Methods of producing autologous t-cells useful for the treatment of b-cell malignancies and other cancers, and compositions thereof

Abstract

To provide a method for producing a T cell expressing a cell surface receptor recognizing a specific antigen part on the surface of a target cell.SOLUTION: The method comprises (1) enriching a population of lymphocytes obtained from a donor subject, (2) stimulating the population of lymphocytes with one or more T cell stimulatory agents in a closed system using a serum-free culture medium to produce a population of activated T cells, and (3) transducing the population of activated T cells with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor using a single cycle of transduction to produce a population of transduced T cells. Transduction is performed in a closed system using serum-free culture medium; and (4) expanding the population of transduced T cells over a predetermined time to produce a population of engineered T cells, wherein the expansion is performed in a closed system using serum-free culture medium.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 935,833, filed February 4, 2014, which is incorporated herein by reference in its entirety.

[0002] [Statement of United States Government Interests] This invention was created as a result of a Cooperative Research and Development Agreement with the National Cancer Institute (NCI), an Agency of the Department of Health and Human Services. The United States Government has certain rights in this invention. [Background technology]

[0003] The process of producing engineered autologous T cells for use in cancer treatment is lengthy (10-24 days), involves two cycles of retroviral transduction, and is not well suited for commercial use (see Non-Patent Documents 1 and 2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Kochenderfer et al., Blood. 2012 119: 2709-2720 [Non-patent document 2] Johnson, et al., Blood. 2009; 114(3):535-546 Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, it is desirable to make improvements to the T cell manufacturing process to overcome these limitations. [Means for solving the problem]

[0006] According to certain embodiments described herein, there is provided a method for producing T cells expressing a cell surface receptor that recognizes a specific antigen moiety on the surface of a target cell. In certain embodiments, there is provided a method for producing T cells expressing a cell surface receptor that recognizes a specific antigen moiety on the surface of a target cell, the method comprising: enriching a population of lymphocytes obtained from a donor subject; stimulating the population of lymphocytes with one or more T cell stimulants to produce a population of activated T cells, wherein the stimulation is performed in a closed system using a serum-free culture medium; transducing the population of activated T cells with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor using a single cycle of transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using a serum-free culture medium; and expanding the population of transduced T cells for a predetermined period of time to produce a population of engineered T cells, wherein the expansion is performed in a closed system using a serum-free culture medium. In certain embodiments, the cell surface receptor may be a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In certain embodiments, the target cell may be a cancer cell. In certain embodiments, the cancer cell may be a B-cell malignancy. In certain embodiments, the cell surface receptor may be an anti-CD19 CAR. In certain embodiments, the anti-CD19 CAR may be an FMC63-28Z CAR or an FMC63-CD828BBz CAR. In certain embodiments, the one or more T cell stimulators may be an anti-CD3 antibody and IL-2. In certain embodiments, the viral vector may be a retroviral vector. In certain embodiments, the retroviral vector may be an MSGV1 gamma retroviral vector. In certain embodiments, In certain embodiments, the MSGV1 gammaretroviral vector may be an MSGV-FMC63-28Z or MSGV-FMC63-CD828BBz gammaretroviral vector. In certain embodiments, the predetermined time for expanding the population of transduced T cells may be three days. In certain embodiments, the time from enrichment of the lymphocyte population to production of engineered T cells may be six days. In certain embodiments, the engineered T cells may be used to treat cancer patients. In certain embodiments, the cancer patient and the donor subject may be the same individual. In certain embodiments, the closed system may be a closed-bag system. In certain embodiments, the population of cells may comprise naive T cells. In certain embodiments, between about 35% and 43% of the population of engineered T cells may comprise naive T cells. In certain embodiments, at least about 35% of the population of engineered T cells may comprise naive T cells. In certain embodiments, at least about 43% of the population of engineered T cells may comprise naive T cells.

[0007] According to embodiments described herein, there is provided a population of engineered T cells expressing a cell surface receptor that recognizes a specific antigen moiety on the surface of a target cell, produced by the methods disclosed herein. In certain embodiments, there is provided a method comprising enriching a population of lymphocytes obtained from a donor subject; stimulating the population of lymphocytes with one or more T cell stimulants to produce a population of activated T cells, wherein the stimulation is performed in a closed system using a serum-free culture medium; transducing the population of activated T cells with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor using a single cycle of transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using a serum-free culture medium; and expanding the population of transduced T cells for a predetermined period of time to produce a population of engineered T cells, wherein the expansion is performed in a closed system using a serum-free culture medium. In certain embodiments, the population of engineered T cells may be any of those described herein.

[0008] According to certain embodiments described herein, pharmaceutical compositions comprising a population of engineered T cells are provided. Provided herein, in certain embodiments, are pharmaceutical compositions comprising a population of engineered T cells described herein. In certain embodiments, the pharmaceutical composition may comprise a therapeutically effective dose of the engineered T cells. In certain embodiments, the cell surface receptor may be a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In certain embodiments, the CAR may be an FMC63-28Z CAR or an FMC63-CD828BBZ CAR. In certain embodiments, a therapeutically effective dose may be greater than about 1 million and less than about 3 million engineered T cells per kilogram of body weight (cells / kg). In certain embodiments, a therapeutically effective dose may be about 2 million engineered T cells / kg.

[0009] According to certain embodiments described herein, there is provided a method of producing T cells, comprising obtaining a population of lymphocytes, stimulating the population of lymphocytes with one or more stimulatory agents to produce a population of activated T cells, wherein the stimulation is performed in a closed system using a serum-free culture medium, transducing the population of activated T cells with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor using at least one cycle of transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using a serum-free culture medium, and expanding the population of transduced T cells to produce a population of engineered T cells, wherein the expansion is performed in a closed system using a serum-free culture medium. In certain embodiments, the population of engineered T cells can be any of those described herein. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 illustrates a T cell manufacturing process (the "improved" process) according to certain embodiments described herein. Because T cell doubling times can vary slightly from subject to subject, additional expansion time beyond 72 hours in the bag (i.e., 3-6 days) is considered if the total cell number is insufficient to deliver the desired target dose (see *). [Figure 2] FIG. 1 illustrates an improved process compared to a conventionally used process (the "conventional" process) according to one embodiment. [Figure 3] 1 is a bar graph illustrating culture expansion in an improved process compared to a conventional process according to one embodiment. The y-axis shows the fold expansion of cells in each of the five trials (x-axis). The culture expansion fold is similar for the conventional and improved processes in the large-scale operation trials. [Figure 4]FIG. 4A shows a series of graphs illustrating T cell phenotypes at days 6 and 10 for the conventional and improved processes, according to one embodiment, for CD3+ cell phenotype (FIG. 4A) and CD3+ cell activation (FIG. 4B), with the markers indicated. T cell phenotypes are comparable between the conventional and improved processes, but cells at day 6 are more undifferentiated. Teff = effector T cells, Tem = effector memory T cells, Tcm = central memory T cells. [Figure 5] FIG. 1 shows a series of graphs illustrating cell phenotypes at day 6 for conventional and improved processes, according to one embodiment. [Figure 6] FIG. 1 is a schematic diagram showing daily cell counts during the stimulation, transduction, and expansion phases of the improved process, according to one embodiment. [Figure 7] FIG. 1 shows the nucleic acid sequence of the MSGV1 gammaretroviral backbone (SEQ ID NO: 4), according to one embodiment. [Figure 8] Figure 1 shows transduction efficiency as a function of RetroNectin® concentration used to coat the bags, according to one embodiment. RN = RetroNectin® concentration in μg / mL. Results were measured 6 days after transduction in PL07 bags from two donors. [Figure 9] Figure 1 shows transduction efficiency with and without a washing step, according to one embodiment. Results were measured 6 days after transduction in Origen PermaLife™ bags. [Figure 10] Figure 1 shows the effect of RetroNectin® concentration on transduction efficiency in OpTmizer™ medium, according to one embodiment. RN = RetroNectin® concentration in μg / mL. "Open" indicates conditions in which transduction was performed in plates in AIM V® + 5% human serum. [Figure 11]

[0023] Figure 1 shows the activity of transduced T cells as measured by CD107a and IFN-gamma expression after 4 hours of co-incubation with CD19+Nalm6 cells as assessed by FACS, according to one embodiment. "Open" indicates conditions in which transduction was performed in plates in AIM V® + 5% human serum. Control T indicates a reference sample of frozen CAR-positive transduced PBMCs. [Figure 12] 1 shows the temperature profile (lower line) and product temperature (upper line) of a rate-controlled freezing chamber for an optimized profile. The displayed profile has been shortened to show only the critical region. DETAILED DESCRIPTION OF THE INVENTION

[0011] According to embodiments described herein, methods or processes for producing T cell preparations that may be useful in treating patients with pathological diseases or conditions are provided. In contrast to known production methods for T cell products, the methods and processes described herein can be completed in a significantly shorter time of approximately six days, thereby accelerating the delivery of cells to the clinic. Also provided herein are populations of engineered T cells produced using the methods described herein, and pharmaceutical compositions thereof.

[0012] In certain embodiments, the described methods can be used to produce T cells expressing cell surface receptors that recognize specific antigenic moieties on the surface of target cells. The cell surface receptor can be a wild-type or recombinant T cell receptor (TCR), a chimeric antigen receptor (CAR), or any other surface receptor capable of recognizing antigenic moieties associated with target cells. The forms of the antigenic moieties recognized by CARs and TCRs differ slightly. CARs have a single-chain variable fragment (scFv) as the target-binding domain, allowing the CAR to be expressed as a single-chain protein. This allows the CAR to recognize natural cancer antigens that are part of intact proteins on the surface of target cells. TCRs have two protein chains designed to bind to specific peptides presented by MHC proteins on the surface of certain cells. Because TCRs recognize peptides in the context of MHC molecules expressed on the surface of target cells, TCRs have the potential to recognize not only cancer antigens directly presented on the surface of cancer cells, but also cancer antigens presented by antigen-presenting cells in tumors, inflammatory and infected microenvironments, and secondary lymphoid organs. Antigen-presenting cells are natural immune system cells responsible for amplifying immune responses.

[0013] Thus, according to embodiments described herein, manufactured T cells expressing cell surface receptors may be used to target and kill any target cell, including, but not limited to, infected, damaged, or dysfunctional cells. Examples of such target cells include cancer cells, virally infected cells, bacterially infected cells, dysfunctionally activated inflammatory cells (e.g., inflammatory endothelial cells), and cells involved in dysfunctional immune responses (e.g., cells involved in autoimmune diseases).

