Methods and compositions for the modification and delivery of lymphocytes

Abstract

This disclosure provides methods and compositions for genetically modifying lymphocytes, such as T cells and / or NK cells. [Solution] In some embodiments, the method comprises a reaction mixture produced using whole blood or its components other than PBMCs, and further comprising T cells and recombinant retroviral particles having polynucleotides encoding CARs, as well as the resulting cell preparation. In some embodiments, the modified lymphocytes are reintroduced subcutaneously into the subject. In some embodiments, polynucleotides are provided that confer to the T cells the ability to regulate cell survival and proliferation in response to binding to CARs.

Description

[Technical Field]

[0001] Cross-reference of related applications This application is a continuation of part of International Application PCT / US2019 / 049259 filed on September 2, 2019, and U.S. Provisional Application No. 62 / 894,849 filed on September 1, 2019, U.S. Provisional Application No. 62 / 894,852 filed on September 1, 2019, U.S. Provisional Application No. 62 / 894,853 filed on September 1, 2019, U.S. Provisional Application No. 62 / 894,926 filed on September 2, 2019, U.S. Provisional Application No. 62 / 943,207 filed on December 3, 2019, and U.S. Provisional Application No. 62 / 985 filed on March 5, 2020. Claiming the benefit of ,741, International Application PCT / US2019 / 049259 is a continuation in part of International Application PCT / US2018 / 051392 filed on 17 September 2018, U.S. Provisional Application 62 / 726,293 filed on 2 September 2018, U.S. Provisional Application 62 / 726,294 filed on 2 September 2018, U.S. Provisional Application 62 / 728,056 filed on 6 September 2018, U.S. Provisional Application 62 / 732,528 filed on 17 September 2018, and U.S. Provisional Application 62 / 82 filed on 20 March 2019. Claiming the interests of U.S. Provisional Application No. 1,434 and U.S. Provisional Application No. 62 / 894,853 filed on September 1, 2019, International Application PCT / US2018 / 051392 is a continuation in part of International Application PCT / US2018 / 020818 filed on March 3, 2018, U.S. Provisional Application No. 62 / 560,176 filed on September 18, 2017, U.S. Provisional Application No. 62 / 564,253 filed on September 27, 2017, U.S. Provisional Application No. 62 / 564,991 filed on September 28, 2017, and U.S. Provisional Application No. 62 / 564,991 filed on September 6, 2018 Claiming the benefit of application No. 62 / 728,056, International application No. PCT / US2018 / 020818 is a continuation in part of International application No. PCT / US2017 / 023112 filed on 19 March 2017, a continuation in part of International application No. PCT / US2017 / 041277 filed on 8 July 2017, a continuation in part of US application No. 15 / 462,855 filed on 19 March 2017, and a continuation in part of US application No. 15 / 644,778 filed on 8 July 2017, and US provisional application No. 62 / 467 filed on 3 March 2017,International application PCT / US2017 / 023112 claims the interests of U.S. provisional application No. 62 / 560,176, filed September 18, 2017, U.S. provisional application No. 62 / 564,253, filed September 27, 2017, and U.S. provisional application No. 62 / 564,991, filed September 28, 2017, and U.S. provisional application No. 62 / 564,991, filed September 28, 2017. Claiming the interests of U.S. Provisional Application No. 62 / 360,041 filed on March 3, 2017, and U.S. Provisional Application No. 62 / 467,039 filed on March 3, 2017, International Application PCT / US2017 / 041277 claims the interests of International Application PCT / US2017 / 023112 filed on March 19, 2017, U.S. Patent Application No. 15 / 462,855 filed on March 19, 2017, and U.S. Provisional Application No. 62 / 467,039 filed on March 3 International Application PCT / US2017 / 041277 filed on March 19, 2017, and U.S. Provisional Application No. 15 / 462,855 filed on July 8, 2016 U.S. Application No. 62 / 360,041 and U.S. Provisional Application No. 62 / 467,039 filed on March 3, 2017 are claimed, and U.S. Application No. 15 / 462,855 claims the benefits of U.S. Provisional Application No. 62 / 390,093 filed on March 19, 2016, U.S. Provisional Application No. 62 / 360,041 filed on July 8, 2016, and U.S. Provisional Application No. 62 / 467,039 filed on March 3, 2017. U.S. Patent Application No. 15 / 644,778 is a continuation in part of International Patent Application No. PCT / US2017 / 023112 filed on 19 March 2017, and a continuation in part of U.S. Patent Application No. 15 / 462,855 filed on 19 March 2017, and claims the benefits of U.S. Provisional Application No. 62 / 360,041 filed on 8 July 2016 and U.S. Provisional Application No. 62 / 467,039 filed on 3 March 2017. These applications are incorporated herein by reference in their entirety.

[0002] Sequence List This application incorporates, by reference, the electronic sequence listing data filed concurrently with this application. The electronic sequence listing data was filed on August 31, 2020, as a text (.txt) file titled "F1_003_WO_01_Sequence_Listing," with a file size of 444KB, and is incorporated herein by reference in its entirety.

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

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

[0005] While recombinant retroviruses are effective in infecting non-dividing cells, quiescent 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 by the CAR gene vector can occur. Following stimulation and transduction, the genetically modified cells are enlarged in vitro and then reintroduced into lymphocyte-depleted patients. With antigen involvement in vivo, the intracellular signaling portion of the CAR can initiate activation-related responses and the release of cytolytic molecules in immune cells, thereby inducing targeted cell death.

[0006] Such current methods require extensive manipulation and production of T cells that proliferate outside the body before reinfusion into the patient, as well as lymphocyte depletion chemotherapy that releases cytokines and depletes competing receptors to promote T cell engraftment. Furthermore, such CAR therapies cannot control the in vivo proliferation rate after introduction into the body, nor can they be safely directed to targets expressed outside the tumor. As a result, CAR therapies today typically require 1 × 10⁶ cells. 5 ~1 × 10 8 Cells are injected from cells grown ex vivo for 12–28 days using doses of individual cells / kg, directed towards a target, such as a tumor target, and their extratumor-on-target toxicity is generally acceptable. These relatively long ex vivo growth times present challenges to scalability, as well as issues with cell viability, sterility, and sample identity. Therefore, there is a great need for safer, more effective, and scalable T-cell or NK-cell therapies. In particular, if such methods involve, for example, collecting the subject's blood within an injection center and then reintroducing the cells into the subject on the same day, further reduction of the complexity and time required for such methods would be highly desirable. Furthermore, simpler and faster methods, or methods requiring fewer specialized instruments, could generalize these cell therapy processes, which are currently only routinely performed in highly specialized medical centers.

[0007] Our understanding of the processes driving lymphocyte transduction, proliferation, and survival is central to a variety of potential commercial applications, including immunological processes; therefore, improved methods and compositions for studying lymphocytes are needed. For example, it is useful to identify methods by which lymphocytes can be genetically modified, as well as methods and compositions that can be used to better characterize and understand factors influencing lymphocyte survival and proliferation. Furthermore, it is useful to identify compositions that drive lymphocyte proliferation and survival. Such compositions can be used to study the regulation of such processes. In addition to methods and compositions for studying lymphocytes, improved viral packaging cell lines and methods for producing and using them are needed. For example, such cell lines and methods are useful for analyzing different components of recombinant viruses, such as recombinant retroviral particles, and for methods of using packaging cell lines for the production of recombinant retroviral particles.

[0008] Furthermore, there remains a need for improved compositions and methods to induce the proliferation and / or survival of lymphocytes in blood, organs, and tissues, as well as preferentially and specifically in the tumor microenvironment. Previous methods have used cells with constitutively expressing CARs, which, upon binding to a target antigen, induce the expression of secreted cytokines under the control of a CAR-stimulated promoter. These secreted cytokines nonspecifically bind to and stimulate T cells and NK cells, thus reducing the amount of cytokine available to stimulate CAR T cells or NK cells. Cytokines can also diffuse away, further reducing the amount of cytokine available to stimulate CAR T cells or NK cells. These earlier methods typically require multiple transductions 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 is collected to receive genetically engineered blood cells. Previous methods for one-step CAR-T cell transduction using vectors encoding two or more transcription units have resulted in low viral titers and / or low expression of one or more transcription units, each of which is a major obstacle to commercialization as a common therapeutic method. Therefore, there remains a need for more efficient methods for generating CAR-T cells that survive and proliferate in blood, organs, and tissues, as well as preferentially and specifically in the suppressive tumor microenvironment.

[0009] Several groups have attempted to simplify ex vivo preparation for cell therapy by eliminating ex vivo cell augmentation by intravenously injecting viral particles or DNA nanocarriers to transduce or transfect cells in vivo (Agarwal et al. (2019) OncoImmunology. 8(12):e1671761-1-e1671761-7, Smith et al. (2017) Nature Nanotech. 12(8):813-820). However, such methods require large amounts of vectors and carry the risk of particle inactivation by coagulation factors and / or other enzymes present in vivo. Finally, such methods carry the risk of high levels of transduction of non-target cells / organs. [Overview of the Initiative]

[0010] Methods, uses, compositions, and kits are provided herein that simplify and expedite the process of genetically modifying lymphocytes, in exemplary embodiments, T cells and / or NK cells. Several embodiments and models provided herein are well suited to point-of-care cell processing and do not require the transport of 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 carrying out adoptive cell therapy using these cells. Accordingly, several embodiments provide herein methods, compositions, and kits for modifying lymphocytes, particularly T cells and / or NK cells, and for transducing, genetically modifying, and / or regulating the activity of modified T cells and / or NK cells. Such methods, compositions, and kits offer improved efficacy and safety compared to current technologies, particularly with respect to engineered T cell receptors (TCRs), chimeric antigen receptors (CARs), and, in exemplary embodiments, T cells and / or NK cells expressing microenvironment-restricted biological ("MRB") CARs. Transduced and / or modified, and, in exemplary embodiments, genetically modified T cells and / or NK cells produced by and / or used therein by the methods provided herein include, in exemplary embodiments, a combination of functionality and capabilities delivered from a retroviral (e.g., lentiviral) genome via retroviral (e.g., lentiviral) particles, which provides improved features for such cells and methods utilizing such cells, e.g., research methods, commercial production methods, and adoptive cell therapy. For example, such cells have improved growth characteristics that can be produced in shorter time ex vivo and can be better regulated. In exemplary embodiments, such methods, uses, compositions, and kits include, or are adapted for, intramuscular delivery to a subject, or, in further exemplary embodiments, subcutaneous delivery.

[0011] In some embodiments, methods are provided for transducing and / or modifying lymphocytes such as T cells and / or NK cells, and, in exemplary embodiments, for genetic modification, as well as, in exemplary embodiments, ex vivo methods for transducing, genetically modifying, and / or modifying resting T cells and / or NK cells. Some of these embodiments can be carried out much more rapidly than previous methods and can facilitate more efficient research, more effective commercial production, and improved methods of patient care. The methods, uses, compositions, and kits provided herein may be used as research tools, in commercial production, and in adoptive cell therapy using transduced and / or modified, and, in exemplary embodiments, genetically modified T cells and / or NK cells expressing TCRs or CARs.

[0012] With regard to methods, uses, and compositions provided herein relating to the transduction of lymphocytes such as T cells and / or NK cells, methods including transduction reactions of enriched PBMCs, TNCs, or transduction reactions without prior cell enrichment, such as in whole blood, as well as related uses and compositions, are provided herein, which are simplified and faster methods for carrying out ex vivo cell treatment, for example, for CAR-T therapy. Such methods require less specialized measurement and training. Furthermore, such methods reduce the risk of non-target cell transduction compared to in vivo transduction methods. In addition, methods, uses, and compositions including embodiments of the methods immediately above are provided herein, which, in combination with any other embodiments provided herein at the optional discretion, can provide a powerful method, use, and composition for driving the proliferation of lymphocytes, particularly T cells and / or NK cells, in vitro, ex vivo, and in vivo, including specific targeted inhibitory RNAs, activating elements, polypeptide lymphoproliferative elements, pseudotyping elements, and artificial antigen-presenting cells. In some embodiments, modified lymphocytes can engraft in a lymphocyte-rich environment. In some embodiments, the patient or subject is not lymphocyte-depleted prior to the reinfusion of modified and / or genetically modified T cells and / or NK cells.

[0013] In some aspects and embodiments, gene constructs particularly suited to providing genetically modified T cells and / or NK cells with the ability to survive and proliferate in a more controllable manner are provided herein. In contrast to lymphoproliferative 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 lymphoproliferative elements that, when induced by CAR binding to their targets, can induce the proliferation of T cells and / or NK cells, such as those present in the tumor microenvironment.

[0014] Further details relating to aspects and embodiments of this disclosure are provided throughout this patent application. Sections and section headings are for readability purposes only and are not intended to limit any combination of disclosures, such as methods, compositions, and kits, or functional elements within them across sections. [Brief explanation of the drawing]

[0015] [Figure 1A-1B] Figures 1A–1D are flowcharts of exemplary cell processing workflows, not limited to these. Figure 1A is a flowchart of the process using a system with PBMC isolation before contacting T cells and NK cells in PBMCs with retroviral particles. Before PBMC isolation, any steps to deplete unwanted cells may be initiated. Figure 1B is a flowchart of the process performing total nucleated cell (TNC) isolation before contacting T cells and NK cells in total nucleated cells with retroviral particles. As discussed herein, TNC isolation in exemplary embodiments is performed using a leukocyte-removing filter assembly. After TNC isolation and before optional PBMC isolation, any steps to deplete unwanted cells may be initiated. [Figure 1C-1D] Figures 1A–1D are flowcharts of exemplary cell processing workflows, not limited to these. Figure 1C is a flowchart of a process in which blood cells are not fractionated or concentrated before contact with retroviral particles in whole blood, and PBMC isolation is performed after contact and optional incubation. Before PBMC isolation, an optional step to deplete unwanted cells may be initiated. Figure 1D is a flowchart of a process in which blood cells are not fractionated or concentrated before contact with retroviral particles in whole blood, and after contact and optional incubation, TNC isolation / concentration is performed using filtration, for example, using a leukocyte removal filter assembly, in an exemplary embodiment. Before the TNC isolation / concentration step, an optional step to deplete unwanted cells, followed by a filtration process, may be performed. [Figure 1E] Figure 1E is a flowchart of the process for performing TNC isolation before "cold contact" of T cells and NK cells in all nucleated cells with retroviral particles. Prior to TNC isolation, an optional step may be initiated to deplete unwanted cells. Another optional step is a secondary incubation, optionally combined with coarse filtration to capture lymphocyte aggregates and / or remove unwanted cells. [Figure 1F] Figure 1F is a flowchart of the process for performing TNC isolation before "cold contact" between T cells and NK cells in all nucleated cells and retroviral particles. Before TNC isolation, any step to deplete unwanted cells may be initiated. Another optional step is secondary incubation. One or more of the washing steps are optional. Each of these cell processing workflows can be used in rPOC cell therapy. [Figure 2] This figure shows an exemplary leukocyte removal filter assembly (200) that includes a filter enclosure (210) containing associated blood processing bags, tubing, valves, and a leukocyte removal filter set. [Figure 3A] Histograms of experimental results using different pseudotyping elements are shown. Figure 3A shows a histogram of the total number of viable cells per well on day 6 after transduction. [Figure 3B] Histograms of experimental results using different pseudotyping elements are shown. Figure 3B shows a histogram of the percentage of transduced CD3+ cells measured by eTAG expression. [Figure 4A] Histograms of experimental results using whole blood, lentiviral particles, and a transduction reaction mixture containing the anticoagulant EDTA or heparin, without PBMC enrichment before the formation of the reaction mixture, are shown. This process was carried out by contacting whole blood with the indicated lentiviral particles F1-3-23G or F1-3-23GU for 4 hours, followed by a density gradient centrifugation-based PBMC enrichment procedure. Figure 4A shows a histogram of the absolute number of cells per uL of the live lymphocyte population. [Figure 4B] Histograms of experimental results using whole blood, lentiviral particles, and a transduction reaction mixture containing the anticoagulant EDTA or heparin, without PBMC enrichment before the formation of the reaction mixture. This process was carried out by contacting whole blood with the indicated lentiviral particles F1-3-23G or F1-3-23GU for 4 hours, followed by a density gradient centrifugation-based PBMC enrichment procedure. Figure 4B shows a histogram of the percentage of CD3+eTag+ cells in the viable lymphocyte population 6 days post-transduction. [Figure 5] Contour FACS plots of CD3 and eTag expression on a live lymphocyte population 7 days after 4 hours of whole blood transduction in F1-3-23GU, followed by isolation of all nucleated cells by TNC filtration using an exemplary leukocyte-removing filter assembly. [Figure 6] This shows the number of CD3+eTAG+CAR-T cells per 60 μL of peripheral blood in individual mice 7, 14, and 21 days after intravenous CAR-T administration. The administered cells were either untransduced or transduced at F1-3-247GU at the indicated MOI. [Figure 7] This shows the number of CD3+eTAG+CAR-T cells per 60 μL of peripheral blood in individual mice 8, 14, and 21 days after subcutaneous CAR-T administration. The administered cells were either untransduced or transduced at F1-3-247GU at the indicated MOI. [Figure 8] The graph shows the mean tumor volume of Raji tumors in B-NDG mice that received intravenous administration on day 0 of either untransduced (UNT) or transduced (TRNSD) PBMCs by 4 hours of exposure to F1-3-247GU at the indicated MOI. Each group of mice received either 1 million or 5 million PBMCs as instructed. [Figure 9]The graph shows the mean tumor volume of Raji tumors in B-NDG mice that received subcutaneous administration on day 0 of either untransduced (UNT) or transduced (TRNSD) PBMCs by 4 hours of exposure to F1-3-247GU at the indicated MOI. Each group of mice received either 1 million or 5 million PBMCs as instructed. [Figure 10] A schematic diagram of a specific genome plasmid used in the example is shown. [Figure 11A] The graphs show the titers of recombinant lentiviral virus particles with various transcription units, under the control of either the forward (F1-0-03) or reverse (F1-0-03RS) EF1-a, PGK, SV40hCD43, or MSCVU3 promoter, or without the reverse (F1-0-03RS-ΔEF1a) promoter. [Figure 11B] This graph shows GFP expression levels in transiently transfected Lenti-X® 293T cells, expressed as mean fluorescence intensity (MFI) as determined by FACS. GFP expression was controlled by either forward (F1-0-03) or reverse (F1-0-03RS) EF1-a, PGK, SV40hCD43, or MSCVU3 promoters. [Figure 12A]A schematic diagram of an exemplary bicistronic lentiviral genome vector with branched transcription units is shown. Under transcriptional regulation of the NFAT-responsive minimal IL-2 promoter (6×NFAT), the first transcription unit, containing an eTag-tagged lymphoproliferative element (eTag:LE) followed by a polyadenylated sequence (PolyA), is encoded in the reverse direction. Optionally, an insulator element (Ins) separates the first and second transcription units. The second transcription unit encodes a CAR (CAR) under transcriptional regulation of the constitutive promoter (promoter) and is encoded in the forward direction. The dashed triangles represent three possible locations where one or more miRNAs can be optionally inserted into the vector. The dotted triangle represents one possible location in an exon within a promoter, such as EF1-a, where one or more miRNAs can be optionally inserted into the vector. "SA" and "SD" correspond to splice donor and splice acceptor sites. [Figure 12B] The identity, characteristics, and overall size of each lentiviral genome vector tested in Example 7 are shown. [Figure 13] The graph shows the percentage of CD19 CAR+ Jurkat cells expressing eTag. Jurkat cells were transduced with a specified bicistronic lentiviral genome construct, and eTag expression was measured by flow cytometry 24 hours after the samples were stimulated (or left unstimulated) with 20 nM PMA and 1 ug / mL ionomycin. [Figure 14] This graph shows the mean fluorescence intensity (MFI) of eTag expression on the surface of CD19 CAR+ Jurkat cells. Jurkat cells were transduced with a specified bicistronic lentiviral genome construct, and eTag expression was measured by flow cytometry 24 hours after stimulation (or leaving unstimulated) of the samples with 20 nM PMA and 1 ug / mL ionomycin. [Figure 15]The graph shows the percentage of Jurkat cells expressing the CD19 CAR. Jurkat cells were transduced with a specified bicistronic lentiviral genome construct, and CAR expression was measured by flow cytometry 24 hours after the samples were stimulated (or left unstimulated) with 20 nM PMA and 1 ug / mL ionomycin. [Figure 16] This graph shows the mean fluorescence intensity (MFI) of CD19 CAR expression on the surface of Jurkat cells. Jurkat cells were transduced with a designated bicistronic lentiviral genome construct, and CAR expression was measured by flow cytometry 24 hours after the samples were stimulated (or left unstimulated) with 20 nM PMA and 1 ug / mL ionomycin. [Figure 17] The graph shows the percentage of CD3+CAR+PMBCs expressing eTag. PBMCs were transduced with the indicated bicistronic lentiviral genome construct and supplied with CD19-expressing Raji cells every other day starting on day 7, or left unsupplied in the absence of exogenous cytokines. CD3+CAR+ cells were assayed daily for eTag expression by flow cytometry as indicated. [Figure 18A-B] The graph shows the growth rate of CD3+CAR+PMBCs. PBMCs were transduced with lentiviral genome construct F1-3-635 (Figure 18A) or F1-3-637 (Figure 18B), and CD19-expressing Raji cells were supplied every other day starting on day 7, or they were left without supply in the absence of exogenous cytokines. CD3+CAR+ cells were detected by flow cytometry. [Figure 18C-D] The graph shows the growth rate of CD3+CAR+PMBCs. PBMCs were transduced with lentiviral genome constructs F1-3-23 (Figure 18C) or F1-3-247 (Figure 18D), and CD19-expressing Raji cells were supplied every other day starting on day 7, or they were left without supply in the absence of exogenous cytokines. CD3+CAR+ cells were detected by flow cytometry. [Figure 19]The graph shows the growth rate of CD3+CAR+PMBCs. PBBCs were transduced with lentiviral genome constructs F1-3-635, F1-3-637, F1-3-23, or F1-3-635, and after 7 days, they were cultured without cytokine supply in the absence of cytokines. [Figure 20] The graph shows the survival rate of CD3+CAR+PMBCs. PBMCs were transduced with lentiviral genome constructs F1-3-635, F1-3-637, F1-3-23, or F1-3-635, and after 7 days, they were cultured without cytokine supply in the absence of cytokines. [Figure 21] The graphs show the total luminous flux [p / sec] of Raji-luciferase-disseminated tumor volume in NSG-(KbDb)null(IA)null mice that were either not transduced (G1) or transduced by a 4-hour exposure to whole blood F1-3-637GU (G2) or F1-4-713GU (G3) lentiviral particles followed by a PBMC enrichment procedure, administered subcutaneously on day 0. G4 mice were treated with half the dose of PBMCs from G2 and G3. The F1-3-637GU and F1-4-713GU genomic vectors encode self-driven CARs for CD19 and CD22, respectively. [Figure 22] Figure 21 shows a graph of the survival probability of mice over 8 weeks. [Figure 23] After 6 days of culture in CTS medium supplemented with rhIL-2, transduced TNCs with F1-3-637GU exhibited total cell recovery and cell surface marker expression. The contact step of the rPOC cell process was performed as shown in either Figure 1D (whole blood) or Figure 1B (on filter). [Figure 24] Figure 23 shows graphs of IFN gamma production (pg / mL) by cells, measured by ELISA, either with cells left untreated (NA) or after being treated with CHO-S, Raji, or PMA + ionomycin for 16 hours. [Figure 25]The graph shows the total luminous flux [p / sec] of Raji-luciferase-disseminated tumor volume in NSG null mice that were subcutaneously administered on day 0 cells transduced by a 4-hour exposure to PBS (G1), TNC (G2), PBMC (G3), or whole blood F1-3-637GU lentivirus particles, followed by the TNC enrichment procedure shown in Figure 1D (G4) or the PBMC enrichment procedure shown in Figure 1C (G5). [Modes for carrying out the invention]

[0016] definition As used herein, the terms “chimeric antigen receptor” or “CAR” or “CARs” refer to engineered receptors that transfer antigen specificity to cells, such as T cells, NK cells, macrophages, and stem cells. The CARs of the present invention comprise at least one antigen-specific targeting region (ASTR), a transmembrane domain (TM), and an intracellular activation domain (IAD), and may include a stalk and one or more costimulatory domains (CSD). In another embodiment, the CAR is a bispecific CAR that is specific to two different antigens or epitopes. After the ASTR specifically binds to the target antigen, the IAD activates intracellular signaling. For example, the IAD can leverage the antigen-binding properties of an antibody to redirect the specificity and reactivity of T cells toward a selected target in an MHC-independent manner. MHC-independent antigen recognition gives T cells expressing the CAR the ability to recognize antigens independently of antigen processing, thereby bypassing the primary mechanism of tumor escape. Furthermore, when expressed in T cells, CARs do not favorably dimerize with endogenous T cell receptor (TCR) alpha and beta chains.

[0017] As used herein, the term “constitutive T cell or NK cell promoter” refers to a promoter that, when operably linked to a polynucleotide encoding or identifying a gene product, causes a cell to produce a gene product under most or all physiological conditions of the cell.

[0018] As used herein, the terms “inducible promoter” or “activatable promoter” refer to a promoter that, when operably linked to a polynucleotide encoding or identifying a gene product, causes a cell to produce a gene product only when substantially promoter-specific inducers are present in the cell. Inducible promoters have little to no basic transcriptional activity, but transcriptional activity is sometimes significantly increased in the presence of an inducing signal.

[0019] As used herein, the term “insulator” refers to a cis-regulatory element that mediates intrachromosomal and interchromosomal interactions and can block interactions between enhancers and promoters. Typically, an insulator is 200–2000 base pairs long and contains clustered binding sites for sequence-specific DNA-binding proteins.

[0020] As used herein, the term “microenvironment” means any part or region of tissue or body that has a constant or transient physical or chemical difference from other areas of tissue or body. For example, as used herein, “tumor microenvironment” refers to the environment in which a tumor resides, which includes non-cellular areas within the tumor and the area immediately outside the tumor tissue, but does not relate to the intracellular compartments of the cancer cells themselves. The tumor microenvironment may refer to any conditions of 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 may include, but is not limited to, changes in conditions such as pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, concentration of electrolytes, concentration of glucose, concentration of hyaluronan, concentration of lactate or lactate, concentration of albumin, level of adenosine, level of R-2-hydroxyglutarate, concentration of pyruvate, concentration of oxygen, and / or the presence of oxidizing agents, reducing agents, or cofactors, as well as other conditions understood by those skilled in the art.

[0021] As used interchangeably herein, the terms “polynucleotide” and “nucleic acid” refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Therefore, these terms include, but are 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, unnatural, or derivatized nucleotide bases.

[0022] As used herein, the term “antibody” includes polyclonal and monoclonal antibodies, which include intact antibodies and fragments of antibodies that retain specific binding to an antigen. Antibody fragments may include, but are not limited to, fragment antigen-binding (Fab) fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fab'-SH fragments, (Fab')2Fv fragments, Fd fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments including single-chain variable fragments (scFv), bivalent scFv, trivalent scFv, and single-domain antibody fragments (e.g., sdAb, sdFv, nanobodies). This term includes genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptidebodies, chimeric antibodies, single-chain antibodies, fully human antibodies, humanized antibodies, fusion proteins containing antigen-specific targeting regions of antibody and non-antibody proteins, heteroconjugate antibodies, multispecific antibodies, such as bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise specified, the term “antibody” should be understood to include its functional antibody fragment. This 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.

[0023] As used herein, the term “antibody fragment” includes a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments called “Fab” fragments (each fragment having a single antigen-binding site) and residual “Fe” fragments (a name reflecting their ability to readily crystallize). Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites and still capable of crosslinking antigens.

[0024] Where used interchangeably in this specification, the terms "single-stranded Fv", "scFv", or "sFv" antibody fragment refer to the V of the antibody. H and V L It contains domains, and these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide is V H Domain and V L The sFv may further contain a polypeptide linker or spacer between the domain and the sFv, thereby enabling the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0025] As used herein, “spontaneous” VH and VL domains refer to VH and VL domains isolated from a host without further molecular evolution to alter their affinity when generated in scFv form under specific conditions, as disclosed in U.S. Patent No. 8,709,755 B2 and application WO / 2016 / 033331A1.

[0026] As used herein, the term "affinity" refers to the equilibrium constant of the reversible binding of two agents and is expressed as the dissociation constant (Kd). The affinity is at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater, or more, than the affinity of the antibody for an irrelevant amino acid sequence. The affinity of the antibody for the target protein may be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM), or more. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation upon dilution. The terms "immunoreactive" and "preferentially binds" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0027] As used herein, the term "binding" refers to the direct association between two molecules by covalent interactions, electrostatic interactions, hydrophobic interactions, and ionic and / or hydrogen bond interactions, including interactions such as salt bridges and water bridges. Nonspecific binding refers to binding with an affinity of less than about 10 -7 M, for example, binding with an affinity of 10 -6 M, 10 -5 M, 10 -4 M, etc.

[0028] As used herein, "cell surface expression system" or "cell surface presentation system" refers to the presentation or expression of a protein or a portion thereof on the surface of a cell. Typically, cells are produced that express the target protein fused to a cell surface protein. For example, the protein is expressed as a fusion protein with a transmembrane domain.

[0029] As used herein, the term “element” includes polypeptide fusions, polypeptide regions, and polypeptides comprising functional variants or fragments thereof, as well as polynucleotides comprising microRNA and shRNA, and polynucleotides comprising functional variants or fragments thereof.

[0030] As used herein, the term “region” refers to any segment of a polypeptide or polynucleotide.

[0031] As used herein, “domain” refers to a region of a polypeptide or polynucleotide having functional and / or structural properties.

[0032] As used herein, the terms “stalk” or “stalk domain” refer to a flexible polypeptide connector region that provides structural flexibility and spacing to adjacent polypeptide regions, and may consist of natural or synthetic polypeptides. A stalk may originate from the hinge or hinge region of an immunoglobulin (e.g., IgG1), which is generally defined as extending from Glu216 to Pro230 in human IgG1 (Burton (1985) Molec. Immunol., 22:161-206). Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy-chain disulfide (SS) bond in the same position. A stalk may be spontaneous or non-spontaneous, including but not limited to modified hinge regions, as disclosed in U.S. Patent No. 5,677,425. A stalk may include a complete hinge region derived from any class or subclass of antibody. Stork may also include regions derived from CD8, CD28, or other receptors that provide similar functionality in offering flexibility and spacing to adjacent regions.

