specific antigens, or are expressed on cancer cells by other means, such as, for example, circulating tumor cells. In some embodiments, surface molecules include Axl, ROR1, ROR2, Her2 (ERBB2), 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, chromogranin, protein melan-A (a melanoma antigen recognized by T lymphocytes;MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), MAGE-AI, high molecular weight-melanoma-associated antigen (HMW-MAA), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, dimeric form of pyruvate kinase isoenzyme type M2 (tumor M2-PK), CD 19, CD20, CD22, CD23, CD24, CD27, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD45, CD70, CD99, CD117, CD123, CD138, CD171, GD2 (ganglioside G2), EphA2, CSPG4, FAP (fibroblast activation protein), kappa, lambda, 5T4, αvβ6 integrin, integrin αvβ3 (CD61), galactin, K-Ras (V-Ki-ras2) Kirsten rat sarcoma virus oncogene), Ral-B, B7-H3, B7-H6, CAIX, EGFR, EGP2, EGP40, EpCAM, fetal AchR, FRα, GD3, HLA-A1+MAGE1, HLA -A1+NY-ESO-1, HLA-DR, IL-11Rα, L-13Rα2, Lewis-Y, Muc16, NCAM, NKG2D ligand, PRAME, Survivin, TAG72, TEMs, VEGFR2, EGFRv III (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 (prostatic six-transmembrane epithelial antigen 1), abnormal ras protein, or abnormal p53 protein, New York esophageal squamous cell carcinoma antigen (NYESO1), or PDL-1. In further exemplary embodiments, the surface molecule is a blood cancer antigen such as CD19, CD20, CD22, CD25, CD32, CD34, CD38, CD123, BCMA, TACI, or TIM3.

[0245] In some embodiments, unwanted cells can be depleted from cell mixtures, such as whole blood, PBMCs, or TNCs, by bead- or column-based separation. In these embodiments, a ligand or antibody to a cell surface molecule is attached to the bead or column. In some embodiments, the antibody attached to the bead can bind to the same antigen as a CAR expressed by, for example, T cells and / or NK cells, used in the method to remove unwanted cells. In some embodiments, the antibody attached to the bead can bind to a different epitope of the same antigen as a CAR that will later be expressed in the patient. In an exemplary embodiment, the antibody attached to the bead can bind to the same epitope of the same antigen as a CAR. In some embodiments, beads can have two or more attached antibodies that bind to antigens on the surface of unwanted cells. In some embodiments, beads with different antibodies attached can be used in combination. In some embodiments, the beads can be magnetic beads. In some embodiments, unwanted cells can be depleted by magnetic separation after incubation of the cell mixture with magnetic beads to which antibodies are attached. In some embodiments, the beads are not magnetic beads.

[0246] In some embodiments, unwanted cells expressing one or more surface molecules are depleted and separated by size from a cell mixture, such as whole blood, PBMCs, or TNCs, using antibody-coated beads. In some embodiments, the beads are polystyrene. In exemplary embodiments, the beads are at least about 30 μm, about 35 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, or about 80 μm in diameter. In some embodiments, the antibody-coated beads are added to a cell mixture during which a recombinant nucleic acid vector, in exemplary embodiments, a replication-incompetent recombinant retroviral particle, is incubated with the cell mixture. In these embodiments, a reaction mixture is formed comprising: (A) a cell mixture, such as from whole blood, enriched TNCs, or enriched PBMCs; (B) a recombinant nucleic acid vector, such as a replication-incompetent recombinant retroviral particle, encoding a transgene of interest, such as a CAR; and (C) antibody-coated beads that bind to one or more surface molecules or antigens expressed on the surface of unwanted cells. In some embodiments, the reaction mixture can be incubated for less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 45 minutes, or for less than 1, 2, 3, 4, 5, 6, 7, or 8 hours. In some embodiments, after incubation, a density gradient centrifugation-based cell enrichment procedure can be performed to enrich for total mononuclear cells, depleted of unwanted cells complexed to the pelleted antibody-coated beads. In other embodiments, the reaction mixture can be passed through a larger-diameter mesh prefilter to deplete unwanted cells complexed to the antibody-coated beads. In some embodiments, the filter can have a pore size (approximately) 5 μm, (approximately) 10 μm, or (approximately) 15 μm smaller than the diameter of the beads. In other embodiments, the beads can be magnetic beads, and the prefilter can be a magnet. Such a filter can capture unwanted cells bound to the beads and allow the desired cells to flow downstream to a leukocyte depletion filter assembly with a smaller pore size.

