Methods of making chimeric antigen receptor-expressing cells

Abstract

To provide methods of making immune effector cells (e.g., T cells, NK cells) that can be engineered to express a chimeric antigen receptor (CAR).SOLUTION: A method comprises: a) providing a frozen input sample comprising immune effector cells, b) thawing the frozen input sample to produce a thawed sample, and c) performing elutriation on the thawed sample and collecting immune effector cells, thereby producing an output sample comprising immune effector cells that are suitable for expression of a chimeric antigen receptor (CAR).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Patent Application No. 62 / 271,695, filed December 28, 2015, and U.S. Patent Application No. 62 / 431,204, filed December 7, 2016, the contents of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on December 27, 2016, is named N2067-7100WO_SL.txt and is 257,808 bytes in size.

[0003] The present invention relates generally to methods for producing immune effector cells (e.g., T cells, NK cells) engineered to express chimeric antigen receptors (CARs), as well as compositions comprising them. [Background technology]

[0004] Adoptive cell transfer (ACT) therapy using autologous T cells, particularly those transduced with chimeric antigen receptors (CARs), has shown promise in clinical trials for several hematological cancers.

[0005] The production of genetically modified autologous T cells is currently a complex process that begins with patient-derived material (e.g., obtained from leukapheresis) from which engineered therapeutic T cells expressing a CAR are obtained. Patient leukapheresis material can have a high level of variability in its cellular composition. This starting material can vary greatly in cellular composition from patient to patient and within a single disease state. Cellular contaminants can include granulocytes, monocytes, erythrocytes, circulating blast cells, and platelets. Autologous cell therapy product manufacturing processes must also address the unique treatment histories and disease states of each patient, which further impact the cellular content of the starting material (Burger et al. 2014, Kaiser et al. 2015, Ramos et al. 2009). Furthermore, such contaminants from the starting material can adversely affect the manufacturing process, the quality of the final product, and the therapeutic efficacy of the product. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there is a need for methods and processes that result in more consistent production of CAR-expressing cell therapy products, thereby simplifying the manufacturing process, improving product quality, and maximizing the therapeutic efficacy of the product. [Means for solving the problem]

[0007] The present disclosure relates to methods of making immune effector cells (e.g., T cells, NK cells) that can be engineered to express a CAR, as well as compositions comprising them.

[0008]

[0006] Accordingly, in one aspect, the disclosure features a method of generating or enriching a population of immune effector cells (e.g., T cells) that can be engineered to express a chimeric antigen receptor (CAR), the method comprising performing elutriation. The method comprises providing a frozen input sample comprising immune effector cells, thawing the frozen input sample to generate a thawed sample, performing elutriation on the thawed sample, and collecting the immune effector cells, thereby generating an output sample comprising immune effector cells that are suitable for expression of the CAR.

[0009] In one embodiment, the frozen input sample is a plasma apheresis sample.

[0010] In one embodiment, the method comprises: i) depleting CD19+ cells under flow conditions; ii) performing density centrifugation using a medium containing iodixanol, e.g., a 60% iodixanol solution in water (e.g., Optiprep medium) or a medium having a higher density than Ficoll (e.g., higher than 1.077 g / ml, e.g., about 1.32 g / ml); iii) performing a washing step (e.g., on the thawed sample) with a buffer containing dextrose and / or sodium chloride, e.g., D5 1 / 2 NS medium (5% dextrose and 0.45% sodium chloride), e.g., a washing step carried out using a cell processing device, e.g., a cell washing device or a device for density gradient centrifugation, e.g., a CS5 (CellSaver5+) device; and iv) performing positive selection of CD3 / CD28+ cells under flow conditions; and further including one, two, three or all of:

[0011] In one embodiment, the method further comprises adjusting the viscosity of the thawed sample, for example, by adding an isotonic solution, such as PBS, to the thawed sample.

[0012] In one embodiment, elutriation is performed using a flow rate of about 30-82 mL / min or 50-80 mL / min and / or the collection volume is about 250-1250 mL or 300-1000 mL for each fraction. In one embodiment, elutriation is performed using a flow rate of about 30 mL / min, 40 mL / min, 50 mL / min, 60 mL / min, 70 mL / min, 72 mL / min, or 82 mL / min, e.g., about 70 or 72 mL / min. In one embodiment, elutriation is performed using a flow rate of about 30-40 mL / min, 40-50 mL / min, 50-60 mL / min, 60-70 mL / min, 70-72 mL / min, 70-82 mL / min, or 72-82 mL / min. In one embodiment, elutriation is performed using a harvest volume of about 250 mL, 400 mL, 500 mL, 900 mL, or 975 mL, e.g., about 400 or 975 mL. In one embodiment, elutriation is performed using a harvest volume of about 250-400 mL, 400-500 mL, 500-900 mL, 900-1000 mL, or 1000-1259 mL. In one embodiment, elutriation is performed at about 2400 rpm. In one embodiment, elutriation is performed at about 2000-2800 rpm, 2200-2600 rpm, or 2300-2500 rpm.

[0013] In one embodiment, the input sample comprises at least 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 35%, or 40% monocytes. In one embodiment, the input sample comprises less than 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% T cells. In one embodiment, the input sample comprises at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% B cells.

[0014] In one embodiment, the output sample contains less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, or 0.1% monocytes. In one embodiment, the output sample contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% T cells. In one embodiment, the output sample contains less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, or 0.1% B cells. In one embodiment, the output sample comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% CD4+CD25+ cells. In one embodiment, the output sample comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% CD8+CD25+ cells.

[0015] In one embodiment, the method results in a T cell yield recovery of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% T cells.

[0016] In one embodiment, the output sample is contacted with a nucleic acid encoding a CAR. In one embodiment, after contacting the output sample with a nucleic acid encoding a CAR, the output sample comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CAR+ cells. In such an embodiment, the output sample comprises at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CAR+CD4+ central memory cells. In such an embodiment, the output sample comprises at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CAR+CD8+ central memory cells.

[0017] In one embodiment, after contacting the output sample with a nucleic acid encoding a CAR, the output sample produces less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 pg of IFN-gamma (IFN-γ) per CAR-expressing cell, e.g., per transduced cell. IFN-gamma (IFN-γ) release assays are described herein, e.g., in the Examples. In one embodiment, after contacting the output sample with a nucleic acid encoding a CAR, the output sample produces a cytotoxicity level (e.g., EC 50 Cytotoxicity assays are described herein, for example, in the Examples.

[0018] In another aspect, the disclosure features a method of generating or enriching a population of immune effector cells (e.g., T cells) that can be engineered to express a CAR, the method including performing density gradient centrifugation (also referred to herein as density centrifugation). The method includes providing an input sample containing immune effector cells and performing density centrifugation using a medium containing iodixanol, e.g., a 60% aqueous solution of iodixanol, e.g., Optiprep medium, or a medium having a higher density than Ficoll (e.g., greater than 1.077 g / ml, e.g., about 1.32 g / ml), thereby generating an output sample containing immune effector cells that are suitable for expression of a CAR.

[0019] In one embodiment, the density gradient centrifugation method described herein comprises: i) depleting CD19+ cells under flow conditions; ii) performing elutriation on the input sample, which may be a thawed input sample; iii) performing a washing step (e.g., prior to density centrifugation) with a buffer containing dextrose and / or sodium chloride, e.g., D5½NS medium (5% dextrose and 0.45% sodium chloride), e.g., a washing step performed using a CS5 (CellSaver5+) device, and performing positive selection of CD3 / CD28+ cells under flow conditions. The method further includes performing one, two, three or all of the following:

[0020] In one embodiment, the density gradient centrifugation method described herein does not include one or more of: the use of a solution containing glycol, e.g., Ficoll solution; or performing a washing step using a buffer containing dextrose and / or sodium chloride, e.g., D5 1 / 2 NS medium, e.g., performed using a CS5 apparatus; or performing a positive selection step.

[0021] In one embodiment, density centrifugation is performed using a cell separation device, for example, a Sepax2 device.

[0022] In one embodiment, the input sample contains less than 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, or 15% T cells. In one embodiment, the input sample contains at least 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% monocytes. In one embodiment, the input sample comprises at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% B cells.

[0023] In one embodiment, the output sample comprises at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% T cells. In one embodiment, the output sample comprises less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.01% B cells.

[0024] In one embodiment, the density gradient centrifugation methods described herein result in a T cell yield recovery of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% T cells.

[0025] In another aspect, the disclosure features a method of generating a population of immune effector cells (e.g., T cells) that can be engineered to express a CAR, the method including a negative selection step to remove cancer-associated antigen-expressing cells, e.g., CD19-expressing (CD19+) cells. The method includes providing an input sample containing immune effector cells and removing CD19+ cells from the input sample under flow conditions, e.g., using a flow-through device, e.g., a cell processing system described herein, thereby generating an output sample containing immune effector cells that are suitable for expression of a CAR. In one embodiment, the CD19+ cells comprise B cells. In one embodiment, the CD19+ cells comprise lymphoblasts.

[0026] In one embodiment, the negative selection method described herein comprises: i) performing elutriation on the input sample, which may be a thawed input sample; ii) performing a density centrifugation step using a medium containing iodixanol, e.g., a 60% iodixanol solution in water (e.g., Optiprep medium) or a medium having a higher density than Ficoll (e.g., higher than 1.077 g / ml, e.g., about 1.32 g / ml); iii) performing a washing step (e.g., before removing CD19+ cells and / or after thawing the input sample) with a buffer containing dextrose and / or sodium chloride, e.g., D5 1 / 2 NS medium (5% dextrose and 0.45% sodium chloride), e.g., a washing step performed using a CS5 (CellSaver5+) device; and iv) Performing positive selection of CD3 / CD28+ cells under flow conditions The method further includes performing one, two, three or all of the following:

[0027] In one embodiment, the negative selection methods described herein do not involve performing elutriation or density centrifugation.

[0028] In one embodiment, CD19+ cells are removed from the input sample by magnetic separation. In one embodiment, magnetic separation comprises contacting the cells with a separation reagent. In one embodiment, the separation reagent comprises a magnetic or paramagnetic member and a CD19-binding member. In one embodiment, magnetic separation comprises flow cytometry or FACS. In one embodiment, CD19+ cells are removed by FACS. In one embodiment, magnetic separation comprises use of a magnetic cell separation device, e.g., a CliniMACs device. In one embodiment, CD19+ cells are removed by a CliniMACs device. In one embodiment, CD19+ cells are removed by a flow-through device as described herein, e.g., a cell processing system as described herein.

[0029] In one embodiment, the input sample contains at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% CD19+ cells. In one embodiment, the output sample contains less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.01% CD19+ cells. In one embodiment, the output sample contains less than 50%, 45%, 40%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 2%, 2% or 1% of the CD19+ cells compared to the input sample.

[0030] In one embodiment, the input sample comprises at least 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 35%, or 40% monocytes. In one embodiment, the input sample comprises less than 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% T cells. In one embodiment, the input sample (e.g., the input sample after washing) comprises at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% B cells.

[0031] In one embodiment, the output sample contains less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, or 0.1% monocytes. In some embodiments, the output sample contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% T cells. In one embodiment, the output sample contains less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.01% B cells.

[0032] In one embodiment, the negative selection methods described herein result in a T cell yield recovery of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% T cells.

[0033] In another aspect, the disclosure features a method of generating a population of immune effector cells (e.g., T cells) that can be engineered to express a CAR, the method including positive selection. The method includes providing an input sample that includes immune effector cells and positively selecting CD3+ / CD28+ cells from the input sample under flow conditions, thereby generating an output sample that includes immune effector cells that are suitable for expression of a CAR, e.g., the positive selection is performed under flow conditions.