[0033] As used herein, the term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it occurs spontaneously). For example, a spontaneously 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 material coexisting in a natural system is isolated. Such polynucleotides may be part of a vector, and / or such polynucleotides or polypeptides may be part of a composition, and such vectors or compositions can still be isolated in that they are not part of their natural environment.

[0034] As used herein, "polypeptide" is a single chain of amino acid residues linked by peptide bonds. Polypeptides do not fold into a fixed structure and have no post-translational modifications. "Protein" is a polypeptide that folds into a fixed structure. "Polypeptide" and "protein" are used interchangeably herein.

[0035] Where used herein, polypeptides may be "purified" to remove contaminants from the natural environment of the polypeptide, such as enzymes, hormones, and other proteinaceous or non-proteinaceous solutes, which may interfere with the diagnostic or therapeutic use of the polypeptide. Polypeptides may be purified (1) to more than 90%, 95%, or 98%, for example, more than 99%, of the antibody as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spinning cup sequencer, 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.

[0036] As used herein, the term “immune cells” generally includes leukocytes derived from hematopoietic stem cells (HSCs) produced in the bone marrow. “Immune cells” also include, for example, lymphocytes (T cells, B cells, natural killer (NK) cells) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells).

[0037] As used herein, "T cell" refers to a T helper cell (CD4 + cells), cytotoxic T cells (CD8 + This includes all types of immune cells that express CD3, including T cells, regulatory T cells (Treg), and gamma-delta T cells.

[0038] As used herein, “cytotoxic cells” means CD8 +T cells, natural killer (NK) cells, NK-T cells, γδT cells, CD4 + This includes subpopulations of cells, and neutrophils, which are cells capable of mediating cytotoxic responses.

[0039] As used herein, the term “stem cells” generally includes pluripotent or multipotent stem cells. “Stem cells” include, for example, embryonic stem cells (ES), mesenchymal stem cells (MSC), induced pluripotent stem cells (iPS), and committed progenitor cells (such as hematopoietic stem cells (HSC) and bone marrow-derived cells).

[0040] As used herein, terms such as “treatment” and “to treat” refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it partially or completely cures a disease and / or adverse effects resulting from the disease. As used herein, “treatment” covers any treatment of a disease in a mammal, e.g., a human, and includes: (a) preventing the development of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., preventing its onset; and (c) alleviating the disease, i.e., causing its regression.

[0041] Where used interchangeably in this specification, the terms “individual,” “subject,” “host,” and “patient” refer to mammals, including but not limited to humans, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), etc.

[0042] As used herein, the terms “therapeutically effective amount” or “efficacious amount” refer to the amount of a drug, or the total amount of two drugs, that, when administered to a mammal or other subject for the treatment of a disease, is sufficient to have an effect on such treatment of the disease. The “therapeutic effective amount” varies depending on the drug, the disease and its severity, as well as the age, weight, etc., of the subject being treated.

[0043] As used herein, the terms “evolve” or “evolve” mean using one or more methods of mutagenesis to produce different polynucleotides that encode different polypeptides, which are themselves improved biomolecules and / or contribute to the creation of another improved biomolecule. “Physiological” or “normal” or “normal physiological” conditions are, but are not limited to, conditions such as pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, electrolyte concentrations, glucose concentration, hyaluronan concentration, lactate or lactate concentration, albumin concentration, adenosine levels, R-2-hydroxyglutarate levels, pyruvate concentration, oxygen concentration, and / or the presence of oxidizing agents, reducing agents, or cofactors, as well as other conditions that are considered to be within the normal range in the tissue or organ of the administration site or site of action to the subject.

[0044] As used herein, “transduced cell” or “stably transduced cell” is a cell containing exogenous nucleic acid integrated into the cell’s genome. As used herein, “genetically modified cell” is a cell containing exogenous nucleic acid, regardless of whether the exogenous nucleic acid is integrated into the cell’s genome or the method used to introduce the exogenous nucleic acid into the cell. Intracellular exogenous nucleic acid that is not integrated into the cell’s genome may be referred to herein as “extrachromosomal.” As used herein, “modified cell” is a cell associated with a recombinant nucleic acid vector, which in exemplary embodiments is a non-replicating recombinant retroviral particle, or a cell genetically modified by the exogenous nucleic acid. Typically, in compositions and methods comprising non-replicating recombinant retroviral particles, the modified cell associates with the non-replicating recombinant retroviral particle through an interaction between proteins on the cell surface and proteins on the surface of the non-replicating recombinant retroviral particle, which includes pseudotyping elements and / or T cell activating elements. In compositions and methods involving the transfection of nucleic acids within lipid-based reagents such as liposome reagents, the lipid-based reagent containing nucleic acids, which are a type of recombinant nucleic acid vector, associates with the lipid bilayer of the modified cells before fusing with or thereby internalizing the modified cells. Similarly, in compositions and methods involving chemical-based transfection of nucleic acids, such as polyethyleneimine (PEI) or calcium phosphate-based transfections, the nucleic acids associate with a positively charged transfection reagent to form a recombinant nucleic acid vector that typically 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, “polypeptide” may include part or whole of a protein molecule, as well as any post-translational or other modifications.

[0045] As used herein, pseudotyping elements may include a “binding polypeptide” comprising one or more polypeptides, typically glycoproteins, that identify and bind to a target host cell, and one or more “fusion polypeptides” that mediate the fusion of a retrovirus and the target host cell membrane, thereby enabling the retroviral genome to enter the target host cell. As used herein, “binding polypeptides” may also be referred to as “T cell and / or NK cell binding polypeptides” or “target-involved elements,” and “fusion polypeptides” may also be referred to as “fusion elements.”

[0046] For example, "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 may include naive T cells that have never encountered a specific antigen, and memory T cells that have been altered by a previous encounter with the antigen. "Resting" lymphocytes may also be referred to as "quiescent" lymphocytes.

[0047] As used herein, “lymphocyte depletion” includes, for example, a method of reducing the number of lymphocytes in a subject by administering a lymphocyte depletion agent. Lymphocyte depletion can also be achieved by fractional radiotherapy of a part or whole body. A lymphocyte depletion 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 lymphocyte depletion agents include, but are not limited to, fludarabine, cyclophosphamide, cladribine, denileukin difutitox, alemtizumab, or combinations thereof.

[0048] 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, by being present in a cell, induces RNAi and results in a reduction in the expression of the transcript targeted by the inhibitory RNA molecule. As used herein, inhibitory RNA molecules have a 5' stem and a 3' stem capable of forming an RNA double helix. Inhibitory RNA molecules may be, for example, miRNA (either endogenous or artificial) or shRNA, a precursor of miRNA (i.e., pre-miRNA or pre-miRNA) or shRNA, or dsRNA that is directly transcribed or introduced as a nucleic acid isolated in a cell or subject.

[0049] As used herein, “double-stranded RNA” or “dsRNA” or “RNA double-stranded” refers to an RNA molecule composed of two strands. Double-stranded molecules include molecules composed of two RNA strands that hybridize to form a double-stranded RNA structure, or single-stranded RNA strands that themselves double to form a double-stranded structure. Most, though not all, bases in a double-stranded region are base pairs. The double-stranded region contains a sequence complementary to the 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 long, or in some embodiments, the lower limit 18, 19, 20, 21, 22, 23, 24, 25 to the upper limit 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and a given range always has a lower limit lower than the upper limit. Such a structure typically includes a 5' stem, loop, and 3' stem, each adjacent to the stem and connected by a loop that is not part of the double helix. In certain embodiments, the loop contains at least 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In other embodiments, the loop contains 2–40, 3–40, 3–21, or 19–21 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, where a given range always has a lower limit lower than the upper limit.

[0050] As used herein, the term “microRNA adjacent sequence” refers to a nucleotide sequence containing microRNA processing elements. MicroRNA processing elements are the smallest nucleic acid sequences that contribute to the production of mature microRNA from precursor microRNA. Often, these elements are located within a 40-nucleotide sequence adjacent to the microRNA stem-loop structure. In some cases, microRNA processing elements are found within a stretch of nucleotide sequences ranging from 5 to 4,000 nucleotides in length, adjacent to the microRNA stem-loop structure.

[0051] When used in reference to multiple inhibitory RNA molecules, the term "linker" refers to the linking mechanism that connects two inhibitory RNA molecules.

[0052] As used herein, “recombinant retrovirus” refers to a retrovirus that is not replicable, or “non-replicable,” unless expressly described as a replicable retrovirus. The terms “recombinant retrovirus” and “recombinant retrovirus particle” are used interchangeably herein. Such retroviruses / retrovirus particles can be any type of retrovirus particle, including, for example, gamma retroviruses, and, in exemplary embodiments, lentiviruses. As is known, such retrovirus particles, e.g., lentivirus particles, are typically formed in packing cells by transfecting the packing cells with a plasmid containing packaging components such as Gag, Pol, and Rev, an envelope or pseudotyping plasmid encoding a pseudotyping element, and an introducer, genome, or retrovirus (e.g., lentivirus) expression vector (which is typically a plasmid encoding the gene or other coding sequence of interest). Thus, the retrovirus (e.g., lentivirus) expression vector contains sequences that facilitate expression and packaging after transfection into cells (e.g., psi packaging elements and 5'LTR and 3'LTR adjacent to the target heterologous coding sequence). The terms "lentivirus" and "lentivirus particle" are used interchangeably herein.

[0053] The “framework” of a miRNA consists of a “5' microRNA flanking sequence” and / or a “3' microRNA flanking sequence” surrounding the miRNA, as well as, in some cases, a loop sequence that separates the stem of a stem-loop structure within the miRNA. In some examples, 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.

[0054] As used herein, the term “miRNA precursor” refers to an RNA molecule of any length that can be enzymatically processed into a miRNA, such as a primary RNA transcript, pre-miRNA, or pre-miRNA.

[0055] As used herein, the term “construction” refers to an isolated polypeptide or an isolated polynucleotide encoding a polypeptide. A polynucleotide construct may encode a polypeptide, for example, a lymphoproliferative element. Those skilled in the art will understand, from the context, whether “construction” refers to an isolated polynucleotide or an isolated polypeptide.

[0056] As used herein, "MOI" refers to the multiplicity ratio of infection, which is equal to the ratio of the number of viral particles used to infect a given number of cells. In some, but not limited to, functional titer measurements of viral particle counts may be performed using FACS and reporter expression.

[0057] Peripheral blood mononuclear cells (PBMCs) are peripheral blood cells with round nuclei, including lymphocytes (e.g., T cells, NK cells, and B cells) as well as monocytes. Several blood cell types that are not PBMCs include red blood cells, platelets, and granulocytes (i.e., neutrophils, eosinophils, and basophils).

[0058] It should be understood that this disclosure, and the aspects and embodiments provided herein, are not limited to the specific examples disclosed and are therefore naturally subject to change. It should also be understood that the terms used herein are intended solely to disclose specific examples and embodiments and are not intended to limit the scope of this disclosure, as it is limited only by the appended claims.

[0059] Where a range of values ​​is provided, unless the context makes it more clearly indicated otherwise, it is understood that each intermediate value between the upper and lower limits of that range, up to one-tenth of the lower limit unit, and any other stated or intermediate values ​​within that stated range are included in this disclosure. The upper and lower limits of these smaller ranges may independently be included in smaller ranges and are included in the invention according to the specifically excluded limits within the stated ranges. Where a stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention. Where multiple lower and multiple higher values ​​are given in overlapping ranges, a person skilled in the art will recognize that the selected range includes lower values ​​smaller than higher values. All headings in this application are for the convenience of the reader and are not limiting.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, but preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference to disclose and explain the methods and / or materials to which the publications refer.

[0061] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple referents unless the context clearly indicates otherwise. Thus, for example, a reference to “a chimeric antigen receptor” includes multiple such chimeric antigen receptors and their equivalents known to those skilled in the art. It should be further noted that claims may be constructed to exclude any optional element. Therefore, this statement is intended to function as an antecedent for using exclusive terms such as “solely,” “only,” or “negative” restrictions in relation to the enumeration of claim elements.

[0062] For clarity, it is understood that certain features of the Invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the Invention described in the context of a single embodiment may also be provided separately or in any preferred partial combination. All combinations of embodiments relating to the Invention are specifically encompassed by the Invention and are disclosed herein as if every possible combination were individually and explicitly disclosed. Furthermore, all partial combinations of various embodiments and their elements are also specifically encompassed by the Invention and are disclosed herein as if every possible such partial combination were individually and explicitly disclosed herein.

[0063] This disclosure overcomes the challenges of the prior art by providing improved methods and compositions for modifying lymphocytes, e.g., NK cells, and, in exemplary embodiments, T cells, and for genetic modification in exemplary embodiments. Some of the methods and compositions herein provide a simplified and faster process for transfecting or inducing lymphocytes, avoiding several steps that require specialized devices. Thus, the methods provide an important step toward the generalization of cell therapies. Exemplary methods and compositions for modifying lymphocytes, e.g., NK cells, and, in exemplary embodiments, T cells are carried out in less time than conventional methods and, in practice, provide rapid point-of-care methods in some embodiments. Furthermore, compositions with many uses (including their use in these improved methods) are provided, including cell formulation compositions adapted for subcutaneous administration. Some of these compositions include modified, modified, and, in exemplary embodiments, genetically modified lymphocytes with improved proliferation and viability characteristics, including in vitro culture in the absence of growth factors. In such modified and exemplary embodiments, genetically modified lymphocytes have practical applications, for example, as research tools for better understanding factors that affect T cell proliferation and survival, and for commercial production for generating specific factors such as growth factors and immunomodulators that can be recovered and tested or used in commercial products. Such modified and genetically modified lymphocytes also have practical applications in the treatment of cancer.

[0064] The exemplary methods and compositions for immunotherapy described herein are adapted for, effective for, and / or adapted for subcutaneous or intramuscular delivery and subcutaneous or intramuscular cell preparations. 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 are further enhanced by the addition of antigens, growth factors, and immunomodulators to the cell preparation or the administration site of the cell preparation.

[0065] Furthermore, methods and compositions for overcoming challenges associated with CAR cancer cell resistance, such as loss of target antigen availability through genetic modification of malignant cells (e.g., epitope or antigen masking), are also provided herein.

[0066] Exemplary cell processing methods for genetically modifying T cells and / or NK cells in the presence of blood or its components. Methods provided in exemplary embodiments of this specification include methods for modifying T cells and / or NK cells, or related methods for producing cell preparations, which include ex vivo contact of blood cells, including lymphocytes (e.g., NK cells and / or T cells), in a reaction mixture with a recombinant vector, such as a polynucleotide encoding a CAR or a non-replicating recombinant retroviral particle containing one. In exemplary embodiments, the reaction mixture includes a T cell activating element in solution or on the surface of the recombinant retroviral particle to facilitate the genetic modification of T cells in the reaction mixture. In the examples of this specification, it has been demonstrated that such a reaction mixture may include unfractionated whole blood or may include all or many cell types found in whole blood, including all nucleated cells (TNCs), and that in exemplary embodiments, the modified T cells are delivered subcutaneously. Figure 1 provides many exemplary, non-limiting workflows of such methods.

[0067] As shown in Figure 1, some of the methods provided herein include an optional step of (110) collecting blood from a subject. As will be discussed in more detail herein, blood may be collected or obtained from a subject by any preferred method known in the art. For example, blood may be collected by venipuncture, apheresis, or any other blood collection method from which a sample of blood is taken. In some embodiments, the amount of blood collected is 1 to 120 mL. In exemplary embodiments, in particular embodiments in which the subject from which blood is collected has normal levels of NK cells, and in exemplary embodiments, T cells, the amount of blood collected is 1 to 25 mL.

[0068] It should be noted that in the modified and exemplary embodiments provided herein, some embodiments of the methods for genetic modification do not involve the step of taking blood from a subject. Whether or not blood is taken from a subject, in embodiments of the exemplary methods provided herein for modifying lymphocytes (e.g., T cells and / or NK cells), the lymphocytes come into contact with non-replicating retroviral particles in a reaction mixture. In exemplary embodiments, this contact and the reaction mixture in which the contact occurs are carried out within a closed cell processing system, as will be discussed in more detail herein. Such closed processing systems and methods used in some embodiments and aspects of the systems and methods provided herein may be any systems and methods known in the art. As an example without limitation, the system or method may be a conventional closed cell processing system and method, or a system or method referred herein to as a “more recent” method or system (see, for example, WO2018 / 136566 and WO2019 / 055946). Conventional closed cell processing methods, particularly those for autologous cell therapy, involving ex vivo genetic modification and / or transduction of lymphocytes, involve numerous steps over several days, including PBMC enrichment, washing, cell activation, transduction, augmentation, harvesting, and, optionally, reintroduction. More recent methods have shortened some of the steps and time involved in this ex vivo cell processing (see, e.g., WO2018 / 136566). Other more recent methods (see Figure 1A) further shorten some of the steps and time involved in this ex vivo cell processing, or eliminate them entirely, for example, the ex vivo augmentation step (see, e.g., WO2019 / 055946). These more recent methods (as well as the further improved cell processing methods provided herein) further utilize rapid ex vivo transduction processes that do not include, or include minimal pre-activation (e.g., exposing lymphocytes such as T cells and / or NK cells to an activator for less than 30, 15, 10, or 5 minutes before contact with retroviral particles).In certain embodiments of such methods, T cell and / or NK cell activators are present in the reaction mixture in which the contact step occurs. In exemplary embodiments, the T cell and / or NK cell activators associate with the surface of retroviral particles present in the reaction mixture. In exemplary embodiments, such methods using rapid ex vivo gene modification without an ex vivo augmentation step are used in rapid point-of-care (rPOC) autologous cell therapies. However, more recent methods still include a PBMC enrichment step / procedure (120A), which typically takes at least about 1 hour in a closed system, followed by cell counting, transduction, and addition of culture medium, which takes at least about 45 minutes in addition before the lymphocytes come into contact with retroviral particles to form a transduction reaction mixture (130A). As discussed in detail herein, following a “viral transduction” step, which is typically a contact step involving incubation, lymphocytes are typically washed away from any remaining retroviral particles in the suspension, for example, using Sepax (140A), collected by resuspending the PBMCs in a delivery solution (150A), and typically forming a cell product in an infusion bag for reinfusion, a syringe for injection, or a cryopreservation vial for storage (160A). As discussed in further detail herein, conventional PBMC enrichment procedures typically involve a Ficol density gradient and centrifugal force (e.g., centrifugation) or centrifugal force (e.g., Sepax), or enrich the PBMCs using leukocyte apheresis.

[0069] In certain sub-embodied embodiments, antibodies directed at antigens on the surface of unwanted cells are added to blood (170A) or TNC (170B) before PBMC isolation and incubated for an effective period of binding to the unwanted cells, as discussed in more detail herein. The antibodies may be conjugated to beads, or additional antibodies may be included in the incubation to form rosettes of unwanted cells with erythrocytes, as described in more detail herein. The unwanted cells are then depleted in the PBMC isolation step, where they are pelleted with erythrocytes.

[0070] As demonstrated in the examples provided herein, it was surprisingly found that lymphocytes (e.g., T cells and / or NK cells) could come into contact with non-replicating retroviral particles in a reaction mixture of unfractionated whole blood containing an anticoagulant, and a significant proportion of the lymphocytes could be modified, genetically altered, and / or transduced. Thus, it was found that effective genetic modification of lymphocytes with recombinant retroviral particles can be carried out in the presence of blood components and blood cells in addition to PBMCs.

[0071] Therefore, in some embodiments, the genetic modification of T cells and / or NK cells, or the resulting modification of T cells or NK cells, is carried out in a reaction mixture containing blood components and blood cells in addition to PBMCs, and such genetic modification occurs by contacting the T cells and NK cells in the reaction mixture with a recombinant nucleic acid vector (in exemplary embodiments, recombinant retroviral particles). In certain exemplary embodiments provided herein (see Figures 1B, 1E, and 1F), instead of a PBMC enrichment procedure (e.g., using a density gradient), a cell processing filter or filter set is used that enriches lymphocytes more than at least one or several other blood cell types (e.g., a leukocyte removal filter assembly configurable for reverse perfusion with a filter set from which leukocytes can be removed by reverse perfusion) (120B, 120E, and 120F), which also enriches cell types other than PBMCs. This step, in certain embodiments, enriches and concentrates the lymphocytes before they come into contact with recombinant retroviral particles to form a transduction reaction mixture (130B, 130E, and 130F). In certain embodiments, the filter may enrich blood cells in addition to PBMCs, for example, the filter may enrich TNCs. Following the “viral transduction” step, which is typically a contact step with optional incubation, as shown in Figure 1B, for example, as discussed in detail herein, the lymphocytes are typically washed away from any retroviral particles remaining in the suspension, for example, by using Sepax or by passing a washing buffer over the cells on a leukocyte-removing filter, and collected by resuspending PBMCs or TNCs in a delivery solution (150B) to form a cell product, the final cell product product typically in an infusion bag for reinfusion, a syringe for injection, or a cryopreservation vial for storage (160B).

[0072] In exemplary embodiments of the methods provided herein, the contact step with optional incubation of the “viral transduction” step is carried out at a temperature of 32°C to 42°C, such as 37°C. In other exemplary embodiments, the contact step with optional incubation of the “viral transduction” step is carried out at a temperature below 37°C, such as 4°C to room temperature (referred herein to as the “low-temperature contact” step) (see Figures 1E and 1F). The optional incubation associated with the low-temperature contact step may be carried out for any length of time considered herein. In exemplary embodiments, the optional incubation associated with the low-temperature contact step is carried out for no more than one hour. Following a cryogenic contact and an optional incubation step, in some embodiments, lymphocytes are washed away from any retroviral particles remaining on the filter by passing a wash buffer over the cells on a leukocyte-removing filter (140E, 140cF), and collected by resuspending the TNCs in a delivery solution (150E, 150bF) to form a cell formulation, the final product typically in an infusion bag for reinjection, a syringe for injection, or a cryopreservation vial for storage (160E, 160F). While not limited by theory, cryogenic contact of TNCs with viral particles expressing activating elements on their surface for a period of less than one hour is thought to result in binding of the viral particles to T and / or NK cells, but with little to no viral internalization. This also results in T and / or NK cell aggregates crosslinked by the viral particles. Furthermore, due to the lower temperatures and shorter incubation times, there is less cell activation compared to cells incubated for longer periods and / or at temperatures closer to 37°C. Activation of T and / or NK cells is thought to lead to their expression of adhesion molecules, which bind to the leukocyte removal filter and hinder its ability to be recovered by reverse perfusion of the filter.

[0073] In certain embodiments including a low-temperature contact step, the “viral transduction” step also includes a secondary incubation (190E, 190F) after the cells have been removed from the leukocyte-removing filter. In some embodiments, the secondary incubation is carried out by suspending the cells in a culture medium such as Complete OpTmizer® CTS® T-Cell Expansion Media. In some embodiments, the secondary incubation is carried out in a delivery solution. In exemplary embodiments, the secondary incubation is carried out in a delivery solution, but without any cryopreservatives. In exemplary embodiments, the secondary incubation is carried out at a temperature of 32°C to 42°C, such as 37°C. Optional secondary incubation may be carried out for any length of time considered herein. In exemplary embodiments, optional secondary incubation is carried out for less than 4 hours. Although not limited by theory, secondary incubation of TNCs with viral particles expressing activating elements on their surface is thought to result in cell activation. Activation of T and / or NK cells causes cell aggregation.

[0074] Therefore, the workflow described in Figure 1 has at least two mechanisms by which T and / or NK cells can form aggregates: (1) surface-bound viral particles crosslink cells, and this activity is enhanced at temperatures of 4°C to room temperature; and (2) activation of T and / or NK cells leads to their aggregation, which is enhanced at temperatures of 32°C to 42°C. Such aggregates formed by either mechanism under different conditions can be captured by a coarse filter, while other debris, including singlet cells such as lymphocytes, monocytes, and granulocytes, which are about 14 μm in size, as well as cell aggregates smaller than the pore size of the coarse filter used, pass through and enter the waste. In some embodiments, a transduction reaction, including incubation at a temperature of around 37°C, passes through a coarse filter and captures aggregated T and / or NK cells (200E). In some embodiments, a transduction reaction at a temperature of 4°C to room temperature or nearby, passes through a coarse filter and captures aggregated T and / or NK cells (200F). Cells on the coarse filter are collected in the delivery solution to form a cell product, typically in an infusion bag for reinjection, a syringe for injection, or a cryopreservation vial for storage (160E and 160F). In exemplary embodiments using a coarse filter to collect T and / or NK cell aggregates, the cell composition of the delivery solution is greater than 40%, 50%, 60%, 70%, 80%, 90%, or 95% T cells.

[0075] In the reaction mixtures, used, modified, and exemplary embodiments provided herein, in certain embodiments of genetically modified T cells or NK cells, or methods for modifying and / or genetically modifying T cells and / or NK cells, the blood sample, and thus the lymphocytes to be modified, genetically modified, and / or transduced, are not subjected to the PBMC enrichment procedure before contact with recombinant retroviral particles. In some such embodiments, a blood sample, e.g., an anticoagulant whole blood sample, is applied to a filter, such as a leukocyte-removing filter, also known as a leukocyte-depleting filter assembly, to obtain total nucleated cells (TNCs), after which a recombinant vector, such as recombinant retroviral particles, is contacted to such TNCs containing lymphocytes derived from the blood sample. The leukocyte-removing filter assembly may include any filter known in the art, e.g., a filter for collecting total nucleated cells (TNCs). In some embodiments, the filter may include a membrane containing polyurethane, cellulose acetate, polyester, combed cotton, PTFE, or GHP. In some embodiments, the leukocyte removal filter assembly may include, for example, a HemaTrate® filter, an Acrodisc® filter, or any leukocyte removal filter available from Pall (e.g., a Leukotrap® filter) or Haemonetics®. In some embodiments, the leukocyte removal filter is a third-generation, fourth-generation, or more advanced leukocyte removal filter (Sharma et al. Asian J Transfus Sci. 2010 Jan;4(1):3-8).

[0076] In some embodiments, the amount of blood sample applied to the leukocyte removal filter is 40–120 mL (Hematrate; Cook Regentec) or 2–12 mL (Acrodisc; Pall, AP-4952). In some embodiments, the pore size of the filter in the leukocyte removal filter assembly is less than 10, 7.5, 5, 4, or 3 μm, or 0.5–4 μm. In some embodiments, the leukocyte removal filter assembly can collect and / or retain at least 90% of the leukocytes in the blood sample, while at least 75% of the non-leukocyte cells pass through the filter and are not collected. In some embodiments, a coarse filter may be physically attached to the leukocyte removal filter assembly. The coarse filter typically has a larger pore size than the filter in the leukocyte removal filter assembly. In some embodiments, the pore size of the coarse filter is at least 15 μm, and in exemplary embodiments, 15–60 μm. In some embodiments, the coarse filter may be used without using the leukocyte removal filter assembly before the contact step. In addition to being used before the contact step of a method for modifying and / or genetically modifying T cells and / or NK cells, a coarse filter may be used after the contact step. In some embodiments, a coarse filter can be used to capture T and / or NK cell aggregates. Such aggregates form when cells are activated and / or crosslinked by viral particles. In some embodiments, the coarse filter is used to remove singlet blood cells, typically containing neutrophils that pass through the filter. In some embodiments, the coarse filter may be used after a secondary incubation, as shown in Figure 1E. In such embodiments, the filtered cells may be collected and introduced or reintroduced into a subject. As discussed elsewhere in this specification, modified and / or genetically modified cells that are part of aggregates are considered to be more advantageously effective in vivo, particularly by subcutaneous administration.

[0077] Furthermore, even when contact is carried out in unfractionated whole blood (also referred to herein as “whole blood”), based on the aforementioned remarkable findings regarding the effective genetic modification of T cells and optionally NK cells by retroviral particles, exemplary embodiments provide a further simplified method in which lymphocytes are modified, genetically modified, and / or transduced (130C) by directly adding non-replicating retroviral particles to whole blood to form a reaction mixture, and the cells in whole blood are contacted with the non-replicating retroviral particles for a contact time accompanied by optionally optimal incubation provided herein. Thus, such a further simplified method in this exemplary embodiment does not include a lymphocyte enrichment step before lymphocytes in whole blood, typically containing an anticoagulant, come into contact with the retroviral particles. This further simplified method, like other cell processing methods herein, is typically carried out in a closed cell processing system and may include little to no pre-activation before lymphocytes come into contact with retroviral particles. These further simplified methods allow lymphocytes in whole blood to come into direct contact with retroviral particles in a blood bag. Following the contact step (130C) in such a method, lymphocytes that have come into contact with retroviral particles can be washed and concentrated using a PBMC concentration procedure (135C). Thus, in such embodiments, the PBMC concentration procedure and lymphocyte concentration filtration are not typically performed before cells in whole blood containing an anticoagulant come into contact with recombinant retroviral particles. However, in the embodiment of Figure 1C, such a PBMC concentration method is performed, for example, using Sepax with a Ficol gradient (135C) after a contact (130C) with optional incubation is performed. Following PBMC concentration, lymphocytes can be further washed away from any retroviral particles that remain unassociated with the cells, optionally using Sepax (140C), and collected by resuspending the PBMCs in a delivery solution (150C) to form a cell product, the final product typically in an infusion bag for reinjection, a syringe for injection, or a cryopreservation vial for storage (160C).

[0078] In a further exemplary embodiment (Figure 1D), the blood sample is not subjected to a PBMC enrichment procedure before recombinant retroviral particles are added to the blood and come into contact with lymphocytes such as T cells and / or B cells. Even after the contact step (i.e., the step in which the recombinant retroviral particles in the reaction mixture come into contact with lymphocytes such as T cells and / or NK cells and are optionally incubated for either the contact and incubation time provided herein), no PBMC enrichment procedure is used at any step of the process. This further simplified method, like other cell processing methods provided herein, is typically performed within a closed cell processing system and does not involve, or involves minimally involves, pre-activation before lymphocytes come into contact with retroviral particles to form a transduction reaction mixture (130D), and therefore, in some lower embodiments, can provide a powerful point-of-care method. In an example of such a further exemplary embodiment, for example, one or more leukocyte-removal cell processing filtrations using a HemaTrate filter (135D) may be performed after the contact step (130D), which includes an optional incubation. Following leukocyte concentration filtration using a leukocyte-removing filter, lymphocytes are optionally further washed away from any remaining retroviral particles by passing PBS containing 2% HSA through the filter (140D), and can be collected by reperfusion with a delivery solution to collect lymphocytes retained on the leukocyte-removing filter in the cell product (150D), for example by eluting and resuspending TNCs (150D), and the final product is typically in syringes for injection, or in infusion bags for delivery to subjects, or in cryopreservation vials for storage (160D).