[0247] In some embodiments, unwanted cells are depleted or removed from a cell mixture containing lymphocytes and red blood cells, e.g., whole blood, by erythrocyte antibody rosetting (EA-rosetting). In EA-rosetting, antibodies that bind to antigens on the cell surface of unwanted cells are incubated with the cell mixture to crosslink the unwanted cells to red blood cells, which are then separated from desired cells by density gradient centrifugation, such as that provided in the RosetteSep™ kit (Stemcell Technologies). In some embodiments, the antibodies that mediate EA-rosetting are added to the cell mixture during the time that a recombinant nucleic acid vector, which in an exemplary embodiment is a replication-incompetent recombinant retroviral particle, is incubated with the cell mixture. In exemplary embodiments, a reaction mixture is formed that includes: (A) a cell mixture of lymphocytes and erythrocytes, such as from whole blood; (B) replication-incompetent recombinant retroviral particles encoding a transgene of interest, and in further exemplary embodiments, a CAR; (C) a first antibody against an antigen on the surface of unwanted cells, e.g., a major antigen such as the blood cancer antigens CD19, CD20, CD22, CD25, CD32, CD34, CD38, CD123, BCMA, TACI, or TIM3; (D) a second antibody against an antigen on the surface of erythrocytes, such as glycophorin A; and (E) a third antibody that crosslinks the first and second antibodies. In further exemplary embodiments, the reaction mixture may include antibodies against more than one antigen on the surface of unwanted cells. In some embodiments, the antibodies may bind to the same antigen as the CAR. In some embodiments, the reaction mixture is incubated for less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 45 minutes, or for less than 1, 2, 3, 4, 5, 6, 7, or 8 hours. In an exemplary embodiment, after incubation, a density gradient centrifugation-based PBMC enrichment procedure is performed to isolate total PBMCs minus the population depleted or removed by EA-rosetting, which pellets with red blood cells.

[0248] As described above, genetic modification of cancer cells with recombinant nucleic acid vectors encoding engineered T cell receptors or CARs can be minimized during cell processing by including a positive selection step to enrich for T cells and / or NK cells prior to formulation and / or delivery to a subject, or by depleting cancer cells from the cell mixture by negative selection in the methods provided herein. Several additional methods for reducing the potential effects of cancer cells genetically modified with engineered T cell receptor or CAR constructs are disclosed herein. For example, a T cell-specific promoter (disclosed elsewhere herein) can be used to express the CAR, which can help prevent non-T cells containing exogenous nucleic acid encoding the CAR from actually expressing the CAR. Thus, the antigen is not masked by the CAR expressed in cis, and CAR-T cells can bind to and kill target cells containing exogenous nucleic acid encoding the CAR. Furthermore, the use of a T cell-specific promoter to express the engineered T cell receptor or CAR serves to reduce, minimize, or in exemplary embodiments substantially eliminate or even eliminate expression of the engineered T cell receptor or CAR in the encapsulated nucleic acid vector, e.g., an RIR retroviral particle or virus-like particle, such that expression of the engineered T cell receptor or CAR in the cell line used to generate the encapsulated nucleic acid vector is reduced, low, negligible, substantially absent, or absent. In exemplary embodiments, such expression is reduced, substantially eliminated, or eliminated on the surface of the encapsulated nucleic acid vector (e.g., an RIR particle or virus-like particle).