[0014] In some embodiments, the antigenic moiety is associated with cancer or cancer cells. Such antigenic moieties include 707-AP (707 alanine proline), AFP (alpha(a)-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen; b-catenin / m, b-catenin / mutant), BCMA (B cell maturation antigen), Bcr-abl (breakpoint cluster region-Abelson), CAIX (carbonic anhydrase IX), CD19 (cluster of differentiation 19), CD20 (cluster of differentiation 20), CD22 (cluster of differentiation 22), CD30 (cluster of differentiation 30), CD33 (cluster of differentiation 33), CD44v7 / 8, CD44v9, CD44v10, CD44v11, CD44v12, CD44v13, CD44v14, CD44v15, CD44v16, CD44v17, CD44v18, CD44v19, CD44v20, CD44v21, CD44v22, CD44v23, CD44v24, CD44v25, CD44v26, CD44v27, CD44v28, CD44v29, CD44v30, CD44v31, CD44v32, CD44v33, CD44v34, CD44v35, CD44v36, CD44v37, CD44v38, CD44v39, CD44v40, CD44v41, CD44v42, CD44v43, CD44v44, CD44v51, CD44v52, CD44v53, CD44v54, CD44v55, CD44v56, CD44v57, CD44v58, CD44 (cluster of differentiation antigen 44, exon 7 / 8), CAMEL (antigen recognized by CTL on melanoma), CAP-1 (carcinoembryonic antigen peptide-1), CASP-8 (caspase-8), CDC27m (cell division cycle protein 27 variant), CDK4 / m (cyclin-dependent kinase 4 variant), CEA (carcinoembryonic antigen), CT (cancer / testis (antigen)), Cyp-B (cyclophilin B), DAM (differentiation antigen melanoma), EGFR (epidermal growth factor receptor), EGFRvIII (epidermal growth factor receptor, variant III), EGP-2 (epithelial glycoprotein 2), EGP-40 (epithelial glycoprotein 40), Erbb2, 3, 4 (erythroblastic leukemia viral oncogene homolog-2, -3, -4), ELF2M (elongation factor 2 mutant), ETV6-AML1 (Ets variant gene 6 / acute myeloid leukemia 1 gene ETS), FBP (folate binding protein), fAchR (fetal acetylcholine receptor), G250 (glycoprotein 250), GAGE ​​(G antigen), GD2 (disialoganglioside 2), GD3 (disialoganglioside 3), GnT-V (N-acetylglucosaminyltransferase V), Gp 100 (glycoprotein 100kD), HAGE (helicase antigen), HER-2 / neu (human epidermal growth factor receptor-2 / neuronal; also known as EGFR2), HLA-A (human leukocyte antigen-A), HPV (human papillomavirus), HSP70-2M (heat shock protein 70-2 variant), HST-2 (human signet ring tumor-2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxylesterase), IL-13R-a2 (interleukin-13 receptor subunit alpha-2), KIAA02 05, KDR (kinase insert domain receptor), kappa-light chain, LAGE (L antigen), LDLR / FUT (low-density lipid receptor / GDP-L-fucose:bD-galactosidase 2-aL fucosyltransferase), LeY (Lewis Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), mesothelin, murine CMV-infected cells, MART-1 / Melan-A (melanoma antigen recognized by T cells 1 / melanoma antigen A), MC1R (melanocortin 1 receptor), myosin / m (myosin variant), MUC1 (mucin 1), MUM-1, -2, -3 (melanoma ubiquitous variants 1, 2, 3), NA88-A (NA of patient M88) cDNA clone), NKG2D (natural killer group 2, member D) ligand, NY-BR-1 (New York breast differentiation antigen 1), NY-ESO-1 (New York esophageal squamous cell carcinoma-1), carcinoembryonic antigen (h5T4), P15 (protein 15), p190 minor bcr-abl (190KD bcr-abl protein), Pml / RARa (promyelocytic leukemia / retinoic acid receptor a), PRAME (preferentially expressed antigen in melanoma), PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), RAGE (kidney antigen), RU1 or RU2 (renal ubiquitous type 1 or 2), SAGE (sarcoma antigen), SART-1 or SART-3 (tumor rejection squamous antigen 1 or 3), SSX1, -2, -3, -4 (synovial sarcoma X1, -2, -3, -4), T These may include, but are not limited to, AA (tumor-associated antigen), TAG-72 (tumor-associated glycoprotein 72), TEL / AML1 (translocation Ets family leukemia / acute myeloid leukemia 1), TPI / m (triosephosphate isomerase mutant), TRP-1 (tyrosinase-related protein 1, or gp75), TRP-2 (tyrosinase-related protein 2), TRP-2 / INT2 (TRP-2 / intron 2), VEGF-R2 (vascular endothelial growth factor receptor 2), or WT1 (Wilms tumor gene).

[0015] In some embodiments, the cell surface receptor is selected from the group consisting of anti-707-AP TCR, anti-AFP TCR, anti-ART-4 TCR, anti-BAGE TCR, anti-Bcr-abl TCR, anti-CAMEL TCR, anti-CAP-1 TCR, anti-CASP-8 TCR, anti-CDC27m TCR, anti-CDK4 / m TCR, anti-CEA TCR, anti-CT TCR, anti-Cyp-B TCR, anti-DAM TCR, anti-TCR, anti-EGFRvIII TCR, anti-ELF2M TCR, anti-ETV6-AML1 TCR, anti-G250 TCR, GAGE ​​TCR, anti-GnT-V TCR, anti-Gp100 TCR, anti-HAGE TCR, anti-HER-2 / neu TCR, anti-HLA-A TCR, anti-HPV TCR, anti-HSP70-2M TCR, anti-HST-2 TCR, anti-hTERT TCR or anti-hTRT TCR, anti-iCEA T ... TCR, anti-KIAA0205, anti-LAGE (L antigen), anti-LDLR / FUT TCR, anti-MAGE TCR, anti-MART-1 / Melan-A TCR, anti-MC1R TCR, anti-myosin / m TCR, anti-MUC1 TCR, anti-MUM-1, -2, -3 TCR, anti-NA88-A TCR, anti-NY-ESO-1 TCR, anti-P15 TCR, anti-p190 minor bcr-abl TCR, anti-Pml / RARa TCR, anti-PRAME TCR, anti-PSA TCR, anti-PSMA TCR, anti-RAGE TCR, anti-RU1 TCR or anti-RU2 TCR, anti-SAGE TCR, anti-SART-1 TCR or anti-SART-3 TCR Any TCR that recognizes a specific antigen moiety on cancer cells, including, but not limited to, anti-SSX1, -2, -3, -4 TCR, anti-TEL / AML1 TCR, anti-TPI / m TCR, anti-TRP-1 TCR, anti-TRP-2 TCR, anti-TRP-2 / INT2 TCR, or anti-WT1 TCR.

[0016] In other embodiments, the cell surface receptor is any CAR that can be expressed by T cells and recognizes a specific antigen moiety on cancer cells. Certain CARs include an antigen binding domain (e.g., scFv) and a signaling domain (e.g., CD3 zeta chain). Other CARs include an antigen binding domain (e.g., scFv), a signaling domain (e.g., CD3 zeta chain), and a costimulatory domain (e.g., CD28). Still other CARs include: It contains an antigen-binding domain (e.g., scFv), a signaling domain (e.g., CD3 zeta chain), and two costimulatory domains (e.g., CD28 and 4-1BB). Examples of surface receptor CARs that can be expressed by T cells generated according to the methods described herein include, but are not limited to, anti-BCMA CAR, anti-CAIX CAR, anti-CD19 CAR, anti-CD20 CAR, anti-CD22 CAR, anti-CD30 CAR, anti-CD33 CAR, anti-CD44v7 / 8 CAR, anti-CEA CAR, anti-EGFRvIII, anti-EGP-2, anti-EGP-40 CAR, anti-Erbb2, 3, 4 CAR, anti-FBP CAR, anti-fAchR CAR, anti-GD2 CAR, anti-GD3 CAR, anti-HER2 / neu CAR, anti-IL-13R-a2 CAR, anti-KDR CAR, anti-κ-light chain CAR, anti-LeY CAR, anti-L1CAM CAR, anti-MAGE-A1 CAR, anti-mesothelin CAR, anti-CAR for murine CMV-infected cells, anti-MUC1 CAR, anti-NKG2D ligand CAR, and anti-NY-BR-1. Examples of the cell surface receptor include an anti-CD19 CAR, an anti-h5T4 CAR, an anti-PSCA CAR, an anti-PSMA CAR, an anti-TAA CAR, an anti-TAG-72 CAR, or an anti-VEGF-R2 CAR. In one embodiment, the cell surface receptor is any anti-CD19 CAR. In one embodiment, the anti-CD19 CAR includes an extracellular scFv domain, an intracellular and / or transmembrane portion of a CD28 molecule, any extracellular portion of a CD28 molecule, and an intracellular CD3 zeta domain. The anti-CD19 CAR may also include additional domains such as CD8 extracellular and / or transmembrane regions, an extracellular immunoglobulin Fc domain (e.g., IgG1, IgG2, IgG3, IgG4), or one or more additional signaling domains such as 41BB, OX40, CD2, CD16, CD27, CD30, CD40, PD-1, ICOS, LFA-1, a receptor for IL-2, an Fc gamma receptor, or other costimulatory domains containing an immunoreceptor tyrosine-based activation motif.

[0017] In certain embodiments, the cell surface receptor is FMC63-28Z CAR or FMC63-28Z CAR, as described in Kochenderfer et al., J Immunother. 2009 September; 32(7): 689-702, "Construction and Pre-clinical Evaluation of an Anti-CD19 Chimeric Antigen Receptor." and anti-CD19 CARs such as FMC63-CD828BBZ CAR, the subject matter of which is incorporated herein by reference for the purposes of providing methods for constructing vectors used to generate T cells expressing the FMC63-28Z CAR or FMC63-CD828BBZ CAR.

[0018] In other embodiments, the antigenic portion is associated with a virus-infected cell (i.e., a viral antigenic portion). Such antigenic portions include Epstein-Barr virus (EBV) antigens (e.g., EBNA-1, EBNA-2, EBNA-3, LMP-1, LMP-2), hepatitis A viral antigens (e.g., VP1, VP2, VP3), hepatitis B viral antigens (e.g., HBsAg, HBcAg, HBeAg), hepatitis C viral antigens (e.g., envelope glycoproteins E1 and E2), herpes simplex types 1, 2, or 8 (HSV1, HSV2, or HSV8) viral antigens (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM, UL20, UL32, US43, UL45, UL49A), cytomegalovirus (CMV) viral antigens, and the like. The cell surface receptor may include, but is not limited to, a viral antigen (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM, or other envelope proteins), a viral antigen of human immunodeficiency virus (HIV) (glycoproteins gp120, gp41, or p24), an influenza viral antigen (e.g., hemagglutinin (HA) or neuraminidase (NA)), a viral antigen of measles or mumps, a viral antigen of human papillomavirus (HPV) (e.g., L1, L2), a viral antigen of parainfluenza virus, a viral antigen of rubella virus, a viral antigen of respiratory syncytial virus (RSV), or a viral antigen of varicella-zoster virus. In such embodiments, the cell surface receptor may be any TCR or any CAR that recognizes any of the above-mentioned viral antigens on target virus-infected cells.

[0019] In other embodiments, the antigenic moiety is associated with cells having immune or inflammatory dysfunction. Such antigenic moieties may include, but are not limited to, myelin basic protein (MBP), myelin proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), carcinoembryonic antigen (CEA), proinsulin, glutamic acid decarboxylase (GAD65, GAD67), heat shock proteins (HSPs), or any other tissue-specific antigen involved in or associated with a pathogenic autoimmune process.

[0020] In some embodiments, the methods described herein may include enriching a population of lymphocytes obtained from a donor subject. The donor subject may be a cancer patient treated with a population of cells produced by the methods described herein (i.e., an autologous donor), or may be an individual providing a lymphocyte sample to be used in the treatment of a different individual or cancer patient by producing a cell population produced by the methods described herein (i.e., an allogeneic donor). The lymphocyte population may be obtained from the donor subject by any suitable method used in the art. For example, the lymphocyte population may be obtained by any suitable ex vivo method, venipuncture, or other blood collection method that obtains a sample of blood and / or lymphocytes. In one embodiment, the lymphocyte population is obtained by apheresis.

[0021] Enrichment of lymphocyte populations may be achieved by any suitable separation method, including, but not limited to, the use of separation media (e.g., Ficoll-Paque®, RosetteSep® HLA Total Lymphocyte enrichment cocktail, Lymphocyte Separation Medium (LSA) (MP Biomedical, Part Number 0850494X), etc.), cell size, shape or density separation by filtration or elutriation, immunomagnetic separation (e.g., magnetic activated cell sorting system, MACS), fluorescence separation (e.g., fluorescence activated cell sorting system, FACS), or bead-based column separation.

[0022] In some embodiments, the methods described herein may include stimulating a population of lymphocytes with one or more T cell stimulatory agents to produce a population of activated T cells. Any combination of one or more suitable T cell stimulatory agents that can be used to generate a population of activated T cells includes, but is not limited to, an antibody or functional fragment thereof that targets a T cell stimulatory or costimulatory molecule (e.g., an anti-CD2 antibody, an anti-CD3 antibody, an anti-CD28 antibody, or a functional fragment thereof), a T cell cytokine (e.g., interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 7 (IL-7), interleukin 15 (IL-15), tumor necrosis factor alpha (TNFα)), or any other suitable mitogen (e.g., tetradecanoylphorbol acetate (TPA), phytohemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen (PWM)), or a natural ligand for a T cell stimulatory or costimulatory molecule.