[0079] As described above, the workflow of the embodiment of the method shown in Figure 1 provides modified T cells and / or NK cells suspended in a cell preparation. Using the delivery solution provided herein, the cells can be eluted, resuspended, and collected from the filter in a manner in which PBMCs or lymphocytes are filtered, and / or in particular, modification, genetic modification, and / or transduction are carried out on the filter, to form a cell preparation having a volume suitable for administration to a subject, particularly subcutaneous or intramuscularly, as provided herein. Such a delivery solution may also be used for the optional washing described above before the cells are resuspended, eluted, and / or collected by other means for administration. Finally, an additional optional step, e.g., removal of unwanted cell types (e.g., any cell type other than T cells and / or NK cells) such as B cells and / or cancer cells by negative selection in a closed system, as disclosed in more detail herein, may be carried out in any of the embodiment of the method workflow in Figure 1. EA rosette formation can be carried out, for example using anti-CD19, as described in more detail herein, to complex B cells with erythrocytes (170A, 170B, or 170C), which then pellet away from the PBMCs in the density gradient PBMC isolation step. Similarly, B cells can be complexed with beads coated with an antibody against CD19, for example (170A, 170B, or 170C), which then pellet away from the PBMCs in the density gradient PBMC isolation step. Alternatively, a filtration step may be used. Such a filtration step can be used to remove cells complexed with beads (180D) or to capture aggregated lymphocytes, such as T and / or NK cells, which are activated and / or crosslinked by recombinant retroviral particles as described herein. In some embodiments, additional washing steps may be performed. In some embodiments, any one or more of the washing steps shown in or described in Figure 1 for the cell process workflow may be omitted.

[0080] The cell filtration process using a leukocyte removal filtration assembly as shown in Figure 2 is faster than conventional PBMC enrichment procedures, particularly those involving density gradient centrifugation (Ficoll-Paque). Therefore, any embodiment of Figures 1D-F provides an even faster method for obtaining enriched preparations of modified, genetically modified, and / or transduced lymphocytes from whole blood, since time-consuming PBMC enrichment procedures are not performed in any step of such a method, either before or after transduction. In exemplary embodiments, the method is carried out in a closed cell processing system and therefore provides a powerful method for very rapid and relatively simple lymphocyte processing as a point-of-care CAR-T method, overcoming, for example, many complications and the excessive time limitations of current methods.

[0081] As provided in the examples herein, subcutaneous administration has shown remarkable results, with increased engraftment of modified and / or genetically modified lymphocytes compared to modified and / or genetically modified lymphocytes introduced via intravenous infusion. This has resulted in more effective reduction and elimination of CAR-dependent tumors in animals. In exemplary embodiments, modified lymphocytes (e.g., T cells and / or NK cells) are introduced in solution and, in exemplary embodiments, are reintroduced into the subject by subcutaneous administration, delivery, or injection. In some examples of these embodiments, which include contacting lymphocytes in a reaction mixture with retroviral particles, such as those illustrated in Figure 1, including exemplary embodiments that include at least several other blood components not typically present after the lymphocytes are isolated in a PBMC enrichment procedure, the resulting cell preparation, which is a distinct aspect provided herein, is optionally administered to the subject (e.g., re-administered). In exemplary embodiments where a PBMC enrichment procedure is not used after the lymphocytes have contacted the retroviral particles (Figure 1D), the cell preparation produced therein can be reintroduced back into the subject using subcutaneous or intramuscular administration. Accordingly, as will be discussed in more detail herein, some embodiments provided herein are cell preparations, as well as delivery solutions (i.e., excipients) for preparing such cell preparations, which in exemplary embodiments are adapted for subcutaneous delivery and effective for subcutaneous delivery, and in further exemplary embodiments are adapted for subcutaneous delivery. Although not limited by theory, the presence of additional blood cells, particularly neutrophils, in a process using only cell processing filters for concentrating and / or washing lymphocytes, such as HemaTrate filters, is considered to make the cell preparation more suitable for subcutaneous delivery in order to avoid some additional risks if these other blood cell types, particularly neutrophils or agglutinated T cells, are to be directly injected and returned into the patient's bloodstream. For example, a subcutaneous preparation of retroviruses reconstituted with all nucleated cells on a lymphocyte-removing filter may contain neutrophils (or more generally granulocytes) in addition to lymphocytes.In exemplary embodiments, the cell preparation comprises neutrophils, B cells, monocytes, erythrocytes, basophils, eosinophils, and / or macrophages, along with modified T cells (CAR-T cells) and / or NK cells (CAR-NK cells). Subcutaneous or intramuscular preparations and administrations are advantageous over intravenous preparations and administrations because the retrovirus preparation (suspension) reconstituted with lymphocytes may further contain cell aggregates that express adhesion receptors that can induce pulmonary congestion upon intravenous delivery.

[0082] Methods for subcutaneous administration are well known in the art and typically involve administration into the fat layer beneath the skin. It should be noted that any embodiment of this specification involving subcutaneous delivery is intended to be intramuscular delivery or intratumoral delivery, which is delivery into the muscle. In some embodiments, subcutaneous administration may be performed in the upper thigh, upper arm, abdomen, or upper buttocks of the subject. Subcutaneous administration can be distinguished from intraperitoneal administration, which penetrates the fat layer used in subcutaneous administration and delivers the formulation or drug into the peritoneum of the subject.

[0083] In such embodiments, if cells are introduced or reintroduced into a subject by subcutaneous administration (also referred to herein as subcutaneous injection or delivery) in a larger volume of excipients to facilitate such subcutaneous administration, hyaluronidase may be added to an isolated modified, genetically modified, and / or transduced lymphocyte preparation containing lymphocytes that have come into contact with recombinant retrovirus, or may be subcutaneously injected at or near the same site of a series of deliveries of the isolated modified, genetically modified, and / or transduced lymphocyte preparation. In exemplary embodiments, an effective amount of hyaluronidase is used in embodiments in which a cell preparation of lymphocytes that have come into contact with retroviral particles, such as modified NK cells, and in exemplary embodiments, a cell preparation containing T cells, is subcutaneously reintroduced into a subject, particularly in amounts greater than 1 or 2 mL (e.g., 2-1,000 mL, 2-500 mL, 2-100 mL, 2-50 mL, 2-10 mL, 2-5 mL, 5-1,000 mL, 5-50 mL, 5-10 mL). While not limited by theory, hyaluronidases, such as recombinant human hyaluronidase, enhance the dispersion and absorption of other injected therapeutics and potentially improve the pharmacokinetic profile of co-injected therapeutics, particularly by enabling subcutaneous delivery of large volumes beyond the typically administered volumes of 2 mL or less (see, for example, Bookbinder LH, et al. “A recombinant human enzyme for enhanced interstitial transport of therapeutics.” J.Control Release (2006) Aug 28;114(2):230-41. Epub 2006 Jun 7 (the entire article is incorporated herein by reference) and Frost, GI, et al. “Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration.” Expert Opinion Drug Delivery (2007) Jul;4(4);427-440 (the entire article is incorporated herein by reference)).Fluid dispersion in cell mixtures can be promoted in larger volumes while minimizing vascular compression at the injection site. Hyaluronidase (e.g., recombinant human hyaluronidase PH20 enzyme (rHuPH20), or Hylenex® 150 USP units) is available from Halozyme Therapeutics, Inc. (San Diego, CA). In some embodiments, rHuPH20 in concentrations of 50–5000 or 1,000–3,000 units / mL may be delivered with modified, genetically modified, and / or transduced lymphocytes, for example, in quantities of 1–50 mL, 2–25 mL, 2–20 mL, 2–10 mL, 2–5 mL, 2–4 mL, 2.5–25 mL, 2.5–20 mL, 2.5–10 mL, 2.5–5 mL, 5–20 mL, or 5 mL–10 mL, or such delivery of hyaluronidase and lymphocytes may be sequential. Additional hyaluronidase enzymes may be found, for example, in U.S. Patent No. 7,767,429 (which is incorporated herein by reference in its entirety).

[0084] Figure 2 provides an exemplary, not limited, cell-processed leukocyte-removal filtration assembly (200) for concentrating nucleated cells, which can be used as a leukocyte-removal filter in the method of Figure 1. In the exemplary embodiment, the exemplary leukocyte-removal filtration assembly (200), which is a single-use filtration assembly, comprises a leukocyte-depleted medium (e.g., filter set) in a filter enclosure (210) having an inlet (225) and an outlet (226), as well as a configuration of bags, valves, and / or channels / tubes that provide the ability to concentrate, concentrate, wash, and collect retained leukocytes or nucleated hematopoiesis using perfusion and back perfusion (see, for example, EP2602315A1, which is incorporated herein in its entirety by reference). In the exemplary embodiment, the leukocyte-removal filtration assembly (200) is a commercially available HemaTrate filter (Cook Regenetec, Indianapolis, IN). Leukocyte-removed filtration assemblies can be used to increase the concentration of total nucleated cells (TNCs), including granulocytes, which are removed in the PBMC enrichment procedure of a closed cell processing system. Filter assemblies comprising leukocyte-depleting media such as HemaTrate filters and the exemplary leukocyte-removed filter assembly in Figure 2 do not remove granulocytes, and therefore they are not considered PBMC enrichment assemblies or filters herein, nor are the methods of incorporating them considered PBMC enrichment procedures or steps herein.

[0085] The leukocyte removal filter assembly (200) in Figure 2 is a single-use sterile assembly comprising various tubes and valves, typically needle-free valves, that enable the isolation of leukocytes from hematopoietic preparations including whole blood and leukocytes, as well as rapid washing and high-concentration of leukocytes. In this exemplary assembly, as disclosed in detail herein, after the reaction mixture is subjected to a contact step with an optional incubation, a 500 mL PVC bag containing a blood bag (215), for example, a transduction / contact reaction mixture containing about 120 mL of whole blood, anticoagulant, and retroviral particles, is connected to the assembly (200) at a first assembly opening (217) of an inlet tube (255). Lymphocytes, including several modified T cells and / or NK cells accompanied by associated retroviral particles, as well as several lymphocytes that may be genetically modified at this point, as well as other blood cells and components in the whole blood reaction mixture, and an anticoagulant, enter the inlet tube (255) through the first assembly opening (217) by gravity when the clamp on the first inlet tube (255) is released. The modified and / or genetically modified T cells and / or NK cells pass through the inlet valve (247) and collection valve (245) and enter the filter enclosure (210) through the filter enclosure inlet (225) and come into contact with the leukocyte removal IV filter set (e.g., SKU J1472A Jorgensen Labs) inside the filter enclosure (210). Nucleated blood cells, including white blood cells, are retained by the filter, while other blood components pass through the filter and enter the outlet tube (256) from the filter enclosure outlet (226), then through the outlet valve (247), and are collected in a waste collection bag (216), which may be, for example, a 2L PVC waste collection bag.

[0086] An optional buffer washing step can be performed by switching the inlet valve (247) to the washing position. In this optional washing step, a buffer bag (219), for example, a 500 mL saline washing bag, is connected to the second assembly opening (218) of the inlet tube (255). The buffer moves into the inlet tube (255) through the second assembly opening (218) by gravity when the clamp on the inlet tube (255) is released. The buffer passes through the inlet valve (247) and the collection valve (245), enters the filter enclosure (210) through the filter enclosure inlet (225), and passes through the leukocyte removal filter set in the filter enclosure (210) to rinse the lymphocytes retained on the filter. The buffer moved from the filter enclosure outlet (226) into the outlet tube (256), then through the outlet valve (247), and was collected in a waste collection bag (216), which may be the same waste collection bag used to collect the reaction mixture components that passed through the filter in the previous step, or a new waste collection bag that replaced the first waste collection bag. After this, the buffer could enter the second assembly opening (218). An optional washing step may be performed multiple times by repeating the above process with additional buffer. Furthermore, in some embodiments, the optional washing step may be performed at least partially using an eluent / delivery solution.

[0087] Once the entire or substantially entire volume of the reaction mixture in the blood bag (215) has passed through the filter (210) and an optional washing step has been optionally performed, a reverse perfusion process is initiated to move the fluid in the opposite direction of the assembly (200) to collect the lymphocytes retained on the filter set in the filter enclosure (210). Exemplary embodiments of the leukocyte removal filter assembly herein are adaptable to reperfusion. Before initiating the reverse perfusion process in the exemplary assembly (200), the outlet valve (247) is switched to the reperfusion position and the collection valve (245) is switched to the collection position. To initiate reperfusion, in some embodiments, a delivery solution, which may be a buffer (e.g., PBS) and may also be an eluent in a syringe (266), which may be, for example, a 25 mL syringe, is passed into the outlet tube (256) by injection using the syringe (266). Next, the delivery solution enters the filter enclosure (210) through the filter enclosure outlet (226), suspending the lymphocytes held on the filter set in the cell preparation, and moving the cell preparation from the filter enclosure (210) through the filter enclosure inlet (225) into the inlet tube (255). The cell preparation containing modified lymphocytes, including several T cells and / or NK cells with associated retroviral particles (some of which may be genetically modified and / or transduced at this point), is then collected in a cell sample collection bag (265), which may be, for example, a 25 mL cryopreservation bag, after passing through the collection valve (245). The collected cell preparation may optionally be administered to the subject, for example, by subcutaneous administration.

[0088] Self-driving CAR method and composition In certain embodiments, the Specified Provisions provide a polynucleotide, referred to herein as a “self-driven CAR,” which encodes a membrane-bound lymphoproliferative element whose expression in T cells or NK cells is under the control of an inducible promoter induced by the binding of an antigen to an extracellular binding pair member polypeptide that is functionally linked to either an intracellular activation domain, such as the CD3 zeta intracellular activation domain or an intracellular activation domain disclosed elsewhere herein. In exemplary embodiments, such a binding pair member polypeptide is a CAR. In other embodiments, such a binding pair member polypeptide is a TCR. Thus, in certain embodiments, the Specified Provisions provide a polynucleotide comprising an inducible promoter operably linked to a nucleic acid encoding a membrane-bound lymphoproliferative element, which is induced by CAR binding to its target. Expression of the lymphoproliferative element can induce proliferation of T cells or NK cells. In certain embodiments, the Specified Provisions provide genetically modified or transduced T cells, referred to as “self-driven CAR-T cells,” which contain a self-driven CAR. Any embodiment including self-driven CAR-T cells may also include "self-driven CAR NK cells," which are genetically modified or transduced NK cells containing self-driven CARs. In some embodiments, self-driven CAR NK cells are present in addition to self-driven CAR-T cells. In other embodiments, self-driven CAR NK cells are present instead of self-driven CAR-T cells. While not limited by theory, these self-driven CARs and self-driven CAR-T cells respond to the binding of the CAR to its target antigen, resulting in a signaling cascade that produces one or more inductive signals that increase the transcription of one or more lymphoproliferative elements. This CAR-stimulated transcription is achieved via downstream transcription factors, such as nuclear factor (NFAT) on activated T cells, activating transcription factor 2 (ATF2), activating protein 1 (AP-1), and nuclear factor kappa light chain enhancer (NF-κB) on activated B cells. In exemplary embodiments, CAR-stimulated transcription is achieved via NFAT, and the inductive promoter that modulates the expression of lymphoproliferative elements is an NFAT-responsive promoter.

[0089] Accordingly, provided herein are isolated polynucleotides comprising, in certain embodiments, one or more first transcription units operably linked to an inducible promoter in at least one of T cells or NK cells (at least one of the one or more first transcription units comprises a first polynucleotide sequence encoding a first polypeptide comprising a lymphoproliferative element), and, in exemplary embodiments, a first sequence comprising a second transcription unit encoding a chimeric antigen receptor (CAR) (the CAR comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain). In certain exemplary embodiments, the lymphoproliferative element is constitutively active in at least one of T cells or NK cells, and the lymphoproliferative element comprises a transmembrane domain. In exemplary embodiments, one or more first transcription units do not encode a polypeptide comprising a signal peptide sequence comprising a signal peptidase cleavage site, or other sequences resulting in an encoded polypeptide that, once expressed, is secreted from T cells or NK cells or otherwise released.

[0090] In another embodiment of a self-propelled CAR, an isolated polynucleotide is provided herein, comprising a reversed first sequence including one or more first transcription units operably linked to an inducible promoter in at least one of T cells or NK cells, further comprising a forward second sequence including 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 one or more first transcription units and the 5' end of one or more second transcription units is less than the number of nucleotides between the 3' end of one or more first transcription units and the 3' end of one or more second transcription units, at least one of the one or more first transcription units encoding a lymphoproliferative element, and at least one of the one or more second transcription units encoding a chimeric antigen receptor (CAR), the CAR comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. The distance between the 5' end of one or more first transcription units and the 5' or 3' end of one or more second transcription units can be measured, for example, as the number of nucleotides between the 5' nucleotide of one or more first transcription units and the 5' or 3' nucleotide of one or more second transcription units. In some embodiments, one or more first transcription units and one or more second transcription units are transcribed in a branched manner, and such transcription units are said to be branched, i.e., arranged in opposite directions, such that the 3' ends of one or more first transcription units and one or more second transcription units are further apart from each other than the 5' ends of one or more first transcription units and one or more second transcription units. A polynucleotide or vector containing two transcription units, i.e., one or more first and second transcription units, may be referred to herein as a bicistronic polynucleotide or vector. A branched bicistronic polynucleotide may encode two, three, four, or more polypeptides and / or inhibitory RNAs. As will be discussed in more detail herein, polynucleotides typically contain an inductive promoter operably linked to a lymphoproliferative element, a constitutive promoter operably linked to a CAR, and any cell tag separated by a ribosome skipping sequence.The binding of the target antigen to the CAR generates an inductive signal that promotes the transcription of transcription units operably linked to an inductive promoter.

[0091] In another embodiment, genetically modified lymphocytes, in exemplary embodiments, genetically modified T cells and / or NK cells, transduced and / or genetically modified with the polynucleotides disclosed above, are provided herein. In yet another embodiment, the use of non-replicating recombinant retroviral particles in the manufacture of a kit for genetically modifying and / or transducing target lymphocytes, in exemplary embodiments, T cells and / or NK cells, the use of which comprises transducing and / or genetically modifying T cells or NK cells with the polynucleotides disclosed above. In yet another embodiment, a method for administering genetically modified lymphocytes to a target is provided herein, the genetically modified lymphocytes are produced by transducing and / or genetically modifying lymphocytes with the polynucleotides disclosed above in this self-driven CAR section. In some embodiments, the administration of genetically modified lymphocytes may be carried out by intravenous injection, subcutaneous administration, or intramuscular administration. In some embodiments, the modified lymphocytes introduced into the target may be allogeneic lymphocytes. In such embodiments, the lymphocytes are derived from different individuals, and the lymphocytes derived from the subject are not modified. In some embodiments, no blood is taken from the subject to recover the lymphocytes. Embodiments provided herein, including polynucleotides disclosed in this section of Self-Driven CAR Methods and Compositions, methods for transducing and / or genetically modifying lymphocytes with Self-Driven CAR polynucleotides, the use of such methods in the manufacture of kits, reaction mixtures formed in such ways, genetically modified lymphocytes produced by such methods, and methods for administering genetically modified lymphocytes produced by such methods, are referred to herein as “embodiments of compositions and methods comprising Self-Driven CAR.”

[0092] In any exemplary embodiment of any aspect of the composition and method for transducing lymphocytes with a self-driven CAR, the polynucleotide may comprise a constitutive T cell or NK cell promoter. Constitutive T cell or NK cell promoters that constitutively express a polynucleotide in T cells or NK cells are known in the art. In some embodiments, the transcription unit is a constitutive expression unit or construct that encodes the CAR in an exemplary embodiment of the aspect of the self-driven CAR. The constitutive expression construct may comprise regulatory sequences such as start and end codons for transcription and translation. In some embodiments, such regulatory sequences are specific to the type of cell into which the constitutive promoter is introduced, i.e., T cells and / or NK cells. The constitutive expression construct may comprise a native or non-native promoter operably ligated to the nucleotide sequence of interest. Preferably, the promoter is functional in lymphocytes, particularly T cells and / or NK cells. Exemplary constitutive promoters include, for example, the CMV, E1F, VAV, TCRvbeta, MCSV, and PGK promoters. The operable ligation of promoters to nucleotide sequences is within the scope of the art. In some embodiments, the constitutive expression construct is or a part thereof of a recombinant expression vector as described herein.

[0093] Constitutive T cell or NK cell promoters can transcribe target sequences in T cells or NK cells at relatively consistent rates, although their activity can vary in accordance with the cell's metabolic activity. In some embodiments, transcription of a target sequence from a single constitutive promoter is limited to a maximum of 2x, 1.5x, 1.45x, 1.4x, 1.35x, 1.3x, 1.25x, 1.2x, 1.15x, 1.1x, 1.05x, or at least 0.5x, 0.55x, 0.6x, 0.65x, 0.7x, 0.75x, 0.8x, 0.85x, 0.9x, or 0.95x of the target sequence transcription under most or all physiological conditions of the cell. In some embodiments, the constitutive T cell or NK cell promoter remains within the range of 0.5x, 0.55x, 0.6x, 0.65x, 0.7x, 0.75x, 0.8x, 0.85x, 0.9x, and 0.95x to 1.05x, 1.1x, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, and 1.5x of the transcription number at the upper limit of the range under most or all physiological conditions of the cell. In some embodiments, the constitutive T cell or NK cell promoter may be any constitutive promoter known in the art. In some embodiments, the constitutive T cell or NK cell promoter may be the EF1-a promoter, PGK promoter, CMV promoter, MSCV-U3 promoter, SV40hCD43 promoter, VAV promoter, TCRbeta promoter, or UBC promoter. In some embodiments, the constitutive T cell or NK cell promoter may include the EF1-a promoter nucleotide sequence (SEQ ID NO: 350), the PGK promoter nucleotide sequence (SEQ ID NO: 351), or a functional portion or variant thereof. In some embodiments, the constitutive T cell or NK cell promoter may include a promoter other than the EF1-a promoter.

[0094] In any exemplary embodiment of the composition and method for transducing lymphocytes with a self-propelled CAR, the polynucleotide comprises an inducible or activatable promoter. In exemplary embodiments, the inducible or activatable promoter is an NFAT-responsive promoter. In some embodiments, the inducible or activatable promoter can increase the transcription of the target sequence in the presence of an inducible signal by at least 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, or 1,000-fold higher than the transcription of the target sequence in the absence of an inducible signal. In some embodiments, inducible or activatable promoters can increase the transcription of a target sequence in the presence of an inducible signal by 5, 6, 7, 8, 9, 10, 20, 25, 50, 100, and 250 times the lower limit of the range of transcription of the target RNA in the absence of an inducible signal, and 10, 20, 25, 50, 100, 250, 500, and 1,000 times the upper limit of the range. In some embodiments, the transcription unit is an inducible expression unit or construct, which may encode a lymphoproliferative element in exemplary embodiments of a self-driven CAR. The inducible expression construct may include regulatory sequences such as start and end codons for transcription and translation. In some embodiments, such regulatory sequences are specific to the type of cell into which the inducible promoter is introduced, i.e., T cells and / or NK cells. The inducible expression construct may include a native or non-native promoter operably ligated to the nucleotide sequence of interest. Preferably, the promoter is functional in lymphocytes, particularly T cells and / or NK cells. In some embodiments, the inducible or activatable promoter may be an NFAT-responsive, ATF2-responsive, AP-1-responsive, or NF-κB-responsive promoter.Other promoters that are induced upon T cell activation and can be used as inducible promoters in the embodiments herein, particularly in the embodiments of self-driven CARs, include the IL-2 promoter, IFNg promoter, CD25 promoter, CD40L promoter, CD69 promoter, CD107a promoter, TNF promoter, VLA1 promoter, LFA1 promoter, or functional and inducible fragments of any of these promoters. As discussed herein, such inducibility may arise from the presence of one or more NFAT binding elements.

[0095] In any exemplary embodiment of the composition and method for transducing lymphocytes with a self-propelled CAR, the first sequence may be reversed and the second sequence may be forward. The orientations of the first and second sequences are relative to the 5'-to-3' direction established by the 5'LTR and 3'LTR of the polynucleotide when present in recombinant retroviral particles capable of genetically modifying T cells or NK cells. Thus, sequences whose 5' end is closer to the 5'LTR than their 3' end is closer to the 5'LTR, such as transcription units, promoters, coding sequences, and miRNAs, are forward, and sequences whose 3' end is closer to the 5'LTR than their 5' end is closer to the 5'LTR, are reversed. The distance between the end of a sequence and the 5'LTR is typically measured, for example, as the number of nucleotides between the 5' or 3' nucleotide of the sequence and the 3' nucleotide of the 5'LTR. In some embodiments, the polynucleotide may further include a reversed riboswitch, as disclosed elsewhere herein.

[0096] In some embodiments, the inducible promoter may be an NFAT-responsive promoter, an ATF2-responsive promoter, an AP-1-responsive promoter, or an NF-κB-responsive promoter. Examples of the NFAT family of transcription factors include NFATc1, NFATc2, NFATc3, NFATc4, and NFAT5. While not limited by theory, calcium signaling initiated by CAR antigen binding and the resulting signaling is thought to activate the transcriptional activity of NFATc1, NFATc2, NFATc3, and NFATc4 in exemplary embodiments. As cellular calcium concentrations increase, it binds to calmodulin, which then activates the phosphatase calcineurin. Dephosphorylation of cytoplasmic NFAT family members by calcineurin leads to their nuclear localization. Once in the nucleus, NFATs bind to bZIP proteins, such as activator protein 1 (AP-1), forming a complex that binds to and activates the NFAT-responsive promoter.

[0097] Various aspects of the tumor microenvironment, including acidic pH and the presence of antiproliferative cytokines, are inhibitory to CAR-T cell proliferation. While not limited by theory, non-secretory and constitutively active lymphoproliferative elements may stimulate CAR-T cell proliferation in tumor microenvironments with high localized concentrations of inhibitory signals. Expression of these lymphoproliferative elements solely by CAR-T cells with active CAR signaling, as in self-driven CARs, may limit CAR-T cell growth in the absence of antigen binding. Furthermore, after successful tumor treatment, self-driven CAR-T cells do not proliferate significantly in the absence of antigens.

[0098] In exemplary embodiments of the self-driven CAR, the inducible promoter is an NFAT-responsive promoter. NFAT transcription factors generally have weak binding, and multiple NFAT binding sites may be used in the inducible promoter. In some embodiments, the inducible or activatable promoter may be an NFAT-responsive promoter and include one or more NFAT binding sites. In some embodiments, one or more NFAT binding sites may be derived from promoters known in the art to be NFAT-responsive promoters. For example, one or more NFAT binding sites may be derived from the IL-2 promoter, the IL-4 promoter, and / or the IL-8 promoter. In exemplary embodiments, one or more NFAT binding sites may be derived from the IL-2 promoter. In some embodiments, an NFAT-responsive promoter may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 NFAT binding sites, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 NFAT binding sites, or 1-12, 2-12, 2-10, 3-10, or 4-8 NFAT binding sites. In exemplary embodiments, an NFAT-responsive promoter may include 4, 6, or 9 NFAT binding sites. In some embodiments, the NFAT binding sites of an NFAT-responsive promoter may include functional sequence variants that retain the ability to bind to NFAT in order to avoid exact repeats. In some embodiments, an NFAT-responsive promoter is responsive to NFATc1, NFATc2, NFATc3, NFATc4, and / or NFATc5. In some embodiments, an NFAT-responsive promoter includes one or more NFAT binding sites of SEQ ID NO: 352. In some embodiments, the interval between copies of the NFAT binding site may be between 3 nucleotides at the lower limit of the range and 60 nucleotides at the upper limit of the range. In exemplary embodiments, the interval between copies of the NFAT binding site may be between 6 nucleotides at the lower limit of the range and 20 nucleotides at the upper limit of the range.In an exemplary embodiment, the NFAT-responsive promoter comprises six NFAT binding sites, and the nucleotide sequence comprises or consists of SEQ ID NO: 353 or a functional portion or functional variant thereof.

[0099] Although not limited by theory, the binding of a CAR to its antigen induces a signaling cascade via the CAR's CD3Z intracellular domain, resulting in an influx of calcium ions that leads to calcineurin activation, which dephosphorylates NFAT, which can then translocate to the nucleus and bind to an NFAT-responsive promoter to activate transcription. In exemplary embodiments, CAR-T cells contain a CAR with a CD3Z intracellular domain, and the inducible promoter for one or more transcription units containing lymphoproliferative elements is an NFAT-responsive promoter.

[0100] In some embodiments, the transcription unit encoding the lymphoproliferative element includes a minimal constitutive promoter having an upstream NFAT binding site for generating an inducible or activatable promoter with low levels of transcription even in the absence of an inductive signal. In some embodiments, in the absence of an inductive signal, the low levels of transcription of the lymphoproliferative element from such an inductive promoter may be less than 1 / 2, 1 / 4, 1 / 5, 1 / 10, 1 / 25, 1 / 50, 1 / 100, 1 / 200, 2 / 250, 1 / 500, or 1 / 1,000 of the transcription level of the CAR from the constitutive promoter. In some embodiments, the minimal constitutive promoter may include a minimal IL-2 promoter, a minimal CMV promoter, or a minimal MHC promoter. In exemplary embodiments, the minimal promoter may be a minimal IL-2 promoter (SEQ ID NO: 354) or a functional part or functional variant thereof. In certain embodiments, one or more NFAT binding sites are upstream of the minimal IL-2 promoter. In an exemplary embodiment, the NFAT-responsive promoter includes six NFAT binding sites upstream of the minimal IL-2 promoter, and the nucleotide sequence includes or consists of SEQ ID NO: 355 or its functional portion or functional variant.