[0034] In one embodiment, the positive selection method described herein comprises: i) depleting CD19+ cells, for example under flow conditions; performing elutriation on the input sample, which may be a thawed input sample; ii) performing density centrifugation using a medium containing iodixanol, e.g., a 60% iodixanol solution in water (e.g., Optiprep medium) or a medium having a higher density than Ficoll (e.g., higher than 1.077 g / ml, e.g., about 1.32 g / ml); and iii) performing a washing step (e.g., before removing CD19+ cells and / or after thawing the input sample) with a buffer containing dextrose and / or sodium chloride, e.g., D5 1 / 2 NS medium (5% dextrose and 0.45% sodium chloride), e.g., a washing step performed using a CS5 (CellSaver5+) device; The method further includes performing one, two, three or all of the following:

[0035] In one embodiment, the positive selection method described herein further comprises performing elutriation on the input sample (e.g., when the input sample is a thawed input sample). Optionally, elutriation is performed in conjunction with one or more (e.g., one, two, or all) of depleting CD19+ cells (e.g., as described in (i) above), performing density centrifugation (e.g., as described in (ii) above), and performing a washing step (e.g., as described in (iii) above).

[0036] In one embodiment, the positive selection method described herein further comprises performing elutriation, a washing step (if desired), and density centrifugation (e.g., using Ficoll or OptiPrep medium) prior to performing the positive selection. In one embodiment, the positive selection method described herein further comprises performing a washing step (if desired) and density centrifugation (e.g., using Ficoll or OptiPrep medium) prior to performing the positive selection. In one embodiment, the negative selection method described herein does not comprise performing elutriation. In one embodiment, the positive selection method described herein further comprises performing a wash using a buffer containing dextrose and / or sodium chloride, e.g., D5 1 / 2 NS buffer, e.g., using a CS5+ apparatus.

[0037] In one embodiment, positive selection involves contacting the input sample with a separation reagent comprising a magnetic or paramagnetic member and a CD3- and / or CD28-binding member. In one embodiment, positive selection of CD3+ / CD28+ cells involves incubating the input sample with the separation reagent for about 10-90 minutes, about 10-60 minutes, about 10-45 minutes, about 12-90 minutes, about 12-60 minutes, about 12-45 minutes, about 15-90 minutes, about 15-60 minutes, about 15-45 minutes, e.g., about 30 minutes or about 20 minutes. In one embodiment, the separation reagent comprises beads coupled (e.g., covalently or noncovalently) to anti-CD3 and / or anti-CD28 antibodies. In one embodiment, positive selection utilizes a ratio of magnetic separation members (e.g., beads) to T cells of about 3:1.

[0038] In one embodiment, positive selection involves flowing a fluid containing immune effector cells and magnetic separation members through a closed system, e.g., a chamber or bag, in which magnetic separation occurs. In one embodiment, flowing is performed at a speed that results in magnetic separation of the members (which may be bound to immune effector cells). In one embodiment, positive selection of CD3+ / CD28+ cells involves a separation or residence time of less than about 6, 5, 6, 3, 2, or 1 minute, or less than about 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 second.

[0039] In one embodiment, positive selection is performed using a magnetic device, such as a Dynamag CTS, a flow-through device containing a magnetic element as described herein, or other apparatus for magnetic elements.

[0040] In one embodiment, positive selection is performed using a device comprising at least one cell suspension module; at least one flow-through magnetic separation / debeading module; at least one non-magnetic output module; at least one magnetic output module; optionally, at least one magnetic component external to the magnetic separation / debeading module, the magnetic component generating a magnetic force and / or a magnetic field gradient; and optionally, at least one buffer module. In one embodiment, the device further comprises at least one magnetic component external to the magnetic separation / debeading module, the magnetic component generating a magnetic force and / or a magnetic field gradient. In one embodiment, the device further comprises at least one buffer module. In one embodiment, the magnetic separation / debeading module comprises a chamber defined by walls and having an x-direction, a y-direction, and a z-direction; an inlet and an outlet disposed at opposite ends of the chamber, e.g., in the x-direction, the y-direction, or the z-direction; and at least two magnets adjacent to or proximate walls of the chamber, positioned to establish a zero gradient line between the inlet and the outlet within the chamber. In one embodiment, the immune effector cells flow through a chamber, where each location within the chamber is within 2 cm of a magnet.

[0041] In one embodiment, the positive selection method includes contacting the immune effector cells with a solution containing dextrose and / or sodium chloride, e.g., D5 1 / 2 NS medium (5% dextrose and 0.45% sodium chloride), optionally at ambient temperature, e.g., about 20-25°C (e.g., between steps a) and b)). In one embodiment, the immune effector cells are present in a flexible container, e.g., a bag, e.g., during steps a) and b). In one embodiment, the method includes placing the bag on top of multiple layers of insulating material, e.g., paper, such as paper towels or wipes (e.g., between steps a) and b), e.g., after contacting the immune effector cells with saline). In some embodiments, the method includes incubating the cells at about 37°C for about 10 minutes (e.g., after step b)). In some embodiments, the method includes incubating the cells at about 36-38°C, 35-39°C, or 34-40°C for about 10 minutes (e.g., after step b)). In certain embodiments, the incubation step lasts for about 8-12 minutes, 5-15 minutes, or 5-20 minutes. In certain embodiments, the incubation is carried out in a Plasmatherm device.

[0042] In certain embodiments of the positive selection method, the input sample containing immune effector cells comprises at least 20% monocytes, hi certain embodiments, the input sample containing immune effector cells comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60% monocytes.

[0043] In one embodiment, the positive selection method comprises: a) thawing a frozen input sample (e.g., a leukapheresis sample) containing immune effector cells from a patient with a hematological malignancy, which sample may contain >20% lymphoblasts; b) washing the immune effector cells with a washing solution called "modified medium" (MM), e.g., X-VIVO15 medium (Lonza), at ambient temperature, e.g., 20-25°C; c) contacting the input sample with a separation reagent comprising a magnetic or paramagnetic member and a CD3 and / or CD28 binding member; d) rotating the input sample and separation reagent using a rotator, for example, at 2-6 rpm, for example, at 4 rpm (wherein the rotation lasts, for example, 10-30 minutes, for example, 20 minutes); and e) performing positive selection, e.g., for 30 seconds to 2 minutes, e.g., 1 minute, to enrich for cells that bind to the separation reagent; The term "amino acid sequence" includes one or more (e.g., two, three, four, or all) of the following, e.g., in the order listed:

[0044] In one embodiment, the positive selection method comprises: a) thawing a frozen input sample (e.g., a leukapheresis sample) containing immune effector cells from a patient with a hematological malignancy, which may contain >20% monocytes; b) washing the immune effector cells with a washing solution, e.g., a washing solution comprising about 5% dextrose and 0.45% sodium chloride, e.g., D5 1 / 2NS, e.g., at ambient temperature, e.g., 20-25°C; c) placing the flexible container containing the cells on top of a thermal insulating material, e.g., multiple layers including paper, e.g., paper towels or wipes; d) contacting the input sample with a separation reagent comprising a magnetic or paramagnetic member and a CD3 and / or CD28 binding member; e) incubating the input sample and separation reagent, e.g., at 37°C, for e.g., 5 to 15 minutes, e.g., 10 minutes; f) rotating the input sample and separation reagent using a rotator, for example, at 2-6 rpm, for example, at 4 rpm (wherein the rotation lasts, for example, 10-30 minutes, for example, 20 minutes); and g) performing positive selection, e.g., for 30 seconds to 2 minutes, e.g., 1 minute, to enrich for cells that bind to the separation reagent; The term "amino acid sequence" includes one or more (e.g., 2, 3, 4, 5, 6, or all) of the following, e.g., in the order listed:

[0045] In one embodiment, the sample, e.g., the input sample, is from a patient with a hematological malignancy, e.g., a hematological malignancy described herein, e.g., ALL or DLBCL.

[0046] In one embodiment, the input sample contains about 1 x 10 nucleated cells. 5 cells / ml, nucleated cells 2×10 5 cells / ml, 5 x 10 nucleated cells 5 cells / ml, nucleated cells 7×10 5 cells / ml, nucleated cells 1×10 6 cells / ml, nucleated cells 2×10 6 cells / ml, 5 x 10 nucleated cells 6 cells / ml, nucleated cells 7×10 6 cells / ml, nucleated cells 1×10 7 cells / ml, nucleated cells 2×10 7 cells / ml, 5 x 10 nucleated cells 7 cells / ml, nucleated cells 7×10 7 cells / ml, nucleated cells 1×10 7 cells / ml, nucleated cells 2×10 8 cells / ml, 5 x 10 nucleated cells 8 cells / ml and nucleated cells 7 × 10 8 In one embodiment, the input sample contains about 1-1.5 x 10 T cells / ml. 7 Includes pieces.

[0047] In one embodiment, the input sample comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% monocytes. In one embodiment, the input sample comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% tumor cells, e.g., lymphoblasts. In one embodiment, the input sample comprises less than 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% immune effector cells, e.g., T cells. In one embodiment, the input sample comprises at least about 5%, 10%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% B cells, e.g., CD45+CD19+ B cells. In one embodiment, the input sample comprises at least about 5%, 10%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% B cells, e.g., CD45-CD19+ B cells.

[0048] In one embodiment, the output sample contains less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, or 0.1% monocytes. In one embodiment, the output sample contains less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, or 0.1% tumor cells. In one embodiment, the output sample contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9% immune effector cells, e.g., T cells. In one embodiment, the output sample comprises at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% T cells, e.g., CD3+CD45+ T cells. In one embodiment, the output sample comprises less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% B cells, e.g., CD45+CD19+ B cells. In one embodiment, the output sample comprises less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% B cells, e.g., CD45-CD19+ B cells. In certain embodiments, the output sample contains at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or 20% T cells.

[0049] In another aspect, the present disclosure provides a method of generating a population of immune effector cells (e.g., T cells) that can be engineered to express a chimeric antigen receptor (CAR), comprising: i) providing an input sample comprising immune effector cells, e.g., a frozen input sample or a fresh input sample, and optionally, if the input sample is a frozen input sample, thawing the frozen input sample to generate a thawed sample; ii) performing a concentration step on the input sample, which may be a thawed input sample, comprising performing elutriation or performing a density centrifugation step using a medium comprising iodixanol, for example a 60% iodixanol solution in water, such as an Optiprep medium, or a medium having a higher density than Ficoll (for example higher than 1.077 g / ml, for example about 1.32 g / ml); iii) performing a selection step, wherein the selection is a positive selection, e.g., a positive selection of CD3 / CD28+ cells, or a negative selection, e.g., a negative selection of CD19+, CD25+, or CD14+ cells. thereby generating an output sample comprising immune effector cells that are suitable for expression of a CAR.

[0050] In another aspect, the present disclosure provides a method of generating a population of immune effector cells (e.g., T cells) that can be engineered to express a chimeric antigen receptor (CAR), comprising: i) providing an input sample containing immune effector cells, e.g., a frozen input sample or a fresh input sample; ii) optionally, if the input sample is a frozen input sample, thawing the frozen input sample to produce a thawed sample; iii) 1) performing elutriation on the input sample, which may be a thawed input sample; or 2) performing a density centrifugation step using a medium containing iodixanol, e.g., a 60% iodixanol solution in water, e.g., Optiprep medium, or a medium having a higher density than Ficoll (e.g., higher than 1.077 g / ml, e.g., about 1.32 g / ml); carrying out a concentration step comprising: iv) performing a selection step, wherein the selection is a positive selection, e.g., a positive selection of CD3 / CD28+ cells, or a negative selection, e.g., a negative selection of CD19+, CD25+, or CD14+ cells. thereby generating an output sample comprising immune effector cells that are suitable for expression of a CAR.