[0023] In some embodiments, stimulating a population of lymphocytes described herein may include stimulating the population of lymphocytes with one or more T cell stimulatory agents at a predetermined temperature, for a predetermined time, and / or in the presence of a predetermined level of CO2. In certain embodiments, the predetermined temperature for stimulation may be about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, or about 39°C. In certain embodiments, the predetermined temperature for stimulation may be about 34°C-39°C. In certain embodiments, the predetermined temperature for stimulation may be about 35°C-37°C. In certain embodiments, a preferred predetermined temperature for stimulation may be about 36°C-38°C. In certain embodiments, the predetermined temperature for stimulation may be about 36°C-37°C, or more preferably about 37°C. In certain embodiments, In some embodiments, stimulating the lymphocyte population comprises stimulating the lymphocyte population with one or more T cell stimulatory agents for a predetermined time period. In certain embodiments, the predetermined time period for stimulation may be about 24 to 72 hours. In certain embodiments, the predetermined time period for stimulation may be about 24 to 36 hours, about 30 to 42 hours, about 36 to 48 hours, about 40 to 52 hours, about 42 to 54 hours, about 44 to 56 hours, about 46 to 58 hours, about 48 to 60 hours, about 54 to 66 hours, or about 60 to 72 hours. In certain embodiments, the predetermined time period for stimulation may be about 48 hours or at least about 48 hours. In certain embodiments, the predetermined time period for stimulation may be about 44 to 52 hours. In certain embodiments, the predetermined time period for stimulation may be about 40 to 44 hours, about 40 to 48 hours, about 40 to 52 hours, or about 40 to 56 hours. In certain embodiments, stimulating the lymphocyte population may include stimulating the lymphocyte population with one or more T cell stimulatory agents in the presence of a predetermined level of CO2. In certain embodiments, the predetermined level of CO2 for stimulation may be about 1.0% to 10% CO2. In certain embodiments, the predetermined level of CO2 for stimulation may be about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In certain embodiments, the predetermined level of CO2 for stimulation may be about 3% to 7% CO2. In certain embodiments, the predetermined level of CO2 for stimulation may be about 4% to 6% CO2. In certain embodiments, the predetermined level of CO2 for stimulation may be about 4.5% to 5.5% CO2. In certain embodiments, the predetermined level of CO2 for stimulation may be about 5% CO2. In some embodiments, stimulating the population of lymphocytes may include stimulating the population of lymphocytes with one or more T cell stimulatory agents at any combination of predetermined temperatures, for predetermined times, and / or in the presence of predetermined levels of CO2.For example, in one embodiment, stimulating the population of lymphocytes may include stimulating the population of lymphocytes with one or more T cell stimulatory agents at a predetermined temperature of about 36°C to 38°C for a predetermined time period of about 44 hours to 52 hours in the presence of a predetermined level of CO2 of about 4.5% to 5.5% CO2.

[0024] In certain embodiments, the population of lymphocytes used in the steps of stimulating a population of lymphocytes described herein may be at a predetermined concentration of lymphocytes. In certain embodiments, the predetermined concentration of lymphocytes is about 0.1 to 10.0 x 10 6 In certain embodiments, the predetermined concentration of lymphocytes may be about 0.1-1.0 x 10 cells / mL. 6 cells / mL, 1.0~2.0×10 6 cells / mL, approximately 1.0~3.0×10 6 cells / mL, approximately 1.0~4.0×10 6 cells / mL, approximately 1.0~5.0×10 6 cells / mL, approximately 1.0~6.0×10 6 cells / mL, approximately 1.0~7.0×10 6 cells / mL, approximately 1.0~8.0×10 6 cells / mL, 1.0~9.0×10 6 cells / mL, or approximately 1.0–10.0 × 10 6 In certain embodiments, the predetermined concentration of lymphocytes may be about 1.0-2.0 x 10 cells / mL. 6 In certain embodiments, the predetermined concentration of lymphocytes may be about 1.0-1.2 x 10 cells / mL. 6 cells / mL, approximately 1.0~1.4×10 6 cells / mL, approximately 1.0~1.6×10 6 cells / mL, approximately 1.0~1.8×10 6 cells / mL, or approximately 1.0-2.0 x 10 6 In certain embodiments, the predetermined concentration of lymphocytes may be at least about 0.1 x 10 cells / mL. 6 cells / mL, at least approximately 1.0 x 10 6 cells / mL, at least approximately 1.1 x 10 6cells / mL, at least approximately 1.2 x 10 6 cells / mL, at least approximately 1.3 x 10 6 cells / mL, at least approximately 1.4 x 10 6 cells / mL, at least approximately 1.5 x 10 6 cells / mL, at least approximately 1.6 x 10 6 cells / mL, at least approximately 1.7 x 10 6 cells / mL, at least approximately 1.8 x 10 6 cells / mL, at least approximately 1.9 x 10 6 cells / mL, at least approximately 2.0 x 10 6 cells / mL, at least approximately 4.0 x 10 6 cells / mL, at least approximately 6.0 × 10 6 cells / mL, at least approximately 8.0 × 10 6 cells / mL, or at least about 10.0 x 10 6 It may be cells / mL.

[0025] In some embodiments, an anti-CD3 antibody (or a functional fragment thereof), an anti-CD28 antibody (or a functional fragment thereof), or a combination of an anti-CD3 antibody and an anti-CD28 antibody may be used in accordance with the step of stimulating a lymphocyte population. Any soluble or immobilized anti-CD2, anti-CD3, and / or anti-CD28 antibody or functional fragment thereof may be used (e.g., clone OKT3 (anti-CD3), clone 145-2C11 (anti-CD3), clone UCHT1 (anti-CD3), clone L293 (anti-CD28), clone 15E8 (anti-CD28)). In some aspects, the antibodies may be commercially purchased from sources known in the art, including, but not limited to, Miltenyi Biotec, BD Biosciences (e.g., MACS GMP CD3 pure 1 mg / mL, Part No. 170-076-116), and eBioscience, Inc. Furthermore, those skilled in the art will understand how to produce anti-CD3 and / or anti-CD28 antibodies using standard methods. Any antibodies used in the methods described herein must be manufactured under Good Manufacturing Practice (GMP) to comply with relevant agency guidelines for biopharmaceuticals. In some embodiments, one or more T cell stimulatory agents that may be used in the step of stimulating a lymphocyte population include an antibody or functional fragment thereof that targets a T cell stimulatory or costimulatory molecule in the presence of a T cell cytokine. In one aspect, the one or more T cell stimulatory agents include an anti-CD3 antibody and IL-2. In certain embodiments, the T cell stimulatory agent may include an anti-CD3 antibody at a concentration of about 20 ng / mL to 100 ng / mL. In certain embodiments, the concentration of the anti-CD3 antibody may be about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL. In certain embodiments, the concentration of anti-CD3 antibody may be about 50 ng / mL. In alternative embodiments, T cell activation is not required.In such an embodiment, the step of stimulating the population of lymphocytes to produce a population of activated T cells is omitted from the above method, and the population of lymphocytes that can be enriched for T lymphocytes is transduced according to the following steps:

[0026] In some embodiments, the methods described herein may include transducing a population of activated T cells with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor using a single-cycle transduction to produce a population of transduced T cells. Several recombinant viruses have been used as viral vectors to deliver genetic material to cells. The viral vector that can be used in connection with the transduction step may be any ecotropic or amphotropic viral vector, including, but not limited to, recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, and recombinant adeno-associated viral (AAV) vectors. In one aspect, the viral vector used to transduce the population of activated T cells is an MSGV1 gammaretroviral vector. In one aspect, such an MSGV1 gammaretroviral vector may comprise a backbone nucleic acid sequence shown in FIG. 6 (SEQ ID NO: 4), wherein a nucleic acid fragment comprising the sequence of a cell surface receptor (e.g., a CAR or TCR) is linked to a nucleic acid fragment comprising the sequence of the MSGV1 gammaretroviral vector. In certain embodiments, the viral vector used to transduce the population of activated T cells may be the MSGV-FMC63-28Z retroviral vector or the MSGV-FMC63-CD828BBZ retroviral vector described in Kochenderfer et al., J Immunother. 2009 September; 32(7): 689-702, the subject matter of which is incorporated herein by reference for the purpose of providing methods for constructing retroviral vectors presented in the "Materials and Methods" section of that publication under "Construction of the MSGV-FMC63-28Z and MSGV-FMC63-CD828BBZ Recombinant Retroviral Vectors." According to one aspect of this embodiment, the viral vector is propagated in culture in a medium specific for viral vector production. In a viral vector inoculum according to the methods described herein, Any suitable growth medium and / or supplements for propagating the viral vector may be used in the transduction step. According to some embodiments, the viral vector may then be added to a serum-free medium as described below during the transduction step.

[0027] In certain embodiments, the step of transducing a population of activated T cells described herein may be performed for a predetermined time, at a predetermined temperature, and / or in the presence of a predetermined level of CO2. In certain embodiments, the predetermined temperature for transduction may be about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, or about 39°C. In certain embodiments, the predetermined temperature for transduction may be about 34°C-39°C. In certain embodiments, the predetermined temperature for transduction may be about 35°C-37°C. In certain embodiments, a preferred predetermined temperature for transduction may be about 36°C-38°C. In certain embodiments, the predetermined temperature for transduction may be about 36°C-37°C, or more preferably about 37°C. In certain embodiments, the predetermined time for transduction may be about 12 hours-36 hours. In certain embodiments, the predetermined time for transduction may be about 12 to 16 hours, about 12 to 20 hours, about 12 to 24 hours, about 12 to 28 hours, or about 12 to 32 hours. In certain embodiments, the predetermined time for transduction may be about 20 hours or at least about 20 hours. In certain embodiments, the predetermined time for transduction may be about 16 to 24 hours. In certain embodiments, the predetermined time for transduction may be at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, or at least about 26 hours. In some embodiments, transducing the population of activated T cells may include transducing the population of activated T cells with the viral vector at a predetermined level of CO2. In certain embodiments, the predetermined level of CO2 for transduction may be about 1.0% to 10% CO2. In certain embodiments, the predetermined level of CO2 for transduction can be about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In certain embodiments, the predetermined level of CO2 for transduction can be about 3%-7% CO2.In certain embodiments, the predetermined level of CO2 for transduction may be about 4% to 6% CO2. In certain embodiments, the predetermined level of CO2 for transduction may be about 4.5% to 5.5% CO2. In certain embodiments, the predetermined level of CO2 for transduction may be about 5% CO2. In some embodiments, the step of transducing a population of activated T cells described herein may be performed for any combination of predetermined time periods, at a predetermined temperature, and / or in the presence of a predetermined level of CO2. For example, in one aspect, the step of transducing a population of activated T cells may include a predetermined temperature of about 36°C to 38°C, a predetermined time period of about 16 hours to about 24 hours, and in the presence of a predetermined level of CO2 of about 4.5% to about 5.5% CO2.

[0028] In some embodiments, the methods described herein may include expanding the population of transduced T cells for a predetermined time to produce a population of engineered T cells. The predetermined time for expansion may be any suitable time that allows for the production of (i) a sufficient number of cells in the population of engineered T cells for at least one dose to be administered to a patient, (ii) a population of engineered T cells that contains a favorable proportion of immature cells compared to typical longer processes, or (iii) both (i) and (ii). This time will depend on the cell surface receptors expressed by the T cells, the vector used, the dose required to have a therapeutic effect, and other variables. Thus, in some embodiments, the predetermined time for expansion may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or more than 21 days. In some aspects, the predetermined time for expansion is shorter than expansion methods known in the art. For example, the predetermined time for expansion is at least The expansion time may be reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or more than 75%. In one aspect, the predetermined time for expansion is about 3 days. In this aspect, the time from enrichment of the population of lymphocytes to production of engineered T cells is about 6 days. In certain embodiments, the step of expanding the population of transduced T cells may be performed at a predetermined temperature and / or in the presence of a predetermined level of CO2. In certain embodiments, the predetermined temperature may be about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, or about 39°C. In certain embodiments, the predetermined temperature may be about 34°C to 39°C. In certain embodiments, the predetermined temperature may be about 35°C to 37°C. In certain embodiments, the preferred predetermined temperature may be about 36°C to 38°C. In certain embodiments, the predetermined temperature may be about 36°C to 37°C, or more preferably about 37°C. In some embodiments, expanding the population of transduced T cells comprises expanding the population of transduced T cells in the presence of a predetermined level of CO2. In certain embodiments, the predetermined level of CO2 may be 1.0% to 10% CO2. In certain embodiments, the predetermined level of CO2 may be about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In certain embodiments, the predetermined level of CO2 may be about 4.5% to 5.5% CO2. In certain embodiments, the predetermined level of CO2 may be about 5% CO2. In certain embodiments, the predetermined level of CO2 may be about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, or about 6.5% CO2. In some embodiments, the step of expanding the population of transduced T cells may be performed at any combination of predetermined temperatures and / or in the presence of predetermined levels of CO2.For example, in one embodiment, the step of expanding the population of transduced T cells may include at a predetermined temperature of about 36°C to 38°C and in the presence of a predetermined level of CO2 of about 4.5% to 5.5% CO2.