[0101] Inducible and constitutive promoters in the polynucleotides disclosed above, having a reversed first sequence and a forward second sequence, can interfere with each other in unpredictable ways, particularly in the presence of potent constitutive promoters such as EF1-a, CMV, and CAG promoters. Promoter interference can result in an increase or decrease in transcription from one or both promoters. Promoter interference can also result in a decrease in the dynamic range of the inducible promoter. In some embodiments, the insulator is located between branched transcription units. In some embodiments, the insulator is located between the inducible promoter and the constitutive promoter. In some embodiments, the insulator may be a chicken HS4 insulator, a Kaiso insulator, a SAR / MAR element, a chimeric chicken insulator-SAR element, a CTCF insulator, a gypsy insulator, or a β-globin insulator, or a fragment thereof known in the art. In some embodiments, the insulator may be a β-globin polyA spacer B (SEQ ID NO: 356), a β-globin polyA spacer A (SEQ ID NO: 357), a 250 cHS4 insulator v1 (SEQ ID NO: 358), a 250 cHS4 insulator v2 (SEQ ID NO: 359), a 650 cHS4 insulator (SEQ ID NO: 360), a 400 cHS4 insulator (SEQ ID NO: 361), a 650 cHS4 insulator and a β-globin polyA spacer B (SEQ ID NO: 362), or a β-globin polyA spacer B and a 650 cHS4 insulator (SEQ ID NO: 3). In some embodiments, the insulator may be oriented forward. In other embodiments, the insulator may be oriented reverse. In exemplary embodiments, a forward-coded EF1-a promoter is separated from an NFAT-inducible minimal IL-2 promoter coded by a reverse-coded 650cHS4 insulator, a reverse-coded β-globin polyA spacer A, or a forward-coded one. Those skilled in the art will understand how to incorporate insulators between promoters to prevent or reduce promoter interference.

[0102] In some embodiments, the polynucleotide may contain a number of adenosine nucleotides known as polyadenylated sequences following the 3' end of a sequence encoding a reverse lymphoproliferative element. In some embodiments, the polyadenylated sequence may be used with an insulator. In other embodiments, the polyadenylated sequence may be used in the absence of an insulator. In some embodiments, the polyadenylated sequence may be derived from a β-globin polyadenylated sequence. In some embodiments, the polyadenylated sequence may be derived from an hGH polyadenylated sequence. In some embodiments, the polyadenylated sequence may be synthetic. In some embodiments, the polyadenylated sequence may contain one or more sequences selected from hGH polyA (SEQ ID NO: 316), SPA1 (SEQ ID NO: 317), or SPA2 (SEQ ID NO: 318). In some embodiments, the polynucleotide does not contain an exogenous splice site. In exemplary embodiments, the polynucleotide does not contain a forward or reverse exogenous splice site.

[0103] In any embodiment of a composition and method for transducing lymphocytes with a self-propelled CAR, the polynucleotide may comprise one or more inhibitory RNA molecules, such as miRNA or shRNA, as disclosed elsewhere herein. In some embodiments, the inhibitory RNA molecule may be encoded within an intron, for example, an EF1-a intron. In exemplary embodiments, the inhibitory RNA molecule may target any of the targets identified herein, including but not limited to the section on inhibitory RNA molecules herein.

[0104] In any embodiment of a composition and method for transducing lymphocytes with a self-driven CAR, the inducible promoter can drive the expression of a lymphoproliferative element, as disclosed elsewhere herein. In exemplary embodiments, the lymphoproliferative element is a non-secretory and constitutively active lymphoproliferative element.

[0105] Cell preparations and methods of administration 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 method for modification according to this specification are present in the cell product, including at the time of an 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 method for modification according to this specification are present in the cell product, including at the time of an 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 method for modification herein are present in the cell preparation, including at the time of an optional delivery step.

[0106] In some embodiments, and in exemplary embodiments in which the cell preparation is administered subcutaneously or intramuscularly, the volume of the cell preparation containing the modified lymphocytes is smaller than that of conventional CAR-T methods, which are typically infusion delivery methods, and may be less than (approximately) 1 mL, (approximately) 2 mL, (approximately) 3 mL, (approximately) 4 mL, (approximately) 5 mL, (approximately) 10 mL, (approximately) 15 mL, (approximately) 20 mL, or (approximately) 25 mL.

[0107] The favorably short time between blood collection and reintroduction of modified lymphocytes into the subject means, in some embodiments, that some lymphocytes have associated with recombinant nucleic acid vectors, in exemplary embodiments, with non-replicating recombinant retroviral particles, and are not yet 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 polynucleotides that are either extrachromosomal or integrated into the genome. In some embodiments, the polynucleotides may be extrachromosomal in at least 5% of the modified lymphocytes. In some embodiments, at least 5% of the modified lymphocytes are not transduced.

[0108] Short contact times in certain embodiments also result in many of the modified lymphocytes in the cell preparation herein having conjugating polypeptides, fusion polypeptides, and, in some embodiments, T cell activating elements formed on the surface of the retroviral particles, either through association with recombinant retroviral particles or by fusion of the retroviral envelope with the plasma membrane, including at the time of an 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 modified lymphocytes in the cell preparation contain pseudotyping elements and / or T cell activating elements, such as T cell activating antibodies. In some embodiments, pseudotyping elements and / or T cell activating elements can bind to the surface of modified lymphocytes via, for example, T cell receptors, CD28, OX40, 4-1BB, ICOS, CD9, CD53, CD63, CD81, CD82, and / or the pseudotyping elements and / or T cell activating elements can be present in the plasma membrane of the modified lymphocytes.

[0109] For example, cell formulations comprising T cells and / or NK cells are provided herein. Such formulations are provided by methods provided herein in exemplary embodiments. Any of the cell formulations provided herein may comprise self-driven CAR-T cells. In one embodiment, a cell formulation is provided herein comprising a population of self-driven CAR-T cells, such as modified, genetically modified, transcribed, transfected, and / or stably incorporated self-driven CAR-T cells in a delivery solution.

[0110] For advantageously short time, lymphocytes come into contact with a 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 T and NK cells have not yet expressed the recombinant nucleic acid or have not yet incorporated the recombinant nucleic acid into the cell genome, and some of the retroviral particles in embodiments including these may associate with, but not fuse with, the target cell membrane before being used or contained in any of the methods or compositions provided herein or before being used to prepare a cell preparation, including, but not limited to, being introduced into or reintroduced back into the target. Accordingly, various forms and embodiments of cell preparations that can be produced from these exemplary methods provided herein, such as rapid point-of-care methods, for example, involving subcutaneous administration in exemplary embodiments, are provided herein. Such cell preparations, including but not limited to those described immediately below this specification and in the section of exemplary embodiments, may be present at the time of collection of cells after they have come into contact with a recombinant retroviral vector and optionally been rinsed, and may be present until (including) administration to a subject, or subcutaneously in exemplary embodiments.

[0111] In some embodiments, cell formulations comprising T cells and / or NK cells are provided herein, wherein 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 10%, or less than 5% of the cells in the cell formulation are T cells and / or NK cells. In some embodiments, cell formulations comprising lymphocytes, NK cells, and / or T cells are provided, 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 T cells in the cell formulation, in exemplary embodiments, are modified cells. In some embodiments, the lower limit of the lymphocyte range is modified by 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and 70%, while the upper limit of the lymphocyte range is modified by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%, for example, 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, the lower limit of the range of modified lymphocytes is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and 70%, and the upper limit of the range of modified lymphocytes is 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 range of modified lymphocytes is not genetically modified, transduced, or stably transfected, for example, 5%-95%, 10%-90%, 25%-75%, and 25%-95%. In some embodiments, the polynucleotides of genetically modified lymphocytes may be extrachromosomal or integrated into the genome in these cell formulations formed after contact and incubation, and at optional administration. In some embodiments of these cell formulations, 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, the lower limit of the range of modified or genetically modified lymphocytes is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and 70%, and the upper limit of the range of modified or genetically modified lymphocytes is 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 range of modified or genetically modified lymphocytes is 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 transfected or stably transfected in these cell preparations as a result of, for example, a method for genetically modifying T cells and / or NK cells provided herein. In some embodiments, the lower limit of the range of modified or genetically modified lymphocytes is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, and 70%, and the upper limit of the range of modified or genetically modified lymphocytes is 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 them are not transfected or stably transfected, for example, 5%-95%, 10%-90%, 25%-75%, and 25%-95%.

[0112] In certain embodiments disclosed herein, including subcutaneous delivery of solutions and cell preparations adapted for subcutaneous delivery, fewer modified or genetically modified lymphocytes can engraft when delivered intravenously compared to when delivered subcutaneously. In some embodiments, the number of lymphocytes that engraft when delivered intravenously compared to when delivered subcutaneously is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% less.

[0113] In some embodiments, a cell preparation (including such preparations in which cells are present at the time of collection of cells after contact with a recombinant retroviral vector and optionally rinsed, and present until (including) the time of administration to a subject) comprises at least two of unmodified lymphocytes, modified lymphocytes, and genetically modified lymphocytes. In some embodiments, such a cell preparation comprises more unmodified lymphocytes than modified lymphocytes. In some embodiments of such cell preparations produced by the methods provided herein, the proportion 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 provided herein for transducing lymphocytes in whole blood, lymphocytes in whole blood, which are added to or used to create the reaction mixture in some embodiments, such as 1% to 20%, or 5% to 20%, or 1% to 15%, or 5% to 15%, or 7% to 12%, or about 10%, of which are 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 modified and have not come into contact with recombinant nucleic acid vectors such as recombinant retroviral particles that lack the ability to replicate. In certain exemplary embodiments, the lymphocytes are tumor-infiltrating lymphocytes.

[0114] In some embodiments, cell formulations are provided herein, in which at least 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the modified T and / or NK cells in the cell formulation do not express CAR, or in certain embodiments, transposase, and / or do not have CAR associated with their cell membrane. In other embodiments, cell formulations are provided herein, in which at least 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the modified T and / or NK cells in the cell formulation contain recombinant viral reverse transcriptase or integrase. While not limited by theory, in exemplary methods provided herein, unlike conventional CAR-T cell processing methods in which cells are cultured ex vivo for several days or weeks, and many cell divisions, T cells and / or NK cells come into contact with retroviral particles within a few hours of delivery, some or most of the reverse transcriptase and integrase present in the retroviral particles that migrate into the T cells and / or NK cells after fusion 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 formulations 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 formulation do not express recombinant mRNA (e.g., encoding CAR and / or recombinant transposase). In some embodiments, cell formulations are provided herein, in which 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 formulation do not have recombinant nucleic acids stably incorporated into their genomes.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.

[0115] In further embodiments, cell preparations comprising 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 a contact step in which they come into contact with a recombinant nucleic acid vector, e.g., a naked DNA vector, or, in exemplary embodiments, a recombinant letovirus particle that lacks replication ability. In some embodiments, at least a portion of the B cells, but less than or equal to 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 preparation that can be optionally administered or readministered. In exemplary embodiments, a portion of the B cells are 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 B cells are not modified in such formulations and methods. Thus, in some embodiments, modified lymphocytes are present in the cell formulation together with unmodified lymphocytes, which are optionally delivered intramuscularly or subcutaneously to the subject. In some embodiments, the modified lymphocytes present in the cell formulation and optionally introduced into the subject may be allogeneic lymphocytes. In such embodiments, the lymphocytes are derived from different individuals, and the lymphocytes derived from the subject are unmodified. In some embodiments, no blood is taken from the subject to recover the lymphocytes.

[0116] In exemplary embodiments, neutrophils are present in cell formulations for subcutaneous delivery of modified T cells and / or NK cells, in concentrations that are too high for intravenous delivery when considering the safety of the subject to whom the cell formulation is administered. Intravenous injection or delivery of neutrophils may result in lung injury, for example, as a consequence of transfusion-associated acute lung injury (TRALI) and / or acute respiratory distress syndrome (ARDS), although this is not limited to theory and will be discussed elsewhere in this specification. For example, this situation may occur if the method for producing modified lymphocytes does not involve a PBMC enrichment step before the cell formulation containing the modified lymphocytes is prepared and before the solution is optionally delivered subcutaneously to the subject. Therefore, in some embodiments, neutrophils are present in the cell formulation, for example, at the time of an 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 method for modification herein are present in the cell preparation, including at the time of an 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 are neutrophils, including at the time of an optional delivery step. In some embodiments, including at the time of an optional delivery step, 5%, 10%, 15%, 20%, 25%, 30%, or 40% of the lower limit of the range of cells present in the cell product are neutrophils, and 30%, 40%, 50%, 60%, 70%, or 75% of the upper limit of the range of cells present in the cell product are neutrophils, for example, including at the time of an optional delivery step, 5% to 50%, 20% to 50%, 30% to 75%, or 50% to 75% of the cells present in the cell product are neutrophils.

[0117] 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 modification method herein are present in the cell preparation, including at the time of an 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 modification method herein are present in the resulting cell preparation, including at the time of an optional delivery step. In some embodiments, the cell preparation may include 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% of the modified T and NK cells in the cell preparation are genetically modified.

[0118] The volume of the cell preparation or other solution administered varies depending on the route of administration, as provided elsewhere herein. Cell preparations administered by subcutaneous or intramuscular injection typically have a smaller volume than those delivered by infusion. In some embodiments, and in exemplary embodiments, the volume of the cell preparation or other solution containing a suspension of modified, and genetically modified, lymphocytes, 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 cell preparation or other solution containing the modified lymphocyte suspension may be from 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 at the lower end of the range to 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, or 50 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 75 mL, 100 mL, 125 mL, 250 mL, 500 mL, or 1000 mL. Therefore, in non-limiting examples, the volume may be 0.2 mL to 10 mL, 0.5 mL to 10 mL, 0.5 mL to 2 mL, 1 mL to 250 mL, 1 mL to 100 mL, 10 mL to 100 mL, or 1 mL to 10 mL. In certain exemplary embodiments, a cell preparation containing modified lymphocytes in a delivery solution is administered subcutaneously or intramuscularly in volumes of less than 10 mL, 1 mL to 25 mL, and in exemplary embodiments, 1 mL to 3 mL, 1 mL to 5 mL, or 1 mL to 10 mL. In exemplary embodiments, the volume of the solution containing modified lymphocytes may be 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 to 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, a 70kg object is 7.0 × 10 7 By subcutaneously administering 1 mL of a T cell delivery formulation containing 1.0 × 10 cells / mL, 6The T cells are administered at a dose of 1 / kg. In some embodiments, the solution 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 where the lymphocytes are filtered after being particularly modified and / or transduction is particularly carried out on the filter, the delivery solution can be used to resuspend and / or elute the cells from the filter in the volume which may be that provided above. Thus, in some embodiments, the delivery solution provided herein is an elution solution.

[0119] In some embodiments, modified, and in exemplary embodiments, genetically modified lymphocytes are introduced or reintroduced into a target by intratumoral or intramuscular administration, or, in exemplary embodiments, by subcutaneous administration using a cell preparation present in a subcutaneous delivery device such as a sterile syringe adapted for subcutaneous delivery of the solution. In some embodiments, a subcutaneous delivery device is used that holds a solution (e.g., the cell preparation as herein) and administers the solution (e.g., the cell preparation) subcutaneously from the liquid-holding portion of the device, having an open end or a retractable end, which in exemplary embodiments is the open end of a needle. Such a subcutaneous delivery device is effective for subcutaneous delivery and, in exemplary embodiments, adapted for subcutaneous delivery or effective for subcutaneous injection or adapted for subcutaneous injection. Not limited examples of subcutaneous delivery devices adapted for subcutaneous delivery of a solution include, for example, indwelling subcutaneous catheters such as Insuflon® (Becton Dickinson), and subcutaneous catheters such as needleless closed indwelling subcutaneous catheter systems with wings, such as Saf-T-Intima® (Becton Dickinson). In some embodiments, the delivery device may include a pump, e.g., an infusion pump or a peristaltic pump. In some embodiments, the cell formulation is fluidly connected to one of the needles disclosed herein, e.g., a needle suitable for subcutaneous delivery, effective for subcutaneous delivery, adapted for subcutaneous delivery, or adapted to or effective for subcutaneous delivery. In exemplary embodiments, the needle may have a gauge of 26 to 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 in a housing, and may include a needle guard. An example of such a pen is a pen used to deliver sumatriptan.In some embodiments, the cell preparation is present in a subcutaneous delivery device, e.g., a syringe, having a needle positioned with its open end in the subcutaneous tissue of the subject (i.e., the subject receiving the subcutaneous injection is the source of the autologous cells being injected), and in some embodiments, the modified T cells and / or NK cells are present in the modified cells present in the syringe, penetrating the skin of the subject (i.e., the subject receiving the subcutaneous injection is the source of the autologous cells being injected), and in some embodiments, the subcutaneous delivery device, e.g., a syringe, having a needle positioned with its open end in the subcutaneous tissue of the subject. 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, for example, a 16-gauge needle may be used. Intramuscular, and in exemplary embodiments, delivery devices such as syringes suitable for subcutaneous delivery are any delivery device (e.g., a syringe) that can be successfully used for intramuscular or subcutaneous delivery, and include delivery devices (e.g., syringes) that are effective and adapted for intramuscular or subcutaneous delivery, as well as general-purpose syringes, and in at least some embodiments, syringes specifically designed for other purposes that can be successfully used for intramuscular or subcutaneous delivery. As is known, with respect to subcutaneous injection, in exemplary embodiments using a syringe, the needle is inserted through the skin at an angle of 45 to 90 degrees. Thus, some embodiments involve injecting cell preparations subcutaneously at an angle of 45 to 90 degrees to the skin, as well as cell preparations contained within a syringe or other subcutaneous delivery device having a needle at an angle of 45 to 90 degrees to the skin of target. A syringe that is effective for intramuscular, and in exemplary embodiments, subcutaneous delivery, or effective for intramuscular or subcutaneous injection, is typically a syringe having parameters that are effective for intramuscular or subcutaneous delivery; for example, a needle with a gauge of 20 to 22 and a length of 1 inch to 1.5 inches is typically effective for intramuscular delivery, and a needle with a gauge of 26 to 30 and a length of 0.5 inches to 0.625 inches is typically effective for subcutaneous delivery. A syringe adapted for subcutaneous delivery, or adapted to be used for subcutaneous injection, is any syringe specifically made for subcutaneous delivery.One such syringe adapted for subcutaneous delivery uses a core annular flow that enables subcutaneous delivery of highly concentrated biological agents 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). Yet 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 up to 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 length of the needle is 1 inch or 0.5 inches or less. In exemplary embodiments, the needle is a 26, 26s, 27, 28, 29, or 30 gauge needle, and the length of the needle is 0.5 inches to 0.625 inches. In some embodiments, the needle may be a winged infusion set, also known as a winged needle or scalp venous needle. In some embodiments, introduction or reintroduction may be performed using a subcutaneous catheter.

[0120] While not limited by theory, in contrast to intravenous delivery in which the cells and other components of a cell therapy are rapidly dispersed, the subcutaneous and intramuscular delivery methods provided herein allow the cells and components of the cell therapy to remain in close proximity within the target for up to several days as a controlled release, for example in exemplary embodiments, while creating a local environment for cell activation and proliferation that maintains properties similar to those encountered by T and NK cells in lymphoid organs such as the spleen or lymph nodes. Absorption of large protein molecules greater than 20 kDa, such as antibodies, from a subcutaneous site is absorbed into the bloodstream via lymphatic vessels over 24–72 hours, whereas controlled release of T or NK cells from a local injection site using the subcutaneous and intramuscular methods provided herein is thought to involve both migration from the injection site after the initial proliferation phase before the modified cells are generally detected in circulation. In some embodiments, local injection controlled release results in genetically modified cells appearing in circulation 1–2 weeks later. In some embodiments, the cell therapy is adapted, or further adapted for, subcutaneous or intramuscular delivery to maintain a locally aggregated state of cells, enabling controlled release of cells into circulation. In some embodiments, the concentration of cells in cell formulations for subcutaneous or intramuscular delivery is typically higher than that for intravenous delivery. In some embodiments, the concentration of leukocytes in cell formulations for subcutaneous or intramuscular delivery is (approximately) 1.5 × 10⁻⁶ 8 cells / mL, (approx.) 5 × 10 8 cells / mL, (approx.) 1 × 10⁶ 9 Cells per mL ~ 1.2 × 10⁻⁶ 9 Larger than individual cells / mL.

[0121] In exemplary embodiments, cells, for example, a mixture of modified and unmodified lymphocytes as described herein, are formulated in a delivery solution, which as a result can be administered subcutaneously or intramuscularly, is effective for that purpose, and is adapted for that purpose. In fact, certain embodiments of the commercial containers and kits provided herein are containers of sterile subcutaneous and / or intramuscular delivery solutions, which in some embodiments are stored under refrigeration. Such delivery solutions can be administered subcutaneously or intramuscularly, and in exemplary embodiments, subcutaneously, is effective for that purpose, and in further exemplary embodiments, is adapted for that purpose. To achieve this, such delivery solutions and the resulting cell formulations typically have a pH and ionic composition that provides an environment in which the cells to be administered can survive, for example, for at least 1 hour, and typically for at least 4 hours, until administration. Such pH is typically pH 6.5–8.0 or 7.0–8.0 or 7.2–7.6 and can be maintained by a buffer such as phosphate buffer or bicarbonate present at a concentration effective to maintain the pH within the target range. The ionic composition of such formulations may include, for example, a saline composition containing salts, such as 0.8–1.0 or about 0.9 or 0.9 percent sodium chloride. In some embodiments, the delivery solution is or contains PBS. In some embodiments of the delivery solution and the resulting cell formulations described herein, Na + The concentration is 110 mM to 204 mM, and Cl - The concentration is 98 mM to 122 mM, and / or K + The concentration is 3 mM to 6 mM.

[0122] In exemplary embodiments, the delivery solution and the cell preparation containing it contain calcium and / or magnesium. The concentration of calcium may be, for example, 0.5 mM to 2 mM. The concentration of magnesium may be, for example, 0.5 mM to 2 mM. In some embodiments, the delivery solution does not contain calcium and magnesium.

[0123] In some embodiments, the delivery solution and cell formulation contain human serum albumin and / or heparin. In some embodiments, the delivery solution and cell formulation contain up to 5% HSA. In some embodiments, the delivery solution is PBS containing 2% HSA. In some embodiments, the delivery solution is DPBS containing 2% HSA. In some embodiments, the delivery solution is saline containing 30–100 U / mL, 40–100 U / mL, 30–60 U / mL, or 60–80 U / mL of heparin, and containing or not containing 0.5–5%, 1–5%, or 1–2.5% HSA. The herein considerations regarding the concentration of heparin in the reaction mixture embodiment apply equally to the delivery solution and cell formulation embodiment.

[0124] In some embodiments, the delivery solution is or comprises multiple electrolyte solutions suitable for injection into a subject. For example, the delivery solution may be or comprises a sterile, nonpyrogenic isotonic solution in a container such as a single-dose container. Such solutions in certain embodiments are suitable for or adapted for intravenous and subcutaneous and / or intramuscular administration. In some embodiments, the delivery solution may comprise a multianalyte solution for injection into a subject, each 100 mL containing 526 mg of sodium chloride, USP (NaCl), and 502 mg of sodium gluconate (C6H 11 The solution contains NaO7, 368 mg of sodium acetate trihydrate USP(C2H3NaO2·3H2O), 37 mg of potassium chloride USP(KCl), and 30 mg of magnesium chloride USP(MgCl2·6H2O) (pH adjusted to 7.4 (6.5-8.0)). In exemplary embodiments, the delivery solution does not contain an antimicrobial agent. The pH is adjusted with sodium hydroxide. As an example, the polyelectrolyte injection may be PLASMA-LYTE A Injection pH7.4, which is available from various commercial suppliers.

[0125] In exemplary embodiments, the cell formulation is never frozen. In exemplary embodiments, the cell formulation contains less than 7%, 6%, 5%, 4%, 3%, 2%, or 1% DMSO(v / v). In further exemplary embodiments, the cell formulation does not contain DMSO.

[0126] Homogeneous single-cell suspensions are ideal for intravenous delivery but are 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, and herein, the delivery solution used to prepare such depot cell formulations includes auxiliary components that provide depot properties. In some embodiments, cells may aggregate in the formulation, for example, 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, when cells, for example modified lymphocytes provided herein, are formulated in a delivery solution containing an agglutinant to produce the formulation. In some embodiments, at least 10%, 20%, 25%, 50%, 75%, 90%, 95%, or 99% of the cells in the cell formulation provided herein are aggregated. Such aggregation may be determined, for example, by using a microscopic count of cells associated with at least one other cell in individual cell pairs, or by counting the average number of cells associated in the formulation. In some embodiments, cell formulations are designed for controlled or delayed release, accompanied by tissue expansion to accommodate cell growth.

[0127] 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.

[0128] Therefore, in some embodiments, the delivery solution or cell formulation 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 formulation. One class of promers that may be used to produce gel-forming biomaterials and may be included in the delivery solutions and cell formulations provided herein consists of poly(ethylene glycol) (PEG) and its copolymers and aliphatic polyesters such as poly(lactic acid) (PLA), poly(D,L-lactic acid-coglycolic acid) (PLGA), poly(ε-caprolactone) (PCL), and polyphosphazene. Other polymers that may be used include thermosensitive triblock copolymers based on poly(N-(2-hydroxypropyl methacrylamide lactate) and poly(ethylene glycol)(p(HPMAm-lac)-PEG) that can spontaneously self-assemble in a physiological environment (Vermonden et al. 2006, Langmuir 22:10180-10184).

[0129] In some embodiments, the hydrogels used in the delivery solutions or cell formulations herein contain hyaluronic acid (HA). Such HA may have carboxylic acid groups that can react with proteins, peptides, polymers, and amine groups on linkers, such as those found on modified lymphocytes provided herein, preferably in the presence of N-hydroxysuccinimide, after being modified with 1-ethyl-3-(3-dimethylaminopropyl)-1-carbodimide hydrochloride. Antibodies, cytokines, and peptides can be chemically conjugated to HA using such methods to produce hydrogels for co-injection as cell emulsions in some embodiments of the cell formulations provided herein. Furthermore, in some embodiments, the HA in the delivery solutions and cell formulations is a polymer (e.g., Healon) and / or is crosslinked via its -OH groups with agents such as glutaraldehyde (e.g., Restylane (Abbive / Allergan)), for example, lightly crosslinked, to reduce local catabolism 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 may 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 hydrogel substrates used in the cell formulations herein, may be configured or adapted to allow cell migration through the substrate. The degree of hydrogel substitution and the concentration at crosslinking affect the porous swelling rate and Young's modulus (or stiffness). For example, the initial 1% substitution of HA with 1 mg / mL tyrosine, when subsequently crosslinked in the presence of a peroxide, results in a hydrogel with higher porosity and lower stiffness than a 3% substitution and a 5 mg / mL solution.Reducing the shear modulus is desirable in some situations to reduce the shear force during injection and to ensure sufficient porosity and half-life for cells to proliferate subcutaneously into the matrix over a period of 1-2 weeks. In some embodiments, the shear modulus is approximately 2.5 kPa, 3 kPa, 3.5 kPa, or 4 kPa.

[0130] In some embodiments, the delivery solution or cell preparation contains cytokines such as IL-2, IL-7, IL-15, and IL-21. In some embodiments, the cell preparation contains antibodies or polypeptides that can bind to CD3, CD28, OX40, 4-1BB, ICOS, CD9, CD53, CD63, CD81, and / or CD82. An EDC-NHS reaction may be used to link such proteins to HA or via other intermediates as described 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 before injection. In other embodiments, these cytokines, antibodies, or polypeptides are in solution.

[0131] The proliferation and survival of genetically modified T cells and / or NK cells expressing CARs are promoted by CAR-mediated signaling when they bind to their homologous antigens under appropriate conditions. In some embodiments, antigens can be added to or co-administered with modified and / or genetically modified T cells and / or NK cells. In some embodiments, antigens may be soluble. In some embodiments, antigens can be immobilized on the surface of an artificial substrate such as a hydrogel. In exemplary embodiments, antigens can be expressed on the surface of cells, such that the cells are target cells. In some embodiments, such target cells are abundant in whole blood and naturally present in cell preparations without the need for addition. For example, B cells are present in whole blood, in isolated TNCs and isolated PBMCs and naturally present in cell preparations and can act as target cells for T cells and / or NK cells expressing CARs directed to CD19 or CD22 (both expressed on B cells as an example, not limited to) In some embodiments, target cells may be isolated or concentrated from a subject, such as from a tumor sample, using methods known in the art. In other embodiments, cells from a subject are modified to express a suitable antigen. In exemplary embodiments, the antigen expressed on target cells may comprise all or part of the antigen-containing protein. In further exemplary embodiments, the antigen expressed on target cells may comprise all or part of the extracellular domain of the antigen-containing protein. In some embodiments, the antigen expressed on target cells may be fused with a transmembrane domain that fixes it to the cell surface. Any of the transmembrane domains disclosed elsewhere in this specification may be used. In some embodiments, the antigen expressed on target cells may be fused with a stalk domain. Any of the stalk domains disclosed elsewhere in this specification may be used. In exemplary embodiments, the antigen may be fused with a CD8 stalk and a transmembrane domain (SEQ ID NO: 24).

[0132] 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 herein to as “artificial antigen-presenting cells” or “aAPCs,” and cells in a separate second cell mixture from the same subject are modified to express an antigen-binding CAR. In some embodiments, if the modified cells, modified with a vector encoding a target antigen, are T cells, the cells may be referred herein to as “T-APCs.” Such modified T-APCs may be produced using a method provided herein, wherein the reaction mixture for modification (e.g., transduction) includes a T cell-binding polypeptide such as a polypeptide directed to CD3. In further exemplary embodiments, the cell mixtures are whole blood, isolated TNCs, and isolated PBMCs. For example, the first cell mixture may 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 the second cell mixture may be modified with a recombinant nucleic acid vector encoding a CAR directed to HER2. The cells can then be formulated in a delivery solution or administered to the target by other means at different CAR effector cell-to-target cell ratios. In some embodiments, the effector-to-target ratio at formulation or administration is approximately 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In exemplary embodiments, the target cells are co-administered subcutaneously or intramuscularly with modified T and / or NK cells.