[0051] In another aspect, the present disclosure provides a method of generating a population of immune effector cells (e.g., T cells) that can be engineered to express a chimeric antigen receptor (CAR), comprising: i) providing an input sample containing immune effector cells, e.g., a frozen input sample or a fresh input sample; ii) performing a concentration step, which includes performing elutriation or density centrifugation (e.g., using Ficoll or Optiprep media); iii) performing a selection step, wherein the selection is a positive selection, e.g., a positive selection of CD3 / CD28+ cells, or a negative selection, e.g., a negative selection of CD19+, CD25+, or CD14+ cells. thereby generating an output sample containing immune effector cells that are suitable for expression of a CAR. In one embodiment, the selection step is performed under flow conditions, for example, by using a flow-through device.

[0052] Further features or embodiments of any of the methods or compositions described herein include one or more of the following.

[0053] In any of the embodiments of any of the methods described herein, the input sample is a biological sample from a subject containing immune effector cells, such as T cells and / or NK cells. In some embodiments, the input sample is a blood sample, e.g., a whole blood sample. In some embodiments, the input sample is an apheresis sample, e.g., a leukapheresis sample. In one embodiment, the input sample is a fresh sample obtained from a subject and processed using any of the methods described herein within 1, 2, 5, or 7 days of obtaining it from the subject. In one embodiment, the input sample is a frozen or cryopreserved sample, e.g., frozen at -20°C or in liquid nitrogen, or frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0054] In embodiments of any of the methods described herein, the input sample comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% monocytes (and, optionally, 40%, 70%, or 95% or less monocytes). In embodiments of any of the methods described herein, the input sample comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% tumor cells, e.g., lymphoblasts (and, optionally, 50% or less 95% monocytes). In embodiments of any of the methods described herein, the input sample comprises less than 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% immune effector cells, e.g., T cells (and optionally, more than 20% T cells).

[0055] In embodiments of any of the methods described herein, the output sample contains less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, or 0.1% monocytes (and optionally, greater than 1% or 0.1% monocytes). In embodiments of any of the methods described herein, the output sample contains less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, or 0.1% tumor cells (and optionally, greater than 1% or 0.1% tumor cells). In embodiments of any of the methods described herein, the output sample comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9% immune effector cells, e.g., T cells (and, optionally, 60% or less than 95% T cells).

[0056] In embodiments of any of the methods described herein, the output sample contains less than 50%, 45%, 40%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 2%, 2%, or 1% of monocytes compared to the input sample. In embodiments of any of the methods described herein, the output sample contains less than 50%, 45%, 40%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 2%, 2%, or 1% of tumor cells compared to the input sample. In embodiments of any of the methods described herein, the output sample contains at least 50%, 45%, 40%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 2%, 2%, or 1% of immune effector cells, e.g., T cells, compared to the input sample.

[0057] In embodiments of any of the methods described herein, the method further includes introducing, e.g., by transduction, a nucleic acid encoding a CAR into one or more immune effector cells in the output sample. Other methods of introducing a nucleic acid encoding a CAR are described herein.

[0058] In embodiments of any of the methods described herein, the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, including, for example, a primary signaling domain and / or a costimulatory signaling domain.

[0059] In embodiments of any of the methods described herein, the method further comprises assaying transduction efficiency. In embodiments of any of the methods described herein, the transduction results in a transduction efficiency of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

[0060] In embodiments of any of the methods described herein, the method further includes performing a wash step on the input sample with a buffer containing dextrose and / or sodium chloride, e.g., D5 medium, e.g., using a CS5+ instrument.

[0061] In embodiments of any of the methods described herein, the immune effector cells are human immune effector cells.

[0062] In embodiments of any of the methods described herein, the output sample comprises CD8+ T cells. In embodiments of any of the methods described herein, the output sample comprises CD4+ T cells.

[0063] In embodiments of any of the methods described herein, the input sample is from a patient having a disease associated with a tumor antigen, e.g., a tumor antigen described herein, e.g., CD19, wherein the disease is selected from a proliferative disease such as a cancer or malignancy, or a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or a preleukemia, or a non-cancer-related indication associated with expression of a tumor antigen described herein. In one embodiment, the disease is a cancer described herein, e.g., a cancer described herein as being associated with a target described herein. In one embodiment, the hematological cancer is leukemia. In one embodiment, the cancer is one or more acute leukemias, including but not limited to B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL); one or more chronic leukemias, including but not limited to chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL); one or more chronic leukemias, including but not limited to B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma,

[0013] The disease associated with expression of a tumor antigen described herein is selected from the group consisting of marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and additional blood cancers or hematological conditions including "preleukemia," which are a diverse collection of hematological conditions combined with ineffective production (or dysplasia) of blood cells in the bone marrow. The disease associated with expression of a tumor antigen described herein includes, but is not limited to, atypical and / or atypical cancers, malignancies, precancerous conditions, or proliferative disorders that express a tumor antigen described herein, and any combination thereof. In another embodiment, the disease associated with a tumor antigen described herein is a solid tumor, e.g., a solid tumor described herein, e.g., prostate, colorectal, pancreatic, cervical, gastric, ovarian, head, or lung cancer.

[0064] In embodiments of any of the methods described herein, the input sample may be one or more acute leukemias, including but not limited to, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL); one or more chronic leukemias, including but not limited to, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL); one or more chronic leukemias, including but not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell lymphoma, or leukemia. and from patients with cancer selected from the group consisting of an additional blood cancer or hematological condition, including leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, preleukemia, atypical and / or atypical cancers, malignancies, precancerous conditions or proliferative disorders, and any combination thereof.

[0065] In embodiments of any of the methods described herein, the input sample is from a patient with ALL.

[0066] In embodiments of any of the methods described herein, the method further comprises assaying one or more cell surface markers on cells in the output sample, for example, CD45, CD19, CD3, CD28, CD25, or CD14.

[0067] In embodiments of any of the methods described herein, the method further includes stimulating the output sample with an agent that stimulates the proliferation of immune effector cells, e.g., an agent that stimulates CD3 / TCR complex-associated signals, and / or a ligand that stimulates costimulatory molecules on the surface of T cells, e.g., an anti-CD3 antibody and an anti-CD28 antibody.

[0068] In embodiments of any of the methods described herein, the method further includes introducing, e.g., by transduction, transfection, or electroporation, a nucleic acid encoding a CAR.

[0069] In another aspect, the disclosure features a reaction mixture produced by a method disclosed herein, eg, a method disclosed above.

[0070] In another aspect, the disclosure features a reaction mixture that includes at least 80%, 85%, 90%, or 95% T cells and less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% monocytes, where the total number of cells in the reaction mixture totals 100%. In one embodiment, the reaction mixture includes at least 1 x 10 total T cells. 6 pieces, 2×10 6 pieces, 5×10 6 pieces, 1×10 7 pieces, 2×10 7 pieces, 5×10 7 pieces, 1×10 8 pieces, 2×10 8 pcs or 5 x 10 8 In one embodiment, the reaction mixture contains less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% B cells. In one embodiment, the reaction mixture contains less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% cancer cells, e.g., lymphoblasts.

[0071] In any of the reaction mixtures described herein, one or more of the T cells express a CAR, e.g., a CAR described herein.

[0072] In any of the reaction mixtures described herein, the reaction mixture further includes a nucleic acid encoding a CAR, e.g., a nucleic acid that is located within or outside the T cell.

[0073] Although suitable methods and materials are described below, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references (e.g., sequence database reference numbers) mentioned herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences referenced herein, for example, in any table herein, are incorporated by reference. Unless otherwise specified, the sequence accession numbers listed herein, including those in any table herein, refer to database entries as of December 28, 2015. When multiple sequence accession numbers are listed for a gene or protein, all sequence variants thereof are included.

[0074] Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting. Headings, subheadings, or numbered or lettered groupings of elements, e.g., (a), (b), (i), etc., are presented merely for ease of reading. The use of headings or numbered or lettered groupings of elements in this document does not require that the steps or elements be performed in alphabetical order or that the steps or elements are necessarily discrete from one another. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0075] [Figure 1A] FIG. 1A is a schematic diagram of a cell processing system with a flow-through magnetic separation / de-beading module.

[0076] [Figure 1B] FIG. 1B is a schematic diagram of a cell processing system with multiple flow-through magnetic separation / de-beading modules in parallel.

[0077] [Figure 1C]FIG. 1C is a schematic diagram of a cell processing system with multiple flow-through magnetic separation / de-beading modules in series.

[0078] [Figure 1D] FIG. 1D is a schematic diagram of a cell processing system with a return loop.

[0079] [Figure 1E] FIG. 1E is a schematic diagram of a cell processing system with a cell suspension module and a buffer module separately connected to a flow-through magnetic separation / de-beading module.

[0080] [Figure 1F] FIG. 1F is a schematic diagram of a cell processing system equipped with a spinning membrane de-beading module.

[0081] [Figure 1G] FIG. 1G is a schematic diagram of a cell processing system comprising multiple flow-through magnetic separation / de-beading modules and multiple spinning membrane de-beading modules.

[0082] [Figure 1H] FIG. 1H is another schematic diagram of a cell processing system with a flow-through magnetic separation / de-beading module.

[0083] [Figure 2A] FIG. 2A is a cross-sectional schematic of a flow-through magnetic separation / de-beading module in an x-oriented magnet configuration.

[0084] [Figure 2B] FIG. 2B is a semi-transparent three-dimensional schematic of the flow-through magnetic separation / de-beading module of FIG. 2A.

[0085] [Figure 3] Figure 3 is a side cross-sectional schematic diagram of a flow-through magnetic separation / de-beading module with membrane and sub-membrane fluid injection ports.

[0086] [Figure 4A] FIG. 4A is a cross-sectional schematic of a flow-through magnetic separation / de-beading module in a zero gradient configuration.

[0087] [Figure 4B] FIG. 4B is a semi-transparent three-dimensional schematic of the flow-through magnetic separation / de-beading module of FIG. 4A.

[0088] [Figure 4C] FIG. 4C is a top-view vertical cross-sectional schematic of a flow-through magnetic separation / de-beading module in a zero gradient configuration to create a zero gradient filter.

[0089] [Figure 4D] FIG. 4D is a top-view longitudinal cross-sectional schematic of a flow-through magnetic separation / de-beading module in a multiple zero gradient configuration to create a zero gradient filter.

[0090] [Figure 5A] FIG. 5A is a schematic longitudinal cross-sectional view of a spinning membrane de-beading module.

[0091] [Figure 5B] FIG. 5B is a cross-sectional schematic diagram of a rotating membrane de-beading module having a first magnet configuration.

[0092] [Figure 5C] FIG. 5C is a cross-sectional schematic diagram of a rotating membrane de-beading module with a second magnet configuration.

[0093] [Figure 6] FIG. 6 is a diagram of the fluid and magnetic forces on the cells.

[0094] [Figure 7A] 7A, 7B, and 7C are diagrams of the flow-through magnetic separation / de-beading module of FIG. 2B with cells undergoing magnetic separation. [Figure 7B] Same as above. [Figure 7C] Same as above.

[0095] [Figure 8A] 8A and 8B are diagrams of the flow-through magnetic separation / de-beading module of FIG. 2B with cells undergoing magnetic de-beading. [Figure 8B] Same as above.

[0096] [Figure 9] Figure 9 is a graph comparing the de-beading results using the flow-through magnetic separation / de-beading module of Figures 2A and 2B with the results using a conventional stop-flow module (boxes represent quartiles and median values).

[0097] [Figure 10A] FIG. 10A is a schematic diagram of the top vertical xy cross section of a flow-through magnetic separation / de-beading module, with paramagnetic particle-bound cells present near the module inlet (left) and near the module outlet (right).

[0098] [Figure 10B] FIG. 10 is a schematic diagram of a side longitudinal section of a flow-through magnetic separation / de-beading module, with paramagnetic particle-bound cells present near the module inlet (left) and near the module outlet (right).

[0099] [Figure 11] FIG. 11 is a diagram of a flow-through magnetic separation / de-beading module similar to that of FIG. 4A during magnetic separation of paramagnetic particle-bound and unbound cells.