[0029] In some aspects, each step of the methods described herein is performed in a closed system. In certain embodiments, the closed system is a closed-bag culture system using any suitable cell culture bag (e.g., Miltenyi Biotec's MACS® GMP cell differentiation bag or Origen Biomedical's PermaLife® cell culture bag). In some embodiments, the cell culture bag used in the closed-bag culture system is coated with recombinant human fibronectin protein during the transduction process. In certain embodiments, the cell culture bag used in the closed-bag culture system is coated with a recombinant human fibronectin protein fragment during the transduction process. The recombinant human fibronectin fragment may comprise three functional domains: a central cell-binding domain, a heparin-binding domain II, and a CS1 sequence. Recombinant human fibronectin protein or a fragment thereof may be used to increase the gene efficiency of retroviral transduction of immune cells by assisting in colocalization of target cells and viral vectors. In certain embodiments, the recombinant human fibronectin fragment is RetroNectin® (Takara Bio Inc.; Japan). In certain embodiments, the cell culture bag may be coated with the recombinant human fibronectin fragment at a concentration of about 1 μg / mL to 60 μg / mL, preferably about 1 μg / mL to 40 μg / mL, hi certain embodiments, the cell culture bag may be coated with the recombinant human fibronectin fragment at a concentration of about 1 μg / mL to 20 μg / mL, 20 μg / mL to 40 μg / mL, or about 40 μg / mL to 60 μg / mL. In certain embodiments, the cell culture bag comprises a 0.1 μg / mL or 0.2 μg / mL or 0.3 μg / mL or 0.4 μg / mL or 0.5 μg / mL or 0.6 μg / mL or 0.7 μg / mL or 0.8 μg / mL or 0.9 μg / mL or 10 μg / mL or 11 μg / mL or 12 μg / mL or 13 μg / mL or 14 μg / mL or 15 μg / mL or 16 μg / mL or 17 μg / mL or 18 μg / mL or The cell culture bag may be coated with about 19 μg / mL or about 20 μg / mL of the recombinant human fibronectin fragment. In certain embodiments, the cell culture bag may be coated with about 2 μg / mL to 5 μg / mL, about 2 μg / mL to 10 μg / mL, about 2 μg / mL to 20 μg / mL, about 2 μg / mL to 25 μg / mL, about 2 μg / mL to 30 μg / mL, about 2 μg / mL to 35 μg / mL, about 2 μg / mL to 40 μg / mL, about 2 μg / mL to 50 μg / mL, or about 2 μg / mL to 60 μg / mL of the recombinant human fibronectin fragment. In certain embodiments, the cell culture bag may be coated with at least about 2 μg / mL, at least about 5 μg / mL, at least about 10 μg / mL, at least about 15 μg / mL, at least about 20 μg / mL, at least about 25 μg / mL, at least about 30 μg / mL, at least about 40 μg / mL, at least about 50 μg / mL, or at least about 60 μg / mL of recombinant human fibronectin fragment. In certain embodiments, the cell culture bag may be coated with at least about 10 μg / mL of recombinant human fibronectin fragment. In certain embodiments, the cell culture bag used in the closed bag culture system may be blocked with human albumin serum (HSA) during the transduction process. In alternative embodiments, the cell culture bag is not blocked with HSA during the transduction process. In other embodiments, serum-free culture medium to which serum has not been added is used to perform one or more of the steps of (a) stimulating a population of lymphocytes, (b) transducing a population of activated T cells, and (c) expanding the population of transduced T cells. In another embodiment, the steps of (a) stimulating a population of lymphocytes, (b) transducing a population of activated T cells, and (c) expanding the population of transduced T cells are each performed using serum-free culture medium. The term "serum-free medium" or "serum-free culture medium" referred to herein means that the growth medium used is not supplemented with serum (e.g., human serum or bovine serum). In other words, serum is not added to the culture medium as a separate, distinct component for the purpose of supporting the viability, activation, and proliferation of the cultured cells.According to the methods described herein, any suitable culture medium, such as T cell growth medium, may be used for culturing cells in suspension. For example, T cell growth medium may include, but is not limited to, a sterile, low-glucose solution containing appropriate amounts of buffer, magnesium, calcium, sodium pyruvate, and sodium bicarbonate. In one embodiment, the T cell growth medium is OpTmizer™ (Life Technologies), although one of skill in the art would understand how to make a similar medium. In contrast to typical methods for producing engineered T cells, the methods described herein use culture medium that is not supplemented with serum (e.g., human or bovine).

[0030] According to embodiments described herein, a method of producing T cells expressing a cell surface receptor that recognizes a specific antigen moiety on the surface of a target cell may include: (1) enriching a population of lymphocytes obtained from a donor subject; (2) stimulating the population of lymphocytes with one or more T cell stimulants to produce a population of activated T cells, wherein the stimulation is performed in a closed system using a serum-free culture medium; (3) transducing the population of activated T cells with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor using a single cycle of transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using a serum-free culture medium; and (4) expanding the population of transduced T cells for a predetermined period of time to produce a population of engineered T cells, wherein the expansion is performed in a closed system using a serum-free culture medium.

[0031] In another embodiment, a method for producing T cells expressing a cell surface receptor that recognizes a specific antigen moiety on the surface of a target cell includes: (1) enriching a population of lymphocytes obtained from a donor subject; (2) transducing the population of lymphocytes with a viral vector comprising a nucleic acid molecule encoding the cell surface receptor using a single cycle of transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium; and (3) continuing the transduction for a predetermined period of time to produce a population of engineered T cells. and expanding the population of introduced T cells, wherein the expansion is carried out in a closed system using serum-free culture medium.

[0032] In one embodiment, the process or method may include, but is not limited to, (1) collection of an apheresis product from a patient and isolation of mononuclear cells in a closed system; (2) stimulation of the mononuclear cell population with an antibody against CD3 in the presence of IL2 to stimulate T cell proliferation in a closed system; (3) introduction or transduction of a novel cell surface receptor gene using a gammaretroviral vector in a closed system, enabling T cells to recognize specific antigenic moieties on the surface of cancer target cells; (4) expansion of the transduced T cells in a closed system; and (5) washing and preparation of the closed-system expanded autologous T cells for re-administration to the cancer patient. In some embodiments, the expansion step takes three days, allowing the entire manufacturing process to be completed in less than one week. Process steps 2 through 4, during which T cells are actively proliferating, are performed in a defined cell culture medium that does not contain human serum (i.e., serum-free medium). The T cells produced by this process are bioactive, become activated by target antigens on the surface of cancer cells, and produce gamma interferon in response. In some aspects of the methods described herein, The process is carried out in a closed system that minimizes the possibility of contamination during T cell manufacturing. The above process is suitable for the production of T cells for clinical use. growing the cells in human serum-free cell culture medium; Transduction of receptor genes into T cells in a closed-bag system. The cells can be made for clinical use in just six days, and The cells exhibit biological activity indicative of in vivo biological activity, Examples include:

[0033] These aspects offer several differences and / or improvements over methods currently used in the art, as follows: The use of human serum-free cell culture media minimizes the chance of introducing human pathogens from raw materials in the process and avoids the use of raw materials that may not be readily available in the future. Furthermore, such use supports GMP compliance, as different serum lots require significant culture testing and release to ensure reproducibility and process reliability. The expansion of T cells in serum-free media has been reported previously (Carstens et al., ISCT meeting in San Diego, 2012; Zuliani, 2011) and has previously been incorporated into processes to produce T cells for clinical use to treat cancer. However, previous studies have not demonstrated robust T cell activation, transduction, and expansion in serum-free media. In the improved process explored in the studies described herein, the use of anti-CD3 monoclonal antibody and IL2 was maintained for stimulation of T cell populations. Furthermore, cell culture in closed bags offers the significant advantage of preventing potential contamination during cell culture and may provide a simplified and shortened process suitable for cGMP manufacturing and product commercialization. The importance of this practical application is significant because many processes described in the literature for T cell expansion are not suitable for widespread commercial application.

[0034] Previously, viral transduction with gammaretroviral vectors was inefficient, and an open process called "spinoculation" was performed in microtiter plates, in which virus and cells were centrifuged into the bottom of RetroNectin®-coated wells. This process is typically repeated twice daily to maximize transduction efficiency. According to some embodiments of the methods described herein, this transduction step has been modified so that transduction is performed in a closed bag system using RetroNectin®-coated bags (rather than plates), and the process is performed once rather than twice. Additionally, the methods described herein may involve growing cells in closed cell culture bags rather than open flasks, which have traditionally been used in the art. Several publications (Lamers et al., Cytotherapy 2008, 10: 406-416; Tumanin et al., J. Immunol. 2009, 10: 407-418; Although other studies (e.g., I et al., Cytotherapy 2013, 11, 1406-1415) include reports of transduction in a bag system, these cases, unlike the methods described herein, involve at least two transductions completed in serum-containing cell culture medium and an expansion time of at least 9 days. Development studies in embodiments described herein demonstrate that transduction in a bag in serum-free medium is not only feasible, but that transduction levels are acceptable for further clinical development after a single transduction and only 3 days of expansion.

[0035] In some embodiments, the population of engineered T cells produced by the above-described methods may optionally be cryopreserved so that the cells can be used at a later date. Accordingly, methods for cryopreserving a population of engineered T cells are provided herein. Such methods may include washing and concentrating the population of engineered T cells with a diluent. In some aspects, the diluent is saline, 0.9% saline, PlasmaLyte A (PL), 5% dextrose / 0.45% NaCl saline solution (D5), human serum albumin (HSA), or a combination thereof. In some aspects, HSA may be added to the washed and concentrated cells to improve cell viability and post-thaw cell recovery. In another aspect, the wash solution is saline, and the washed and concentrated cells are supplemented with HSA (5%). The above-described methods may also include creating a cryopreservation mixture, wherein the cryopreservation mixture comprises the population of cells diluted in a diluent and a suitable cryopreservation solution. In some embodiments, the cryopreservation solution may be any suitable cryopreservation solution, including but not limited to CryoStor10 (BioLife Solutions), and may be used to dilute the engineered T cells. The cryopreservation mixture may be mixed with the solution in a 1:1 or 2:1 ratio. In certain embodiments, HSA may be added to the cryopreservation mixture to give a final concentration of about 1.0% to 10% HSA. In certain embodiments, HSA may be added to the cryopreservation mixture to give a final concentration of about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% HSA. In certain embodiments, HSA may be added to the cryopreservation mixture to give a final concentration of about 1% to 3% HSA, about 1% to 4% HSA, about 1% to 5% HSA, about 1% to 7% HSA, about 2% to 4% HSA, about 2% to 5% HSA, about 2% to 6% HSA, or about 2% to 7% HSA. In certain embodiments, HSA may be added to the cryopreservation mixture to provide a final concentration of about 2.5% HSA. For example, in certain embodiments, cryopreserving a population of engineered T cells may include washing the cells with 0.9% saline, adding a final concentration of 5% HSA to the washed cells, and diluting the cells 1:1 with CryoStor™ CS10 (for a final concentration of 2.5% HSA in the final cryopreservation mixture). In some embodiments, the method also includes freezing the cryopreservation mixture. In one aspect, about 1 x 10 6 HSA per mL of cryopreservation mixture is added. 6 ~Approx. 1.5×10 7 and freezing the cryopreservation mixture in a controlled rate freezer using a prescribed freezing cycle at a cell concentration of 0.05%. The method may also include storing the cryopreservation mixture in vapor phase liquid nitrogen.