[0133] The proliferation and survival of genetically modified T cells and / or NK cells expressing CARs can also be promoted by CAR signaling initiated by crosslinking CARs through interactions other than those mediated by the ASTR of CARs that bind to their congener antigens. In some embodiments, small molecules or proteins can crosslink and activate CARs on the cell surface. In exemplary embodiments, antibodies can crosslink and activate CARs on the cell surface. In further exemplary embodiments, antibodies recognize epitopes in the extracellular domains of CARs, such as stalk or spacer domains. 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 the HisX6 tag (SEQ ID NO: 77). In some embodiments, the CAR may 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, such as scFv, bound to an epitope tag, also referred to herein as a feeder cell, on its surface. In some embodiments, scFv associates with the cell membrane via a GPI anchor. In exemplary embodiments, scFv associates with the cell membrane via a transmembrane domain. In further exemplary embodiments, a stalk or spacer separates the scFv from the transmembrane domain.In some embodiments, the same feeder cells, for example, feeder cells expressing anti-HisX6 scFv attached to the CD8a stalk and transmembrane domain, may be used together with cells expressing CARs that bind to different antigens but have an ASTR containing the HisX6 epitope tag in their stalk. These feeder cells, which may be used together with cells expressing different CARs containing a common epitope tag, are also referred to herein as universal feeder cells. In universal feeder cells, it is not necessary to generate different feeder cells expressing homologous antigens of CARs containing different ASTRs, provided that the CAR contains the epitope tag. The epitope tag on the CAR-expressing cell is cross-linked by the universal feeder cell and participates in the clustering and proliferation of the CAR. For example, anti-HisX6 universal cells may be used together with cells expressing a CAR that binds to Her2 and contains the HisX6 epitope tag, and may also be used together with cells expressing a CAR that binds to Axl and contains the HisX6 epitope tag. The combination of feeder cells and CARs can enable CAR-T proliferation before the cells engage their homologous antigen. Furthermore, if the ASTR of a CAR is restricted in the microenvironment, the use of antigen-binding feeder cells may allow it to proliferate outside of that restrictive environment.

[0134] In another embodiment, a cell preparation comprising aggregates of T cells and / or NK cells is provided herein, wherein the T cells and / or NK cells are modified with polynucleotides comprising one or more transcription units, each of which is operably linked to an active promoter in the T cells and / or NK cells, and one or more transcription units encode a first polypeptide comprising a chimeric antigen receptor (CAR) in a solution, in an exemplary embodiment, in a delivery solution, and further, the aggregate comprises at least 4, 5, 6, or 8 T cells and / or NK cells, the cell aggregate is at least 15 μM in its minimum dimension, and / or the cell aggregate is held by a coarse filter having a diameter of at least 15 μm or a coarse filter having a diameter of 15 μm to 60 μm.

[0135] Recombinant retrovirus particles For example, recombinant retroviral particles for modifying T cells and / or NK cells to produce genetically modified and / or transduced T cells and / or NK cells are disclosed in the methods and compositions provided herein. Recombinant retroviral particles themselves constitute an embodiment of the present invention. Typically, recombinant retroviral particles included in the embodiments provided herein are non-replicating, meaning that they cannot replicate once they leave the packaging cell. In practice, unless otherwise indicated herein, retroviral particles are non-replicating, and if such retroviral particles contain nucleic acids in their genome that are not native to the retrovirus, they are “recombinant retroviral particles.” In exemplary embodiments, the recombinant retroviral particles are lentiviral particles.

[0136] In some embodiments, non-replicating recombinant retroviral particles for use in transduction of cells, typically lymphocytes, and, in exemplary embodiments, T cells and / or NK cells are provided herein. The non-replicating recombinant retroviral particles may include any of the pseudotyping elements discussed elsewhere herein. In some embodiments, the non-replicating recombinant retroviral particles may include any of the activating elements discussed elsewhere herein. In one embodiment, non-replicating recombinant retroviral particles comprising polynucleotides including: one or more transcription units operably linked to an active promoter in AT cells and / or NK cells, encoding a chimeric antigen receptor (CAR); and a pseudotyping element and a T cell activating element on its surface, wherein the T cell activating element is not encoded by the polynucleotide in the non-replicating recombinant retroviral particle. In some embodiments, the T cell activating element may be any of the activating elements discussed elsewhere herein. In exemplary embodiments, the T cell activating element may be anti-CD3 scFvFc. In another embodiment, a non-replicating recombinant retroviral particle is provided herein, comprising a polypeptide containing one or more transcription units operably linked to an active promoter in T cells and / or NK cells, wherein the one or more transcription units encode a first polypeptide containing a chimeric antigen receptor (CAR) and a second polypeptide containing a lymphoproliferative element. In some embodiments, the lymphoproliferative element may be a chimeric lymphoproliferative element. In exemplary embodiments, the lymphoproliferative element does not contain IL-7 linked to an IL-7 receptor alpha chain or a fragment thereof. In some embodiments, the lymphoproliferative element does not contain IL-15 linked to an IL-2 / IL-15 receptor beta chain.

[0137] In some embodiments, non-replicating recombinant retroviral particles comprising polypeptides are provided herein, each comprising one or more transcription units operably linked to an active promoter in T cells and / or NK cells, wherein the one or more transcription units encode a first polypeptide comprising a chimeric antigen receptor (CAR), and a second polypeptide comprising a chimeric lymphopregenerative element, e.g., a constitutively active chimeric lymphopregenerative element. In exemplary embodiments, the chimeric lymphopregenerative element does not contain cytokines linked to its homologous receptor or to fragments of its homologous receptor.

[0138] In some embodiments, recombinant retroviral particles are provided herein, comprising: (i) a pseudotyping element capable of binding to T cells and / or NK cells and promoting membrane fusion of recombinant retroviral particles to them; (ii) a polynucleotide having one or more transcription units operably linked to a promoter active in T cells and / or NK cells (one or more transcription units encoding 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 lymphoproliferative element, the expression of the first engineered signaling polypeptide and / or the second engineered signaling polypeptide being regulated by an in vivo regulatory element); and (iii) an activating element on its surface capable of binding to T cells and / or NK cells and an activating element not encoded by the polynucleotide in the recombinant retroviral particle. In some embodiments, the promoter active in T cells and / or NK cells is inactive in the packaging cell line or is only inactive in the packaging cell line in an inducible manner. In any of the embodiments disclosed herein, either the first or second engineered signaling polypeptide may have a chimeric antigen receptor, and the other engineered signaling polypeptide may have at least one lymphoproliferative element.

[0139] In some embodiments, non-replicating recombinant retroviral particles comprising polynucleotides encoding self-propelled CARs are provided herein. Details relating to embodiments of such non-replicating recombinant retroviral particles, as well as compositions and methods comprising self-propelled CARs, are disclosed herein in more detail, for example, in the sections on self-propelled CAR methods and compositions, and on exemplary embodiments.

[0140] Various elements and combinations of elements contained in non-replicating recombinant retroviral particles, such as pseudotyping elements, activating elements, and membrane-bound cytokines, as well as nucleic acid sequences contained in the genome of non-replicating recombinant retroviral particles, such as, but not limited to, nucleic acids encoding CARs, nucleic acids encoding lymphoproliferative elements, nucleic acids encoding regulatory elements such as riboswitches, promoters, particularly promoters that are constitutively active or inducible in T cells, and nucleic acids encoding inhibitory RNA molecules, are provided throughout this disclosure. Furthermore, various embodiments provided herein, such as methods for producing recombinant retroviral particles, methods for carrying out adoptive cell therapy, and methods for transducing T cells, produce and / or include non-replicating recombinant retroviral particles. The non-replicating recombinant retrovirus itself produced and / or included in such a manner forms a separate embodiment of the present invention as a non-replicating recombinant retroviral particle composition, which may be in isolated form. Such a composition may be in a dry (e.g., lyophilized) form, or may be a suitable solution or medium known in the art for the storage and use of retroviral particles.

[0141] Accordingly, in a non-limiting example, in another embodiment, a non-replicating recombinant retroviral particle is provided herein having a polynucleotide in its genome having one or more nucleic acid sequences operably linked to an active promoter in T cells and / or NK cells, which in some cases comprises a first nucleic acid sequence encoding one or more (e.g., two or more) inhibitory RNA molecules targeting one or more RNA targets, and a second nucleic acid sequence encoding a chimeric antigen receptor, or CAR, as described herein. In other embodiments, there is a third nucleic acid sequence that is not an inhibitory RNA molecule and encoding at least one lymphoproliferative element as described herein. In certain embodiments, the polynucleotide includes one or more riboswitches, as presented 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 a construct, the expression of one or more inhibitory RNAs, CARs, and / or one or more lymphoproliferative elements that are not inhibitory RNAs is controlled by the riboswitches. In some embodiments, 2 to 10 inhibitory RNA molecules are encoded by the first nucleic acid sequence. In further embodiments, 2 to 6 inhibitory RNA molecules are encoded by the first nucleic acid sequence. In exemplary embodiments, 4 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 certain embodiments, the intron comprises all or part 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 an active promoter in T cells and / or NK cells. In some embodiments, the intron is EF1-α intron A.

[0142] Embodiments of recombinant retroviral particles described herein include those in which the retroviral particle comprises a genome containing one or more nucleic acids encoding one or more inhibitory RNA molecules. Various alternative embodiments of such nucleic acids encoding inhibitory RNA molecules that may be contained in the genome of a retroviral particle (including combinations of such nucleic acids with other nucleic acids encoding lymphoproliferative elements other than CARs or inhibitory RNA molecules) are, for example, included in the section on inhibitory RNA provided herein, as well as in various other paragraphs combining these embodiments. Furthermore, various alternatives of such non-replicating recombinant retroviruses may be identified by exemplary nucleic acids disclosed within embodiments of packaging cell lines 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 of 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 that focus on inhibitory RNA molecules and the nucleic acids encoding these molecules. Furthermore, various embodiments of specific inhibitory RNA molecules provided in other sections of this specification may be used in the recombinant retroviral particles of this disclosure.

[0143] The necessary elements of recombinant retroviral vectors, such as lentiviral vectors, are known in the art. These elements are provided in the sections on packaging cell lines and examples and are included in the details for producing non-replicating recombinant retroviral particles as shown in WO2019 / 055946. For example, a lentiviral particle typically includes packaging elements REV, GAG, and POL that can be delivered to a packaging cell line via one or more packaging plasmids, pseudotyping elements that can be delivered to a packaging cell line via pseudotyping plasmids (various examples thereof are provided herein), and a genome produced by polynucleotides delivered to a host cell via a transplasmid. These polynucleotides typically include a 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 transplasmid may be self-inactivated, for example, by removing the U3 region of the 3'LTR. In some non-limited embodiments, Vpu, such as a Vpu-containing polypeptide (sometimes referred to herein as a “Vpu polypeptide”), including but not limited to Src-FLAG-Vpu, is packaged within retroviral particles with respect to any aspects and embodiments of any composition or method provided herein that comprises retroviral particles. In some non-limited embodiments, Vpx, such as Src-FLAG-Vpx, is packaged within retroviral particles. Although not limited by theory, during T cell transduction, Vpx enters the cell cytosol, promotes the degradation of SAMHD1, and increases the pool of cytoplasmic dNTPs available for reverse transcription. In some non-limited embodiments, Vpu and Vpx are packaged within retroviral particles with respect to any aspects and embodiments of any composition or method provided herein that comprises retroviral particles.

[0144] Retroviral particles (e.g., lentiviral particles) included in various embodiments of the present invention are non-replicating, particularly for safety reasons in exemplary embodiments, especially embodiments involving the introduction of transduced cells with such retroviral particles. When cells are transduced using non-replicating retroviral particles, no retroviral particles are produced from the transduced cells. Modifications to retroviral genomes to ensure that retroviral particles containing genomes are non-replicating are known in the art. However, it will be understood that in some embodiments of any of the embodiments provided herein, reproducible recombinant retroviral particles may be used.

[0145] Those skilled in the art will recognize that the functional elements considered herein can be delivered to packaging cells and / or T cells using different types of vectors, such as expression vectors. Exemplary embodiments of the present invention utilize retroviral vectors, and in some particularly exemplary embodiments, lentiviral vectors. Specific embodiments of this specification can be achieved using other suitable expression vectors. Such expression vectors include viral vectors (e.g., vaccinia virus-based viral vectors, poliovirus, adenovirus (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)); and adeno-associated viruses (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.See PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; or retroviral vectors (e.g., mouse leukemia virus, splenic necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary gland tumor virus), such as gamma retrovirus; or human immunodeficiency virus (e.g., see Miyoshi et al., PNAS 94:10319 23, 1997, Takahashi et al., J Virol 73:7812 7816, 1999), but are not limited to these.

[0146] As disclosed herein, non-replicating recombinant retroviral particles are a common tool for gene delivery (Miller, Nature (1992) 357:455-460). Due to their ability to deliver unreconstructed nucleic acid sequences to a wide range of rodent, primate, and human somatic cells, non-replicating recombinant retroviral particles are highly suitable for introducing genes into cells. In some embodiments, non-replicating recombinant retroviral particles may originate from the genera alpha-retrovirus, beta-retrovirus, gamma-retrovirus, delta-retrovirus, epsilon-retrovirus, lentivirus, or supmavirus. Many retroviruses exist that are suitable for use in the methods disclosed herein. For example, mouse 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's mouse leukemia virus (Mo-MLV), FBR mouse osteosarcoma virus (FBR MSV), Moloney's mouse sarcoma virus (Mo-MSV), Abelson's mouse leukemia virus (A-MLV), avian myelocytosis virus-29 (MC29), and avian erythroblastosis virus (AEV) may 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 structures of some retroviruses can be found in the relevant field. For example, details about HIV can be found in the NCBI Genbank (i.e., genome accession number AF033819).

[0147] In exemplary embodiments, non-replicating recombinant retroviral particles may originate from the lentivirus genus. In some embodiments, non-replicating recombinant retroviral particles may originate from HIV, SIV, or FIV. In further exemplary embodiments, non-replicating recombinant retroviral particles may originate from the lentivirus genus human immunodeficiency virus (HIV). Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural functions. Due to their higher complexity, lentiviruses can modulate their life cycle, such as during the process of latent infection. A typical lentivirus is human immunodeficiency virus (HIV), the virulence factor of AIDS. In vivo, HIV can infect cells that rarely divide and eventually differentiate, such as lymphocytes and macrophages.

[0148] In exemplary embodiments, the non-replicating recombinant retroviral particles provided herein contain a Vpx polypeptide.

[0149] In some embodiments, the non-replicating recombinant retroviral particles provided herein include and / or contain the Vpu polypeptide.

[0150] In exemplary embodiments, the retroviral particle is a lentiviral particle. Such a retroviral particle typically contains a retroviral genome within a capsid located within the viral envelope.

[0151] In some embodiments, DNA-containing viral particles are used instead of recombinant retroviral particles. Such viral particles may be adenoviruses, adeno-associated viruses, herpesviruses, cytomegaloviruses, poxviruses, avipoxviruses, influenza viruses, varicella stomatitis viruses (VSV), or Sindbisviruses. 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 may contain functional units to induce the integration of all or part of the DNA genome of the viral particle into the genome of T cells transduced with such a virus.

[0152] In some embodiments, the polynucleotide regions encoding HIV RRE and HIV Rev may be replaced with polynucleotide regions encoding an N-terminal RGG box RNA-binding motif and ICP27. In some embodiments, the polynucleotide region encoding HIV Rev may be replaced with one or more polynucleotide regions encoding adenovirus E1B 55-kDa and E4 Orf6.

[0153] In certain embodiments, non-replicating recombinant retroviral particles may include nucleic acids encoding self-propelled CARs, as disclosed elsewhere herein. As an example not limited to this, such embodiments are retroviral particles whose genome comprises one or more first transcription units operably ligated to an inducible promoter in at least one of T cells or NK cells, and one or more second transcription units operably ligated to a constitutive T cell or NK cell promoter, wherein the number of nucleotides between the 5' end of one or more first transcription units and the 5' end of one or more second transcription units is less than the number of nucleotides between the 3' end of one or more first transcription units and the 3' end of one or more second transcription units. a. At least one of the one or more first transcription units encodes a lymphoproliferative element, b. At least one of the one or more second transcription units encodes a first chimeric antigen receptor (CAR), the CAR comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain.

[0154] In some embodiments, recombinant retroviral particles lacking replication ability may further present T cell activating elements.

[0155] While not limited by theory, transduced T cells that come into contact with these non-replicating recombinant retroviral particles containing nucleic acids encoding self-driven CARs can receive initial transcription enhancement from CAR-stimulated promoters, as T cell activators can stimulate the induction signal of CAR-stimulated promoters. Binding of T cell activators may induce calcium ion influx, leading to dephosphorylation of NFAT and subsequent nuclear translocation and binding to NFAT-responsive promoters. Lymphoproliferative elements transcribed and translated from these CAR-stimulated promoters may result in an initial increase in proliferation in these cells. In exemplary embodiments, the T cell activator may be a membrane-bound anti-CD3 antibody, which may be GPI-conjugated or otherwise presented on the virus. In some embodiments, a membrane-bound anti-CD3 antibody may be fused to a viral envelope protein such as MuLV or VSV-G.

[0156] In some embodiments, the isolated, non-replicating retroviral particles are in a large batch contained in a large container. Such a large batch is, for example, 10 6 ~10 8 The titer in TU / mL, and 1 × 10⁻⁶ 10 TU~1×10 13 TU, 1×10 11 TU~1×10 13 TU, 1×10 12 TU~1×10 13 TU, 1×1010 TU~5×10 12 TU, or 1 × 10 11 TU~5×10 12 The total batch size of TU can be such that the retroviral particles relating to any aspect or embodiment provided herein are substantially pure, as will be discussed in more detail herein.

[0157] Retrovirus genome size In the methods and compositions provided herein, recombinant retroviral genomes, and in exemplary examples not limited to lentiviral genomes, have 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 a cleavage or other deletion that retains 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 for generating two or more functional polypeptides from one fusion polypeptide and one promoter. Furthermore, some functions that are not required after the initial ex vivo transduction are not contained in the retroviral genome and rather reside on the surface of the non-replicating recombinant retroviral particle via the packaging cell membrane. These various strategies are used herein to maximize the functional elements packaged within the non-replicating recombinant retroviral particle.

[0158] In some embodiments, the packaged recombinant retroviral genome may be between 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, and 8,000 nucleotides at the lower end of the range and between 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, and 11,000 nucleotides at the upper end of the range. The packaged retroviral genome includes one or more polynucleotide regions encoding first and second operational 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. The functions that can be packaged, as discussed elsewhere in this specification, include retroviral sequences necessary for retroviral assembly and packaging, such as the retroviral rev, gag, and pol coding regions, as well as the 5'LTR and 3'LTR, or their active cleaved fragments, nucleic acid sequences encoding retroviral cis-active RNA packaging elements, and cPPT / CTS elements. Furthermore, in exemplary embodiments, the non-replicating recombinant retroviral particles herein may include one or more or all of the following, which in some embodiments are oriented in the opposite direction to the 5'-to-3' direction established by the retroviral 5'LTR and 3'LTR (as shown in WO2019 / 055946 as an example not limited to them): one or more polynucleotide regions encoding first and second manipulated signaling polypeptides (at least one of which includes at least one lymphoproliferative element); a second manipulated signaling polypeptide which may include a chimeric antigen receptor; regulatory elements such as miRNAs, riboswitches (which typically regulate the expression of the first and / or second manipulated signaling polypeptides); safety switch polypeptides, introns, promoters active in target cells such as T cells, 2A cleavage signals, and / or IRESs.

[0159] Kits and commercial products In one embodiment, a container, such as a commercial container or package, or a kit containing retroviral particles according to any embodiment or configuration of non-replicating recombinant retroviral particles provided herein is provided herein. In a non-limiting example, the retroviral particles may contain polynucleotides in their genome, 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 sequences of one or more nucleic acid sequences may encode lymphoproliferative elements and / or chimeric antigen receptors (CARs) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. In some embodiments, the nucleic acid sequences of one or more nucleic acid sequences may encode one, two, or more inhibitory RNA molecules targeting one or more RNA targets.

[0160] Containers containing recombinant retroviral particles in any aspect or embodiment, including commercial containers and kits, may be tubes, vials, plate wells, or other tanks for storing retroviral particles. In practice, several aspects provided herein include containers containing retroviral particles, such retroviral particles containing any nucleic acids or other components disclosed herein. Such containers in exemplary embodiments contain substantially pure, non-replicating recombinant retroviral particles, which may be referred to herein as substantially pure retroviral particles for the sake of brevity. Typically, preparations and / or containers of substantially pure retroviral particles are sterilized and negative for mycoplasma, retroviruses of the same type with replicating ability, and exogenous viruses according to standard protocols (see, for example, “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 retroviral particles are provided in the examples herein. For such methods, the viral supernatant was purified by a combination of deep filtration, TFF, benzonase treatment, diafiltration, and formulation. In certain exemplary embodiments, substantially pure retroviral particles, based on quality control test results, meet all of the following characteristics: a. Negative for Mycoplasma b. Endotoxin less than 25 EU / mL, and in certain further exemplary embodiments, less than 10 EU / mL. c. No retroviruses of the same type as the one intentionally detected (e.g., lentivirus) are present in the detected container. d. No foreign viruses were detected. e. Host cell DNA / virus TU less than 1 pg, and less than 0.3 pg / TU in certain further exemplary embodiments. f. Less than 100 residual plasmid copies / virus TU, and in certain further exemplary embodiments, less than 10 copies / virus TU of any plasmid used to produce recombinant retroviral particles. g. Less than 1 ng of HEK protein / TU, and in certain further exemplary embodiments, less than 50 pg of HEK protein / TU. h.P24 protein greater than 100 TU / ng, and in certain further exemplary embodiments, P24 protein greater than 10,000 TU / ng.

[0161] Retroviral particles are typically tested against shipping specifications, including some or all of those provided above, before being shipped to the customer. The titer of each particle may be defined based on the measurement of p24 viral capsid protein by ELISA, viral RNA genome copy by q-RT PCR, and reverse transcriptase activity by qPCR-based product-enhanced RT (PERT) assay, but all can be converted to infectious titer by measuring the transduction units (TUs) of functional gene transfection in bioassays.

[0162] Determination of the infectivity titer of purified bulk retrovirus material and final product by bioassay and qPCR is an exemplary analytical test method for determining the infectivity titer of retroviruses. An indicator cell bank (e.g., F1XT) may be grown, for example, in serum-free medium, seeded at 150,000 cells per well, and subsequently exposed to serial dilutions of retrovirus products. Dilutions of purified retrovirus particles are performed on indicator cells, for example, at 1:200 to 1:1,600. A reference standard virus may be added for system compatibility. After incubation with retrovirus for 4 days, cells are harvested and DNA is extracted and purified. For example, standard curves of 100 to 10,000,000 copies / well of human genome and intrinsic retrovirus genome sequence plasmid pDNA amplicons are used, followed by the addition of genomic DNA from cell samples exposed to retrovirus particles. For each PCR reaction, the Cq values ​​of both the retroviral amplicon and the endogenous control, such as hRNAseP, are extrapolated back to the copy number per reaction. From these values, the incorporated genome copy number is calculated. In some cases, since indicator cells such as 293T are characterized as triploid, a single copy gene with 3 copies per cell should be used for the calculation. The transduction units (TU) per mL of retroviral particles may be determined using the initial viable cell count per well, the volume of retrovirus added to the cells, and the genome copy number ratio.

[0163] Titer testing may include titer testing against shipping specifications using purity and specific activity. For example, shipping testing of the titer of the final product may compare the number of transduction units (TUs) with viral particle count (e.g., by performing p24 capsid protein ELISA using a p24 cutoff of at least 100, 1,000, 2,000, or 2,500 TU / ng, for example, by performing ELISA against lentiviral viral proteins), and CAR functionality by measuring interferon-gamma release by reporter cell lines exposed to genetically modified cells.

[0164] In any embodiment of the kits or isolated recombinant retroviral particles of the Specified Spectrum (including containers of such retroviral particles), sufficient recombinant retroviral particles are present in the container to achieve an MOI (number of transduction units, or TU applied per cell) in a reaction mixture prepared using retroviral particles 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 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 transduction units of the viral particles provided in the kit should allow for the use of MOIs that prevent the production of too many implements in individual cells, on average, fewer than 3 lentigenome copies per cell genome, and more preferably 1 copy per cell. For embodiments of the kit and isolated retroviral particles, such an MOI should be 1 × 10⁻⁶. 6 Assuming a target cell count of 1 × 10¹¹ / mL, for example in whole blood, 6 Assuming a blood concentration of 1 PBMC / mL, the reaction mixture can be based on 1, 2.5, 5, 10, 20, 25, 50, 100, 250, 500, or 1,000 mL. Therefore, the container for retrovirus particles can be 1 × 10⁶ 5 ~1 × 10 9 , 1 x 10 5 ~1 × 10 8 , 1 x 10 5 ~5×10 7 , 1 x 10 5 ~1 × 10 7 , 1 x 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 x 10 7 ~5×10 7 , 1 x 10 6 ~1 × 10 7 , and 1 × 10 6 ~5×10 6 Includes TU. In a particular exemplary embodiment, the container is 1 × 10 7 ~1 × 10 9 , 5×10 6 ~1 × 10 8 , 1 x 10 6 ~5×10 7 , 1 x 10 6 ~5×10 6 , or 5×10 7 ~1 × 10 8 The method may contain retroviral transduction units. While not limited by theory, the number of such particles supports 1 to 100 mL of blood with an MOI of 1 to 10. In some exemplary embodiments as shown herein, as little as 10 mL, 5 mL, 3 mL, or even 2.5 mL of blood may be processed for T cell and / or NK cell modification, as well as for subcutaneous and / or intramuscular administration methods provided herein by option. Therefore, an advantage of this method is that, in some exemplary embodiments, it requires far fewer retroviral particle transduction units than conventional methods involving nucleic acids encoding CARs, such as CAR-T methods.

[0165] Each container containing retrovirus particles may contain, for example, volumes of 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 mL to 5 mL, or 0.25 mL to 2 mL.

[0166] 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 regulatory bodies), or products of retroviral manufacturing steps carried out using a GMP system. Such retroviral particles are typically produced using Good Manufacturing Practices (GMP) of the U.S. FDA (i.e., U.S. GMP or U.S. cGMP), EMA (i.e., EMA GMP or EMA cGMP), or the National Medical Products Administration (NMPA) of China (i.e., the Chinese FDA) (i.e., NMPA GMP or NMPA cGMP), for example, using a GMP quality system and GMP procedure control. These products are typically produced in facilities that meet GMP or cGMP requirements. Such products are typically manufactured under a strict quality control system based on GMP or cGMP regulations. GMP-grade retroviral particles are typically sterile. This can be achieved, for example, by filtering retroviral particles, such as 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 manufacturing control test specifications for titer, quality, and safety.

[0167] In some embodiments, a solution containing retroviral particles in a container does not contain detectable bovine proteins and may be referred to as “bovine-free.” For example, bovine proteins such as bovine serum proteins are not used to culture packaging cells during retroviral production, so such a solution of retroviral particles can be bovine-free. In some embodiments, the solution of retroviral particles is GMP grade and bovine-free. Substantially pure nucleic acid solutions are typically bovine-free and are prepared in bovine-free broth.

[0168] In some embodiments, kits for modifying NK cells and / or T cells in exemplary embodiments are provided herein. Such a kit in a particular embodiment comprises one or more containers containing polynucleotides, typically substantially pure polynucleotides, including one or more first transcription units (one or more first transcription units encoding a first chimeric antigen receptor (CAR) (sometimes referred to as the first CAR)) operably linked to an active promoter in T cells and / or NK cells, and one or more containers of accompanying components (also referred herein as accompanying kit components). The polynucleotides (e.g., retroviral particles) can be cryopreserved, for example, at -70°C or below (e.g., -80°C).

[0169] In exemplary embodiments, the polynucleotide encoding the CAR is located in the genome of a retroviral particle, typically a substantially pure retroviral particle, according to any embodiment and configuration of the non-replicating recombinant retroviral particle provided herein. In exemplary embodiments, the non-replicating recombinant retroviral particle in the kit comprises a polynucleotide comprising one or more transcription units operably linked to an active promoter in T cells and / or NK cells, wherein one or more first transcription units encode a first polypeptide comprising a first chimeric antigen receptor (CAR), according to any embodiment provided herein, and optionally encode a second polypeptide comprising a lymphoproliferative element.

[0170] The included kit components may include one or more of the following: a. One or more containers containing a delivery solution suitable for subcutaneous and / or intramuscular administration as provided herein, in exemplary embodiments, that is effective therefor, and in further exemplary embodiments, that is adapted therefor. b. One or more containers of hyaluronidase provided herein, c. In exemplary embodiments, one or more blood bags, such as a blood collection bag, a blood processing buffer bag, a blood processing waste collection bag, and a blood processing cell sample collection bag, containing an anticoagulant in a bag or separate container, Suitable for subcutaneous or intramuscular delivery of dT cells and / or NK cells, in exemplary embodiments, one or more sterile syringes effective therefor, and in further exemplary embodiments, adapted therefor. e. T cell activating elements disclosed in detail herein, for example, in a solution in a container containing retroviral particles, or in a separate container, or anti-CD3 provided in exemplary embodiments, associate with the surface of retroviral particles that lack replication ability. f. One or more leukocyte removal filtration assemblies, In exemplary embodiments, one or more containers containing a solution or medium suitable for transduction of gT cells and / or NK cells, effective therefor, and in further exemplary embodiments, adapted therefor; In exemplary embodiments, one or more containers containing a solution or culture medium suitable for rinsing hT cells and / or NK cells, effective therefor, and / or in further exemplary embodiments, adapted therefor; i. One or more containers containing pH adjusting agents, One or more vessels containing polynucleotides comprising one or more second transcription units operably linked to an active promoter in jT cells and / or NK cells, typically substantially pure polynucleotides (e.g., found in recombinant retroviral particles according to any embodiment herein), wherein one or more second transcription units encode, in exemplary embodiments, polypeptides comprising second CARs that target different target epitopes found on the same target cancer cells (e.g., B cells), and in specific embodiments, polypeptides that target different antigens. k. One or more containers containing a homologous antigen of a first CAR and / or a second CAR encoded by a nucleic acid (e.g., a retroviral particle), and l. Instructions for use, either physically or digitally, associated with other kit components, for, for example, modifying T cells and / or NK cells, for subcutaneous or intramuscular delivery of modified T cells and / or NK cells to a target, and / or for treating tumor growth or cancer in a target.