[0100] [Figure 12] FIG. 12 is a diagram of the flow-through magnetic separation / de-beading module of FIG. 4C with cells undergoing magnetic separation of paramagnetic particle-bound and unbound cells.

[0101] [Figure 13]FIG. 13 is a diagram of the flow-through magnetic separation / de-beading module of FIG. 4C with cells undergoing magnetic separation of paramagnetic particles from de-beaded unbound cells.

[0102] [Figure 14] FIG. 14 is a diagram of the spinning membrane de-beading module of FIG. 5 with cells present during de-beading.

[0103] [Figure 15] Figures 15A and 15B are photographs showing cells after Sepax SmartWash and resuspension in modified medium (Figure 15A) or D51 / 2NS aqueous solution (Figure 15B).

[0104] [Figure 16] Figures 16A and 16B are graphs comparing cell recovery rates after different washing protocols. Figure 16A shows a comparison of cell recovery rates after the CS5+ wash procedure and the Sepax SmartWash procedure, both of which were performed in a D5 1 / 2NS aqueous solution. Figure 16B shows a comparison of cell recovery rates after a CS5 wash and 2-hour incubation for cells in modified medium compared to cells in a D5 1 / 2NS aqueous solution.

[0105] [Figure 17] FIG. 17 shows a comparison of the steps in Process B compared to the new OptiPrep process.

[0106] [Figure 18] Figure 18 is a schematic diagram showing how the Sepax 2 NeatCell bag and tubing kit works.

[0107] [Figure 19-1]Figures 19A, 19B, 19C, 19D, and 19E are graphs showing a phenotypic comparison of products produced by Process B (density gradient centrifugation using Ficoll product) or the novel OpriPrex-based Sepax method (OptiPrep product). Figure 19A shows T cell yield. Figure 19B shows B cell yield. Figure 19C shows monocyte yield. Figure 19D shows a relative comparison of the final product phenotypes. Figure 19E shows an absolute comparison of the final product phenotypes. [Figure 19-2] Same as above.

[0108] [Figure 20] FIG. 20 is a schematic diagram showing a sterile bag kit for optimized positive selection (FAST).

[0109] [Figure 21] FIG. 21 is a schematic diagram showing an improved lid for optimized positive selection (FAST).

[0110] [Figure 22] Figure 22 is a graph showing viable cell counts for clinical lots to date. The black line represents lots processed by Process B. The colored line represents lots processed by the FAST method.

[0111] [Figure 23A] Figures 23A, 23B, and 23C show details for Experiment 1 of Example 5. Figure 23A is a schematic overview and summary of the results. Figure 23B is a series of pre- and post-FAST enrichment plots for experimental arm 1. Figure 23C is a series of pre- and post-FAST enrichment plots for experimental arm 2. [Figure 23B] Same as above. [Figure 23C] Same as above.

[0112] [Figure 24A]Figures 24A, 24B, and 24C show details for Experiment 2 of Example 5. Figure 24A is a schematic overview and summary of the results. Figure 24B is a series of pre- and post-FAST enrichment plots for experimental arm 1. Figure 24C is a series of pre- and post-FAST enrichment plots for experimental arm 2. [Figure 24B] Same as above. [Figure 24C] Same as above.

[0113] [Figure 25A] Figures 25A and 25B show details for Experiment 3 of Example 5. Figure 25A is a schematic overview and summary of the results. Figure 25B is a series of pre- and post-FAST enrichment plots. [Figure 25B] Same as above.

[0114] [Figure 26A] Figures 26A and 26B are tables showing results from a CD19 negative selection run. Figure 26A shows the feasibility of CD19+ cell depletion in apheresis samples from healthy donors and ALL patients. Figure 26B shows the distribution of different cell types, T cells, monocytes, and CD19 B cells, before and after different enrichment protocols (TR149, TR150, and TR151). [Figure 26B] Same as above.

[0115] [Figure 27] Figure 27 is a schematic diagram showing the steps for Process B. "APH Sample" represents the input apheresis sample, which may be fresh or frozen. "CS5" represents a wash step, e.g., using a cell washing or cell processing system such as the CS5+ device. "Sepax" represents density gradient separation using Ficoll. "Positive Selection" represents Dynabeads-based "static" separation, e.g., using CD3 / CD28 Dynabeads.

[0116] [Figure 28]Figure 28 is a schematic diagram showing current "static" magnetic separation (left) and "dynamic" magnetic separation, e.g., under flow conditions (right). The "selection" (center) schematic shows the separation of a magnetic separation agent associated with its target molecule, which is attracted to a magnetic element, thereby separating the target molecule from undesired molecules.

[0117] [Figure 29] Figures 29A, 29B, and 29C show the results of an IFNγ release assay after stimulation with CD19 (Figure 29A), PMA (Figure 29B), and mesothelin (Figure 29C). The immune effector cell samples tested were prepared using different protocols, listed along the X-axis.

[0118] [Figure 30] Figures 30A, 30B, and 30C show the results of cytotoxicity assays. Figures 30A and 30B show the percentage of specific lysis for each immune effector sample prepared using the different protocols listed along the X-axis. Figure 30C shows the cytotoxicity results from Figures 30A and 30B as reciprocal EC50 units.

[0119] [Figure 31] 1 is a graph of recovery yield as a function of input cell number for a non-flow-through de-beading process and a flow-through de-beading process.

[0120] [Figure 32] 1 is a binned graph of recovery yield as a function of input cell number for the non-flow-through de-beading process and the flow-through de-beading process. DETAILED DESCRIPTION OF THE INVENTION

[0121] definition Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0122] The terms "a" and "an" refer to one or to more than one (i.e., at least one) of the grammatical object article. By way of example, "an element" means one element or more than one element.

[0123] The term "about," when referring to a measurable value such as an amount, duration, etc., is meant to encompass a ±20% variation, or in some cases a ±10% variation, or in some cases a ±5% variation, or in some cases a ±1% variation, or in some cases a ±0.1% variation from the stated value, since such variations are appropriate for performing the disclosed methods.

[0124] The term "chimeric antigen receptor" or alternatively "CAR" refers to a set of polypeptides, typically two polypeptides in the simplest embodiment, that, when present in an immune effector cell, confers specificity for a target cell, typically a cancer cell, and the generation of an intracellular signal to that cell. In some embodiments, a CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and an intracytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and / or costimulatory molecule, as defined below. In some embodiments, the set of polypeptides is in the same polypeptide chain (e.g., comprising a chimeric fusion protein). In some embodiments, the set of polypeptides is not contiguous with each other, e.g., in different polypeptide chains. In some embodiments, the set of polypeptides includes a dimerization switch that can couple the polypeptides to each other in the presence of a dimerization molecule, e.g., couple the antigen-binding domain to the intracellular signaling domain. In one embodiment, the stimulatory molecule of the CAR is a zeta chain associated with the T cell receptor complex. In one aspect, the intracytoplasmic signaling domain comprises a primary signaling domain (e.g., the primary signaling domain of CD3-zeta). In one embodiment, the intracytoplasmic signaling domain further comprises one or more functional signaling domains of at least one costimulatory molecule as defined below. In one embodiment, the costimulatory molecule is a costimulatory molecule described herein, e.g., 4-1BB (i.e., CD137), CD27, ICOS, and / or CD28. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain of a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain of a costimulatory molecule and a functional signaling domain of a stimulatory molecule.In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains of one or more costimulatory molecules and a functional signaling domain from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains of one or more costimulatory molecules and a functional signaling domain from a stimulatory molecule. In one embodiment, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, where the leader sequence may be cleaved from the antigen-binding domain (e.g., scFv) during intracellular processing and localization of the CAR to the cell membrane.

[0125] A CAR that includes an antigen-binding domain (e.g., an scFv or TCR) that targets a specific tumor antigen X, such as those described herein, is also referred to as an XCAR. For example, a CAR that includes an antigen-binding domain that targets CD19 is referred to as a CD19CAR.

[0126] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting intracellular signals to regulate cellular activity through defined signaling pathways by generating second messengers or functioning as an effector in response to such messengers.

[0127] The term "antibody," as used herein, refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural or recombinant sources. An antibody may also be a tetramer of immunoglobulin molecules.

[0128] The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv); Fd fragments consisting of the VH and CH1 domains; linear antibodies; single-domain antibodies, such as sdAb (either VL or VH); camelized VHH domains; multispecific antibodies formed from antibody fragments, such as bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments may also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes minibodies of fibronectin polypeptides).

[0129] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short, flexible polypeptide linker, so that the fusion protein can be expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it was derived. Unless otherwise specified, scFv, as used herein, may have the VL and VH variable regions in either order, e.g., with respect to the N- and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or VH-linker-VL.

[0130] The portion of the CAR comprising an antibody or antibody fragment thereof can exist in various forms in which the antigen-binding domain is expressed as part of a continuous polypeptide chain, including, for example, single-domain antibody fragments (sdAbs), single-chain antibodies (scFvs), and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the antigen-binding domain of the CAR comprises an antibody fragment. In a further embodiment, the CAR comprises an antibody fragment comprising an scFv.

[0131] As used herein, the term "binding domain" or "antibody molecule" refers to a protein, e.g., an immunoglobulin chain or fragment thereof, that contains at least one immunoglobulin variable domain sequence. The term "binding domain" or "antibody molecule" encompasses antibodies and antibody fragments. In certain embodiments, an antibody molecule is a multispecific antibody molecule, e.g., it contains multiple immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the multiple immunoglobulin variable domain sequences has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the multiple immunoglobulin variable domain sequences has binding specificity for a second epitope. In one embodiment, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which usually determines the class to which the antibody belongs.

[0132] The portion of the CAR of the present invention comprising an antibody or antibody fragment thereof may exist in various forms, where the antigen-binding domain is expressed as part of a continuous polypeptide chain, such as, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), a humanized antibody, or a bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the antigen-binding domain of the CAR composition of the present invention comprises an antibody fragment. In a further embodiment, the CAR comprises an antibody fragment comprising an scFv.

[0133] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which usually determines the class to which the antibody belongs.

[0134] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0135] The term "complementarity determining region" or "CDR" as used herein refers to the amino acid sequence in an antibody variable region that confers antigen specificity and binding affinity.For example, there are generally three CDRs (e.g., HCDR1, HCDR2, and HCDR3) in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, and LCDR3) in each light chain variable region.The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, such as those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme), or a combination thereof. According to the Kabat numbering scheme, in some embodiments, the amino acid residues of the CDRs in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the amino acid residues of the CDRs in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). According to the Chothia numbering scheme, in some embodiments, the amino acid residues of the CDRs in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues of the CDRs in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both.For example, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in a VH, e.g., a mammalian VH, e.g., a human VH; and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in a VL, e.g., a mammalian VL, e.g., a human VL.

[0136] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed in a bacteriophage or yeast expression system. The term shall also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody (which DNA molecule expresses the antibody protein) or an amino acid sequence specifying the antibody, where the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequence technology available and well known in the art.

[0137] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve either antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can serve as an antigen. Furthermore, antigens may be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response will thereby encode an "antigen," as the term is used herein. Those skilled in the art will also understand that an antigen is not necessarily encoded solely by the full-length nucleotide sequence of a gene. The present invention encompasses, but is not limited to, the use of partial nucleotide sequences from more than one gene, and that these nucleotide sequences may be arranged in various combinations to encode a polypeptide that elicits the desired immune response. Those skilled in the art will also understand that an antigen does not necessarily have to be encoded by a "gene." It will also be readily understood that an antigen may be generated or synthesized, derived from a biological sample, or may be a macromolecule other than a polypeptide. Such biological samples may include, but are not limited to, tissue samples, tumor samples, and fluids containing cells or other biological components.

[0138] The term "autologous" refers to any material derived from the same individual into which it is later reintroduced.

[0139] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to one another if their genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species may be genetically distinct enough to interact antigenically.

[0140] The term "xenogenic" refers to any material derived from an animal of a different species.