[0036] In certain embodiments, the population of engineered T cells produced by the methods described herein may be cryopreserved at a predetermined dose. In certain embodiments, the predetermined dose may be a therapeutically effective dose, or any of the therapeutically effective doses provided below. The predetermined dose of engineered T cells may depend on the cell surface receptors expressed by the T cells (e.g., the affinity and density of the cell surface receptors expressed on the cells), the type of target cell, the characteristics of the disease or condition being treated, or a combination of both. In certain embodiments, the cell surface receptors expressed by the engineered T cells are FMC63-28Z CAR or FM CAR, as described in Kochenderfer et al., J Immunother. 2009 September; 32(7): 689-702. The subject matter may be an anti-CD19 CAR, such as an FMC63-28Z CAR or an anti-CD19 CAR, such as an FMC63-CD828BBZ CAR, and the subject matter is incorporated herein by reference for purposes of providing methods for constructing vectors used to generate T cells expressing the FMC63-28Z CAR or the FMC63-CD828BBZ CAR. and form part of the present specification. In certain embodiments, a predetermined dose of engineered T cells expressing an FMC63-28Z CAR or an FMC63-CD828BBZ CAR may be greater than about 1 million and less than about 3 million transduced engineered T cells / kg. In certain embodiments, a predetermined dose of engineered T cells expressing an FMC63-28Z CAR or an FMC63-CD828BBZ CAR may be greater than about 1 million and up to about 2 million transduced engineered T cells per kilogram of body weight (cells / kg). In certain embodiments, a predetermined dose of engineered T cells expressing an FMC63-28Z CAR or an FMC63-CD828BBZ CAR may be greater than about 1 million and up to about 2 million transduced engineered T cells per kilogram of body weight (cells / kg). In certain embodiments, a predetermined dose of engineered T cells expressing an FMC63-28Z CAR or an FMC63-CD828BBZ CAR may be at least about 2 million to less than about 3 million transduced engineered T cells / kg. In certain embodiments, a preferred predetermined dose of engineered T cells expressing an FMC63-28Z CAR or an FMC63-CD828BBZ CAR may be about 2 million transduced engineered T cells / kg. In certain embodiments, a predetermined dose of engineered T cells expressing an FMC63-28Z CAR or an FMC63-CD828BBZ CAR may be at least about 2 million transduced engineered T cells / kg. In certain embodiments, the predetermined dose of engineered T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR may be about 2 million, about 2.1 million, about 2.2 million, about 2.3 million, about 2.4 million, about 2.5 million, about 2.6 million, about 2.7 million, about 2.8 million, or about 2.9 million transduced engineered T cells / kg. In certain embodiments, the population of engineered T cells may be cryopreserved at a predetermined dose of about 1 million engineered T cells per kilogram of body weight (cells / kg). In certain embodiments, the population of engineered T cells may be cryopreserved at a predetermined dose of about 0.5 million to about 1 million engineered T cells / kg.In certain embodiments, the population of engineered T cells may be cryopreserved at a predetermined dose of at least about 1 million, at least about 2 million, at least about 3 million, at least about 4 million, at least about 5 million, at least about 6 million, at least about 7 million, at least about 8 million, at least about 9 million, at least about 10 million engineered T cells / kg. In other aspects, the population of engineered T cells may be cryopreserved at a predetermined dose of less than 1 million cells / kg, 1 million cells / kg, 2 million cells / kg, 3 million cells / kg, 4 million cells / kg, 5 million cells / kg, 6 million cells / kg, 7 million cells / kg, 8 million cells / kg, 9 million cells / kg, 10 million cells / kg, more than 10 million cells / kg, more than 20 million cells / kg, more than 30 million cells / kg, more than 40 million cells / kg, more than 50 million cells / kg, more than 60 million cells / kg, more than 70 million cells / kg, more than 80 million cells / kg, more than 90 million cells / kg, or more than 100 million cells / kg. In certain embodiments, the population of engineered T cells may be cryopreserved at a predetermined dose of about 1 million to about 2 million engineered T cells / kg. In other embodiments, the population of engineered T cells may be cryopreserved at a predetermined dose of about 1 million cells / kg to about 2 million cells / kg, about 1 million cells / kg to about 3 million cells / kg, about 1 million cells / kg to about 4 million cells / kg, about 1 million cells / kg to about 5 million cells / kg, about 1 million cells / kg to about 6 million cells / kg, about 1 million cells / kg to about 7 million cells / kg, about 1 million cells / kg to about 8 million cells / kg, about 1 million cells / kg to about 9 million cells / kg, or about 1 million cells / kg to about 10 million cells / kg. In certain embodiments, the predetermined dose of the population of engineered T cells may be calculated based on the subject's body weight. In certain embodiments, the population of engineered T cells may be cryopreserved in about 0.5 mL to 200 mL of cryopreservation medium.In certain embodiments, the population of engineered T cells may be cryopreserved in about 0.5 mL, about 1.0 mL, about 5.0 mL, about 10.0 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 70 mL, about 80 mL, about 90 mL, or about 100 mL of cryopreservation medium. In some embodiments, the population of engineered T cells may be cryopreserved in about 10 mL to 30 mL, about 10 mL to 50 mL, about 10 mL to 70 mL, about 10 mL to 90 mL, about 50 mL to 70 mL, about 50 mL to 90 mL, about 50 mL to 110 mL, about 50 mL to 150 mL, or about 100 mL to 200 mL of cryopreservation medium. In certain embodiments, the population of engineered T cells may be cryopreserved in preferably about 50 mL to 70 mL of cryopreservation medium.

[0037] The methods described herein are used to produce a population of engineered T cells that can be used to treat a disease or condition in a subject having the disease or condition by administering a therapeutically effective amount or dose of the engineered T cells to the subject. In this manner, a population of engineered T cells that expresses a cell surface receptor that recognizes a specific antigenic moiety on the surface of a target cell is produced by the methods provided herein. Conditions that can be treated with the engineered T cells produced by the methods described herein include, but are not limited to, cancer, viral infection, acute or chronic inflammation, autoimmune disease, or any other immune dysfunction. Examples of methods for treating patients with the administration of engineered T cells are described in Kochenderfer, et al., J Clin Oncol. 2014 Aug 25. pii: JCO.2014.56.2025 (entitled "Chemotherapy - Refractory Diffuse Large B-Cell Lymphoma and Indolent B-Cell Malignancies Can Be Effectively Treated With Autologous T Cells Expressing an Anti-CD19 Chimeric Antigen Receptor") and Kochenderfer et al. Blood. 2012 Mar 22;119(12):2709-20 (entitled "B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells"). and "T cell therapy," the subject matter of which is incorporated herein by reference in its entirety as if fully set forth herein for the purpose of providing details regarding standard practice in treating patients with engineered T cell medications.

[0038] According to some embodiments, the population of engineered T cells produced by the methods described above may comprise one or more subpopulations of cells. In certain embodiments, the one or more subpopulations of cells may include, but are not limited to, naive T cells, effector T cells, effector memory T cells, and / or central memory T cells. As presented in Example 2 below, it was unexpected that using the methods described herein, in addition to simply reducing the culture period from 10 or more days to 6 days, resulted in a distribution of more immature T cells while increasing the representation of naive T cells and decreasing the presence of differentiated effector T cells. In certain embodiments, the population of engineered T cells may comprise a subpopulation of naive T cells. In certain embodiments, about 34% to 43% of the population of engineered T cells may comprise a subpopulation of naive T cells. In certain embodiments, at least about 35% of the population of engineered T cells may comprise a subpopulation of naive T cells. In certain embodiments, at least about 40% of the population of engineered T cells may comprise a subpopulation of naive T cells. In certain embodiments, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, or at least about 44% of the population of engineered T cells may comprise a subpopulation of naive T cells. In certain embodiments, the population of engineered T cells may comprise a subpopulation of central memory T cells. In certain embodiments, no more than about 15% of the population of engineered T cells may comprise a subpopulation of central memory T cells. In certain embodiments, no more than about 15%, no more than about 14%, no more than about 13%, no more than about 12%, or no more than about 11% of the population of engineered T cells may comprise a subpopulation of central memory T cells.

[0039] As referred to herein, "cancer" includes acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenocortical carcinoma, AIDS-related cancer, anal Cancer of the central nervous system, B-cell leukemia, lymphoma or other B-cell malignancies, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brain stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphoma Myeloid leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative syndrome, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, germinoma, central nervous system, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, nasal neuroblastoma, Ewing's sarcoma family tumors, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, eye cancer, fibrous histiocytoma of bone, malignant and osteosarcoma, Gallbladder cancer, gastric (abdominal) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), soft tissue sarcoma, germ cell tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, cardiac cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (pancreatic endocrine gland), Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, lymphoma, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma and osteosarcoma of bone, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell neck cancer with mediastinal tumor of unknown primary involving the NUT gene, oral cancer cancer), multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myeloma, multiple myeloproliferative syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, Pancreatic cancer, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, gestational breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureteral transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, small cell lung cancer, small intestine cancer The cancer may be any cancer associated with a surface antigen or cancer marker, including, but not limited to, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma of the cervix, abdominal (gastric) cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, skin, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0040] In some embodiments, the cancer is a B-cell malignancy. Examples of B-cell malignancies include, but are not limited to, non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), small lymphocytic lymphoma (SLL / CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), extranodal (MALT lymphoma), nodal (monocytoid B-cell lymphoma), splenic diffuse large cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, Burkitt's lymphoma, and lymphoblastic lymphoma.

[0041] A "viral infection" as referred to herein may be an infection caused by any virus that causes a disease or pathology in a host. Examples of viral infections that can be treated with the engineered T cells produced by the methods described herein include, but are not limited to, viral infections caused by Epstein-Barr virus (EBV), viral infections caused by hepatitis A virus, hepatitis B virus, or hepatitis C virus, viral infections caused by herpes simplex 1 virus, herpes simplex 2 virus, or herpes simplex 8 virus, viral infections caused by cytomegalovirus (CMV), viral infections caused by human immunodeficiency virus (HIV), viral infections caused by influenza virus, viral infections caused by measles or mumps virus. viral infections include those caused by human papillomavirus (HPV), parainfluenza virus, rubella virus, respiratory syncytial virus (RSV), or varicella-zoster virus. In some embodiments, the viral infection may lead to or result in the development of cancer in a subject with the viral infection (e.g., HPV infection may cause or be associated with the development of several cancers, including cervical, vulvar, vaginal, penile, anal, and oropharyngeal cancers, and HIV infection may cause the development of Kaposi's sarcoma).

[0042] Examples of chronic inflammatory diseases, autoimmune diseases or any other immune dysfunction that can be treated with the engineered T cells produced by the methods described herein include, but are not limited to, multiple sclerosis, lupus and psoriasis.

[0043] The terms "treat," "treating," or "treatment" as used herein with respect to a condition or disease may refer to preventing the condition or disease, slowing the onset or rate of onset of the condition or disease, reducing the risk of onset of the condition or disease, preventing or delaying the onset of symptoms associated with the condition or disease, reducing or terminating symptoms associated with the condition or disease, causing complete or partial regression of the condition or disease, or any combination thereof.