[0249] Another method for reducing the potential effect of CAR cancer cells is to use two or more separate CARs, and in an exemplary embodiment, two CARs expressed in two cell populations, to kill target cells that may mask one of the epitopes. Cell populations, such as blood cells or PBMCs, are genetically modified separately, so that each population expresses either the first CAR or the second CAR. In an exemplary embodiment, target cells expressing the first or second CAR do not mask the epitope bound by the second and first CARs, respectively. Thus, target cells expressing the first or second CAR can be killed by effector T cells or NK cells expressing the second or first CAR, respectively. In some embodiments, the first and second CARs can bind to different epitopes of the same antigen expressed on the target cell. In other embodiments, the first and second CARs can bind to different antigens expressed on the same target cell, including any of the antigens disclosed elsewhere herein. In some embodiments, the first and second CARs may bind to different epitopes of, or different antigens selected from, CD19, CD20, CD22, CD25, CD32, CD34, CD38, CD123, BCMA, TACI, or TIM3. In a further exemplary embodiment, the first CAR may bind to CD19 and the second CAR may bind to CD22, both of which are expressed on B cells. In other embodiments, the CAR may be an extracellular ligand for a cancer antigen. In exemplary embodiments, the modified cell populations are formulated separately. In some embodiments, the separate cell preparations are introduced or reintroduced into the subject at different sites within the body. In some embodiments, the separate cell preparations are introduced or reintroduced into the subject at the same site. In other embodiments, the modified cell populations are optionally combined into a single preparation that is introduced or reintroduced into the subject together at the same site. In exemplary embodiments in which the cell populations are combined, the cell populations are not combined until after a wash step in which the cells are washed away from the recombinant nucleic acid vectors.By this method, which uses two or more different CARs, CAR cancer cells expressing a first or second CAR that binds and masks its cognate epitope in cis are killed by CAR-T cells expressing the second or first CAR, respectively.

[0250] In some embodiments, unwanted cells may be depleted, such that unwanted cells comprise at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the cells in a cell mixture, cell preparation, or reaction mixture. In some embodiments, unwanted cells may be depleted, such that unwanted cells comprise 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, or 40% at the lower end of the range of cells in the cell mixture, cell preparation, or reaction mixture, and 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% at the upper end of the range of cells in the cell mixture, cell preparation, or reaction mixture. In some embodiments, unwanted cells may be depleted, such that unwanted cells comprise 10% to 90%, 20% to 90%, 30% to 90%, 40% to 90%, 40% to 80%, 45% to 75%, 1% to 14%, 2% to 14%, 3% to 14%, 4% to 14%, 5% to 14%, 5% to 13%, 5% to 12%, 5% to 11%, or 5% to 10% of the cells in the cell mixture, cell preparation, or reaction mixture.

[0251] Membrane-bound cytokines Some embodiments of the aspects of the methods and compositions provided herein include a membrane-bound cytokine, typically associated with and / or located on the outer membrane or surface of a RIP, or located on the cell membrane of an engineered, genetically engineered, and / or transduced T cell and / or NK cell, or population thereof, or comprising a polynucleotide encoding the membrane-bound cytokine, and in exemplary embodiments, comprising the polynucleotide within the RIP, e.g., in the genome of a RIP or a population of RIPs, or within a T cell and / or NK cell, or population thereof. Cytokines are typically, but not always, secreted proteins. Naturally secreted cytokines can be engineered as membrane-bound fusion proteins. Membrane-bound cytokine fusion polypeptides are included in the methods and compositions disclosed herein and are also an aspect of the present invention. In some embodiments, a RIP has a membrane-bound cytokine fusion polypeptide on its surface that can bind to and promote the proliferation and / or survival of T cells and / or NK cells, or that can attract or retain immune cells. Such RIPs can be used, for example, in RIP formulations provided herein for administration to a subject. Typically, the membrane-bound polypeptide is incorporated into the membrane of a replication-incompetent recombinant retroviral particle, and when a cell is transduced by the replication-incompetent recombinant retroviral particle, fusion of the retroviral membrane with the host cell membrane occurs, resulting in the production of a polypeptide, such as a cytokine fusion polypeptide, that is bound to the membrane of the transduced cell. In some embodiments, the RIP formulation and / or delivery solution contains a membrane-bound cytokine associated with the RIP surface. In some embodiments, a RIP formulation or delivery solution comprising a RIP having a membrane-bound cytokine associated with its surface as disclosed herein is administered in vivo to a subject in need thereof. In some embodiments, a RIP having a membrane-bound cytokine associated with its surface as disclosed herein contacts T cells and / or NK cells in vivo (direct RIP administration).In some embodiments, a RIP having a membrane-bound cytokine associated with its surface as disclosed herein is contacted with T cells and / or NK cells ex vivo.