[0141] The term "cancer" refers to a disease characterized by the uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Various examples of cancer are described herein, including, but not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid and liquid tumors, e.g., diffuse or circulating tumors. The terms "cancer" or "tumor," as used herein, encompass precancerous as well as malignant cancers and tumors.

[0142] "Derived from," as the term is used herein, refers to the relationship between a first molecule and a second molecule. This generally refers to the structural similarity between the first molecule and the second molecule and does not imply or include a limitation on the process or source of the first molecule from which the second molecule is derived. For example, in the case of an intracellular signaling domain derived from a CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta structure to have the required function, i.e., the ability to generate a signal under appropriate conditions. This does not imply or include a limitation to the particular process by which the intracellular signaling domain is produced, e.g., it does not mean that one must start with the CD3 zeta sequence and delete or mutate unnecessary sequences to provide the intracellular signaling domain.

[0143] The phrase "disease associated with expression of a tumor antigen as described herein" includes, but is not limited to, a disease associated with expression of a tumor antigen as described herein or a condition associated with cells expressing a tumor antigen as described herein, including, for example, a proliferative disease such as a cancer or malignant tumor, or a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or preleukemia; or a non-cancer-related indication associated with cells expressing a tumor antigen as described herein. In one embodiment, a cancer associated with expression of a tumor antigen as described herein is a hematological cancer. In one embodiment, a cancer associated with expression of a tumor antigen as described herein is a solid cancer. Furthermore, diseases associated with expression of a tumor antigen as described herein include, but are not limited to, for example, atypical and / or atypical cancers, malignancies, precancerous conditions, or proliferative diseases associated with expression of a tumor antigen as described herein. Non-cancer-related indications associated with expression of a tumor antigen as described herein include, but are not limited to, for example, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, the tumor antigen-expressing cells express, or have at some time expressed, mRNA encoding the tumor antigen. In some embodiments, tumor antigen-expressing cells produce tumor antigen proteins (e.g., wild-type or mutant), which may be present at normal or reduced levels. In some embodiments, tumor antigen-expressing cells produced detectable levels of tumor antigen proteins at one time, but subsequently produced substantially no detectable tumor antigen proteins.

[0144] The phrase "diseases associated with CD19 expression" includes, but is not limited to, diseases associated with CD19 expression or conditions associated with cells expressing CD19, including, for example, proliferative diseases such as cancer or malignant tumors, or precancerous conditions such as myelodysplasia, myelodysplastic syndrome, or preleukemia; or non-cancer-related symptoms associated with cells expressing CD19. In one embodiment, the cancer associated with CD19 expression is a blood cancer. In one embodiment, the blood cancer is leukemia or lymphoma. In one embodiment, the cancer associated with CD19 expression includes, but is not limited to, cancers and malignancies such as, for example, but not limited to, one or more acute leukemias, such as, for example, acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL); and one or more chronic leukemias, such as, but not limited to, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL). Additional cancers or hematological conditions associated with CD19 expression include, but are not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, These include Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, myeloproliferative neoplasms; histiocytic disorders (e.g., mast cell disorders or blastic plasmacytoid dendritic cell neoplasm); mast cell disorders, such as systemic mastocytosis or mast cell leukemia; B-cell prolymphocytic leukemia, plasma cell myeloma, and "preleukemia," a diverse collection of hematological conditions combining ineffective production (or dysplasia) of blood cells in the bone marrow. Additionally, diseases associated with the expression of CD19 expression include, but are not limited to, atypical and / or atypical cancers, malignancies, precancerous conditions, or proliferative disorders associated with the expression of CD19.Non-cancer-related indications associated with CD19 expression include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, tumor antigen-expressing cells express or have at some time expressed mRNA encoding the tumor antigen. In certain embodiments, tumor antigen-expressing cells produce tumor antigen proteins (e.g., wild-type or mutant), and the tumor antigen proteins may be present at normal or reduced levels. In certain embodiments, tumor antigen-expressing cells produced detectable levels of tumor antigen proteins at one time, but subsequently did not produce substantially detectable tumor antigen proteins. In other embodiments, the disease is CD19-negative cancer, e.g., CD19-negative recurrent cancer. In some embodiments, tumor antigen (e.g., CD19)-expressing cells express or have at some time expressed mRNA encoding the tumor antigen. In some embodiments, tumor antigen (e.g., CD19)-expressing cells produce tumor antigen protein (e.g., wild-type or mutant), which may be present at normal or reduced levels. In some embodiments, tumor antigen (e.g., CD19)-expressing cells produced detectable levels of tumor antigen protein at one time, but subsequently produced substantially no detectable tumor antigen protein.

[0145] The phrase "diseases associated with B cell antigen expression" includes, but is not limited to, diseases associated with the expression of one or more of CD19, CD20, CD22, or ROR1, or conditions associated with cells that express, or have at any time expressed, one or more of CD19, CD20, CD22, or ROR1, including, for example, proliferative diseases such as cancer or malignant tumors, or precancerous conditions such as myelodysplasia, myelodysplastic syndrome, or preleukemia; or non-cancer-related indications associated with cells that express one or more of CD19, CD20, CD22, or ROR1. For the avoidance of doubt, diseases associated with B cell antigen expression may include conditions associated with cells that do not currently express a B cell antigen but that have expressed this antigen at some time, for example, because antigen expression has been downregulated due to treatment of B cells with a molecule that targets the B cell antigen, e.g., a CAR. The phrase "diseases associated with B cell antigen expression" includes diseases associated with CD19 expression, as described herein. In embodiments, the CAR-expressing cells are used to treat diseases associated with B cell antigens. In embodiments, the CAR produced by the methods herein comprises an antigen-binding domain that targets a B cell antigen.

[0146] The term "relapse," as used herein, refers to the reappearance of a disease (e.g., cancer) after a period of initial response (e.g., complete response or partial response), e.g., after a previous treatment associated with a therapy, e.g., cancer therapy. The period of initial response can include a decrease in the level of cancer cells below a certain threshold, e.g., below 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. Reappearance can include a rise in the level of cancer cells above a certain threshold, e.g., above 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, in the context of B-ALL, reappearance can include the reappearance of blasts in the blood, bone marrow (>5%), or any extramedullary site, e.g., after a complete response. A complete response in this context can include <5% BM blasts. More generally, in certain embodiments, a response (e.g., complete response or partial response) can include the absence of detectable MRD (minimal residual disease). In certain embodiments, the initial response period lasts for at least 1, 2, 3, 4, 5 or 6 days, at least 1, 2, 3 or 4 weeks, at least 1, 2, 3, 4, 6, 8, 10 or 12 months, or at least 1, 2, 3, 4 or 5 years.

[0147] "Refractory," as used herein, refers to a disease, e.g., a cancer, that does not respond to treatment. In embodiments, a refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, a refractory cancer may become resistant during treatment. A refractory cancer is also referred to as a resistant cancer.

[0148] The term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing that amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody or antibody fragment of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CARs described herein can be replaced with other amino acid residues from the same side chain family, and the altered CARs can be tested using the functional assays described herein.

[0149] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex or a CAR) with its cognate ligand (or tumor antigen in the case of a CAR), thereby mediating a signaling event, such as, but not limited to, signaling through the TCR / CD3 complex or through the appropriate NK receptor or signaling domain of the CAR. Stimulation may also mediate altered expression of certain molecules.

[0150] The term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides a cytoplasmic signaling sequence that regulates immune cell activation in a stimulatory manner with respect to at least some aspects of an immune cell signaling pathway. In one embodiment, the signal is a primary signal initiated, for example, by binding of the TCR / CD3 complex to a peptide-presenting MHC molecule, which results in mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. The primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif, known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs containing cytoplasmic signaling sequences that have particular use in the present invention include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In certain CARs of the invention, the intracellular signaling domain in any one or more CARs of the invention comprises an intracellular signaling sequence, such as the primary signaling sequence of CD3-zeta. In certain CARs of the invention, the primary signaling sequence of CD3-zeta is the sequence provided as SEQ ID NO: 9 (mutant CD3 zeta) or the equivalent residues from a non-human species, such as mouse, rodent, monkey, ape, etc. In certain CARs of the invention, the primary signaling sequence of CD3-zeta is the sequence provided as SEQ ID NO: 10 (wild-type human CD3 zeta) or the equivalent residues from a non-human species, such as mouse, rodent, monkey, ape, etc.

[0151] The term "antigen-presenting cell" or "APC" refers to immune system cells such as accessory cells (e.g., B cells, dendritic cells, etc.) that present foreign antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells may recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0152] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain can generate a signal that promotes the immune effector function of a cell containing a CAR, e.g., a CART cell. Examples of immune effector functions, for example, in a CART cell, include cytolytic activity and helper activity, such as cytokine secretion. In embodiments, the intracellular signaling domain is a portion of a protein that transmits an effector function signal and causes the cell to perform a specialized function. While the entire intracellular signaling domain may be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, as long as it transmits the effector function signal. Thus, the term intracellular signaling domain is meant to encompass any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0153] In some embodiments, the intracellular signaling domain may comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules involved in primary or antigen-dependent stimulation. In some embodiments, the intracellular signaling domain may comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules involved in costimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain may comprise a cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain may comprise a cytoplasmic sequence from a co-receptor or costimulatory molecule.

[0154] The primary intracellular signaling domain may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI, and CD66d, CD32, DAP10, and DAP12.

[0155] The terms "zeta" or alternatively "zeta chain," "CD3-zeta," or "TCR-zeta" are defined as the protein provided under GenBank Accession No. BAG36664.1 or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and "zeta stimulatory domain" or alternatively "CD3-zeta stimulatory domain" or "TCR-zeta stimulatory domain" are defined as the amino acid residues from the cytoplasmic domain of the zeta chain sufficient to functionally transmit the initial signal necessary for T cell activation. In one embodiment, the zeta cytoplasmic domain comprises residues 52 to 164 of GenBank Accession No. BAG36664.1 or the equivalent residues from a non-human species that is a functional ortholog, e.g., mouse, rodent, monkey, ape, etc. In one embodiment, the "zeta stimulatory domain" or "CD3-zeta stimulatory domain" is the sequence provided as SEQ ID NO:9. In one embodiment, the "zeta stimulatory domain" or "CD3-zeta stimulatory domain" is the sequence provided as SEQ ID NO:10.

[0156] The term "costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1B. B (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, IT GA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2 , CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile) , CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0157] A costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule. The intracellular signaling domain may comprise the entire intracellular portion, or the entire native intracellular signaling domain of the molecule from which it is derived, or a functional fragment thereof.

[0158] The intracellular signaling domain may comprise the entire intracellular portion of the molecule from which it is derived or the entire native intracellular signaling domain, or a functional fragment thereof.

[0159] The term "4-1BB" refers to a member of the TNFR superfamily having the amino acid sequence provided as GenBank Accession No. AAA62478.2 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and a "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Accession No. AAA62478.2 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc. In one embodiment, the "4-1BB costimulatory domain" is the sequence provided as SEQ ID NO: 7 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc.

[0160] "Immune effector cells," as the term is used herein, refer to cells that are involved in immune responses, for example, promoting immune effector responses. Examples of immune effector cells include T cells, such as alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0161] "Immune effector function or immune effector response," as used herein, refers to, for example, a function or response of an immune effector cell that enhances or promotes immune attack of a target cell. For example, immune effector function or response refers to a property of a T or NK cell that promotes killing or inhibiting the growth or proliferation of a target cell. In the case of T cells, primary stimulation and costimulation are examples of immune effector functions or responses.

[0162] The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity such as cytokine secretion.

[0163] The terms "depletion" or "depleting," when used interchangeably herein, refer to a reduction or decrease in the level or amount of cells, proteins, or macromolecules in a sample after a process, e.g., a selection step, e.g., negative selection, has been performed. Depletion may be a complete or partial depletion of cells, proteins, or macromolecules. In certain embodiments, depletion is at least a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% reduction or decrease in the level or amount of cells, proteins, or macromolecules compared to the level or amount of cells, proteins, or macromolecules in the sample before the process has been performed.