[0171] In some embodiments, a blood bag may hold 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, or 500 mL of blood. In some embodiments, a blood 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, multiple blood bags may hold 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, a blood bag may hold blood in amounts 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, a blood bag may hold blood in amounts 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 may contain heparin. In other embodiments, the blood bag does not contain heparin.

[0172] In some embodiments, the kit may be a single-use pack / kit, while in other embodiments, the kit is a multi-use pack or multi-use kit for processing two or more blood samples from contact with a nucleic acid encoding a CAR via subcutaneous administration, optionally. Typically, the container of nucleic acid encoding a CAR within the kit (and optionally, in certain embodiments, a paired container of nucleic acid encoding a second CAR) is used for modifying T cells and / or NK cells and for a single implementation of the method for subcutaneous administration, optionally. The containers containing the CAR and optionally the nucleic acid encoding a second CAR are typically stored and shipped frozen. Accordingly, the kit may include a sufficient number of containers (e.g., vials) of CAR-encoding nucleic acids (and optionally, in certain embodiments, paired containers encoding a second CAR) for 1, 2, 3, 4, 5, 6, 10, 12, 20, 24, 50, and 100 executions of the method for modifying T cells and / or NK cells provided herein, and thus may include 1, 2, 3, 4, 5, 6, 10, 12, 20, 24, 50, and 100 containers (e.g., vials) of CAR-encoding nucleic acids (e.g., retroviral particles), and similarly, 1, 2, 3, 4, 5, 6, 10, 12, 20, 24, 50, and 100 packs, executions, doses, or X kits, respectively. Similarly, the accompanying components within the kit are provided for a similar number of executions of the method for modifying T cells and / or NK cells and, optionally, for subcutaneous administration, using the kit.

[0173] If one or more leukocyte-removing filtration assemblies are present in such a kit, they typically include one or more leukocyte-removing filters or leukocyte-removing filter sets (each typically housed in a filter enclosure), as illustrated by the exemplary assembly in Figure 2, adapted for use in a single-use closed blood processing system, as well as a plurality of connected sterile tubes connected to or adapted to be connected thereto, and a plurality of valves connected to or adapted to be connected thereto. Typically, one leukocyte-removing filtration assembly is present 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 leukocyte-removing filtration assemblies. In some embodiments, the kits herein comprise one or more containers containing nucleic acids and one or more leukocyte-removing filtration assemblies. Such kits may optionally be intended for use for administration to a subject via any route, such as infusion, or intramuscular delivery in an exemplary embodiment, and / or subcutaneous delivery in a further exemplary embodiment. Thus, such kits may optionally include other accompanying components intended for use in such routes of administration. One or more containers for subcutaneous or intramuscular delivery solutions, which are discussed in more detail herein, are typically sterile 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 the exemplary embodiment, there is one container of delivery solution for each container of nucleic acid encoding a CAR in the kit. Thus, the 20-pack kit in the exemplary embodiment includes 20 vials of nucleic acid encoding a CAR and 20 containers of sterile delivery solution.

[0174] In certain embodiments of the kit, embodiments are provided herein in which either or both of the containers containing the nucleic acid encoding a first CAR and, optionally, the nucleic acid encoding a second CAR are nucleic acids according to one of the embodiments of the self-driving CAR provided herein. In such embodiments, the accompanying 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 administration as provided herein, and b. Instructions for use, either physically or digitally, associated with other kit components, for example, for intravenous delivery targeting modified T cells and / or NK cells.

[0175] In certain embodiments, the use of non-replicating recombinant retroviral particles in the manufacture of a kit for modifying T cells or NK cells is provided herein, the use of the kit comprising ex vivo contact of T cells or NK cells with non-replicating recombinant retroviral particles, the non-replicating recombinant retroviral particles comprising surface pseudotyping elements and surface T cell activating elements, and such contact promotes transduction of T cells or NK cells by the non-replicating recombinant retroviral particles, thereby genetic modification, and in exemplary embodiments, the production of genetically modified T cells or NK cells.

[0176] In some embodiments, embodiments are provided herein that include the use of non-replicating recombinant retroviral particles in the manufacture of kits for modifying T cells or NK cells. Details relating to polynucleotides and non-replicating recombinant retroviral particles containing such polynucleotides are disclosed in more detail herein and in the sections on exemplary embodiments. In some embodiments, the T cells or NK cells may be derived from a subject. In some embodiments, the T cell activating element may be membrane-bound. In some embodiments, contact may be carried out for 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 upper end of the range, for example, 1 to 12 hours. Non-replicating recombinant retroviral particles for use in the manufacture of kits may include any of the embodiments, models, or sub-embodiments discussed elsewhere herein.

[0177] Furthermore, in another embodiment, a container, e.g., a commercial container or package, or a kit comprising isolated packaging cells, isolated from a packaging cell line in an exemplary embodiment, in any embodiment of the packaging cells and / or packaging cell lines provided herein, is provided herein. In some embodiments, the kit includes an additional container comprising additional reagents, such as buffers or reagents used in the methods provided herein. Furthermore, in certain embodiments, the use of any non-replicating recombinant retroviral particles provided herein in any embodiment for modifying T cells or NK cells, and in exemplary embodiments, for the manufacture of a kit for genetic modification is provided herein. Furthermore, in certain embodiments, the use of any packaging cells or packaging cell lines provided herein in any embodiment for the manufacture of a kit for producing non-replicating recombinant retroviral particles in any embodiment provided herein is provided herein.

[0178] In another aspect, pharmaceutical compositions for treating or preventing cancer or tumor growth are provided herein that include replication-incompetent recombinant retroviral particles as an active ingredient. In another aspect, infusion compositions or other cell preparations for treating or preventing cancer or tumor growth that include replication-incompetent recombinant retroviral particles are provided herein. The replication-incompetent recombinant retroviral particles of the pharmaceutical or infusion composition can include any of the aspects, embodiments, or sub-embodiments discussed above or elsewhere in this specification.

[0179] Compositions and methods for transducing lymphocytes in additional blood components In certain embodiments, methods are provided herein for transducing, genetically modifying, and / or modifying peripheral blood mononuclear cells (PBMCs) or lymphocytes, typically T cells and / or NK cells, and in certain exemplary embodiments, resting T cells and / or resting NK cells in a reaction mixture comprising contacting lymphocytes with non-replicating recombinant retroviral particles in the reaction mixture. Such reaction mixtures themselves represent distinct embodiments provided herein. The reaction mixture in the exemplary embodiments comprises lymphocytes and non-replicating recombinant retroviral particles, T cell activating elements, and one or more additional blood components described below, present in the exemplary embodiments so that the reaction mixture contains at least 10% whole blood, the non-replicating recombinant retroviral particles typically include binding polypeptides and fusion polypeptides, and in exemplary embodiments, pseudotyping elements on their surface. In such a manner, contact (and incubation under contact conditions) promotes the association of lymphocytes with recombinant retroviral particles that lack the ability to replicate, and the recombinant retroviral particles genetically modify and / or transduce the lymphocytes. The reaction mixture in these methods or embodiments of the reaction mixture contains at least 10% unfractionated whole blood (e.g., at least 10%, 20%, 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% whole blood) and, optionally, an effective amount of anticoagulant, or the reaction mixture further contains at least one additional blood or hematopoietic component that is not a PBMC, for example, the reaction mixture contains an effective amount of anticoagulant and one or more hematopoietic components that are not PBMCs. The percentage of whole blood is the volume percentage of the reaction mixture prepared using unfractionated whole blood. For example, if the reaction mixture is formed by adding whole blood, and in exemplary embodiments, unfractionated whole blood, to recombinant retrovirus particles that lack replication ability, the proportion of whole blood in the reaction mixture is the volume of whole blood divided by the total volume of the reaction mixture and multiplied by 100.In an exemplary embodiment, such blood or blood preparation components that are not PBMCs are one or more (e.g., at least 1, 2, 3, 4, or 5) or all of the following additional components: a) Red blood cells (where the red blood cells constitute 1 to 60% of the volume of the reaction mixture), b) Neutrophils (where the neutrophils constitute at least 10% of the white blood cells in the reaction mixture or the reaction mixture contains at least 10% of the same number of neutrophils as T cells), c) Basophils (where the basophils constitute at least 0.05% of the white blood cells in the reaction mixture), d) Eosinophils (where the reaction mixture contains at least 0.1% of the white blood cells in the reaction mixture), e) Plasma (where the plasma constitutes at least 1% of the volume of the reaction mixture), and f) Anticoagulant, (Such blood or blood preparation components a - f above are referred to herein as ("non - PBMC blood or blood preparation components of interest")).

[0180] In any of the embodiments disclosed herein that include a proportion of whole blood, the proportion is volume - based. For example, in a particular embodiment, at least 25% of the volume of the reaction mixture may be whole blood. Thus, in such an embodiment, at least 25 mL out of 100 mL of such a reaction mixture is whole blood.

[0181] One or more additional blood components other than PBMCs as seen in certain embodiments of this specification are present in certain exemplary embodiments of the reaction mixture (including, in the relevant uses, cell preparations, modifications, and exemplary embodiments provided herein, genetically modified T cells or NK cells, or methods for modifying T cells and / or NK cells) for the reason that in these exemplary embodiments the reaction mixture contains at least 10% whole blood, and in certain exemplary embodiments, at least 25%, 50%, 75%, 90%, or 95% whole blood, or, for example, 25% to 95% whole blood. In these exemplary embodiments, such a reaction mixture is formed by combining whole blood with an anticoagulant (for example, by collecting whole blood in a blood collection tube containing an anticoagulant) and by adding a recombinant retrovirus solution to the blood together with the anticoagulant. Thus, in exemplary embodiments, the reaction mixture contains an anticoagulant as described in more detail herein, for example, in the section on exemplary embodiments. In some embodiments, whole blood is not umbilical cord blood, or does not contain umbilical cord blood.

[0182] The reaction mixture in exemplary embodiments of these aspects is formed by a certain amount of whole blood, which is added directly to other reaction mixture components for forming the reaction reaction. Thus, the reaction mixture in such embodiments is typically formed by a method that does not involve a PBMC concentration procedure. Thus, typically, such a reaction mixture contains additional components listed in a) to f) above, which are not PBMCs. Furthermore, in exemplary embodiments, the reaction mixture is substantially whole blood or contains whole blood, and therefore the reaction mixture contains all of the additional components listed in a) to e) above. "Substantially whole blood" means blood that has been separated from an individual, has not been subjected to a PBMC concentration procedure, and has been diluted by less than 50% with other solutions. For example, this dilution may be by the addition of an anticoagulant, as well as the addition of a certain amount of liquid containing retroviral particles. Further embodiments of reaction mixtures for methods and compositions relating to the transduction of lymphocytes in whole blood are provided herein.

[0183] In yet another embodiment, the use of non-replicating recombinant retroviral particles in the manufacture of a kit for modifying target lymphocytes, in exemplary embodiments T cells and / or NK cells, the use of the kit comprising the above method of transducing, genetically modifying, and / or modifying lymphocytes in whole blood. In yet another embodiment, a method for administering the modified lymphocytes to a target, the modified lymphocytes being produced by the above method of transducing, genetically modifying, and / or modifying lymphocytes in whole blood. Embodiments provided herein, including such methods of transducing, genetically modifying, and / or modifying lymphocytes in whole blood, the use of such methods in the manufacture of a kit, reaction mixtures formed by such methods, cell preparations produced by such methods, modified lymphocytes produced by such methods, and modifications produced by such methods, and in exemplary embodiments, a method for administering genetically modified lymphocytes, are referred to herein as “embodiments of compositions and methods for transducing lymphocytes in whole blood.” Exemplary embodiments of such embodiments include contacting T cells and / or NK cells with retroviral particles in whole blood, but it should be noted that such embodiments also include other embodiments in which one or more of the above additional components a-f are present in the transduction reaction mixture at concentrations higher than typical concentrations after the PBMC enrichment procedure. For example, such embodiments occur when the blood is fractionated using a filter that separates the blood into components containing T cells and / or NK cells, as well as additional blood components not present in the PBMC preparation, for example, the use of a leukocyte removal filter, and the presence of neutrophils in the cell fraction containing T cells and NK cells that are retained by the filter.

[0184] Various elements or steps of embodiments of such methods for transducing lymphocytes in whole blood and reaction mixtures containing whole blood or one or more components thereof are provided herein, for example, in this section and the section of exemplary embodiments, and such methods include embodiments provided throughout this specification, as will be further discussed herein. Those skilled in the art will recognize that many embodiments provided anywhere herein may be applied to any of embodiments of compositions and methods for transducing lymphocytes in whole blood. For example, any embodiment of any embodiment of compositions and methods for transducing lymphocytes in whole blood provided in this section and / or the section of exemplary embodiments may include any embodiment of non-replicating recombinant retroviral particles provided herein, which include one or more polypeptide lymphoproliferative elements, inhibitory RNA, CAR, pseudotyping elements, riboswitches, activating elements, membrane-bound cytokines, miRNA, Kozak-type sequences, WPRE elements, triple stop codons, and / or other elements disclosed herein, and may be combined with the methods herein for producing retroviral particles using packaging cells. Furthermore, any aspect and embodiment of the composition (e.g., reaction mixture) and method for transducing lymphocytes in whole blood may be combined with any aspect of the composition and method including self-driving CAR provided herein. Details regarding any aspect of the composition and method including self-driving CAR are disclosed herein in more detail, for example, in the sections on self-driving CAR methods and compositions, and on exemplary embodiments.

[0185] In certain exemplary embodiments, the retroviral particles are lentiviral particles. Such methods for modifying lymphocytes, such as T cells and / or NK cells, in whole blood, and, in exemplary embodiments, for genetic modification, can be carried out in vitro or ex vivo.

[0186] Anticoagulants are included in the reaction mixtures of certain embodiments of the compositions (e.g., reaction mixtures) and methods for transducing lymphocytes in whole blood provided herein. In some exemplary embodiments, blood is collected with the anticoagulant present in a collection tank (e.g., a tube or bag) using, for example, a standard blood collection protocol known in the art. Anticoagulants that may be used in the compositions and methods for transducing lymphocytes in whole blood provided herein include compounds or biological agents that block or limit the thrombin coagulation cascade. The anticoagulant comprises a metal chelating agent, preferably a calcium ion chelating agent such as a citrate (e.g., containing free citrate ions), which includes citric acid, sodium citrate, phosphate, adenine, and monosaccharides or polysaccharides, such as dextrose, oxalate, and EDTA; heparin and heparin analogs, such as unfractionated heparin, low molecular weight heparin, and other synthetic sugars; and a solution of citrate containing one or more components such as a vitamin K antagonist, such as coumarin. Exemplary citrate compositions include acidic dextrose citrate (ACD) (also called anticoagulant dextrose citrate solution A and solution B (United States Pharmacopeia 26, 2002, pp 158)); and citrate phosphate dextrose (CPD) solution (which can also be prepared as CPD-A1, as is known in the art). Thus, the anticoagulant composition may also contain phosphate ions or monobasic phosphate ions, adenine, and monosaccharides or polysaccharides.

[0187] Such anticoagulants may be present in the reaction mixture at concentrations (i.e., effective doses) that are effective in preventing blood coagulation, as is known in the art, or at concentrations of, for example, 2x, 1.5x, 1.25x, 1.2x, 1.1x, or 9 / 10, 4 / 5, 7 / 10, 3 / 5, 1 / 2, 2 / 5, 3 / 10, 1 / 5, or 1 / 10 of the effective concentration. The effective concentrations of many different anticoagulants are known and can be readily determined empirically by analyzing different concentrations for their ability to physically prevent blood coagulation. Numerous coagulation meters are commercially available for measuring coagulation, and various sensor technologies, such as QCM sensors, may be used (see, for example, Yao et al., “Blood Coagulation Testing Smartphone Platform Using Quartz Crystal Microbalance Dissipation Method,” Sensors (Basel). 2018 Sep;18(9):3073). The effective concentration includes the concentration of any commercially available anticoagulant in a commercially available tube or bag after the anticoagulant has been diluted with the blood volume for the tube or bag. For example, the concentration of acidic dextrose citrate (ACD) in the reaction mixture in certain embodiments of the compositions and methods for transducing lymphocytes in whole blood provided herein may be 0.1 to 5 times, or 0.25 to 2.5 times, 0.5 to 2 times, 0.75 to 1.5 times, 0.8 to 1.2 times, 0.9 to 1.1 times, about 1 time, or 1 time of the concentration of ACD in a commercially available ACD blood collection tube or bag. For example, in a standard process, blood may be collected in a tube or bag containing 3.2% (10⁹ mM) sodium citrate (10⁹ mM) in a ratio of 9 parts blood to 1 part anticoagulant. Therefore, in a particular exemplary embodiment using a reaction mixture prepared by adding 1-2 parts of retroviral particle solution to this mixture of 1 part anticoagulant to 9 parts blood, the citrate concentration may be, for example, 25% to 0.4%, or 0.30% to 0.35%. In an exemplary standard blood collection embodiment, 15 mL of ACD solution A is present in a blood bag for collecting 100 mL of blood.The ACD before blood addition contains 7.3 g / L (0.73%) of anhydrous citric acid, 22.0 g / L (2.2%) of sodium citrate (dihydrate), and 24.5 g / L [USP] (2.4%) of dextrose (monohydrate). After adding 100 mL of blood to the bag containing the ACD, retroviral particles are added in volumes of, for example, 5 to 20 mL. Therefore, in some embodiments, the concentrations of the ACD components in the reaction mixture may be 0.05 to 0.1%, or 0.06 to 0.08% of anhydrous citric acid, 0.17 to 0.27%, or 0.20 to 0.24% of sodium citrate (dihydrate), or 0.2 to 0.3%, or 0.20 to 0.28%, or 0.22 to 0.26% of dextrose (monohydrate). In certain embodiments, sodium citrate is used in the reaction mixture at a concentration of 0.001 to 0.02 M.

[0188] In some embodiments, heparin is present in the reaction mixture at concentrations of 0.1 to 5 times, or 0.25 to 2.5 times, 0.5 to 2 times, 0.75 to 1.5 times, 0.8 to 1.2 times, 0.9 to 1.1 times, about 1 time, or 1 time compared to, for example, the concentration of heparin in a commercially available heparin blood collection tube. Heparin is a glycosaminoglycan anticoagulant having a molecular weight in the range of 5,000 to 30,000 daltons. In some embodiments, heparin is used in reaction mixture concentrations of about 1.5 to 45, 5 to 30, 10 to 20, or 15 USP units / mL. In some embodiments, the effective concentration of EDTA, for example as K2EDTA, in the reaction mixture herein may be 0.15 to 5 mg / mL, 1 to 3 mg / mL, 1.5 to 2.2 mg / mL of blood, or 1 to 2 mg / mL, or about 1.5 mg / mL. The reaction mixtures in embodiments of compositions and methods for transducing lymphocytes in whole blood provided herein may contain two or more anticoagulants such that their combined effective dose prevents coagulation of the blood before the formation of the reaction mixture and / or the reaction mixture itself.

[0189] In some embodiments, an anticoagulant may be administered to the subject before blood is taken from the subject for ex vivo transduction, so that blood coagulation at the time of blood collection is inhibited at least partially and at least throughout the contact step and a subsequent optional incubation period. In such embodiments, for example, dextrose acidic citrate may be administered to the subject at doses of 80 mg / kg / day to 5 mg / kg / day (mg refers to mg of citrate, and kg applies to the mammal being treated). Heparin may be delivered at doses of, for example, 5 units / kg / hour to 30 units / kg / hour.

[0190] The reaction mixtures in certain exemplary embodiments of this specification may include blood or blood preparation components other than PBMCs, as provided herein. Exemplary concentrations of such components, not limited to these, are provided in the following paragraphs. In exemplary embodiments, it will be understood that the cell preparations resulting from methods using these reaction mixtures include these additional components in the same ratios or proportions to the other cells, as provided below for the reaction mixtures in some embodiments.

[0191] With respect to red blood cells, in some embodiments, red blood cells are present in the reaction mixture and cell preparation herein in relative amounts to T cells that are greater than those after typical PBMC isolation, and in some embodiments, red blood cells may constitute 0.1, 0.5, 1, 5, 10, 25, 35, or 40% of the lower limit of the volume range of the reaction mixture to 25, 50, 60, or 75% of the upper limit of the volume range of the reaction mixture. In exemplary embodiments, red blood cells constitute 1-60%, 10-60%, 20-60%, 30-60%, 40-60%, 40-50%, 42-48%, 44-46%, about 45%, or 45% of the reaction mixture. In some embodiments, there are more red blood cells than T cells in the reaction mixture or cell preparation.

[0192] With respect to neutrophils, in some embodiments, neutrophils are present in the reaction mixtures and cell preparations provided herein in relative amounts to T cells that are greater than those after typical PBMC isolation, and in some embodiments, neutrophils may constitute 25%, 50%, 60%, 70%, 75%, and 80% of the leukocyte range in the reaction mixture or cell preparation, for example, 25% to 70%, or 30% to 60%, or 40% to 60% of the leukocyte range in the reaction mixture or cell preparation. In some embodiments, more neutrophils are present in the reaction mixtures and cell preparations herein than T cells and / or NK cells.

[0193] With respect to eosinophils, in some embodiments, eosinophils are present in the reaction mixture or cell preparation in a relative amount to T cells greater than that after typical PBMC isolation, and in some embodiments, eosinophils may constitute 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8% of the lower limit of the leukocyte range in the reaction mixture or cell preparation to 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.5, 4, 5, 6, 8, and 10% of the leukocyte range in the reaction mixture or cell preparation. In exemplary embodiments, eosinophils constitute 0.05–10.0%, 0.1–9%, 0.2–8%, 0.2–6%, 0.5–4%, 0.8–4%, or 1–4% of the leukocytes in the reaction mixture or cell preparation.

[0194] With respect to basophils, in some embodiments, basophils are present in the reaction mixture or cell preparation in relative amounts to T cells that are greater than those after typical PBMC isolation, and in some embodiments, basophils may constitute 0.05, 0.1, 0.2, 0.4, 0.45, and 0.5% of the lower limit of the leukocyte range in the reaction mixture to 0.8, 0.9, 1.0, 1.1, 1.2, 1.5, and 2.0% of the upper limit of the leukocyte range in the reaction mixture. In exemplary embodiments, basophils constitute 0.05-1.4%, 0.1-1.4%, 0.2-1.4%, 0.3-1.4%, 0.4-1.4%, 0.5-1.4%, 0.5-1.2%, 0.5-1.1%, or 0.5-1.0% of the leukocytes in the reaction mixture.

[0195] With respect to plasma, in some embodiments, the plasma component is present in the reaction mixture or cell preparation, and in some embodiments, the plasma may constitute 0.1, 0.5, 1, 5, 10, 25, 35, or 45% of the lower limit of the volume range of the reaction mixture to 25, 50, 60, 70, and 80% of the upper limit of the volume range of the reaction mixture. In exemplary embodiments, the plasma constitutes 0.1-80%, 1-80%, 5-80%, 10-80%, 30-80%, 40-80%, 45-70%, 50-60%, 52-58%, 54-56%, about 55%, or 55% of the reaction mixture. With respect to platelets, in some embodiments, platelets are present in the reaction mixture or cell preparation in a relative amount to T cells greater than that after typical PBMC isolation, and in some embodiments, platelets are present at a lower limit of 1 × 10⁶ 5 , 1 x 10 6 , 1 x 10 7 , or 1 × 10 8 Reaction mixture of platelets / mL ~ upper limit of range 1 × 10⁶ 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 2×10 13 , or 2 × 10 14 A reaction mixture of 1 × 10 platelets / mL can be formed. In an exemplary embodiment, the platelets are 1 × 10 5 ~1 × 1012 individual platelets, 1×10 6 ~1×10 11 individual platelets, 1×10 7 ~1×10 10 individual platelets, 1×1×10 8 ~1×10 9 individual platelets / mL, or 1×10 8 ~5×10 8 In some embodiments, the reaction mixture of individual platelets / mL, or 1×10~5×10 individual platelets / mL, is in a relative amount to T cells that is greater than after typical PBMC isolation, and in some embodiments, can constitute 0.1% - 9%, 0.1% - 1%, or 1% - 9% of the white blood cells in the reaction mixture or cell preparation.

[0196] Steps and reaction mixtures for methods for modifying and / or genetically modifying lymphocytes In certain embodiments, methods are provided herein for transfecting, transfecting, genetically modifying, and / or modifying lymphocytes such as peripheral blood mononuclear cells (PBMCs), typically T cells and / or NK cells, and in certain exemplary embodiments, resting T cells and / or NK cells (typically populations thereof), the methods comprising contacting lymphocytes with a recombinant nucleic acid vector (typically population thereof), which in exemplary embodiments is a recombinant retroviral particle lacking replication ability, and such contact (and incubation under contact conditions) promotes membrane association, membrane fusion or endocytosis, and optionally, transfection or transfection of resting T cells and / or NK cells by the recombinant nucleic acid vector, thereby producing modified, and in exemplary embodiments, genetically modified T cells and / or NK cells. It should be noted that while many embodiments and models provided herein consider recombinant retroviral particles, many different recombinant nucleic acid vectors, including but not limited to those provided herein, are intended to be used and / or included in such methods and compositions and will be recognized by those skilled in the art. In exemplary embodiments, where the recombinant nucleic acid vector is a non-replicating recombinant retroviral particle, the non-replicating recombinant retroviral particle typically includes fusion and binding elements on its surface, which may be part of the pseudotyping elements. In exemplary embodiments, prior activation of T cells and / or NK cells is not required, and the activating elements, which may be any activating elements provided herein, are present in the reaction mixture in which contact takes place. In further exemplary embodiments, the activating elements are present on the surface of the non-replicating recombinant retroviral particle. In exemplary embodiments, the activating elements are anti-CD3, such as anti-CD3 scFv or anti-CD3 scFvFc.

[0197] Many aspects of the methods provided herein include the following steps: 1) an optional step of collecting blood from a subject; 2) a step of contacting cells, such as NK cells and / or T cells in exemplary embodiments, which may originate from the collected blood, in a reaction mixture with a recombinant vector encoding CAR and / or lymphoproliferative elements (typically many copies thereof), in exemplary embodiments, with recombinant retroviral particles that lack the ability to replicate (contact may include an optional incubation); 3) a step of washing away the unbound recombinant vector from the cells in the reaction mixture, typically; 4) a step of collecting modified cells, such as modified NK cells and / or modified T cells in exemplary embodiments, in a solution, typically, in exemplary embodiments, which may be a delivery solution, to form a cell suspension, which in exemplary embodiments is a cell preparation; and 5) an optional step of delivering the cell preparation to a subject, in exemplary embodiments the subject from which the blood was collected, for example, by injection, or in certain exemplary embodiments intramuscular or intratumor, or in further exemplary embodiments subcutaneously. Notably, in certain exemplary embodiments, the reaction mixture may include unfractionated whole blood or one or more cell types that are not PBMCs, and may include all or many cell types found in whole blood, including totally nucleated cells (TNCs). Notably, in certain embodiments, the recombinant vector may include a self-driven CAR encoding both a CAR and a lymphoproliferative element.

[0198] In some embodiments, not limited to the above, 10–120 mL of blood is collected (or leukocytes are isolated in 10–120 mL by performing leukocyte apheresis on a total blood volume of 0.5–2.0 mL), the collected unfractionated blood / isolated cells are passed through a leukocyte-removing filter to isolate TNCs on the filter, non-replicating recombinant retrovirus particles are added to the TNCs on the leukocyte-removing filter up to a total volume of 500 μL–10 mL of the reaction mixture to form a reaction mixture and initiate contact, the reaction mixture is optionally incubated for any of the contact times provided herein, 1–4 hours in some embodiments, not limited to the above, the non-replicating recombinant retrovirus particles that have not associated are washed away from the cells in the reaction mixture by filtering the reaction mixture with 10–120 mL of washing solution, and the cells, including modified T cells and NK cells, retained on the TNC filter are eluted from the filter using 2 mL–10 mL of delivery solution, thereby forming a cell preparation suitable for introduction or reintroduction into a subject.

[0199] Some embodiments of any method used in any aspect provided herein, which is typically a method for modifying lymphocytes, PBMCs, and, in exemplary embodiments, NK cells, and / or, in further exemplary embodiments, T cells, and, in exemplary embodiments, a method for genetic modification, may include the step of collecting blood from a subject. The blood includes blood components, including blood cells such as lymphocytes (e.g., T cells and NK cells), which may be used in the methods and compositions provided herein. In certain exemplary embodiments, the subject is a human subject suffering from cancer (i.e., a human cancer subject). It should be noted that 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 preferred method known in the art, as will be discussed in more detail herein, and the collected blood or blood-derived components are referred to as “blood-derived products,” and are typically “peripheral blood-derived products,” as they are 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, thereby collecting a sample of unfractionated whole blood into a vessel, e.g., a blood bag, or thereby isolating leukocytes and lymphocytes from the blood by apheresis (e.g., leukocyte apheresis or lymphocyte-plasma apheresis). In some embodiments, the amount of blood collected (e.g., unfractionated whole blood) is 1-5 mL, 5-10 mL, 10-15 mL, 15-20 mL, 20-25 mL, 5-25 mL, 25-250 mL, 25-125 mL, 50-100 mL, or 50-250 mL, 75-125 mL, 90-120 mL, or 95-110 mL.In some embodiments, the amount of blood collected is 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 amount may be 0, or 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 amount 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 amount of blood collected and processed during apheresis (e.g., leukocyte apheresis or lymphocyte-plasma apheresis) is between the total blood volume of the subject at the lower end of the range of 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.25, or 1.5, and the total blood volume of the subject at the upper end of the range of 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, or 2.5, e.g., total blood volume of 0.5–2.5, 0.5–2, 0.5–1.5, or 1–2. The total blood volume of a human is typically in the range of 4.5–6 L, and therefore, much more blood is typically collected and processed during apheresis than would be the case if unfractionated whole blood were collected. Whether target blood cells (e.g., T cells) are obtained by apheresis or unfractionated whole blood is collected, for example, in a blood bag, the target blood cells (e.g., T cells) therein are processed according to the method provided herein, the method intended to result in target blood cells that, in certain exemplary embodiments, are 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 lymphoplasmapak), such cells are resuspended in a solution either directly or after one or more washings, and a recombinant vector encoding a CAR is added thereto to form the reaction mixture provided herein. Such a reaction mixture can be used in any of the methods herein. In some exemplary methods where a subject or a blood sample from the 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.