[0044] A "therapeutically effective amount" or "therapeutically effective dose" is an amount of engineered T cells that produces a desired therapeutic effect in a subject, such as preventing or treating a target condition by killing target cells or alleviating symptoms associated with the condition. The most effective outcome in terms of therapeutic efficacy in a given subject will vary depending on a variety of factors, including, but not limited to, the characteristics of the engineered T cells (including lifespan, activity, pharmacokinetics, efficacy, and bioavailability), the physiological condition of the subject (including age, sex, type and stage of disease, general condition, responsiveness to a given dosage, and type of medication), the characteristics of any pharmaceutically acceptable carrier(s) in any composition used, and the route of administration. The therapeutically effective dose of engineered T cells will also depend on the cell surface receptors expressed by the T cells (e.g., the affinity and density of cell surface receptors expressed on the cells), the type of target cells, the characteristics of the disease or condition being treated, or a combination thereof. Thus, in some aspects, a therapeutically effective dose of transduced engineered T cells is about 1 million to about 2 million transduced engineered T cells per kilogram of body weight (cells / kg). Thus, in some aspects, a therapeutically effective dose of transduced engineered T cells is about 1 million to about 3 million transduced engineered T cells / kg. In certain embodiments, the therapeutically effective dose is about 2 million transduced engineered T cells / kg. In certain embodiments, the therapeutically effective dose is at least about 2 million transduced engineered T cells / kg. In certain embodiments, the therapeutically effective dose is at least about 1 million, at least about 2 million, at least about 3 million, at least about 4 million, at least about 5 million, at least about 6 million, at least about 7 million, at least about 8 million, at least about 9 million, or at least about 10 million engineered T cells / kg.In other embodiments, the therapeutically effective dose may be less than 1 million cells / kg, 1 million cells / kg, 2 million cells / kg, 3 million cells / kg, 4 million cells / kg, 5 million cells / kg, 6 million cells / kg, 7 million cells / kg, 8 million cells / kg, 9 million cells / kg, 10 million cells / kg, more than 10 million cells / kg, more than 20 million cells / kg, more than 30 million cells / kg, more than 40 million cells / kg, more than 50 million cells / kg, more than 60 million cells / kg, more than 70 million cells / kg, more than 80 million cells / kg, more than 90 million cells / kg, or more than 100 million cells / kg. In other embodiments, the therapeutically effective dose may be from about 1 million cells / kg to about 2. million cells / kg, about 1 million cells / kg to about 3 million cells / kg, about 1 million cells / kg to about 4 million cells / kg, about 1 million cells / kg to about 5 million cells / kg, about 1 million cells / kg to about 6 million cells / kg, about 1 million cells / kg to about 7 million cells / kg, about 1 million cells / kg to about 8 million cells / kg, about 1 million cells / kg to about 9 million cells / kg, or about 1 million cells / kg to about 10 million cells / kg. In certain embodiments, the total therapeutically effective dose (transduced cells per patient) is about 1 x 10 6 of transduced cells, approximately 1 x 10 6 ~Approx. 1×10 7 of transduced cells, 1 × 10 7 ~Approx. 1×10 8 of transduced cells, 1 × 10 8 ~Approx. 1×10 9 of transduced cells, approximately 1 x 10 9 ~Approx. 1×10 10 of transduced cells, approximately 1 x 10 10 ~Approx. 1×10 11 of transduced cells, approximately 1 x 10 11 of transduced cells, or approximately 1 x 10 11 In one embodiment, the therapeutically effective dose may reach a number of transduced cells greater than about 1 x 10 8 ~about 2×10 8The therapeutically effective amount may be a transduced cell of the type described above. Those skilled in the clinical and pharmacological fields can determine the therapeutically effective amount through routine experimentation, i.e., by monitoring the subject's response to administration of the compound and adjusting the dosage accordingly. In certain embodiments, the cell surface receptor expressed by the engineered T cell is an anti-CD19 CAR. In certain embodiments, the anti-CD19 CAR may be an FMC63-28Z CAR or an FMC63-CD828BBZ CAR as described in Kochenderfer et al., J Immunother. 2009 September; 32(7): 689-702, the subject matter of which is incorporated herein by reference for the purpose of providing methods for constructing vectors used to generate T cells expressing an FMC63-28Z CAR or an FMC63-CD828BBZ CAR. In certain embodiments, a therapeutically effective dose of engineered T cells expressing an FMC63-28Z CAR or an FMC63-CD828BBZ CAR may be greater than about 1 million and less than about 3 million transduced engineered T cells per kilogram of body weight (cells / kg). In certain embodiments, a therapeutically effective dose of engineered T cells expressing an FMC63-28Z CAR or an FMC63-CD828BBZ CAR may be greater than about 1 million and less than about 2 million transduced engineered T cells per kilogram of body weight (cells / kg). In certain embodiments, a therapeutically effective dose of engineered T cells expressing an FMC63-28Z CAR or an FMC63-CD828BBZ CAR is between about 2 million and less than about 3 million transduced engineered T cells / kg. In certain embodiments, the therapeutically effective dose of engineered T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR is about 2 million, about 2.1 million, about 2.2 million, about 2.3 million, about 2.4 million, about 2.5 million, about 2.6 million, about 2.7 million, about 2.8 million, or about 2.9 million transduced engineered T cells / kg.In certain embodiments, a preferred therapeutically effective dose of engineered T cells expressing an FMC63-28Z CAR or an FMC63-CD828BBZ CAR is about 2 million transduced engineered T cells / kg. In certain embodiments, a therapeutically effective dose of engineered T cells expressing an FMC63-28Z CAR or an FMC63-CD828BBZ CAR is at least about 2 million transduced engineered T cells / kg.

[0045] In some embodiments, a pharmaceutical composition may comprise a population of engineered T cells produced by the methods described herein. In certain embodiments, a pharmaceutical composition may also comprise a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may be a pharmaceutically acceptable material, composition, or vehicle involved in the transport or transfer of cells of interest from one tissue, organ, or body part to another. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of a carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation. Each component of a carrier must also be suitable for contact with any tissue, organ, or body part that the carrier may come into contact with, i.e., without causing toxicity, irritation, allergic reaction, immunogenicity, or other adverse effects that would overwhelm its therapeutic benefit. This means that the risk of any other complication must not exceed the risk of any other complication.

[0046] As used herein, the term "about" means within a stated range of values ​​or within 5% or 10% of a range of values.

[0047] The following examples are intended to illustrate various embodiments of the present invention. Accordingly, the specific embodiments discussed should not be construed as limitations on the scope of the present invention. For example, although the following examples relate to T cells transduced with an anti-CD19 chimeric antigen receptor (CAR), those skilled in the art will understand that the methods described herein can be applied to T cells transduced with any CAR. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present invention, and it is understood that such equivalent embodiments are encompassed herein. Furthermore, all references cited in this disclosure are incorporated by reference in their entirety as if fully set forth herein. [Example]

[0048] Example 1: Generation of ex vivo genetically modified autologous cells An overview of an exemplary T cell manufacturing process (the "improved" process), according to one embodiment, is presented in FIG. 1. This improved process includes improvements over traditionally used T cell manufacturing processes (the "traditional" process) while maintaining the properties of the T cell product (see FIG. 2, which illustrates these improvements). Specifically, and unexpectedly, the improved process is a closed process that can eliminate the use of serum. Furthermore, this improved process produces a population of transduced T cells using a single cycle of transduction. Furthermore, cells that undergo a total of six days of expansion using this process exhibit a more juvenile immunophenotypic profile compared to cells that undergo ten days of expansion. While this process can reproducibly produce a product containing a target number of transformed T cells expressing a chimeric antigen receptor (CAR), for example, for CD19, these methods apply to T cells transduced with any CAR.

[0049] Specifically, the process is designed to be compatible with apheresis products collected using standard apheresis equipment and procedures, enrich a subject's apheresis for lymphocytes, activate the subject's T cells for a defined culture period in the presence of recombinant IL-2 and anti-CD3 antibodies, provide an ex vivo culture environment in which T cells selectively survive and proliferate, transfect the subject's T cells with a retroviral vector engineered to express the CD19 chimeric antigen receptor within a consistent range of transfection efficiencies, reduce product-related impurities to consistent levels (product-related impurities include non-T cells in the subject-derived starting material), and reduce process-related impurities to consistent levels (process-related impurities include growth medium, cytokines, and other process reagents).

[0050] Apheresis Collection. Leukocytes were collected (leukapheresis) using a standard apheresis device such as Cobe® Spectra, Spectra Optia®, Fenwal™ Amicus®, or equivalent. The leukapheresis process typically produces approximately 200 mL to 400 mL of patient-derived apheresis product. The apheresis product may be subjected to on-site manufacturing processing or, optionally, transported at 1°C to 10°C to a facility for manufacturing processing at a different location. Further processing steps may be performed in an ISO7 cell culture process area (or similar cleanroom-type environment), as outlined in Figure 1.

[0051] Volume reduction. Where appropriate, the volume reduction step of the improved process is implemented in Sepax® 2. The procedure was performed using cell processing equipment, such as a laboratory instrument (Biosafe SA, Houston, TX) or equivalent, and was carried out using standard sterile tubing kits. Given the variability in the number of cells derived from each subject and the volume of the incoming feedstock (approximately 200 mL to 400 mL), the volume reduction step was designed to standardize the cell volume to approximately 120 mL. If the apheresis volume is less than 120 mL, the volume reduction step is not necessary, and the cells are transported directly to the lymphocyte enrichment step. The volume reduction step is designed to standardize the volume of cells received from each subject, preserve mononuclear cells, achieve consistent cell yields and high cell viability, and maintain a closed system to minimize contamination risk.

[0052] Lymphocyte Enrichment. Following the volume reduction step, cells were subjected to Ficoll-based separation in a cell processing device such as Sepax® 2 or equivalent using a standard sterile tubing kit and a separation protocol developed and recommended by the device manufacturer (NeatCell Program). The lymphocyte enrichment step reduces product-related impurities such as RBCs and granulocytes, enriches and concentrates mononuclear cells, washes and reduces process-related residues such as Ficoll, and formulates cells in growth medium in preparation for cell activation, as well as achieving consistent cell yields and high cell viability. The closed system minimizes environmental contamination.

[0053] The process may be carried out in an ISO 7 area at ambient temperature, with all connections made using a sterile tubing welder or in an ISO 5 laminar flow hood.

[0054] T Cell Activation. The T cell activation step can be performed using either freshly prepared cells by lymphocyte enrichment or previously cryopreserved cells. If cryopreserved cells are used, the cells may be thawed prior to use using the developed protocol.

[0055] The T cell activation process selectively activates T cells to make them receptive to retroviral vector transduction, depletes the viable population of all other cell types, achieves consistent cell yields and high T cell viability, and maintains a closed system to minimize contamination risk.

[0056] Wash 1. Following the T cell activation step, cells were washed with fresh culture medium in a standard sterile kit using a cell handling device such as Sepax® 2 or equivalent, using a protocol developed by the manufacturer. Optionally, cells were concentrated to a final volume of approximately 100 mL in preparation for transduction with retroviral vectors. The Wash 1 step reduces process-related residues such as anti-CD3 antibodies, spent growth medium, and cell debris, achieving consistent cell yields and high T cell viability, maintaining a closed system to minimize contamination risk, and concentrating and delivering sufficient numbers of viable T cells in a low volume suitable for initiating transduction.

[0057] Retroviral transduction. Activated cells from Wash 1 in fresh cell growth medium were transferred to cell culture bags (PL240 from Origen Biomedical or equivalent) that had been pre-prepared by first coating the bags with recombinant fibronectin or its fragments, such as RetroNectin® (Takara Bio Inc., Japan), and then incubating them with retroviral vectors according to established protocols prior to transduction of the activated cells. Coating with RetroNectin™ (10 μg / mL) was performed for 20 hours ± 4 hours at 2°C to 8°C, followed by washing with dilution buffer and subsequent incubation with thawed retroviral vectors for approximately 180 to 210 minutes at 37°C ± 1°C and 5% ± 0.5% CO2. After adding the cells to the bags, transduction was carried out for 20 hours ± 4 hours at 37°C ± 1°C and 5% ± 0.5% CO2. The retroviral transduction process involves culturing activated T cells in the presence of retroviral vectors under controlled conditions to allow efficient transduction, achieve consistent cell yields and high cell viability, and maintain a closed system to minimize contamination risks.