[0252] In some embodiments, the cytokine fusion polypeptide comprises one or more of IL-1, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, TNFα, IFNγ, GM-CSF, CCL1, CCL2 (MCP-1), CCL3, CCL5, CCL7 (MCP-3), CCL8 (MCP-2), CCL19, CCL20, CCL21, CCL22, CCL28, CXCL1, CXCL9, CXCL10, CXCL11, CXCL12, CXCL14 (BRAK), or CX3CL1, or a variant of any of them, or an active fragment of any of them. In some embodiments, the cytokine fusion polypeptide does not comprise IL-2, IL-7, or IL-15.

[0253] In some embodiments, the cytokine fusion polypeptide comprises a cytokine capable of promoting the proliferation and / or survival of T cells and / or NK cells. In some embodiments, the cytokine fusion polypeptide capable of promoting the proliferation and / or survival of T cells and / or NK cells comprises one or more of IL-2, IL-7, IL-15, IL-21, or variants thereof, or active fragments of any of them.

[0254] In some embodiments, the cytokine fusion polypeptide comprises a cytokine capable of stimulating inflammation, hi some embodiments, the cytokine fusion polypeptide comprises one or more of IL-1, IL-12, IL-18, TNFα, IFNγ, GM-CSF, or a variant thereof, or an active fragment of any of them.

[0255] In some embodiments, the cytokine fusion polypeptide comprises a chemotactic cytokine (e.g., a chemokine). Without being limited by theory, the chemotactic cytokine can attract and optionally retain immune cells (e.g., T cells, NK cells, NK T cells, TILs (tumor-infiltrating T cells), MILs (bone marrow-infiltrating lymphocytes), TINKs (tumor-infiltrating NK cells), or dendritic cells). In some embodiments, the chemotactic cytokine comprises a CC motif. In some embodiments, the chemotactic cytokine comprising a CC motif comprises one or more of CCL1, CCL2 (MCP-1), CCL3, CCL5, CCL7 (MCP-3), CCL8 (MCP-2), CCL19, CCL20, CCL21, CCL22, CCL28, or variants thereof, or active fragments of any of them. In exemplary embodiments, the chemotactic cytokine comprising a CC motif comprises CCL19, CCL21, or a variant thereof, or one or more active fragments of any of the foregoing capable of binding to CCR7 or CXCR3. In some embodiments, the chemotactic cytokine comprises a C-X-C motif. In some embodiments, the chemotactic cytokine comprising a C-X-C motif comprises CXCL1, CXCL9, CXCL10, CXCL11, CXCL12, CXCL14 (BRAK), or a variant thereof, or one or more active fragments of any of the foregoing. In some embodiments, the chemotactic cytokine comprises a C-X-3-C motif. In some embodiments, the chemotactic cytokine comprising a C-X-3-C motif comprises CX3CL1, or a variant thereof, or one or more active fragments of any of the foregoing.