[0200] Whether blood is collected from a subject or blood cells are obtained by apheresis, in any aspect of the methods provided herein for modifying lymphocytes (e.g., T cells and / or NK cells), a population of lymphocytes (e.g., T cells and / or NK cells) is typically brought into contact with many copies of a recombinant vector in a reaction mixture, which in some embodiments are copies of a non-viral vector and, in exemplary embodiments, are recombinant retroviral particles lacking identical replication ability. The contact in any embodiment provided herein may be carried out, for example, in a blood bag, or in a chamber of a closed system adapted for processing blood cells, as will be discussed in more detail herein. In some embodiments, the blood bag may contain 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, or 500 mL or less of blood during contact. In some embodiments, a blood bag may contain at least 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, or 500 mL of blood during contact. In some embodiments, a blood bag may contain 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. For example, a blood bag may contain 1-10 mL, 5-25 mL, 10-50 mL, 25-100 mL, 50-200 mL, or 100-500 mL of blood during contact. In some embodiments, the mixture in the blood bag may contain heparin. In other embodiments, the mixture in the blood bag does not contain heparin. The transduction reaction mixture may include one or more buffers, ions, and culture media.Retroviral particles in certain exemplary reaction mixtures provided herein, and in exemplary embodiments, recombinant retroviral particles having a multiplicity of infection (MOI) 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 1 to less than 6, 11, or 51, or in some embodiments, recombinant retroviral particles having no replication capacity at an MOI of 5 to 10 are present. 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, in certain embodiments, PBMCs are isolated and a higher MOI may be used than the method used in the reaction mixture. For example, an exemplary embodiment of a composition and method for transducing lymphocytes in whole blood is 1×10. 6 Assuming 1×10 PBMCs / mL of blood, retroviral particles having an MOI of 1 to 50, 2 to 25, 2.5 to 20, 2.5 to 10, 4 to 6, or about 5, and in some embodiments, 5 to 20, 5 to 15, 10 to 20, or 10 to 15 can be used.

[0201] In exemplary embodiments, this contact and the reaction mixture in which the contact occurs are carried out within a closed cell processing system, as will be discussed in more detail herein. Packaging cells, and in exemplary embodiments, packaging cell lines, and in particularly exemplary embodiments, packaging cells provided in specific embodiments herein, can be used to produce recombinant retroviral particles that lack the ability to replicate. The cells in the reaction mixture may be PBMCs or TNCs and / or additional blood components, including an anticoagulant and / or additional types of blood cells other than PBMCs, may be present in embodiments of these compositions and methods for transducing lymphocytes in whole blood, as will be discussed herein. In fact, in exemplary embodiments of embodiments of these compositions and methods for transducing lymphocytes in whole blood, the reaction mixture may essentially be whole blood and a relatively small amount of solution typically containing an anticoagulant, retroviral particles, and the retroviral particles delivered to the whole blood.

[0202] In reaction mixtures relating to embodiments of compositions and methods for modifying lymphocytes in whole blood provided herein, lymphocytes, including NK cells and T cells, may be present in lower proportions of blood cells and lower proportions of leukocytes in the reaction mixture than in methods that include a PBMC enrichment step before forming the reaction mixture. For example, in some embodiments of these embodiments, more granulocytes or neutrophils than NK cells or even T cells are present in the reaction mixture. Details regarding the composition of anticoagulants and one or more additional blood components present in the reaction mixtures of embodiments for modifying lymphocytes in whole blood are provided in detail in other sections herein. In some reaction mixtures provided herein, T cells may be, for example, 10, 20, 30, or 40% of the lower limit of the lymphocyte range of the reaction mixture to 40, 50, 60, 70, 80, or 90% of the upper limit of the lymphocyte range of the reaction mixture. In exemplary embodiments, T cells constitute 10–90%, 20–90%, 30–90%, 40–90%, 40–80%, or 45–75% of the lymphocytes. In such embodiments, for example, NK cells may be present at 1, 2, 3, 4, or 5% of the lower limit of the lymphocyte range of the reaction mixture to 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14% of the upper limit of the lymphocyte range of the reaction mixture. In exemplary embodiments, T cells constitute 1–14%, 2–14%, 3–14%, 4–14%, 5–14%, 5–13%, 5–12%, 5–11%, or 5–10% of the lymphocytes of the reaction mixture.

[0203] As disclosed herein, embodiments of compositions and methods for transducing lymphocytes from whole blood typically do not involve blood fractionation, such as a PBMC enrichment step of the blood sample, before lymphocytes derived from a blood sample come into contact with recombinant nucleic acid vectors, such as retroviral particles, in the reaction mixture disclosed herein for those embodiments. Thus, in some embodiments, lymphocytes in unfractionated whole blood come into contact with recombinant retroviral particles. However, in some embodiments, particularly for some embodiments of the self-driven CAR methods and compositions sections herein, neutrophils / granulocytes are separated from other blood cells before the cells come into contact with recombinant retroviral particles that lack the ability to replicate. In some embodiments, peripheral blood mononuclear cells (PBMCs), including peripheral blood lymphocytes (PBLs) such as T cells and / or NK cells, are isolated from other components of the blood sample, for example, using a PBMC enrichment procedure, before being combined with retroviral particles in the reaction mixture. Those skilled in the art will understand that different blood fractions containing T cells and / or NK cells can be enriched using various methods known in the art.

[0204] PBMC enrichment procedures are those which enrich PBMCs at least 25 times, and typically at least 50 times, compared to other blood cell types. For example, PBMCs are thought to constitute less than 1% of blood cells in whole blood. After a PBMC enrichment procedure, at least 30%, and in some cases as much as 70%, of the cells isolated in the PBMC fraction are PBMCs. Even higher enrichments 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 a ficol density gradient centrifugation process that separates major cell populations such as lymphocytes, monocytes, granulocytes, and erythrocytes across a density gradient medium. In such a method, the aqueous medium contains ficol, a hydrophilic polysaccharide that forms a high-density solution. When whole blood is layered on top of or below density gradient medium without mixing the two layers, and then centrifuged, the cells are dispersed according to density, and the PBMC fraction forms a thin white layer at the interface between the plasma and the density gradient medium (see, for example, Panda and Ravindran (2013) Isolation of Human PBMCs. BioProtoc. Vol.3(3)). Furthermore, centripetal force can be used to separate PBMCs from other blood components in Ficol using the rotational force of the Sepax cell processing system.

[0205] In some embodiments, cells such as PBMCs can be isolated using apheresis, for example, leukocyte apheresis. For example, AMICUS RBCX (Fresenius-Kabi) and Trima Accel (Terumo BCT) apheresis devices and kits may be used. Cells isolated by apheresis typically contain T cells, B cells, NK cells, monocytes, granulocytes, other nucleated leukocytes, erythrocytes, and / or platelets. Cells collected by apheresis may be washed to remove the plasma fraction and placed in a suitable buffer or medium, such as phosphate-buffered saline (PBS) or a washing solution, which may be calcium-deficient and possibly magnesium-deficient, or may be deficient in many but not all divalent cations, for subsequent processing steps. In some embodiments, cells collected by apheresis may be genetically modified by any of the methods provided herein. In some embodiments, cells collected by apheresis may be used to prepare any of the cell preparations provided herein. In some embodiments, cells collected by apheresis may be resuspended in various biocompatible buffers, such as Ca-free and Mg-free PBS. Alternatively, unwanted components may be removed from the sample containing cells collected by apheresis, and the cells may be resuspended in culture medium. In some embodiments, cells such as lymphocytes can be isolated using leukocyte apheresis. LeucoPac may be used in any embodiment provided herein that includes PBMCs. BuffyCoat may be used in any embodiment that includes TNCs. Another PBMC enrichment method uses an automated leukocyte apheresis collection system (such as the SPECTRA OPTIA® APHERESIS SYSTEM from Terumo BCT, Inc., Lakewood, CO 80215, USA) to typically separate the whole blood inflow from the target PBMC fraction using high-speed centrifugation, while returning efflux materials such as plasma, erythrocytes, and granulocytes to the donor. 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 the time-consuming purification of PBMCs, and the leukocyte apheresis method requires the presence and participation of a patient during the PBMC enrichment step.

[0206] As further, but not limited, examples of PBMC enrichment procedures, in some embodiments of the transduction, genetic modification, and / or modification methods herein, PBMCs are isolated using a Sepax or Sepax 2 cell processing system (BioSafe). In some embodiments, PBMCs are isolated using a CliniMACS Prodigy cell processor (Miltenyi Biotec). In some embodiments, an automated apheresis separator is used in which blood is taken from a subject, passed through a device that sorts specific cell types (e.g., PBMCs), and the remainder is returned to the subject. Density gradient centrifugation may be performed after apheresis. In some embodiments, PBMCs are isolated using a leukocyte-removing filter assembly. In some embodiments, a specific cell population is then purified from PBMCs, e.g., PBLs or a subset thereof, according to cell phenotype (i.e., positive selection) using magnetic bead-activated cell sorting, and then used in the reaction mixture herein.

[0207] Other purification methods may also be used, such as substrate adhesion, which utilizes a substrate that mimics the environment that T cells encounter during recruitment, in order to purify T cells before adding them to the reaction mixture, or negative selection may be used, in which unwanted cells are targeted for removal using an antibody complex that targets unwanted cells for removal before the reaction mixture is formed in the contact step. In some embodiments, erythrocyte rosette formation can be used to remove erythrocytes before the reaction mixture is formed. In other embodiments, hematopoietic stem cells may be removed before the contact step, and therefore in these embodiments, hematopoietic stem cells are not present during the contact step. In some embodiments herein, particularly with respect to 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 contact with or without optional incubation (i.e., not in the reaction mixture in which contact is made), or at any step of the method.

[0208] In certain exemplary embodiments of any aspect provided herein, lymphocytes are modified, and in exemplary embodiments, genetically modified and / or transduced, without prior activation or stimulation, and / or without the need for prior activation or stimulation, whether in vivo, in vitro, or ex vivo, and / or further, in some embodiments, without ex vivo or in vitro activation or stimulation after initial contact with or without optional incubation, or without the need for ex vivo or in vitro activation or stimulation after initial contact with or without optional incubation. In certain exemplary embodiments, cells are activated during contact and are not activated at all before contact, or are not activated for more than 15 minutes, 30 minutes, 1, 2, 4, or 8 hours. In certain exemplary embodiments, activation by elements not present on the surface of retroviral particles is not required to modify, genetically modify, and / or transduce cells. Thus, no such activating or stimulating elements are required other than retroviral particles before, during, or after contact. Therefore, as will be discussed in more detail herein, these exemplary embodiments, which do not require prior activation or stimulation, provide the ability to rapidly carry out in vitro experiments aimed at better understanding T cells and the biological mechanisms within them. Furthermore, such methods provide much more efficient commercial production of biological products produced using PBMCs, lymphocytes, T cells, or NK cells, and the development of such commercial production methods. Finally, such methods provide a more rapid ex vivo treatment of lymphocytes (e.g., NK cells and especially T cells) for adoptive cell therapy, and fundamentally simplify the provision of such therapies by providing, for example, a rapid point-of-care (rPOC) method. In exemplary embodiments, some, most, at least 25%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%, or all, of the lymphocytes are resting when they combine with retroviral particles to form a reaction mixture, and typically when they come into contact with retroviral particles in the reaction mixture.In methods for modifying lymphocytes such as T cells and / or NK cells in blood or its components, the lymphocytes may be in contact with the typical resting state they are in when present in blood collected in vivo immediately before collection. In some embodiments, T cells and / or NK cells are 95-100% resting cells (Ki-67). - ) consist of. In some embodiments, the T cells and / or NK cells that come into contact with the recombinant retroviral particles lacking replication ability include resting cells at the lower end of the range of 90, 91, 92, 93, 94, and 95% to the upper end of the range of 96, 97, 98, 99, or 100%. In some embodiments, the T cells and / or NK cells include naive cells. In some exemplary embodiments, the sub-embodied embodiments of this paragraph include embodiments of compositions and methods for transducing lymphocytes in whole blood.

[0209] In exemplary embodiments of the embodiments herein, including non-replicating recombinant retroviral particles, contact between T cells and / or NK cells and non-replicating recombinant retroviral particles may facilitate the transduction of T cells and / or NK cells by the non-replicating recombinant retroviral particles. Not limited by theory, during the contact period, the non-replicating recombinant retroviral particles identify and bind to T cells and / or NK cells, and the T cells and NK cells are “modified,” as used herein. At this point, the retrovirus and host cell membrane begin to fuse, and any retroviral pseudotyping elements and / or T cell activating elements, including anti-CD3 antibodies, become incorporated into the surface of the modified T cells and / or NK cells. Subsequently, as the next step in the transduction process, genetic material from the non-replicating recombinant retroviral particles enters the T cells and / or NK cells, at which point the T cells and / or NK cells are “genetically modified,” as used herein. It is noteworthy that such processes can occur several hours or even several 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 T cells and / or NK cells, at which point the T cells and / or NK cells are “transfected,” as this term is used herein. Similarly, cells can be modified, genetically modified, and / or transfected by recombinant vectors other than recombinant retroviral particles that lack the ability to replicate. Cells can also internalize and incorporate the genetic material into the genomic DNA of T cells and / or NK cells after transfection, at which point the T cells and / or NK cells are “stable transfected,” as this 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) as described herein is also a method for transfecting lymphocytes (e.g., T cells and / or NK cells).It is believed that by day 6, either in vivo or ex vivo, after contact has been initiated, the majority of modified and genetically altered cells have been transduced. Methods for transducing lentiviruses are known. Exemplary methods are described, for example, in Wang et al. (2012) J.Immunother. 35(9):689-701, Cooper et al. (2003) Blood. 101:1637-1644, Verhoeyen et al. (2009) Methods Mol Biol. 506:97-114, and Cavalieri et al. (2003) Blood. 102(2):497-505. Throughout this disclosure, transduced, or in some embodiments, stably transduced T cells and / or NK cells, include offspring of ex vivo transduced cells that retain at least a portion of the nucleic acids or polynucleotides that are incorporated into the cell genome during ex vivo transduction. In the methods of this specification that enumerate the “reintroduction” of transduced cells, it will be understood that such cells are typically not transduced when taken from the blood of the subject.

[0210] In exemplary embodiments, T cells and / or NK cells are not activated before contact with recombinant retrovirus in the method herein, but the T cell activating elements in exemplary embodiments are present in the reaction mixture where the initial contact between recombinant retrovirus and lymphocytes occurs. For example, such T cell activating elements may be in solution in the reaction mixture. For example, a soluble anti-CD3 antibody may be present in the reaction mixture at 25-200, 50-150, 75-125, or 100 ng / mL during contact and subsequent optional incubation. In exemplary embodiments, the T cell activating elements are associated with the retrovirus surface. The T cell activating elements may be any T cell activating elements provided herein. In exemplary embodiments, the T cell activating elements may be anti-CD3 such as anti-CD3 scFv or anti-CD3 scFvFc. Thus, in some embodiments, recombinant retrovirus particles without replication ability may further contain T cell activating elements, which in further exemplary examples are associated with the outside of the retrovirus surface.

[0211] The contact step of a method for transducing and / or modifying or genetically modifying lymphocytes in whole blood, as provided herein, typically comprises a first step in which retroviral particles, typically a population of retroviral particles, are brought into contact with a population of blood cells, typically including an anticoagulant and / or additional blood components other than PBMCs that are not present after the PBMC enrichment procedure, while the retroviral particles are suspended in a liquid buffer and / or culture medium to form a transduction reaction. Following this contact, an optional incubation period may follow in this reaction mixture, which includes the retroviral particles and blood cells (e.g., T cells and / or NK cells) in the suspension. In a method for modifying T cells and / or NK cells in blood or its components, the reaction mixture may include at least one, two, three, four, five, or all of the additional blood components disclosed herein, and in exemplary embodiments, one or more anticoagulants.

[0212] In any embodiment provided herein, the transduction reaction mixture may be incubated at 23–39°C, and in some exemplary embodiments at 37°C, in an optional incubation step following the initial contact of retroviral particles and lymphocytes. In certain embodiments, the transduction reaction may be carried out at 37–39°C for faster fusion / transduction. In some embodiments, the contact step is a low-temperature contact step accompanied by an optional incubation step, as discussed elsewhere herein. In some embodiments, the low-temperature contact step is carried out at a temperature below 37°C, such as 1°C–25°C or 2°C–6°C. Optional incubations associated with contact steps at these temperatures may be carried out for any length of time, as discussed herein, for example, in the section on exemplary embodiments. In exemplary embodiments, optional incubations associated with these temperatures are carried out for no more than one hour.

[0213] In some embodiments, including exemplary embodiments in which contact is carried out on a filter, contact is carried out at a lower temperature, e.g., 2°C to 25°C, and is referred herein to as cold contact, and retroviral particles that remain unassociated in the suspension are removed from the reaction mixture by washing the reaction mixture on a filter, such as a leukocyte-removing filter that retains leukocytes, including T cells and NK cells, but does not retain free, unassociated viral particles. Cells and retroviral particles upon contact in the transduction reaction mixture may be immediately processed to remove retroviral particles that remain free in the suspension and are not associated with cells from the cells. Optionally, cells in the suspension, and retroviral particles, whether free in the suspension or associated with cells in the suspension, may be incubated for varying lengths of time as provided herein for the contact step of the methods provided herein. Washing may be carried out before further steps, regardless of whether such cells are studied in vitro, ex vivo, or introduced into a subject. Such suspensions may include allowing cells and retroviral particles to settle, or causing such settling by applying a force, such as centrifugal force, to the bottom of the tank or chamber, as will be discussed in more detail herein. In exemplary embodiments, such g-force is lower than the g-force commonly used in spinoculation procedures. Further contact times and considerations regarding contact and optional incubation are discussed further herein, for example, in the section on exemplary embodiments.

[0214] Current methods require prolonged ex vivo augmentation of genetically modified lymphocytes before formulation and reintroduction into the subject. There has been a long-standing urgent need for an effective point-of-care adoptive cell therapy that would allow the subject to be collected in a single visit and the lymphocytes to be modified and reintroduced. The methods provided herein enable rapid ex vivo processing of lymphocytes, and in certain exemplary embodiments, PBMCs, and in other exemplary embodiments, all nucleated cells (TNCs), without the ex vivo augmentation step, and fundamentally simplify the delivery of adoptive cell therapy by providing such a point-of-care method, and in some exemplary embodiments, in a shorter period of time (rapid point-of-care (rPOC)). Exemplary methods for modifying lymphocytes, particularly NK cells, and in exemplary embodiments, T cells, which are far shorter and simpler than previous methods are disclosed herein. Accordingly, in some embodiments, the contact step in any method provided herein for transducing, genetically modifying, and / or modifying PBMCs or lymphocytes, typically T cells and / or NK cells, may be carried out for any of the periods provided herein, including but not limited to the periods provided in the section on exemplary embodiments. For example, such contact may be less than 24 hours, e.g., less than 12 hours, less than 8 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes, or less than 15 minutes, but in any case, as will be discussed in more detail herein, there is at least an initial contact step in which the retroviral particles and cells come into contact in a suspension in the transduction reaction mixture, thereafter the retroviral particles remaining in the suspension that do not associate with cells are separated from the cells and typically discarded. It should be noted, though not intended to be limited by theory, that contact is typically considered to begin when the retroviral particles and lymphocytes are combined together by adding a solution containing retroviral particles to a solution containing lymphocytes (e.g., T cells and / or NK cells).

[0215] Following the initial contact, which includes the initial cold contact, in some embodiments, there is an incubation of the reaction mixture containing the cells and the recombinant nucleic acid vector (which is retroviral particles in exemplary embodiments) in a suspension for a specified period of time, without removing the recombinant nucleic acid vector (e.g., retroviral particles) that remains free in the solution and is not associated with the cells. This incubation may be referred to herein as an optional incubation. Thus, in exemplary embodiments, the contact (including the initial contact and the optional incubation) may be carried out for 15 minutes to 12 hours, 15 minutes to 10 hours, or 15 minutes to 8 hours, or any of the times included in the section of the exemplary embodiment. In certain embodiments including the cold contact step, the secondary incubation is carried out by suspending the cells after an optional washing step such that the recombinant nucleic acid vector that is not associated with the cells, and retroviral particles in exemplary embodiments, are washed away. In exemplary embodiments, the secondary incubation is carried out at a temperature of 32°C to 42°C, such as 37°C. The optional secondary incubation may be carried out for any length of time considered herein. In exemplary embodiments, an optional secondary incubation is performed for no more than 6 hours. Thus, in exemplary embodiments, contact (including the initial contact and optional incubation) may be performed for 30 seconds at the lower end of the range, or 1, 2, 5, 10, 15, 30, or 45 minutes, or 1, 2, 3, 4, 5, 6, 7, or 8 hours to 10 minutes, 15 minutes, or 30 minutes at the upper end of the range, or 1, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, and 72 hours (as generally shown herein, the lower end of the selected range is smaller than the upper end of the selected range). Thus, in some embodiments, after the time formed by adding retroviral particles to lymphocytes, the reaction mixture may be incubated for 5 minutes at the lower end of the range to 10, 15, or 30 minutes at the upper end of the range, or 1, 2, 3, 4, 5, 6, 8, 10, or 12 hours.In other embodiments, the reaction mixture may be incubated for 15 minutes to 12 hours, 15 minutes to 10 hours, 15 minutes to 8 hours, 15 minutes to 6 hours, 15 minutes to 4 hours, 15 minutes to 2 hours, 15 minutes to 1 hour, 15 minutes to 45 minutes, or 15 minutes to 30 minutes. In other embodiments, the reaction mixture may be incubated for 30 minutes to 12 hours, 30 minutes to 10 hours, 30 minutes to 8 hours, 30 minutes to 6 hours, 30 minutes to 4 hours, 30 minutes to 2 hours, 30 minutes to 1 hour, or 30 minutes to 45 minutes. In other embodiments, the reaction mixture may be incubated for 1 hour to 12 hours, 1 hour to 8 hours, 1 hour to 4 hours, or 1 hour to 2 hours. In another exemplary embodiment, contact is performed only during the initial contact step (without further incubation in the reaction mixture, including the retroviral particles free in the suspension and the cells in the suspension), or during incubation in the reaction mixture for 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 1 hour.

[0216] Following the indicated period for initial contact and optional incubation, which may be part of the contact step, the blood cells in the reaction mixture or their T cell and / or NK cell-containing fractions are separated from retroviral particles that are not associated with such cells. For example, this can be done using a PBMC enrichment procedure (e.g., a Ficol gradient in a Sepax unit) or, in certain exemplary embodiments provided herein, by filtering the reaction mixture over a leukocyte-removing filter set assembly and then collecting the leukocytes, including T cells and NK cells. In another embodiment, this can be done by centrifugation of the reaction mixture with a relative centrifugal force of less than 500 g, e.g., 400 g, or 300–490 g or 350–450 g. Such centrifugation for separating retroviral particles from cells may be performed for, for example, between 5 and 15 minutes or between 5 and 10 minutes. In exemplary embodiments using centrifugal force to separate cells from retroviral particles that are not associated with cells, such g-force is typically lower than the g-force well used in spinoculation procedures.

[0217] In some exemplary embodiments, the methods provided herein in any embodiment do not involve performing spinocuration. In such embodiments, the cells(s) are not subjected to spinocuration of at least 400g, 500g, 600g, 700g, or 800g for at least 15 minutes. In some embodiments, the cells(s) are not subjected to spinocuration of at least 800g for at least 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, or 45 minutes. In some embodiments, spinocuration is included as part of the contact step. In exemplary embodiments, if spinocuration is performed, there is no additional incubation as part of the contact, as the time of spinocuration provides the incubation time for the optional incubation described above. In other embodiments, there is additional incubation after spinocuration of 15 minutes to 4 hours, or 15 minutes to 2 hours, or 15 minutes to 1 hour. Spinoculation may be performed for, for example, 30 to 120 minutes, typically at least 60 minutes, for example 60 to 180 minutes or 60 to 90 minutes. Spinoculation is typically performed in a centrifuge with a relative centrifugal force of at least 800 g, and more typically at least 1200 g, for example 800 g to 2400 g, 800 g to 1800 g, 1200 g to 2400 g, or 1200 g to 1800 g. After spinoculation, such a method typically includes the additional step of resuspending the pelleted cells and retroviral particles, and then removing retroviral particles that have not associated with cells according to the steps above if spinoculation is not performed.

[0218] In embodiments including a contact step with an optional incubation, and spinoculation, the spinoculation may be carried out at 4°C to 42°C or 20°C to 37°C. In certain exemplary embodiments, spinoculation is not performed, and the contact and associated optional incubation are carried out at 20 to 25°C for a period of 4 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, or 15 minutes to 2 hours, 15 minutes to 1 hour, or 15 minutes to 30 minutes.

[0219] Methods for genetically modifying lymphocytes provided herein typically involve the insertion into a cell of a polynucleotide comprising one or more transcription units encoding any transgene, e.g., a CAR or a lymphoproliferative element, or, in exemplary embodiments, both a CAR and a lymphoproliferative element, according to any embodiment of the CAR and lymphoproliferative element provided herein. Such CARs and lymphoproliferative elements may be provided to support shorter and simpler methods provided herein that can support the growth of T cells and / or NK cells after contact and optional incubation modification, genetic modification, and / or transduction. Thus, in exemplary embodiments of any method provided herein, the lymphoproliferative element may be delivered from the genome of a retroviral particle in genetically modified and / or transductioned T cells and / or NK cells, resulting in those cells having the increased proliferation and / or survival characteristics disclosed in the lymphoproliferative element section herein. In exemplary embodiments of any method provided herein, genetically modified T cells or NK cells can be engrafted in vivo in mice and / or enriched in mice in vivo for at least 7, 14, or 28 days. Those skilled in the art will recognize that such mice can be treated or otherwise genetically modified so that any immunological differences between genetically modified T cells and / or NK cells do not result in an immune response induced in mice against any component of lymphocytes transduced by recombinant retroviral particles lacking replication ability.

[0220] In the contact step, for example, when the cells and retroviral particles are first brought into contact, or during a subsequent optional incubation period with the reaction mixture containing the retroviral particles and cells in a suspension in a culture medium in any embodiment provided herein, or during cell culture and / or various washing steps in any embodiment provided herein, the culture medium may include a basic medium such as a commercially available medium for ex vivo T cell and / or NK cell culture. Such culture media include, but are not limited to, X-VIVO® 15 Chemically Defined, Serum-free Hematopoietic Cell Medium (Lonza) (2018 catalog numbers BE02-060F, BE02-00Q, BE-02-061Q, 04-744Q, or 04-418Q), ImmunoCult®-XF T Cell Expansion Medium (STEMCELL Technologies) (2018 catalog number 10981), PRIME-XV® T Cell Expansion XSFM (Irvine Scientific) (2018 catalog number 91141), AIM V® Medium CTS® (therapeutic grade) (Thermo Fisher Scientific (hereinafter referred to as "Thermo Fisher")), or CTS® Optimizer® medium (Thermo This includes Fisher's 2018 catalog numbers A10221-01 (basic medium (bottle)), A10484-02 (supplement), A10221-03 (basic medium (bag)), A1048501 (basic medium and supplement kit (bottle)), and A1048503 (basic medium and supplement kit (bag)). Such media may be serum-free preparations of known composition manufactured in accordance with cGMP, as discussed herein for kit components. The media may be xeno-free and complete. In some embodiments, the basic media are approved by regulatory authorities for use in ex vivo cell processing, such as in FDA 510(k) approved devices.In some embodiments, the medium is a basic medium with or without the accompanying T cell expansion supplement, both available from Thermo Fisher (Waltham, MA) under catalog numbers A1048501 (CTS® OpTmizer® T Cell Expansion SFM, bottle format) or A1048503 (CTS® OpTmizer® T Cell Expansion SFM, bag format) of 2018. Additives such as human serum albumin, human AB+ serum, and / or serum derived from the subject may be added to the transduction reaction mixture. Co-cytokines such as IL2, IL7, or IL15, or those found in human serum, may be added to the transduction reaction mixture. In certain embodiments, dGTP may be added to the transduction reaction product.

[0221] In some embodiments of any method herein that includes the step of genetically modifying lymphocytes (e.g., T cells and / or NK cells), the cells may come into contact with retroviral particles without prior activation. In some embodiments of any method herein that includes the step of genetically modifying T cells and / or NK cells, the T cells and / or NK cells are not incubated on a substrate that adheres to monocytes for more than 4 hours in one embodiment, or more than 6 hours in another embodiment, or more than 8 hours in yet another embodiment, prior to transduction. In one exemplary embodiment, the T cells and / or NK cells are incubated overnight on an adhesive substrate to remove monocytes prior to transduction. In another embodiment, the method may include incubating the T cells and / or NK cells on an adhesive substrate that binds to monocytes for 30 minutes, 1 hour, or 2 hours prior to transduction. In another embodiment, the T cells and / or NK cells are not exposed to a step of removing monocytes by incubation on an adhesive substrate prior to the transduction step. In another embodiment, T cells and / or NK cells are not incubated with or exposed to bovine serum, such as cell culture bovine serum, such as fetal bovine serum, before or during the contact step, and / or the gene modification and / or transduction step.

[0222] Some or all steps of the modification methods provided herein, or the use of such methods, are carried out in a closed system. Thus, the reaction mixture formed by such methods, as well as the modified, genetically modified, and / or transduced lymphocytes (e.g., T cells and / or NK cells) produced by such methods, may be contained within such a closed system. A closed system is a cell processing system that is generally closed or completely sealed off from the environment outside the chamber, such as a room, or even a fume hood, conduits such as tubes in the system through which cells are processed and / or transported. One of the greatest risks to safety and control in cell processing procedures is the risk of contamination due to frequent exposure to the environment, as seen in conventional open cell culture systems. To mitigate this risk, especially in the absence of antibiotics, several commercial processes have been developed that focus on the use of disposable (single-use) equipment. However, even when used under sterile conditions, there is always a risk of contamination by opening flasks to take samples or adding additional growth medium. To overcome this problem, the methods provided herein, typically ex vivo methods, are typically carried out within a closed system. Such processes can be designed and operated so that the products are not exposed to the external environment. Material transfer is carried out via sterile connections, such as sterile tubing and sterile welded connections. Air for gas exchange can be provided via a gas permeable membrane and a 0.2 μm filter to prevent exposure to the environment. In some exemplary embodiments, the method is carried out on T cells, for example, to provide modified, and in exemplary embodiments, genetically modified T cells.