[0058] Wash 2. Following the retroviral transduction step, using a protocol developed by the manufacturer, in a standard sterile kit, cells were washed with fresh growth medium using a cell processing device such as Sepax® 2 or equivalent, and the cells were concentrated to a final volume of approximately 100 mL in preparation for the expansion step. The Wash 2 step is designed to reduce process-related residues such as retroviral vector particles, process residues from vector production, spent growth medium, and cellular debris, achieve consistent cell yields and high cell viability, maintain a closed system to minimize contamination risk, and exchange the spent growth medium for fresh medium containing a target number of cells in a defined volume suitable for the start of the expansion step.

[0059] T Cell Expansion. Cells from Wash 2 were aseptically transferred into culture bags (Origen Biomedical PL325 or equivalent), diluted with fresh cell growth medium, and cultured for approximately 72 hours at 37°C ± 1°C and 5% ± 0.5% CO2. Cell density was measured daily starting on day 5. Because T cell doubling times can vary slightly from subject to subject, additional expansion time beyond 72 hours (i.e., 3 to 6 days) may be required if the total cell number is insufficient to deliver the target dose of CAR-positive T cells / kg of subject body weight. The T cell expansion process is designed to culture cells under controlled conditions to produce a sufficient number of transduced cells for effective dose delivery, maintain a closed system to minimize contamination risk, and achieve consistent cell yield and high cell viability. One such effective dose or target dose was 2 x 10 T cells produced via transduction with either the MSGV-FMC63-28Z or MSGV-FMC63-CD828BBZ retroviral vector, respectively. 6 and FMC63-28Z CAR-positive or FMC63-CD828BBZ CAR-positive T cells / kg (±20%) of subject's body weight, both of which are described in detail in Kochenderfer et al., J Immunother. 2009 September; 32(7): 689-702. No. 6,239,999, the subject matter of which is incorporated herein by reference in its entirety as if fully set forth herein.

[0060] Wash 3 and Concentration. Following the T cell expansion step, cells were washed with 0.9% saline using cell processing equipment such as Sepax™ 2 or equivalent in a standard sterile kit using protocols developed by the manufacturer, and the cells were concentrated to a final volume of approximately 35 mL in preparation for formulation and cryopreservation. The Wash 3 step is designed to reduce process-related residues, such as retroviral production process residues, spent growth medium, and cell debris, achieve consistent cell yields and high cell viability, and maintain a closed system to minimize contamination risk.

[0061] Once the cells are concentrated and washed with 0.9% saline, the appropriate cell dose may be formulated for making the final cryopreserved product. Cells were prepared for cryopreservation and cryopreserved according to the method presented in Example 4 below.

[0062] Example 2: Proliferation results of T cells expanded in cell culture bags The embodiments described herein provide efficient production of engineered autologous T cell therapy over a six-day period. The following improvements over prior art have been achieved: a shortened six-day process instead of either the previously used 24, 14, or 10 days, which reduces the number of tests required for product release (including RCR testing); an improved T cell product containing a higher percentage of immature T cells for increased potency and efficacy; cultures that can be initiated with a larger number of cells to offset the shorter manufacturing time; a closed system for performing the method steps described herein; identification of human serum-free culture conditions that support T cell proliferation; single-cycle retroviral transduction in bags; activation and expansion of cell cultures performed in bags rather than flasks; and provision of frozen products. These improvements have been used in the development and commercialization of novel engineered peripheral blood autologous T cell therapy (eACT) for the treatment of multiple cancer indications. Cells obtained from the development program maintained the same phenotypic and activity profile as cells grown by conventional methods.

[0063] Generation of Engineered T Cells. Figure 2 shows an overview of the T cell manufacturing process, including the improvements described herein in the improved process. Briefly, T cells are generated from peripheral blood mononuclear cells (PBMCs) obtained by apheresis, split from subjects with B cell malignancies. , operated in parallel using conventional techniques and the improved technique described herein. Five studies evaluated all process steps through day 6 expansion, except for the final washing and cryopreservation steps. Two additional studies, again using apheresis product from lymphoma patients, performed the improved process from the initial processing of the apheresis material through the final compounding and freezing steps.

[0064] The apheresis product (i.e., "sample") was enriched for lymphocytes by Ficoll separation of PBMCs using a closed Sepax 2 process, followed by supplementation with an investigational prototype supplement (Life Technologies' T Cell SR Media Supplement). in a serum-free medium (OpTmizer™ from Life Technologies) in a closed culture bag. Lymphocytes were expanded in PBS and stimulated with anti-CD3 antibody and rIL-2 (recombinant IL-2) for 48 hours (days 0-2) for T cell activation. Activated T cells were then washed using a closed Sepax 2 process.

[0065] On days 2-3 of the manufacturing process, activated T cells were transduced with anti-CD19 CAR using a gammaretroviral vector. Transduction was completed in a closed system as follows: Closed cell culture bags (i.e., Origen PermaLife™ PL240 bags) were coated with 2 μg / mL-10 μg / mL RetroNectin®, after which the RetroNectin® was removed and the bags were washed with buffered saline. The gammaretrovirus was then introduced into the closed system bag, followed by an incubation period. Activated T cells were then added directly to the bag containing the retroviral vector and incubated overnight at 37°C. The material from the RetroNectin®-coated culture bag was removed and placed in a separate cell culture bag for cell expansion. An optional washing step may be added prior to cell expansion. The transduced T cells were expanded in an antibiotic-free closed bag system for three days (days 3-6). The resulting engineered T cells were then harvested and cryopreserved (cryopreservation is an optional step).

[0066] Engineered T cell phenotype. Engineered T cells were analyzed by fluorescence-activated cell sorting (FACS) to (i) confirm CAR gene expression, (ii) confirm T cell population purity, and (iii) identify the cell phenotypes present in the engineered T cell population using cell surface expression of T cell subset markers CCR7, CD45RA, CD62L, and functional competitive markers CD27 and CD28.

[0067] The activity of engineered T cells was also analyzed using an in vitro co-culture bioassay to measure interferon gamma (IFNγ) production by engineered T cells after co-culture with antigen (Ag)-positive (i.e., CD19+) target cells. Engineered T cells were also analyzed by FACS for intracellular production of interferon gamma (IFNγ) by engineered T cells and for CD107a expression after co-culture with Ag-positive target cells.

[0068] Proliferation studies. T cell proliferation and viability were assessed in each experiment to ensure cell proliferation was stable and consistent with (or better than) previous studies.

[0069] On the sixth day, the cells were washed with saline, and then HSA was added to the cells until the cell volume reached 5%. was mixed 1:1 with CryoStor™ 10 (BioLife Solutions™). The cells were then frozen in a controlled-rate freezer using a defined freezing cycle and then stored in vapor phase liquid nitrogen. This process resulted in approximately 1 × 10 cells per mL. 6 ~1.5×10 7 We demonstrated that cells could be successfully cryopreserved and thawed in saline containing HSA, which was as good as or better than other solutions tested (e.g., PL / D5), and that HSA improved freeze-thaw recovery.

[0070] Cells were assessed for viability upon thawing by trypan blue exclusion as well as FACS (FACS-based staining for Annexin V as well as 7AAD). Cells retained phenotype and biological function as measured by IFN-gamma after thawing.

[0071] result Growth studies were performed in serum-free medium combined with a medium growth supplement. In these studies, performance was variable, with success defined as a medium that resulted in a phenotype similar to that of medium containing 5% human serum (AIMV). The serum-free medium used in the method described above resulted in superior T cell expansion and a phenotype similar to expansion in AIMV. One unexpected observation was that a cell density of approximately 1.5 x 10 cells was required to achieve superior growth and viability. 6 The main point was that cells had to be passaged when the cell concentration reached approximately 0.4 × 10 / mL. If cells were not passaged at this cell concentration, viability could decrease. 6 / mL ~ 1.5 × 10 6In the range of 0.1% / mL, cells grew well in either flasks or closed bag systems at 37°C with a doubling time of less than 24 hours.

[0072] The process of generating engineered T cells, in which new receptor genes are introduced into T cells using gammaretroviral vectors, requires that the cells be actively proliferating so that transduction can be successful. Here, T cells were transduced with an anti-CD19 CAR; however, the process described herein may be used with any CAR or TCR. We demonstrated that human T cell proliferation can be stimulated in OpTmizer™ medium using anti-CD3 antibody and IL2 in either an open T-flask or closed cell culture bag system. Using FACS, we demonstrated that CFSE-stained cells proliferated equally well in OpTmizer™ medium or AIMV containing 5% human serum during this stimulation. Although T cell proliferation was observed with other incubation times, a two-day incubation with anti-CD3 antibody and IL2 was demonstrated to be optimal for obtaining actively proliferating cells in OpTmizer™ medium.

[0073] Various conditions were evaluated to observe transduction in OpTmizer™ medium in a closed-bag system. One novel aspect of the present invention is the specific sequence of steps developed to achieve transduction in OpTmizer™ medium in a closed-bag system. The described process has the advantage of being operationally simple yet providing transduction frequencies similar to conventionally used conditions. Prior steps included in the transduction protocol (e.g., blocking the coated surface with a protein such as HSA) were found to be unnecessary. Transduction frequencies are not affected by washing of cells after removal from RetroNectin™-coated cell culture bags.

[0074] Phenotypic analysis of the cells either 6 or 10 days after the start of the stimulation process revealed that in OpTmizer™ medium, the cells were nearly identical to cells grown in AIMV medium in similar bag or commonly used plate systems (see Figures 4 and 5). Similarly, cells produced in OpTmizer™ medium in a simple closed-process bag system were able to produce IFN-gamma in response to Ag-positive target cells in an in vitro co-culture assay, demonstrating that T cells produced by this improved process are biologically active.

[0075] Table 1 below shows IFN gamma production (pg / ml) on day 6 using the improved process described herein.

[0076] [Table 1]

[0077] Another unexpected observation was that simply reducing the culture period from 10 days or more to 6 days in accordance with the present invention resulted in a more immature distribution of T cells, with an increased presence of naive cells, central memory cells, and a decreased appearance of differentiated effector T cells (see Figures 4 and 5). This has a favorable impact on the potency and other properties of the product. Specifically, sufficient cells can be recovered from the expanded product after only 3 days of culture. The total time from the start of stimulation to recovery of expanded and transduced cells is 6 days, resulting in approximately 1 x 10 8 ~2×10 8 Maintain a dose of CAR-positive cells (Figure 6). If a larger number of cells is needed, the cells can be reliably continued to grow in the bag and the cell culture can be harvested in 10 days or more. Alternatively, more cells can be used in the starting population to generate a larger cell population within a 6-day period.

[0078] Additionally, two full-scale studies were conducted using apheresis samples from lymphoma patients, in which day 6 cells were further washed with 0.9% saline, combined into the final product formulation, and cryopreserved. Day 6 cell products were evaluated for a number of parameters pre- and post-thaw. There was no significant difference in the percentage of CAR-positive T cells 3 days after thawing compared to pre-freeze levels, suggesting that the cryopreservation protocol did not impair CAR expression. Furthermore, CAR-positive cells were shown to subsequently recognize the CD19-specific antigen after co-culture with CD19-positive targets, as measured by IFN-gamma release. The viability of the cells upon thawing was 90% and 79%, respectively, for the two tested products.

[0079] Example 3: Development of transduction conditions in a closed system Previously, transduction of PBMCs was performed in non-tissue culture treated 6-well plates. Plates were coated with 10 μg / mL RetroNectin® overnight at 2°C-8°C or for 2 hours at room temperature. After incubation, RetroNectin® was removed, and plates were blocked with 2.5% HSA for 30 minutes and then washed with HBSS + 5 mM HEPES. In a plate-based process, retroviral vectors were applied to the coated wells, spun in a centrifuge, and approximately 75% of the viral supernatant was removed before adding cells for transduction by spinoculation.

[0080] As presented herein, three studies were completed to optimize RetroNectin™ concentration for transduction of PBMCs in closed cell culture bags and to determine whether HSA washes and removal of viral supernatants affected transduction. Cell activation, transduction, and expansion were performed in AIM V™ medium + 5% human serum. The first experiment was performed in the rigen PermaLife™ PL07 bag. RetroNectin™ concentrations ranging from 2 μg / mL to 40 μg / mL were evaluated in PBMCs from three separate donors. There was no significant difference between plate-based transduction and bag-based transduction at RetroNectin™ concentrations of 10 μg / mL and 40 μg / mL, or between transduction performed without HSA blocking at the 95% confidence level. However, at the same confidence level, reducing the RetroNectin™ concentration to 2 μg / mL or removing the retroviral vector from the bag prior to transduction revealed a modest decrease in transduction efficiency, as shown in Table 2 below.