[0164] The term "encode" refers to the inherent property and resulting biological properties of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids. Thus, a gene, cDNA, or RNA encodes a protein when a protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and usually shown in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0165] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Also, the phrase "nucleotide sequence encoding a protein or RNA" may include introns to the extent that the nucleotide sequence encoding the protein may contain introns in some cases.

[0166] The term "endogenous" refers to any material derived from or produced within an organism, cell, tissue, or system.

[0167] The term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0168] The term "expression" refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0169] The term "transfer vector" refers to a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides bound to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" encompasses autonomously replicating plasmids or viruses. This term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0170] The term "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating the recombinant polynucleotide.

[0171] The term "lentivirus" refers to a genus of the Retroviridae family.Lentivirus is unique among retroviruses in that it can infect non-dividing cells, and it can deliver a significant amount of genetic information to the DNA of host cells, making it one of the most efficient gene delivery vectors.HIV, SIV and FIV are all examples of lentivirus.

[0172] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, and specifically includes self-inactivating lentiviral vectors such as those described in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used in clinical settings include, but are not limited to, Oxford BioMedica's LENTIVECTOR® gene delivery technology and LENTIMAX™ vector systems from Lentigen. Non-clinical types of lentiviral vectors are also available and are expected to be known in the art.

[0173] The term "homology" or "identity" refers to the identity of subunit sequences between two polymers, for example, between two nucleic acid molecules, for example, between two DNA molecules or two RNA molecules, or between two polypeptide molecules. If a subunit position in both molecules is occupied by the same monomer subunit, for example, if a position in each of two DNA molecules is occupied by adenine, they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in two sequences (e.g., 5 positions in a polymer length of 10 subunits) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, the two sequences are 90% homologous.

[0174] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies and their antibody fragments are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments may comprise residues found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications may further refine and optimize antibody or antibody fragment performance. Generally, a humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a substantial portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0175] "Fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, where the entire molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.

[0176] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide is "isolated" if it is partially or completely separated from the coexisting materials in its natural state. An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-native environment, such as a host cell.

[0177] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0178] The term "operably linked" or "transcriptional control" refers to the operably linking of a regulatory sequence with a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Operatively linked DNA sequences can be contiguous with each other and can be in the same reading frame, for example, when necessary to combine the coding regions of two proteins.

[0179] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection, intratumoral injection or infusion techniques.

[0180] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or combinations of DNA or RNA, and polymers thereof in either single- or double-stranded form. The term "nucleic acid" encompasses genes, cDNAs, or mRNAs. In one embodiment, a nucleic acid molecule is a synthetic (e.g., chemically synthesized) or recombinant nucleic acid molecule. Unless specifically limited, the term encompasses nucleic acids containing analogs or derivatives of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly presented. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with hybrid bases and / or deoxyinosine residues [Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)].

[0181] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may comprise a protein or peptide sequence. A polypeptide encompasses any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins, among others. A polypeptide can be a natural peptide, a recombinant peptide, or a combination thereof.

[0182] The term "promoter" refers to a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence.

[0183] The term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be the core promoter sequence, and in other instances, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be a promoter / regulatory sequence that expresses the gene product in a tissue-specific manner.

[0184] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes a gene product to be produced in a cell under most or all physiological conditions of the cell.

[0185] The term "inducible" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0186] The term "tissue-specific" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes a gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0187] The terms "cancer-associated antigen" or "tumor antigen" refer interchangeably to a molecule (typically a protein, carbohydrate, or lipid) that is preferentially expressed on the surface of cancer cells compared to normal cells, either in whole or as a fragment (e.g., MHC / peptide), and is useful for preferential targeting of pharmaceutical agents to cancer cells. In some embodiments, the tumor antigen is a marker expressed by both normal and cancer cells, such as a lineage marker, e.g., CD19 on B cells. In certain embodiments, the tumor antigen of the present invention is derived from cancer, including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinoma, such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, etc. In some embodiments, the cancer-associated antigen is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more, compared to normal cells. In some embodiments, cancer-associated antigens are cell surface molecules that are inappropriately synthesized in cancer cells, such as molecules that contain deletions, additions, or mutations compared to molecules expressed in normal cells. In some embodiments, cancer-associated antigens are expressed exclusively on the cell surface of cancer cells, either in their entirety or as fragments (e.g., MHC / peptide), and are not synthesized or expressed on the surface of normal cells. In some embodiments, the CARs of the present invention include CARs that contain an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to a peptide presented by MHC. Typically, peptides derived from endogenous proteins fill the pocket of major histocompatibility complex (MHC) class I molecules and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy.TCR-like antibodies that target peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Bood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibodies can be identified by screening libraries such as human scFv phage display libraries.

[0188] The term "flexible polypeptide linker" or "linker," when used in the context of an scFv, refers to a peptide linker consisting of amino acids such as glycine and / or serine residues, used alone or in combination, to link the variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker, having the amino acid sequence (Gly-Gly-Gly-Ser). n (SEQ ID NO: 15), where n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, and n=10. In one embodiment, flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 (SEQ ID NO: 27) or (Gly4Ser)3 (SEQ ID NO: 28). In another embodiment, the linker comprises multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO: 29). Linkers described in WO2012 / 138475, incorporated herein by reference, are also within the scope of the present invention.

[0189] 5' cap (RNA cap, RNA 7-methylguanosine cap or RNAm 7 As used herein, a 5' cap (also referred to as a G-cap) is a modified guanine nucleotide added to the "front" or 5' end of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of a terminal group attached to the first transcribed nucleotide. Its presence is crucial for ribosome recognition and protection from RNases. Capping is coupled with transcription, occurring by co-transcription, each affecting the other. Shortly after transcription initiation, the 5' end of the mRNA being synthesized is bound to a cap-synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions required for mRNA capping. Synthesis proceeds as a multistep biochemical reaction. The capping component may be modified to modulate mRNA functionality, such as its stability or translation efficiency.

[0190] "In vitro transcribed RNA," as used herein, refers to RNA, such as mRNA, synthesized in vitro. Generally, in vitro transcribed RNA is produced from an in vitro transcription vector. The in vitro transcription vector contains a template used to produce the in vitro transcribed RNA.

[0191] As used herein, "poly(A)" is a series of adenosines attached to an mRNA by polyadenylation. In some embodiments of constructs for transient expression, the poly(A) is between 50 and 5000 (SEQ ID NO: 30), e.g., greater than 64, e.g., greater than 100, e.g., greater than 300 or 400. The poly(A) sequence may be chemically or enzymatically modified to modulate mRNA functionality, such as localization, stability, or translation efficiency.

[0192] "Polyadenylation," as used herein, refers to the covalent linkage of a polyadenylyl moiety or its modified variants to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at their 3' ends. The 3' poly(A) tail is a long sequence (often several hundred) of adenine nucleotides added to pre-mRNA via the action of the enzyme polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added to transcripts containing a specific polyadenylation signal. The poly(A) tail and its associated proteins help protect the mRNA from exonuclease degradation. Polyadenylation is also important for transcription termination, mRNA export outside the cell nucleus, and translation. Polyadenylation occurs in the cell nucleus immediately after DNA transcription into RNA, but can also occur later in the cytoplasm. After transcription is terminated, the mRNA strand is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, an adenosine residue is added to the free 3' end at the cleavage site.

[0193] "Transient," as used herein, refers to expression of an unintegrated transgene for a period of hours, days, or weeks, where the period of expression is shorter than the period of expression of the gene when integrated into the genome or contained within a stable plasmid replicon in the host cell.

[0194] Apheresis is a process in which whole blood is removed from an individual, separated into selected components, and the remainder is returned to the circulation. Generally, there are two methods for separating blood components: centrifugal and non-centrifugal. Leukapheresis involves the active selection and removal of a patient's white blood cells.

[0195] As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or amelioration of the progression, severity, and / or duration of a proliferative disorder or an amelioration of one or more symptoms (e.g., one or more discernible symptoms) of a proliferative disorder resulting from the administration of one or more therapies (e.g., one or more therapeutic agents, e.g., a CAR of the invention). In certain embodiments, the terms "treat," "treatment," and "treating" refer to an amelioration of at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which may not necessarily be discernible by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to inhibiting the progression of a proliferative disorder, either physically, e.g., by stabilization of a discernible symptom, physiologically, e.g., by stabilization of a physical parameter, or both. In other embodiments, the terms "treat," "treatment," and "treating" refer to a reduction or stabilization of tumor size or cancer cell number.

[0196] The term "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" encompasses molecules and molecular complexes that can receive a signal and transmit the signal across the cell membrane.

[0197] The term "subject" is intended to include organisms in which an immune response can be elicited (eg, mammals, humans).

[0198] The term "substantially purified" cells refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types that normally coexist with them in their naturally occurring context. In some cases, a population of substantially purified cells refers to a homogenous population of cells. In other instances, the term simply refers to cells that have been separated from the cells that naturally coexist with them in their native state. In some embodiments, such cells are cultured in vitro. In other embodiments, such cells are not cultured in vitro.

[0199] In the context of this invention, a "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen common to a particular hyperproliferative disorder. In certain embodiments, the tumor antigen is derived from a cancer, including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinoma, such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, etc.

[0200] The terms "transfection" or "transformation" or "transduction" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0201] The term "specifically binds" refers to antibodies or ligands that recognize and bind to their cognate binding partner proteins present in a sample, but do not substantially recognize or bind to other molecules in the sample.

[0202] Ranges: Throughout this disclosure, various aspects of the invention may be presented in range format. It will be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range shall be considered to have specifically disclosed all possible subranges, as well as individual numerical values within that range. For example, the description of a range such as 1 to 6 would be considered to have specifically disclosed subranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes something with 95%, 96%, 97%, 98% or 99% identity, and also includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity, regardless of the breadth of the range.

[0203] description Provided herein are methods for producing immune effector cells (e.g., T cells, NK cells) that can be processed with CAR, for example, the CAR described herein, as well as reaction mixtures and compositions containing such cells. Provided herein are methods that improve the yield and quality, for example, purity, of cells suitable for CAR expression. Without wishing to be bound by theory, it is believed that improving the yield and quality of cells that can be engineered to express CAR improves the efficiency of introducing the nucleic acid encoding CAR, and improves the expansion of the resulting CAR-expressing cells. Thus, the methods and compositions described herein provide improved CAR-expressing cell products for use in treating target diseases.

[0204] In one aspect, the present disclosure features methods for removing undesirable substances, non-target cells, or cells that may adversely affect the expression of a CAR or the therapeutic effect of a CAR-expressing cell. For example, the methods featured herein can be used to remove or deplete any one or more of the following: monocytes, granulocytes, red blood cells, platelets, B cells, cancer cells such as lymphoblasts, cryoprotectants (from frozen samples), hemoglobin, or cell debris. For example, the methods featured herein can be used to enrich or increase the number of any one or more of the following: T cells (CD4+ and / or CD8+ T cells), NK cells, dendritic cells. Practice of each method described herein, alone or in any combination with each or all of the methods described herein, results in improved starting material suitable for engineering to express a CAR.