[0223] Such closed-system methods can be carried out using commercially available devices. Different closed-system devices may be used in different steps within the method, and cells may be moved between these devices using tubing and connections such as welds, Luer, spikes, or clave ports to prevent the cells or culture medium from being exposed to the environment. For example, blood may be collected in an IV bag or syringe containing an anticoagulant, and in some embodiments, transferred to a Sepax 2 device (Biosafe) for PBMC enrichment and isolation. In other embodiments, whole blood may be filtered and leukocytes collected using a leukocyte removal filter assembly. The isolated PBMCs or isolated leukocytes may be transferred to a chamber of a G-Rex device for optional activation, transduction, and optional amplification. Alternatively, collected blood may be transduced in a blood bag, e.g., within the bag from which it was collected. Finally, cells may be recovered using a Sepax 2 device and collected in another bag. This method can be carried out with any device or combination of devices adapted for closed-system T cell and / or NK cell production. Examples of such devices, though not limited to them, include the G-Rex device (Wilson Wolf), GatheRex (Wilson Wolf), Sepax 2 (Biosafe), WAVE Bioreactors (General Electric), CultiLife Cell Culture bags (Takara), PermaLife bags (OriGen), CliniMACS Prodigy (Miltenyi Biotec), and VueLife bags (Saint-Gobain). In exemplary embodiments, selective activation, transduction, and selective augmentation may be carried out within the same chamber or cell of a closed system. For example, in exemplary embodiments, the chamber may be the chamber of a G-Rex device, and PBMCs or leukocytes may be transferred to the chamber of the G-Rex device after they have been concentrated and isolated, and may remain within the same chamber of the G-Rex device until they are recovered.

[0224] Methods provided herein may include transferring blood and cells and / or fractions thereof, as well as lymphocytes before or after contact with retroviral particles, between tanks within a closed system, and thus without environmental exposure. The tanks used in the closed system may be, for example, tubes, bags, syringes, or other containers. In some embodiments, the tanks are those used in research facilities. In some embodiments, the tanks are those used in commercial production. In other embodiments, the tanks may be collection tanks used in a blood collection process. Methods for modifications herein typically include a contact step in which lymphocytes come into contact with recombinant retroviral particles that lack the ability to replicate. Contact in some embodiments may be carried out within a tank, for example, within a blood bag. Blood and various lymphocyte-containing fractions may be transferred from one tank to another (e.g., from a first tank to a second tank) within a closed system for contact. The second tank may be a cell processing compartment in a closed device, such as a G-Rex device. In some embodiments, after contact, modification, and in exemplary embodiments, genetically modified (e.g., transduced) cells may be transferred to different tanks within a closed system (i.e., without exposure to the environment). Either before or after this transfer, the cells are typically washed within the closed system to remove substantially all or all retroviral particles. In some embodiments, the processes disclosed herein, from blood collection to contact (e.g., transduced), optional incubation, and post-incubation isolation and optional washing, are carried out for a period of 15 minutes, 30 minutes, or 1, 2, 3, or 4 hours at the lower end of the range to 4, 8, 10, or 12 hours at the upper end of the range.

[0225] Various embodiments of this method, as well as other aspects such as the use of NK cells and T cells produced by such methods, are disclosed in detail herein. Furthermore, various elements or steps of embodiments of such methods for transducing, genetically modifying and / or modifying PBMCs, lymphocytes, T cells and / or NK cells are provided herein, for example in this section and the section on exemplary embodiments, and such methods include embodiments provided throughout this Spec. For example, any embodiment of the embodiments for transducing, genetically modifying and / or modifying PBMCs or lymphocytes, for example NK cells, or, in exemplary embodiments, T cells, provided in this section and the section on exemplary embodiments, may include any embodiment of non-replicating recombinant retroviral particles provided herein, comprising one or more lymphocyte proliferation elements, CARs, pseudotyping elements, riboswitches, activating elements, membrane-bound cytokines, miRNAs, Kozak-type sequences, WPRE elements, triple stop codons, and / or other elements disclosed herein, which may be combined with the method herein for producing retroviral particles using packaging cells. In certain exemplary embodiments, the retroviral particles are lentiviral particles. Such methods for modifying, genetically modifying, and / or transducing PBMCs or lymphocytes such as T cells and / or NK cells may be carried out in vitro or ex vivo. Those skilled in the art will recognize that the details provided herein for transducing, genetically modifying, and / or modifying PBMCs or lymphocytes such as T cells and / or NK cells may be applicable to any embodiment including such steps.

[0226] The modifications in the methods provided herein, and in exemplary embodiments, the administration and re-administration of genetically modified lymphocytes to a subject, also referred to herein, may be via any route known in the art. Such introduction or re-introduction typically involves suspending i) modified, and / or ii) genetically modified, and / or iiia) transduced or iiib) transfected cells in a delivery solution to form a cell preparation that can be introduced or re-introduced to a subject, as will be discussed in more detail herein. For example, introduction or re-introduction may be delivered by injection into the blood vessels of the subject. In some embodiments, modified lymphocytes (e.g., T cells and / or NK cells) are introduced or re-introduced to a subject by intramuscular administration, or, in exemplary embodiments, by subcutaneous administration.

[0227] Some administered cells are modified with nucleic acids encoding lymphoproliferative elements. In exemplary, but not limited, ways, the ex vivo delivery of polynucleotides encoding lymphoproliferative elements to resting T cells and / or NK cells (which may be incorporated into the genome of the T cells or NK cells) provides the cells with an in vivo propensity for growth without requiring the host to lymphocyte depletion. Thus, in exemplary embodiments, the subjects are not exposed to lymphocyte depletion agents within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days from the implementation of contact, or within 1, 2, 3, 3, or 6 months, during contact, 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 modified T cells and / or NK cells have been reintroduced back into the subjects. Furthermore, in exemplary embodiments, the methods provided herein may be carried out without exposing the subject to a lymphocyte depletion agent during the step in which non-replicating recombinant retroviral particles are in contact with the subject's resting T cells and / or resting NK cells, and / or throughout the entire ex vivo method. Thus, methods for modifying a subject in vivo, and in exemplary embodiments, for increasing genetically modified T cells and / or NK cells, are characteristic of some embodiments of the present disclosure. In exemplary embodiments, such methods are ex vivo, or substantially, without reproduction.

[0228] The entire method / process, from blood collection from a subject to modification of the subject after ex vivo transduction of T cells and / or NK cells, and, in exemplary embodiments, reintroduction of genetically modified lymphocytes, may be carried out over a period of less than 48 hours, less than 36 hours, less than 24 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, 2 hours, or less than 2 hours, in any exemplary embodiment, not limited to any aspect, provided herein. In any embodiment disclosed herein, the introduction or reintroduction of modified lymphocytes may be carried out by intravenous injection, subcutaneous administration, or intramuscular administration. In other embodiments, the entire method / process from blood collection / blood sampling from a subject to the reintroduction of modified lymphocytes into the subject after ex vivo transduction of T cells and / or NK cells is carried out over periods of 1 to 12 hours, 2 to 8 hours, 1 to 3 hours, 2 to 4 hours, 2 to 6 hours, 4 to 12 hours, 4 to 24 hours, 8 to 24 hours, 8 to 36 hours, 8 to 48 hours, 12 to 24 hours, 12 to 36 hours, or 12 to 48 hours, over periods of 15, 30, 60, 90, 120, 180, and 240 minutes at the lower end of the range to 120, 180, and 240, 300, 360, 420, and 480 minutes at the upper end of the range. In other embodiments, the entire method / process from blood collection / blood sampling from a subject to ex vivo gene transfer modification of T cells and / or NK cells, and in exemplary embodiments, reintroduction of the genetically modified lymphocytes into the subject, is carried out over a period of 1, 2, 3, 4, 6, 8, 10, and 12 hours at the lower end of the range to 8, 9, 10, 11, 12, 14, 18, 24, 36, or 48 hours at the upper end of the range. In some embodiments, the modified and genetically modified T cells and / or NK cells are isolated from unassociated, non-replicating recombinant retroviral particles after the period in which contact takes place.

[0229] The methods provided herein for modifying lymphocytes, and related methods for carrying out adoptive cell therapy, can be carried out in significantly shorter timeframes than previous methods, thus enabling fundamental improvements in patient care and safety, as well as product manufacturability. Therefore, such processes are expected to be advantageous from the perspective of regulatory bodies responsible for approving such processes when performed in vivo for therapeutic purposes. For example, in an example not limited to any embodiment provided herein, including a subject, the subject may remain in the same building (e.g., an infusion clinic) or room as the equipment processing the blood or sample while the sample is being processed before the modified T cells and / or NK cells are reintroduced into the patient. In an exemplary embodiment not limited to any example, the subject remains within the facility's lines and / or within 100, 50, 25, or 12 feet or arms of the blood or cells being processed throughout the entire method / process from blood collection / blood sampling from the subject to the reintroduction of the blood into the subject after ex vivo transduction of T cells and / or NK cells. In other, not-limited, exemplary embodiments, the subject remains awake and / or at least one person can continue to monitor the subject's blood or cells being treated throughout the entire method / process from blood collection to the reintroduction of blood into the subject after ex vivo transduction of T cells and / or NK cells. With the improvements provided herein, adoptive cell therapy, and / or the entire method / process for transducing resting T cells and / or NK cells from blood collection from the subject to the reintroduction of blood into the subject after ex vivo transduction of T cells and / or NK cells, can be carried out with continuous human monitoring. In other, not-limited, exemplary embodiments, at any point in the entire method / process from blood collection from the subject to the reintroduction of blood into the subject after ex vivo transduction of T cells and / or NK cells, the blood cells are not incubated in a room without human presence.In other, not-limited, exemplary embodiments, the entire method / process from blood collection / blood sampling from the subject to the reintroduction of the blood into the subject after ex vivo transduction of T cells and / or NK cells is carried out next to the subject, and / or in the same room as the subject, and / or next to the subject's bed or chair. Thus, sample misidentification, as well as long and costly incubations lasting several days or weeks, can be avoided. This is further provided by the fact that the method provided herein is readily adaptable to closed and automated blood processing systems in which the blood sample and its components to be reintroduced into the subject come into contact only with disposable, single-use components.

[0230] Methods for modifying, genetically modifying, and / or transducing lymphocytes such as T cells and / or NK cells provided herein may be part of a method for carrying out adoptive cell therapy. Typically, a method for carrying out adoptive cell therapy includes the steps of collecting blood from a subject and returning modified, genetically modified, and / or transduced lymphocytes (e.g., T cells and / or NK cells) to the subject. This disclosure provides various treatment methods using CARs. The CARs of this disclosure, when present in T lymphocytes or NK cells, can mediate cytotoxicity against target cells. The CARs of this disclosure bind to antigens present on the target cells, thereby mediating the killing of target cells by T lymphocytes or NK cells genetically modified to produce CARs. The ASTR of the CAR binds to antigens present on the surface of target cells. This disclosure provides a method for killing or inhibiting the growth of target cells, the method comprising contacting cytotoxic immune effector cells (e.g., cytotoxic T cells or NK cells) that have been genetically modified to produce a target CAR so that T lymphocytes or NK cells recognize an antigen present on the surface of the target cells and mediate the killing of the target cells. The target cells may be, for example, cancer cells, and the autologous cell therapy herein may, in some exemplary embodiments, be a method for treating cancer. In these embodiments, the subject may be an ani...

Claims

1. The use of non-replicating recombinant retroviral particles in the manufacture of a kit for administering a cell therapy, wherein the use of the kit is a) Ex vivo contact of blood cells, including T cells and / or NK cells, with non-replicating recombinant retrovirus particles in a reaction mixture containing a T cell and / or NK cell activating element, wherein the non-replicating recombinant retrovirus particles i) Binding polypeptides and fusion polypeptides on the surface of the non-replicating recombinant retrovirus particles, wherein the binding polypeptides can bind to T cells and / or NK cells, and the fusion polypeptides can mediate the fusion of the retrovirus particle membrane with the T cell and / or NK cell membrane, and ii) A polynucleotide comprising one or more transcription units, each of which is operably linked to an active promoter in T cells and / or NK cells, wherein the one or more transcription units encode a first polypeptide comprising a chimeric antigen receptor (CAR), The aforementioned contact promotes the association of the T cells and / or NK cells with the non-replicating recombinant retroviral particles, and the non-replicating recombinant retroviral particles modify the T cells and / or NK cells, b) Subcutaneously administering the cell preparation to the subject, wherein the cell preparation comprises the modified T cells and / or NK cells, i) The reaction mixture contains at least 25% by volume of unfractionated whole blood, ii) The reaction mixture contains neutrophils and / or iii) Use comprising administering the modified T cells and / or NK cells subcutaneously in a delivery solution together with neutrophils.

2. A cell preparation comprising modified T cells and / or NK cells, wherein the modified T cells and / or NK cells are suspended in a delivery solution, and one or both are a) Genetically modified with polynucleotides containing one or more transcription units, each of which is operably linked to an active promoter in T cells and / or NK cells, or b) Associating with recombinant retroviral particles containing the polynucleotide and lacking replication ability, A cell preparation wherein one or more transcription units encode a first polypeptide comprising a chimeric antigen receptor (CAR), the cell preparation has a volume of 2 mL to 10 mL and further contains neutrophils, and the cell preparation is contained in a syringe.

3. The use of non-replicating recombinant retroviral particles in the manufacture of a kit for administering modified T cells and / or NK cells, wherein the use of the kit is Use comprising subcutaneously administering a cell preparation containing the modified T cells and / or NK cells to the subject, wherein one or both of the modified T cells and / or NK cells are a) genetically modified with a polynucleotide comprising one or more transcription units, each of which is operably linked to an active promoter in the T cells and / or NK cells, or b) associated with a non-replicating recombinant retroviral particle comprising the polynucleotide, wherein the one or more transcription units encode a first polypeptide comprising a chimeric antigen receptor (CAR), and at least one of neutrophils, B cells, monocytes, basophils, and eosinophils is subcutaneously administered in the cell preparation together with the modified T cells and / or NK cells.

4. A method for preparing cell preparations, a) Ex vivo contact of blood cells, including T cells and / or NK cells, with non-replicating recombinant retroviral particles in a reaction mixture containing a T cell and / or NK cell activating element, wherein the non-replicating recombinant retroviral particles i) Binding polypeptides and fusion polypeptides on the surface of the non-replicating recombinant retrovirus particles, wherein the binding polypeptides can bind to T cells and / or NK cells, and the fusion polypeptides can mediate the fusion of the retrovirus particle membrane with the T cell and / or NK cell membrane, and ii) A polynucleotide comprising one or more transcription units, each of which is operably linked to an active promoter in T cells and / or NK cells, wherein the one or more transcription units encode a first polypeptide comprising a chimeric antigen receptor (CAR), The contact described above promotes the association of the T cells and / or NK cells with the non-replicating recombinant retroviral particles, the non-replicating recombinant retroviral particles modify the T cells and / or NK cells, and the reaction mixture contains neutrophils. b) Collecting the modified T cells and / or NK cells in a delivery solution to form a cell preparation containing a suspension of the modified T cells and / or NK cells, c) A method comprising transferring 0.5 mL to 10 mL of the cell preparation into a syringe.

5. Use of a population of modified T cells and / or NK cells in the manufacture of a kit for subcutaneous or intramuscular delivery to a subject, wherein the use of the kit comprises subcutaneous delivery to the subject of 0.2 to 10 mL of a cell preparation containing the modified T cells and / or NK cells, wherein the modified T cells and / or NK cells are genetically modified with polynucleotides comprising one or more transcription units, each of the one or more transcription units being operably linked to an active promoter in the T cells and / or NK cells, and the one or more transcription units encoding a first polypeptide comprising a chimeric antigen receptor (CAR) and a second polypeptide comprising a lymphoproliferative element comprising an intracellular signaling domain from a cytokine receptor.

6. The use, method, or cell preparation according to any one of claims 1 to 5, wherein the one or more transcription units encode a second polypeptide comprising a lymphoproliferative element, the lymphoproliferative element comprising an intracellular signaling domain from a cytokine receptor, and optionally activating the Janus kinase / signaling and transcriptional activator (JAK / STAT) pathway or the tumor necrosis factor receptor (TNF-R)-related factor (TRAF) pathway.

7. The use or method according to claim 6, wherein the lymphoproliferative element is constitutively active and comprises Box1 and Box2 JAK binding motifs, and a STAT binding motif containing a tyrosine residue.

8. The use or method according to claim 6, wherein the lymphoproliferative element does not contain an extracellular ligand-binding domain or a small molecule-binding domain.

9. The use according to claim 1 or 3, wherein neutrophils are present in the cell preparation, and as a result, at least 10% of the administered cells are neutrophils.

10. The use according to any one of claims 1 to 4, wherein the modified T cells and / or NK cells are administered subcutaneously in the presence of hyaluronidase.

11. The use according to claim 1 or 3, wherein the modified T cells and / or NK cells are administered subcutaneously in a volume of 1 mL to 5 mL of the cell preparation.

12. The use according to claim 1 or 3, wherein the modified T cells and / or NK cells are reintroduced into the subject within 14 hours from the time the peripheral blood-derived product containing the T cells and / or NK cells is collected from the subject.

13. The use or method according to claim 1 or 4, wherein the reaction mixture comprises an anticoagulant, and the T cells and / or NK cells are present in the unfractionated whole blood from the subject when they come into contact.

14. 1 x 10 6 ~1 x 10 9 The use or method according to claim 1 or 3, wherein individual modified T cells and / or modified NK cells are delivered subcutaneously to the subject.

15. The use or method according to claim 1 or 4, wherein the reaction mixture contains at least 50% by volume of unfractionated whole blood.

16. The use or method according to claim 1 or 4, wherein the reaction mixture is in a closed cell processing system, the contact occurs when the reaction mixture is in a leukocyte removal filter assembly of the closed cell processing system, and the blood cells in the reaction mixture are totally nucleated cells (TNCs).

17. The use or method according to claim 16, wherein the T cell and / or NK cell activating element is on the surface of the non-replicating recombinant retroviral particles, the contact is performed at 2°C to 15°C and optionally at 2°C to 6°C for less than 1 hour, optionally thereafter the TNC is incubated at 32°C to 42°C for 5 minutes to 4 hours, and optionally thereafter the modified T cells and / or NK cells are collected on a filter to form the cell preparation.

18. The use or method according to claim 1 or 4, wherein the reaction mixture comprises at least 25% by volume of unfractionated whole blood and an effective amount of an anticoagulant.

19. The use or method according to claim 18, wherein the anticoagulant is selected from the group consisting of dextrose acid citrate, EDTA, and heparin.

20. The use or method according to claim 18, wherein the anticoagulant is other than dextrose acid citrate.

21. The use or method according to claim 18, wherein the anticoagulant comprises an effective amount of heparin.

22. The use or method according to claim 1 or 4, wherein the modified cell is a modified T cell, the activating element is a T cell activating element, and the T cell activating element is one or more polypeptides that bind to anti-CD3 antibody, anti-CD28 antibody, or mitogen tetraspanin.

23. The use or method according to claim 22, wherein the T cell activating element is an anti-CD3 antibody, and the anti-CD3 antibody is bound to the membrane of the recombinant retrovirus particle lacking replication ability.

24. The use or method according to claim 23, wherein the membrane-bound anti-CD3 antibody is anti-CD3 scFv or anti-CD3 scFvFc.

25. The use or method according to claim 23, wherein the anti-CD3 antibody is bound to the membrane by a GPI anchor and is a recombinant fusion protein having a MuLV virus envelope protein having or not having a mutation at the furin cleavage site, or the anti-CD3 antibody is a recombinant fusion protein having a VSV virus envelope protein.

26. The use or method according to claim 1 or 4, wherein the recombinant retrovirus particles lacking replication ability are present in the reaction mixture at an MOI of 2.5 to 5.

27. The use or method according to claim 1 or 4, wherein the cells (or cells) are not subjected to spinoculation during the method.

28. The use or method according to claim 1 or 4, wherein the reaction mixture is present in the blood bag during the contact.

29. The use or method according to claim 1 or 4, wherein the blood cells are in contact with a leukocyte removal filter assembly in a closed cell processing system, during the contact which includes optionally incubating in the reaction mixture while the blood cells are in contact with recombinant retroviral particles prior to the contact, and / or after the contact which includes optionally incubating in the reaction mixture.

30. The use or method according to claim 1 or 4, wherein the reaction mixture is in contact with a leukocyte removal filter assembly in a closed cell processing system after the contact.

31. The use or method according to claim 1 or 4, wherein the unfractionated whole blood is other than umbilical cord blood.

32. The use or method according to claim 1 or 4, wherein the contact is carried out for less than 12 hours, and thereafter the retroviral particles remaining in the suspension in the reaction mixture are separated from the cells.

33. The use, method, or cell preparation according to any one of claims 1 to 4, wherein the CAR is MRB-CAR.

34. The promoter operably linked to the first transcription unit is constitutively active, and the non-replication recombinant retroviral particle further comprises a second transcription unit operably linked to an inducible promoter in at least one of T cells or NK cells, wherein the first transcription unit and the second transcription unit are arranged in opposite directions. The use or method according to claim 1 or 4, wherein the second transcription unit encodes a lymphoproliferative element.

35. The use or method according to claim 1 or 4, wherein the recombinant retrovirus particle lacking replication ability is a lentiviral particle, and the modified cell is a modified T cell.

36. The cell preparation according to claim 2, wherein the modified T cells and / or NK cells are genetically modified with the polynucleotide, and the polynucleotide is a nonviral vector.

37. a) At least 25%, or optionally at least 50%, of the modified T cells and / or NK cells in the cell preparation do not express one or more of the CAR or transposases. b) At least 25%, or optionally at least 50%, of the modified T cells and / or NK cells in the cell preparation comprises recombinant viral reverse transcriptase or recombinant viral integrase, c) At least 25%, or optionally at least 50%, of the modified T cells and / or NK cells in the cell preparation lack the polynucleotides stably incorporated into their genomes. d) 1% to 20%, or optionally 5% to 15%, of the T cells and / or NK cells in the cell preparation are genetically modified, and / or e) The cell preparation according to claim 2, wherein at least 25%, or optionally at least 50%, of the modified T cells and / or modified NK cells in the cell preparation are viable.

38. The cell preparation according to claim 2, wherein at least 5% of the modified T cells and / or NK cells in the cell preparation are genetically modified.

39. A kit for modifying NK cells and / or T cells, One or more vessels containing a polynucleotide comprising a first transcription unit operably linked to an active promoter in T cells and / or NK cells, wherein the first transcription unit encodes a first polypeptide comprising a chimeric antigen receptor (CAR), and one or more accessory components, a) One or more containers containing a delivery solution adapted for subcutaneous or intramuscular administration, b) One or more sterile syringes adapted for subcutaneous or intramuscular delivery of T cells and / or NK cells, c) A kit comprising one or more auxiliary components selected from one or more leukocyte removal filtration assemblies.

40. The kit according to claim 39, wherein the polynucleotide in one or more containers containing the polynucleotide encoding the CAR is located within a recombinant retrovirus particle that lacks replication ability.

41. The kit according to claim 40, wherein the non-replicating recombinant retroviral particle comprises a polynucleotide comprising one or more transcription units operably linked to an active promoter in T cells and / or NK cells, the one or more transcription units encoding a first polypeptide comprising the CAR.

42. The kit according to claim 40, wherein a recombinant retrovirus particle lacking replication ability comprises a binding polypeptide and a fusion polypeptide on its surface, the binding polypeptide being able to bind to T cells and / or NK cells, and the fusion polypeptide being able to mediate the fusion of the retrovirus particle membrane with the T cell and / or NK cell membrane.

43. The kit according to claim 42, wherein the surface of the recombinant retroviral particles lacking replication ability further comprises an activating element, the activating element being capable of activating T cells and / or NK cells.

44. The kit according to claim 41, wherein one or more containers containing the non-replicating retrovirus particles contain substantially pure GMP-grade non-replicating retrovirus particles.

45. Each container containing the retrovirus particles that lack replication ability has a volume of 0.1 mL to 10 mL and 1 × 10 6 ~5 x 10 9 The kit according to claim 44, comprising a retroviral particle trait introduction unit.

46. The kit according to claim 45, wherein the kit comprises one or more containers containing a delivery solution adapted for subcutaneous administration.

47. The kit according to claim 39 or 40, wherein the kit comprises one or more leukocyte removal filtration assemblies.

48. The kit according to claim 39 or 40, wherein the kit comprises one or more sterile syringes adapted for subcutaneous delivery of T cells and / or NK cells.

49. The aforementioned kit, a) One or more containers containing a delivery solution adapted for subcutaneous administration, b) The kit according to claim 39 or 40, comprising one or more sterile syringes adapted for subcutaneous delivery of T cells and / or NK cells.

50. The kit according to claim 39 or 40, wherein the polynucleotide comprising a first transcription unit encoding a first polypeptide comprising a CAR further comprises a second transcription unit encoding a second polypeptide comprising a lymphoproliferative element, the lymphoproliferative element comprising an intracellular signaling domain from a cytokine receptor that activates the JAK / STAT pathway and the TRAF pathway.

51. The kit according to claim 50, wherein the lymphoproliferative element is constitutively active and comprises a BOX 1 and BOX 2 JAK binding motif, and a STAT binding motif containing a tyrosine residue.

52. The kit according to claim 51, wherein the lymphoproliferative element does not contain cytokines.

53. An isolated polynucleotide comprising a first transcription unit operably linked to an inducible promoter in at least one of T cells or NK cells, and a second transcription unit operably linked to a constitutive T cell or NK cell promoter, wherein the first transcription unit and the second transcription unit are branched and arranged In the first transcription unit, a lymphoproliferative element is encoded, An isolated polynucleotide comprising, in the second transcription unit, encoding a chimeric antigen receptor (CAR), wherein the CAR includes an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain.

54. Recombinant retroviral particles lacking replication ability, comprising isolated polynucleotides as described in claim 53.

55. The polynucleotide according to claim 53 or the recombinant retroviral particle without replication ability according to claim 54, wherein the insulator is located between the branched transcription units.

56. A container comprising, in a substantially pure formulation, isolated polynucleotides according to claim 53 or recombinant retroviral particles lacking replication ability according to claim 54.

57. The container according to claim 56, and the following accessory components: a) One or more containers containing a delivery solution adapted, suitable for, and / or effective for intravenous, subcutaneous, and / or intramuscular administration. b) One or more containers of hyaluronidase, c) One or more blood bags, d) One or more sterile syringes, e) One or more leukocyte removal filtration assemblies, f) One or more containers containing a solution or culture medium suitable for transduction of T cells and / or NK cells, g) One or more containers containing a solution or culture medium suitable for rinsing T cells and / or NK cells, h) One or more containers containing substantially pure nucleic acids encoding a second CAR directed to different target epitopes on different antigens found on the same target cancer cells as the first CAR, i) One or more containers containing a congener antigen for the first CAR, or j) A kit comprising one or more instructions for the use of other kit components, either physically or digitally, for modifying T cells and / or NK cells.

58. Genetically modified T cells or NK cells, produced by genetically modifying the T cells or NK cells according to a method comprising ex vivo contact of the T cells or NK cells with the isolated polynucleotides described in claim 53 or the recombinant retroviral particles lacking replication ability described in claim 54.

59. The use of non-replicating recombinant retroviral particles in the manufacture of a kit for modifying target T cells and / or NK cells, wherein the use of the kit is The use comprises ex vivo contacting the T cells or NK cells with the non-replicating recombinant retroviral particles described in claim 54, wherein the contact promotes association between the T cells or NK cells and the non-replicating recombinant retroviral particles, thereby modifying the T cells or NK cells.

60. A method for modifying T cells or NK cells, comprising ex vivo contacting the T cells or NK cells with non-replicating recombinant retroviral particles as described in claim 54, wherein the contact promotes association between the T cells or NK cells and the non-replicating recombinant retroviral particles, thereby modifying the T cells or NK cells.

61. The polynucleotide according to claim 53 or the non-replicating recombinant retroviral particle according to claim 54, wherein the constitutive T cell or NK cell promoter comprises an EF-1a promoter, a PGK promoter, a CMV promoter, an MSCV-U3 promoter, an SV40hCD43 promoter, a VAV promoter, a TCRbeta promoter, or a UBC promoter.

62. The polynucleotide according to claim 53 or the recombinant retroviral particle without replication ability according to claim 54, wherein the inducible promoter comprises an NFAT-responsive promoter.

63. The polynucleotide or non-replicating recombinant retroviral particle according to claim 62, wherein the NFAT-responsive promoter comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 NFAT-binding sites, the NFAT-binding sites comprising functional sequence variants that retain the ability to bind NFAT, and the NFAT-responsive promoter comprises a minimal constitutive promoter having an upstream NFAT-binding site that has low levels of transcription even in the absence of an inductive signal.

64. The polynucleotide according to claim 53 or the recombinant retroviral particle without replication ability according to claim 54, wherein the transcription of the lymphoproliferative element is less than 1 / 2, 1 / 4, 1 / 5, 1 / 10, 1 / 25, 1 / 50, 1 / 100, 1 / 200, 1 / 250, 1 / 500, or 1 / 1,000 of the transcription level of the CAR in the absence of an inductive signal.

65. The recombinant retrovirus particle lacking replication ability further comprises on its surface an activated polypeptide, a binding polypeptide, and a fusion polypeptide, wherein the activated polypeptide can activate T cells and / or NK cells, the binding polypeptide can bind to T cells and / or NK cells, and the fusion polypeptide can mediate the fusion of the retrovirus particle membrane with the T cell and / or NK cell membrane, according to claim 54.

66. The polynucleotide according to claim 53 or the non-replicating recombinant retroviral particle according to claim 54, wherein the lymphoproliferative element comprises an intracellular signaling domain from a cytokine receptor that activates the Janus kinase / signaling and transcription activator (JAK / STAT) pathway or the tumor necrosis factor receptor (TNF-R)-related factor (TRAF) pathway.

67. The polynucleotide or non-replicating recombinant retroviral particle according to claim 66, wherein the lymphoproliferative element is constitutively active and comprises Box 1 and Box 2 JAK binding motifs, and a STAT binding motif containing a tyrosine residue.

68. The lymphoproliferative element is a polynucleotide or a recombinant retroviral particle lacking replication ability according to claim 67, wherein the lymphoproliferative element does not contain cytokines.

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