[0081] [Table 2]

[0082] A second study in Origen PermaLife™ PL07 bags in AIM V® medium + 5% human serum using PBMCs from two separate donors confirmed the results from the first study, demonstrating that maximal transduction efficiency in the bag occurred in the range of 1 μg / mL to 20 μg / mL of RetroNectin® (see Figure 8). Furthermore, the HSA blocking step did not enhance the process or increase transduction efficiency (see Figure 9). Furthermore, the study showed that the phenotype (CD45RA / CCR7) of the transduced cells was unaffected by the elimination of the HSA blocking step.

[0083] In a third study, PBMCs from two donors were stimulated for two days in Origen PermaLife™ PL70 bags with either OpTmizer™ + 2.5% supplement or AIM V™ + 5% HSA. On day 2, cells were washed and transduced with retroviral vectors in PL30 or 6-well plates. The cell concentration during transduction was 0.5 x 10 6On day 3, the transduced cells were transferred to either T175 flasks (as a control) or PL30 bags. On day 6 or 7, the cells were analyzed in co-culture assays for potency and by FACS for CAR expression and phenotype. Figure 10 shows the effect of RetroNectin™ concentration on transduction frequency, where RetroNectin™ concentrations above 5 μg / mL had no effect on transduction frequency within the bags. Figure 11 shows that when the criteria of cell activation in response to CD19 antigen recognition on target cells (CD107a expression and IFN-gamma production) were evaluated, the activity of cells tested under these conditions was similar. In this case, transduced cells were incubated with CD19-positive Nalm6 cells for 4 hours and then stained for cell surface expression of CD107a and intracellular IFN-gamma production.

[0084] Thus, according to the methods provided herein, bags are coated with 10 μg / mL RetroNectin™, transduction is performed in bags using OpTmizer™ media + 2.5% supplements, the blocking step with HSA has no effect on transduction efficiency or on cell potency or phenotype, transduction in bags results in a T cell product with a phenotype similar to transduction in plates, and removal of the retroviral vector prior to adding cells to the bag during transduction supports a process that does not increase overall transduction frequency and may slightly decrease it.

[0085] Example 4: Development of a cryopreservation process for manufactured anti-CD19 CAR+ T cells A series of development studies were conducted to determine optimal conditions for cryopreservation of manufactured anti-CD19 CAR T cells. Studies were designed to establish conditions for high viability upon thawing, frozen product formulation, optimized freezing protocols, and to identify the effects of freeze-thawing on cell phenotype and potency. Performance was assessed using the following analytical methods: cell counting by trypan blue exclusion before and after thawing, annexin staining by FACS after thawing, FACS staining to identify CAR+ T cells and phenotype (CCR7, CD45RA), proliferation in culture of cryopreserved cells after thawing, and potency by IFN-gamma production after co-culture with CD19+ cells.

[0086] Retroviral vector-transduced PBMCs were used to assess the performance criteria described above. In the development study, 3 × 10 cells were transduced in a final cryopreservation volume of 20 mL. 6 / mL ~ 12 × 10 6 Cryopreserved cells in a concentration range of 1000 / mL were used. The cell density of the actual clinical product is expected to fall within this range based on subject weight and CAR transduction frequency. Studies were performed in either OriGen's CS50 bag or AFC's KryoSure™ 20-F bag, with no significant difference.

[0087] Transduced cells were washed and resuspended in a solution containing either 0.9% saline, with or without human serum albumin (HSA), or a 1:1 mixture of PLASMA-LYTE® A and D5 1 / 2 saline (5% dextrose / 0.45% NaCl). The cells were then mixed with CryoStor® CS10 at a 1:1 or 1:2 ratio. In various studies, cells were cryopreserved in a controlled-rate freezer (CRF) and stored in vapor-phase LN2 for more than two days before thawing and assessing viability, CAR expression, phenotype, and activity. In some experiments, cells were mixed 1:1 with 80% human AB serum + 20% DMSO as a control.

[0088] Cell recovery upon thawing was enhanced when a final concentration of 2.5% HSA was included in the cryopreserved product (Table 3). Furthermore, cells frozen with HSA maintained higher viability, initiated proliferation more quickly when returned to culture, and regained high cell viability more quickly (Table 4).

[0089] [Table 3]

[0090] [Table 4]

[0091] A study comparing 0.9% saline with PLASMA-LYTE® A / D5 1 / 2 saline showed no significant differences in thaw recovery, cell growth performance, or phenotype in post-thaw culture (Table 5). Furthermore, there was no improvement in these parameters when cells were diluted 1:2 with CryoStor™ CS10 versus 1:1 with CryoStor™ CS10. In most experiments using 5% human serum in T cell dilutions with a final concentration of 2.5% HSA in the cryopreserved product, performance was comparable to that of cells frozen 1:1 in 80% human AB serum / 20% DMSO. Therefore, it was decided to choose the final cryopreserved product, where cells were washed with 0.9% saline, HSA was added to 5%, and then the cells were diluted 1:1 in CryoStor™ CS10.

[0092] [Table 5]

[0093] Freezing cycle development was performed on selected formulations of 0.45% saline, 2.5% HSA, and 50% CryoStor™ CS10, with a final product volume of approximately 50 mL to 60 mL in Origen™ CS250 bags. All trials were performed independently using a single media formulation and volume for each trial. Air was purged from the bag, and product temperature was monitored by attaching a thermocouple to the outer surface of the bag. Custom Individual bags were placed into a freezing cassette designed to accommodate CS250 bags, such as BioGenic Systems™, Part Number ZC021, and placed in the middle of the freezing rack in a controlled rate freezer.

[0094] To help determine where a temperature spike begins, To achieve this, a series of freezing cycles were evaluated to identify the point at which 0.45% saline, 2.5% HSA, and 50% CryoStor™ CS10 spontaneously nucleated. After each trial, modifications to the freezing protocol were made until a satisfactory sample temperature profile was produced. The criterion for producing a satisfactory sample was that the cold spike should increase the heat of fusion to the maximum possible The temperature was adjusted to a degree that offset the freezing rate, and the samples were subjected to a cooling rate of 1°C / min. The optimized protocol for a 50 mL bag in a controlled rate freezer (CRF) is shown in Table 6, and the corresponding temperature profiles of the chamber and product are shown in Figure 12.

[0095] [Table 6]

Claims

1. 1. A method for producing T cells that express a cell surface receptor that recognizes a specific antigen moiety on the surface of a target cell, comprising: Enriching a population of lymphocytes obtained from the donor subject; stimulating said population of lymphocytes with one or more T cell stimulants to produce a population of activated T cells, said stimulation being carried out in a closed system using serum-free culture medium; transducing the population of activated T cells with a viral vector comprising a nucleic acid molecule encoding the cell surface receptor using a single cycle of transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium; expanding the population of transduced T cells for a predetermined period of time to produce a population of engineered T cells, wherein the expansion is performed in a closed system using serum-free culture medium; A method comprising:

2. 2. The method of claim 1, wherein the cell surface receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

3. The method of claim 1 , wherein the target cell is a cancer cell.

4. 4. The method of claim 3, wherein the cancer cells are B-cell malignancies.

5. 4. The method of claim 3, wherein the cell surface receptor is an anti-CD19 CAR.

6. 2. The method of claim 1, wherein the one or more T cell stimulatory agents are an anti-CD3 antibody and IL-2.

7. The method of claim 1 , wherein the viral vector is a retroviral vector.

8. 8. The method of claim 7, wherein the retroviral vector is an MSGV1 gamma retroviral vector.

9. 2. The method of claim 1, wherein the predetermined time period for expanding the population of transduced T cells is 3 days.

10. 10. The method of claim 1, wherein the time from enrichment of the population of lymphocytes to production of the engineered T cells is 6 days.

11. 11. The method of claim 10, wherein the engineered T cells are used to treat a cancer patient.

12. 12. The method of claim 11, wherein the cancer patient and the donor subject are the same individual.

13. The method of claim 1 , wherein the closed system is a closed bag system.

14. The method of claim 1 , wherein the population of cells comprises naive T cells.

15. 15. The method of claim 14, wherein about 35% to 43% of the population of engineered T cells comprises naive T cells.

16. 15. The method of claim 14, wherein at least about 35% of the population of engineered T cells comprises naive T cells.

17. 15. The method of claim 14, wherein at least about 43% of the population of engineered T cells comprises naive T cells.

18. A population of engineered T cells that express cell surface receptors that recognize specific antigenic moieties on the surface of target cells, Enriching a population of lymphocytes obtained from the donor subject; stimulating said population of lymphocytes with one or more T cell stimulants to produce a population of activated T cells, said stimulation being carried out in a closed system using serum-free culture medium; transducing the population of activated T cells with a viral vector comprising a nucleic acid molecule encoding the cell surface receptor using a single cycle of transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium; expanding the population of transduced T cells for a predetermined period of time to produce a population of engineered T cells, wherein the expansion is performed in a closed system using serum-free culture medium; A population produced by a method comprising:

19. 19. The population of claim 18, wherein the cell surface receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

20. 20. The population of claim 18, wherein the target cells are cancer cells.

21. 21. The population of claim 20, wherein the cancer cells are a B-cell malignancy.

22. 19. The population of claim 18, wherein the cell surface receptor is an anti-CD19 CAR.

23. 19. The population of claim 18, wherein the one or more T cell stimulatory agents are an anti-CD3 antibody and IL-2.

24. 19. The collection of claim 18, wherein the viral vector is a retroviral vector.

25. 25. The population of claim 24, wherein the retroviral vector is an MSGV1 gamma retroviral vector.

26. 20. The population of claim 18, wherein the predetermined time for expanding the population of transduced T cells is 3 days.

27. 20. The population of claim 18, wherein the time from enrichment of the population of lymphocytes to production of the engineered T cells is 6 days.

28. 28. The population of claim 27, wherein the engineered T cells are used to treat a cancer patient.

29. 29. The population of claim 28, wherein the cancer patient and the donor subject are the same individual.

30. 19. The population of claim 18, wherein the closed system is a closed bag system.

31. 19. The population of claim 18, wherein the population of engineered T cells comprises naive T cells.

32. 32. The population of claim 31, wherein about 35% to 43% of the population of engineered T cells comprises naive T cells.

33. 32. The population of claim 31 , wherein at least about 35% of the population of engineered T cells comprises naive T cells.

34. 32. The population of claim 31 , wherein at least about 43% of the population of engineered T cells comprises naive T cells.

35. A pharmaceutical composition comprising the population of engineered T cells according to any one of claims 18 to 34.

36. 36. The pharmaceutical composition of claim 35, comprising a therapeutically effective dose of the engineered T cells.

37. 37. The pharmaceutical composition of claim 36, wherein the cell surface receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

38. The pharmaceutical composition of claim 37, wherein the CAR is FMC63-28Z CAR or FMC63-CD828BBZ CAR.

39. 39. The pharmaceutical composition of claim 38, wherein the therapeutically effective dose is greater than about 1 million to less than about 3 million engineered T cells per kg of body weight.

40. 40. The pharmaceutical composition of claim 39, wherein the therapeutically effective dose is about 2 million engineered T cells / kg.

41. 1. A method for producing T cells, comprising: (a) obtaining a population of lymphocytes; (b) stimulating the population of lymphocytes with one or more stimulatory agents to produce a population of activated T cells, wherein the stimulation is performed in a closed system using serum-free culture medium; (c) transducing the population of activated T cells with a viral vector comprising a nucleic acid molecule encoding the cell surface receptor using at least one cycle of transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium; (d) expanding the population of transduced T cells to produce a population of engineered T cells, wherein the expansion is performed in a closed system using serum-free culture medium.

42. 42. The method of claim 41, wherein the cell surface receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

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