[0256] In some embodiments, the cytokine fusion polypeptide comprises one or more polypeptides capable of binding to CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CXCR3, CXCR4, CXCR5, CXCR6, or Cx3cr1. In exemplary embodiments, the cytokine fusion polypeptide comprises one or more polypeptides capable of binding to CCR7, CXCR3, CXCR4, or CXCR6.

[0257] In some embodiments, the cytokine fusion polypeptide comprises one or more polypeptides capable of binding to CCR1, CC42, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CXCR3, CXCR4, CXCR5, or CXCR6.

[0258] Membrane-bound cytokine fusion polypeptides are typically cytokines fused to a heterologous signal sequence and / or heterologous membrane attachment sequence. In some embodiments, the heterologous membrane attachment sequence is a GPI anchor attachment sequence. The heterologous GPI anchor attachment sequence can be derived from any known GPI-anchored protein (Ferguson MAJ, Kinoshita T, Hart GW. Glycosylphosphatidylinositol Anchors. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Discussed in Chapter 11). In some embodiments, the heterologous GPI anchor attachment sequence is a GPI anchor attachment sequence from CD14, CD16, CD48, CD55 (DAF), CD59, CD80, and CD87. In some embodiments, the heterologous GPI anchor attachment sequence is derived from CD16. In exemplary embodiments, the heterologous GPI anchor attachment sequence is derived from the Fc receptor FcγRIIIb (CD16b). In some embodiments, the GPI anchor is the GPI anchor of DAF.

[0259] In exemplary embodiments, membrane-bound cytokine is a cytokine fusion polypeptide that is fused with DAF.DAF is known to accumulate in lipid rafts that are incorporated into the membrane of replication-incompetent recombinant retroviral particles that bud from packaging cells.Therefore, without being limited by theory, it is believed that DAF fusion protein is preferentially targeted to the part of the membrane of packaging cell that becomes part of the recombinant retroviral membrane.

[0260] In a non-limiting exemplary embodiment, the cytokine fusion polypeptide is IL-7 or an active fragment thereof fused to DAF. In a specific non-limiting exemplary embodiment, the fusion cytokine polypeptide comprises, in order, the DAF signal sequence (residues 1-31 of DAF), IL-7 without its signal sequence, and residues 36-525 of DAF.

[0261] In some embodiments, the membrane-bound cytokine fusion polypeptide comprises a cleavage site. In some embodiments, the cleavage site may be within the sequence of the cytokine. In some embodiments, the cleavage site may be within the sequence of the heterologous signal sequence. In some embodiments, the cleavage site may be within the sequence of the heterologous membrane-binding sequence. In some embodiments, the cleavage site may be between the cytokine and the heterologous signal sequence or the heterologous membrane-binding sequence.

[0262] In some embodiments, the membrane-bound cytokine fusion polypeptide may include a linker as otherwise disclosed herein.

[0263] Engineered signaling polypeptides In some embodiments, replication-incompetent recombinant retroviral particles used to contact T cells and / or NK cells, either in vivo (e.g., aspects and embodiments of direct RIP administration) or ex vivo, comprise 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 antibody, 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 lymphoproliferation elements (LEs), such as chimeric antigen receptors (CARs) or chimeric lymphoproliferation elements (CLEs). In some embodiments, at least one, two, or all of the engineered signaling polypeptides are engineered T cell receptors ...

Claims

1. below: a) greater than 10 million transduction units (TU) / ml, wherein said TU are replication-incompetent recombinant retroviral particles (RIPs), wherein said RIPs have the following: i) an activating element on the surface of the RIP; and ii) a polynucleotide comprising one or more transcription units wherein each of the one or more transcription units is operably linked to a promoter, and the one or more transcription units encode a first polypeptide and / or an inhibitory RNA molecule. and b) 0.5% to 10% colloid, wherein the colloid is selected from dextran or hetastarch, and wherein the molecular weight of said colloid is greater than 10 kDa. A subcutaneous formulation comprising:

2. 1. A replication-incompetent recombinant retroviral particle (RIPs) for use in administering a formulation to a subject, the use of the RIPs comprising administering a formulation to a subject, the formulation comprising a transduction unit (TU), the TU being a RIPs, the RIPs comprising: a) a membrane-associated activation element of RIP; and b) a polynucleotide comprising one or more transcription units, each of said one or more transcription units being operably linked to a promoter, and said one or more transcription units encoding a first polypeptide and / or an inhibitory RNA molecule. The replication-incompetent recombinant retroviral particles (RIPs) comprising:

3. 1. A method of administering a formulation to a subject, comprising administering the formulation to a subject, wherein the formulation comprises a plurality of transduction units (TUs), the TUs being replication-incompetent recombinant retroviral particles (RIPs), the RIPs comprising: a) a membrane-associated activation element of RIP; and b) a polynucleotide comprising one or more transcription units, each of said one or more transcription units being operably linked to a promoter, said one or more transcription units encoding a first polypeptide and / or an inhibitory RNA molecule; The method comprising:

4. 4. A RIP for use in administering a formulation to a subject according to claim 2, wherein the formulation comprises greater than 10 million transduction units (TU) / ml, the formulation comprises 0.5% to 10% colloid, the colloid being selected from dextran or hetastarch, and the molecular weight of the colloid being greater than 10 kDa; or the method of claim 3.

5. 10. The subcutaneous formulation of claim 1, the RIP for use in administering the formulation to a subject of claim 2 or 4, or the method of any one of claims 3 to 4, wherein the formulation comprises more than 50 million TU / ml.

6. 6. The subcutaneous formulation according to claim 1 or 5, wherein the molecular weight of the colloid is greater than 40 kDa. A RIP for use in administering a formulation to a subject according to any one of claims 2, 4 to 5, or a method according to any one of claims 3 to 5.

7. A subcutaneous formulation according to any one of claims 1 or 5-6, a RIP for use in administering the formulation to a subject according to any one of claims 2, 4-6, or a method according to any one of claims 3-6, wherein the colloid is dextran.

8. A subcutaneous formulation according to any one of claims 1 or 5 to 6, a RIP for use in administering the formulation to a subject according to any one of claims 2, 4 to 6, or a method according to any one of claims 3 to 6, wherein the colloid is hetastahl.

9. A subcutaneous formulation according to any one of claims 1 or 5 to 8, a RIP for use in administering the formulation to a subject according to any one of claims 2, 4 to 8, or a method according to any one of claims 3 to 8, wherein the formulation comprises less than 50 pg of non-human and non-viral protein / TU.

10. A subcutaneous formulation according to any one of claims 1 or 5-9, a RIP for use in administering the formulation to a subject according to any one of claims 2, 4-9, or a method according to any one of claims 3-9, wherein the formulation comprises 100 pg or less of host cell protein / TU.

11. 11. The subcutaneous formulation, RIP, or method for administering the formulation to a subject of claim 10, wherein the host cell is a HEK 293 cell or a mutant thereof.

12. A subcutaneous formulation according to any one of claims 1 or 5 to 11, a RIP for use in administering the formulation to a subject according to any one of claims 2, 4 to 11, or a method according to any one of claims 3 to 11, wherein the formulation comprises at least 1,000 TU / ng of p24.

13. A subcutaneous formulation according to any one of claims 1 or 5 to 12, a RIP for use in administering the formulation to a subject according to any one of claims 2, 4 to 12, or a method according to any one of claims 3 to 12, wherein the formulation further comprises human serum albumin (HSA).

14. 14. The subcutaneous formulation, RIP, or method of use in administering the formulation to a subject of claim 13, wherein the formulation comprises 0.2% to 5% HSA.

15. A subcutaneous formulation according to any one of claims 1 or 5 to 14, a RIP for use in administering the formulation to a subject according to any one of claims 2, 4 to 14, or a method according to any one of claims 3 to 14, wherein the formulation comprises 1% or less DMSO.