[0205] Fresh apheresis material is often used to produce cells suitable for CAR expression. The use of frozen, e.g., cryopreserved, apheresis material has the advantage that it can be easily transported, eliminating any limitations related to the proximity of patients to CAR-expressing cell product manufacturing facilities, enabling the industrialization of the CAR-expressing cell manufacturing process and enabling greater availability of therapeutic products to patients in need thereof. Currently used methods for producing CAR-expressing cells are optimized for processing fresh apheresis material, and these methods cannot be used to obtain the same quality or yield of cells suitable for CAR expression from frozen apheresis samples. In contrast, the methods described herein can be used to process and produce cells suitable for CAR expression from frozen, e.g., cryopreserved, apheresis samples. In embodiments in which the starting material is frozen, e.g., cryopreserved, the methods described herein optionally include a thawing step in which the frozen cells are left to thaw, e.g., without operator or device intervention to accelerate the thawing process, or a thawing step in which the frozen cells are subjected to a device or process that accelerates the thawing process, e.g., by using a thawing device, e.g., PlasmaTherm. In such embodiments, the thawing material has the same temperature as the ambient environment, e.g., the same temperature as the ambient room temperature, or the same temperature as the buffer to which the thawing material is added, used to wash the thawing material, or incubated with the thawing material. The methods described herein are particularly useful for generating or enriching a population of immune effector cells that can be processed to express a CAR from a frozen or thawed input sample, e.g., a frozen or thawed apheresis sample.

[0206] Process B, as referred to herein, is a currently used standard protocol for enriching immune effector cells that can be engineered to express a CAR. Process B involves performing density gradient purification using Ficoll and positive selection using CD3 / CD28 Dynabeads, where the input sample is fresh apheresis material. The method described herein results in a higher degree of enrichment, improved quality, and yield of desired immune effector cells suitable for expressing a CAR.

[0207] In another aspect, the disclosure features an immune effector cell (e.g., a T cell, an NK cell) engineered to express a CAR, e.g., produced by any of the manufacturing methods described herein, wherein the engineered immune effector cell exhibits anti-tumor properties. In one embodiment, the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. An exemplary antigen is a cancer-associated antigen (i.e., a tumor antigen) described herein. In one aspect, a cell is transformed with a CAR, and the CAR is expressed on the cell surface. In some embodiments, the cell (e.g., a T cell, an NK cell) is transduced with a viral vector encoding the CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell can stably express the CAR. In another embodiment, the cell (e.g., a T cell, an NK cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding the CAR. In some such embodiments, the cell can transiently express the CAR.

[0208] Additionally, the present disclosure provides CAR-expressing cell compositions and uses of the CAR-expressing cell compositions in medicaments or methods for treating, among other diseases, cancer or any malignancy or autoimmune disease involving cells or tissues that express tumor antigens as described herein.

[0209] Elutriation In one aspect, the methods described herein feature an elutriation method that results in improved enrichment of desired immune effector cells suitable for CAR expression by removing undesirable cells, such as monocytes and blasts. In one embodiment, the elutriation method described herein is optimized for enrichment of desired immune effector cells suitable for CAR expression from a previously frozen sample, such as a thawed sample. In one embodiment, the elutriation method described herein provides a preparation of cells with improved purity compared to preparations of cells collected from elutriation protocols known in the art.

[0210] To facilitate manufacturing logistics (remote sample collection, shipping, storage, and production unit scheduling), the cell source is typically cryopreserved whole blood or apheresis material, which must be thawed before manufacturing begins. However, the density and size of cells from previously frozen, thawed material are quite different from those of fresh material. Therefore, standard elutriation protocols commonly used to isolate cells for engineering CAR expression often fail to remove monocytes, granulocytes, or any larger-sized cells from cryopreserved, thawed whole blood or apheresis material. This situation adversely affects the results of subsequent CAR manufacturing steps, resulting in poor yields, product quality concerns, and out-of-specification process drift. While elutriation can remove monocytes, it is ineffective at removing blast cells, which have a density and size similar to T lymphocytes.

[0211] In certain embodiments, the elutriation methods described herein involve the use of an optimal viscosity of the starting sample, e.g., a cell sample, e.g., a lysed cell sample, by dilution with a particular isotonic solution (e.g., PBS) and the use of an optimal combination of flow rate and collection volume for each fraction collected by the elutriation device. An example of an improved elutriation program is described in Example 1.

[0212] Exemplary elutriation setting ranges for the separation of lymphocytes, e.g., T cells, from monocytes are provided in Table 4. Settings for flow rate, centrifugation, and volume for an exemplary elutriation program are also provided in Table 4 in the column labeled "Ex." [Table 1]

[0213] In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more fractions are collected from the elutriation step. In one embodiment, 5 fractions are collected from the elutriation step. In an embodiment where 5 fractions are collected, the third fraction (F3) or the fourth fraction (F4), or a combination of the third and fourth fractions, contains the desired lymphocyte population and has minimal amounts of monocytes, granulocytes, and other non-lymphocyte cells. In one embodiment, each fraction is collected using a different flow rate. In one embodiment, the flow rate for each fraction is increased from the flow rate used to collect the previous fraction. In one embodiment, one or more of the fractions are collected using a different collection volume.

[0214] In one embodiment, elutriation is performed using a flow rate of about 20-90 mL / min, about 30-90 mL / min, about 40-90 mL / min, about 50-90 mL / min, about 60-90 mL / min, about 70-90 mL / min, about 40-85 mL / min, about 50-82 mL / min, about 60-82 mL / min, about 70-82 mL / min, about 50-80 mL / min, about 60-80 mL / min, or about 70-80 mL / min. In one embodiment, elutriation is performed using a flow rate of about 30-82 mL / min or about 50-80 mL / min. In one embodiment, elutriation is performed using a flow rate of about 30, 40, 50, 60, 70, 72, 80, or 82 mL / min. In one embodiment, elutriation is performed using a flow rate of about 70 mL / min or 72 mL / min.

[0215] In one embodiment, the flow rate for one or more fractions containing the desired lymphocyte population and having significant amounts of monocytes, granulocytes, other non-lymphocyte cells, and other undesirable components is about 20-90 mL / min, about 30-90 mL / min, about 40-90 mL / min, about 50-90 mL / min, about 60-90 mL / min, about 70-90 mL / min, about 40-85 mL / min, about 50-82 mL / min, about 60-82 mL / min, about 70-82 mL / min, about 50-80 mL / min, about 60-80 mL / min, or about 70-80 mL / min. In one embodiment, the flow rate for one or more fractions containing the desired lymphocyte populations is about 50-82 mL / min, about 50-80 mL / min, about 60-82 mL / min, about 60-80 mL / min, about 70-82 mL / min, about 70-80 mL / min, about 70-75 mL / min, or about 70-72 mL / min. In one embodiment, the flow rate for one or more fractions containing the desired lymphocyte populations is about 70 mL / min or 72 mL / min.

[0216] In one embodiment, elutriation is performed using a collection volume of about 250 to 1250 mL, about 250 to 1000 mL, about 300 to 1000 mL, about 400 to 1000 mL, about 500 to 1000 mL, about 600 to 1000 mL, about 700 to 1000 mL, about 800 to 1000 mL, about 900 to 1000 mL, about 250 to 975 mL, about 300 to 975 mL, about 400 to 975 mL, about 500 to 975 mL, about 600 to 975 mL, about 700 to 975 mL, about 800 to 975 mL, about 300 to 900 mL, about 300 to 800 mL, about 300 to 700 mL, about 300 to 600 mL, about 300 to 500 mL, or about 300 to 400 mL. In one embodiment, elutriation is performed using a harvest volume of about 250, 400, 500, 900, or 975 mL. In one embodiment, elutriation is performed using a harvest volume of about 400 mL or about 975 mL.

[0217] In one embodiment, the collection volume for one or more fractions containing the desired lymphocyte population and having significant amounts of monocytes, granulocytes, other non-lymphocyte cells, and other undesired components is about 250-1250 mL, about 250-1000 mL, about 300-1000 mL, about 400-1000 mL, about 500-1000 mL, about 600-1000 mL, about 700-1000 mL, about 800-1250 mL, about 900-1000 mL, about 1000-1250 mL, about 1100-1250 mL, about 1250-1000 mL, about 1300-1000 mL, about 1400-1000 mL, about 1500-1000 mL, about 1600-1000 mL, about 170 The collection volume for one or more fractions containing the desired lymphocyte population is about 0 to 1000 mL, about 900 to 1000 mL, about 250 to 975 mL, about 300 to 975 mL, about 400 to 975 mL, about 500 to 975 mL, about 600 to 975 mL, about 700 to 975 mL, about 800 to 975 mL, about 300 to 900 mL, about 300 to 800 mL, about 300 to 700 mL, about 300 to 600 mL, about 300 to 500 mL, or about 300 to 400 mL. In one embodiment, the collection volume for one or more fractions containing the desired lymphocyte population is about 250 mL, 400 mL, 500 mL, 900 mL, or 975 mL. In one embodiment, the collection volume for one or more fractions containing the desired lymphocyte population is about 400 mL or about 975 mL.

[0218] In one embodiment, the elutriation method described herein is performed using an elutriation device. For example, the elutriation device is the Caridian BCT Elutra™ Cell Separation System (Terumo BCT Model 71800). The Caridian BCT Elutra™ Cell Separation System (Terumo BCT Model 71800) is a closed system that utilizes continuous counterflow elutriation technology to separate cells primarily based on size and secondarily on specific gravity. Due to the opposing forces generated by the flow of media into the separation chamber and the sedimentation velocity caused by centrifugal force, cells are sorted by size and density within the separation chamber, where they are automatically siphoned into a collection bag. The dedicated Elutra setup is designed to allow for the distribution of lymphocytes and monocytes combined with granulocytes in different fractions. The Elutra can be operated according to the manufacturer's instructions.

[0219] Density gradient centrifugation The manufacture of adoptive cell therapy products requires the separation of desired cells, such as immune effector cells, from the complex mixture of blood cells and blood components present in the starting material for peripheral blood apheresis. Peripheral blood-derived lymphocyte samples have been successfully isolated using density gradient centrifugation with Ficoll solution. However, Ficoll is not suitable for clinical use and is therefore not a preferred reagent for isolating therapeutic cells. Additionally, Ficoll contains glycol, which may be toxic to cells. Furthermore, Ficoll density gradient centrifugation of thawed apheresis products after cryopreservation results in suboptimal T cell products, as described in the Examples herein. For example, a loss of T cells in the final product, accompanied by a relative increase in non-T cells, particularly undesirable B cells, blast cells, and monocytes, has been observed in cell preparations isolated by density gradient centrifugation with Ficoll solution.

[0220] Without wishing to be bound by theory, it is believed that immune effector cells, such as T cells, dehydrate during cryopreservation, becoming more concentrated than fresh cells. Without wishing to be bound by theory, it is believed that immune effector cells, such as T cells, also remain more concentrated for longer than other blood cells, and therefore are more easily lost than other cells during Ficoll density gradient separation. Therefore, without wishing to be bound by theory, it is believed that a medium with a density higher than Ficoll will result in improved isolation of desired immune effector cells, compared to Ficoll or other media with the same density as Ficoll, for example, 1.077 g / mL.

[0221] In one embodiment, the density gradient centrifugation methods described herein involve the use of a density gradient medium comprising iodixanol, hi one embodiment, the density gradient medium comprises about 60% iodixanol in water.

[0222] In one embodiment, the density gradient centrifugation methods described herein involve the use of a density gradient medium having a density higher than Ficoll. In one embodiment, the density gradient centrifugation methods described herein involve the use of a density gradient medium having a density higher than 1.077 g / mL, e.g., higher than 1.077 g / mL, higher than 1.1 g / mL, higher than 1.15 g / mL, higher than 1.2 g / mL, higher than 1.25 g / mL, higher than 1.3 g / mL, or higher than 1.31 g / mL. In one embodiment, the density gradient medium has a density of about 1.32 g / mL.

[0223] In one embodiment, the density gradient centrifugation method described herein involves the use of a medium containing iodixanol, e.g., about 60% iodixanol in water, having a density higher than that of Ficoll, e.g., higher than 1.077 g / mL, e.g., 1.32 g / mL. In one embodiment, the density gradient centrifugation method described herein involves the use of density gradient medium OptiPrep™ (Sigma). OptiPrep™ is a pre-made, sterile, endotoxin-tested 60% (w / v) iodixanol solution with a density of 1.320±0.001 g / mL. In contrast, Ficoll density gradient solutions have a density of only 1.077 g / mL. Another advantage of OptiPrep™ over Ficoll is that OptiPrep™ is available in GMP grade and is therefore suitable for therapeutic use.