16. A subcutaneous formulation according to any one of claims 1 or 5 to 15, a RIP for use in administering the formulation to a subject according to any one of claims 2, 4 to 15, or a method according to any one of claims 3 to 15, wherein the formulation further comprises hyaluronidase.

17. The formulation further comprises one or more cytokines, wherein the cytokines are IL-1, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, TNFα, IFNγ, GM-CSF, CCL1, CCL2 (MCP-1), CCL3, CCL5, CCL7 (MCP-3), CCL8 (MCP-2), CCL19, CCL20, CCL21, CCL22, CCL28, CXCL1, CXCL9, CXCL10, CXCL11, CXCL12, CXCL14 (BRAK), CX3CL1, and mutants and active fragments thereof; a RIP for use in administering the formulation to a subject according to any one of claims 2, 4 to 16; or a method according to any one of claims 3 to 16.

18. A subcutaneous formulation according to any one of claims 1 or 5 to 17, a RIP for use in administering the formulation to a subject according to any one of claims 2, 4 to 17, or a method according to any one of claims 3 to 17, wherein the formulation further comprises T cells.

19. A subcutaneous formulation according to any one of claims 1 or 5 to 18, a RIP for use in administering the formulation to a subject according to any one of claims 2, 4 to 18, or a method according to any one of claims 3 to 18, wherein the formulation has a volume of 0.5 ml to 20 ml and is contained within a subcutaneous delivery device.

20. The RIP comprises one or more membrane-bound cytokines, the one or more cytokines being IL-1, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, TNFα, IFNγ, GM-CSF, CCL1, CCL2 (MCP-1), CCL3, CCL5, CCL7 (MCP-3), CCL8, (MCP-2), CCL19, CCL20, CCL21, CCL22, CCL28, CXCL1, CXCL9, CXCL10, CXCL11, CXCL12, CXCL14 (BRAK), or CX3CL1; a RIP for use in administering the formulation to a subject according to any one of claims 2, 4 to 19; or a method according to any one of claims 3 to 19.

21. A subcutaneous formulation according to any one of claims 1 or 5 to 20, a RIP for use in administering the formulation to a subject according to any one of claims 2, 4 to 20, or a method according to any one of claims 3 to 20, wherein the activating element comprises one or more of an antibody or antibody mimetic capable of binding to CD3, TCR alpha / beta, CD28, or a proliferative tetraspanin, or wherein the activating element is a mitogenic tetraspanin.

22. The subcutaneous formulation of any one of claims 1 or 5 to 21, the RIP for use in administering the formulation to a subject of any one of claims 2, 4 to 21, or the method of any one of claims 3 to 21, wherein the one or more transcription units encode a first polypeptide, and the first polypeptide is a chimeric antigen receptor (CAR), a recombinant T cell receptor (TCR), or a lymphoproliferative factor.

23. A subcutaneous formulation according to any one of claims 1 or 5 to 21, a RIP for use in administering the formulation to a subject according to any one of claims 2, 4 to 21, or a method according to any one of claims 3 to 21, wherein the RIP encodes one or more inhibitory RNAs.

24. the activating element comprises one or more of an antibody or antibody mimetic capable of binding to CD3; A subcutaneous formulation according to any one of claims 1 or 5 to 23, a RIP for use in administering the formulation to a subject according to any one of claims 2, 4 to 23, or a method according to any one of claims 3 to 23.

25. A subcutaneous formulation according to any one of claims 1 or 5 to 24, a RIP for use in administering the formulation to a subject according to any one of claims 2, 4 to 24, or a method according to any one of claims 3 to 24, wherein the activating element is an anti-CD3 antibody.

26. A RIP for use in administering a formulation to a subject according to any one of claims 2, 4 to 25, or a method according to any one of claims 3 to 25, wherein the formulation is administered subcutaneously.

Images