[0224] Without wishing to be bound by theory, it is believed that the use of an OptiPrep density gradient centrifugation step, for example with thawed apheresis material, is less likely to retain unwanted B cells and monocytes, and therefore further improves the collection of desired target immune effector cells, e.g., T cells, for subsequent activation and transduction steps. Thus, without wishing to be bound by theory, it is believed that the higher OptiPrep density compared to Ficoll allows for both improved purification and recovery of desired immune effector cells, e.g., T cells, and the simultaneous removal of unwanted non-T cell types that may otherwise interfere with the consistent success of CAR-expressing immune effector cells, e.g., T cells, product manufacturing results.

[0225] In one embodiment, density gradient centrifugation is performed using a cell separation device. An example of a cell separation device is Sepax2 (Biosafe). In embodiments in which a washing step, e.g., an improved washing step as described herein, is performed, e.g., before or after the density gradient centrifugation step, the washing step can be performed using the same device used in the density gradient centrifugation step.

[0226] Enrichment by selection Provided herein are methods for selecting specific cells to improve the enrichment of desired immune effector cells suitable for CAR expression. In one embodiment, the selection comprises positive selection, e.g., selection of desired immune effector cells. In another embodiment, the selection comprises negative selection, e.g., selection of undesired cells, e.g., removal of undesired cells. In an embodiment, the positive or negative selection methods described herein are performed under flow conditions, e.g., by using a flow-through device, e.g., a flow-through device described herein.

[0227] Current selection methods, such as positive selection, using, for example, Dynabeads® CD3 / CD28 CTS™, can be further optimized for enrichment. First, the amount of Dynabeads used during selection is typically not based on the percentage of CD45+ / 3+ cells, e.g., CD45+ / 3+ cells present in the sample after density gradient centrifugation, e.g., Sepax-Ficoll, but rather on the percentage of cells, e.g., CD45+ / 3+ cells, present in the original patient-derived material. Given the significant changes in composition caused by the density gradient centrifugation step, e.g., Sepax-Ficoll separation procedure, this prediction typically results in a lower percentage of T cells and an increased monocyte content. Second, the 2-hour incubation time exposes the sample to both nonspecific binding of Dynabeads® to non-target cells (i.e., non-CD3 and / or non-CD28 cells) and bead uptake by non-target cells (e.g., monocytes) via endocytosis, which can compromise T cell yield and purity of the positively selected product. Finally, the magnetic device and operation used for selection can be suboptimal. Currently, magnetic separation is performed in large volumes of fluid (200 ml), resulting in a large distance between the magnetically labeled cells and the magnetic surface. This limits the magnetic force available for separation, thus reducing separation sensitivity and requiring longer separation times. In addition, magnetic separation is currently performed statically, with the sample placed on the magnetic surface for 5 minutes before removal of the negative fraction. Such long separation times are harmful to cells and often negatively impact cell viability due to a "pile-up" effect during the procedure, providing additional opportunities for non-target cells to bind to or internalize the beads.

[0228] In stark contrast to current selection methods, the selection methods described herein involve separations that are performed "dynamically," e.g., under flow conditions, as opposed to current "static" separation procedures. In certain embodiments, separations under flow conditions include magnetic separation reagents, e.g., magnetic beads that selectively bind to target antigens, an input sample, and a magnet, where the magnetic separation reagents and input sample pass, e.g., flow, over the magnet. In certain embodiments, the magnetic separation reagents and input sample continuously pass, e.g., flow, over the magnet. Without being bound by theory, this dynamic technique allows for shorter incubation times (i.e., contact between the sample and separation reagents) and separation times, thus minimizing adverse effects on target cells and significantly reducing the likelihood of nonspecific binding and / or bead uptake by non-target populations. Additionally, the selection methods described herein do not require any changes to the selection reagents (Dynabeads® CD3 / CD28 CTS™) or the amount of reagents used in the selection (a 3:1 bead-to-T cell ratio).

[0229] In one embodiment, separation or selection under flow conditions as described herein comprises a Flow-through Antibody-based Selection Technique (FAST) protocol. Table 12 provides an overview of the parameters that differ between current selection techniques and the FAST protocol. [Table 2]

[0230] In one embodiment, the selection method described herein involves a shorter incubation time of the separation reagents and input sample followed by magnetic separation than current standard protocols. In one embodiment, the incubation time is less than 2 hours, e.g., less than 110 minutes, less than 100 minutes, less than 90 minutes, less than 80 minutes, less than 70 minutes, less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 25 minutes, less than 20 minutes, less than 15 minutes, less than 10 minutes, or less than 5 minutes. In one embodiment, the incubation is performed with gentle rotation.

[0231] An exemplary kit for the selection method described herein is shown in FIG. 20. In one embodiment, the kit consists of a three-bag assembly that can be connected to additional sample / buffer bags by spiking or by sterile welding. In addition, a modified DynaMag lid, shown in FIG. 21, is used in the separation to limit the maximum volume during separation to a low volume, e.g., less than 100 mL, less than 90 mL, less than 80 mL, less than 70 mL, less than 60 mL, less than 50 mL, less than 40 ml, e.g., about 50 ml. Without wishing to be bound by theory, it is believed that the low volume used during separation optimizes the magnetic forces acting during the separation procedure and minimizes separation time. The modified DynaMag lid also limits the maximum distance that the bead:cell conjugates can be separated from the magnet, standardizing the magnetic forces experienced during positive selection.

[0232] In one embodiment, one or more of the bags in the kit described herein are triangular bags. In one embodiment, the selection bag is triangular and has ports on both ends of the selection bag, allowing for magnetic separation in a "flow-through" mode. In such an embodiment, cells can be continuously flowed over a magnetic element (e.g., a magnetic plate such as a DynaMag), thus enabling real-time separation of magnetically labeled particles, while unlabeled cells are not attracted by the magnetic field and will flow through. This flow-through configuration makes the system particularly suitable for automation. This modified separation bag requires the modified lid of Figure 21 to accommodate the additional ports.

[0233] In one embodiment, the selection bag is not a triangular bag. In embodiments where the selection bag is not a triangular bag, the incubation time is less than 2 hours, e.g., less than 110 minutes, less than 100 minutes, less than 90 minutes, less than 80 minutes, less than 70 minutes, less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 25 minutes, less than 20 minutes, less than 15 minutes, less than 10 minutes, or less than 5 minutes.

[0234] positive selection In embodiments, the positive selection methods described herein involve selecting, e.g., enriching, for desired immune effector cells. In one embodiment, the negative selection methods described herein involve selecting for CD3+ / CD28+ cells. In other embodiments, the negative selection methods described herein involve selecting for one or more of the following: CD3+ cells, CD28+ cells, CD4+ cells, CD8+ cells, or CD45+ cells.

[0235] The separation reagent used in the selection methods described herein comprises a magnetic or paramagnetic member and an antigen-binding member. In one embodiment, the separation reagent comprises beads, e.g., beads with magnetic or paramagnetic properties, coupled (e.g., covalently or non-covalently) to an antigen-binding member. In one embodiment, the antigen-binding member is an antibody or antibody fragment thereof. In one embodiment, the separation reagent used for positive selection of CD3+ / CD28+ cells comprises beads coupled (e.g., covalently or non-covalently) to a CD3 and / or CD28 binding member, e.g., an anti-CD3 and / or anti-CD28 antibody or antibody fragment.

[0236] negative selection Also provided herein are negative selection methods that negatively select or deplete an input sample of undesirable cells, such as monocytes, granulocytes, erythrocytes, platelets, and B cells, thereby enriching the resulting output sample for desired immune effector cells, such as T cells. In certain embodiments, the negative selection methods described herein are performed under flow conditions, e.g., using a flow-through device, e.g., a flow-through device described herein.

[0237] In one embodiment, the negative selection methods described herein involve negatively selecting one or more of monocytes, granulocytes, erythrocytes, platelets, B cells, or cancer cells, such as lymphoblasts.

[0238] In embodiments where depletion or removal of one or more of monocytes, granulocytes, erythrocytes, platelets, or B cells is desired, negative selection methods select for cells expressing one or more of the following: CD19, CD25, CD14, or other surface markers or proteins expressed by monocytes, granulocytes, erythrocytes, platelets, or B cells.

[0239] In embodiments in which the subject has a hematological cancer, cancer cells may be present in the apheresis sample, and removal of the cancer cells may be desired. In one embodiment, the negative selection methods described herein involve negatively selecting CD19+ cells, e.g., lymphoblasts. In another embodiment, the negative selection methods described herein involve negatively selecting cancer cells that express one or more of the following: CD19, CD33, CD123, CLL-1, BCMA, ROR1, or FLT3.

[0240] The separation reagent used in the selection methods described herein comprises a magnetic or paramagnetic member and an antigen-binding member. In one embodiment, the separation reagent comprises beads, e.g., beads with magnetic or paramagnetic properties, coupled (e.g., covalently or non-covalently) to an antigen-binding member. In one embodiment, the antigen-binding member is an antibody or antibody fragment thereof. In one embodiment, the separation reagent used for negative selection of CD19+ cells comprises beads coupled (e.g., covalently or non-covalently) to a CD19-binding member, e.g., an anti-CD19 antibody or antibody fragment. In one embodiment, the separation reagent used for negative selection of CD14+ cells comprises beads coupled (e.g., covalently or non-covalently) to a CD14-binding member, e.g., an anti-CD14 antibody or antibody fragment. In one embodiment, the separation reagent used for negative selection of CD25+ cells comprises beads coupled (e.g., covalently or non-covalently) to a CD25-binding member, e.g., an anti-CD25 antibody or antibody fragment.

[0241] Exemplary Flow-Through Devices As described herein, the selection methods can be performed under flow conditions, e.g., by using a flow-through device, also referred to as a cell processing system, to further enrich the cell preparation for desired immune effector cells, e.g., T cells, suitable for CAR expression. Exemplary flow-through devices for use in such selection methods under flow conditions are described in this section.

[0242] In one embodiment, the cell processing system comprises at least one cell suspension module; at least one buffer module; at least one flow-through magnetic separation / de-beading module; at least one non-magnetic output module; and at least one magnetic output module.

[0243] In some variations of this system, the variations can include at least one return loop returning upstream of the at least one flow-through magnetic separation / de-beading module; at least two flow-through magnetic separation / de-beading modules in parallel; at least two flow-through magnetic separation / de-beading modules in series; at least one additional module, or any combination thereof. The at least one additional module can include at least one rotating membrane de-beading module; at least two rotating membrane de-beading modules in parallel; or at least two rotating membrane de-beading modules in series. Any of the rotating membrane de-beading modules can include at least one magnet adjacent to or proximate to the cylindrical sidewall.

[0244] In a more specific variation, the flow-through magnetic separation / de-beading module comprises a chamber defined by walls and having an x-direction, a y-direction, and a z-direction; an inlet and an outlet positioned at opposite ends of the chamber in the y-direction; and at least two magnets adjacent to or proximate to the walls of the chamber and positioned to establish a zero gradient line within the chamber between the inlet and the outlet.

[0245] In another more specific variation, which may be standalone or combined with the first more specific variation, the spinning membrane de-beading module comprises a de-beading chamber partially defined by a cylindrical side wall; a porous spinning membrane having an interior and oriented coaxially with the cylindrical side wall; a sample inlet; a waste output module connected to the interior of the spinning membrane; and a cell output module connected to the de-beading chamber.

[0246] In another embodiment, the flow-through magnetic separation / de-beading module comprises a chamber defined by walls and having an x-direction, a y-direction, and a z-direction; an inlet and an outlet disposed at opposite ends of the chamber in the y-direction; and at least two magnets adjacent to or proximate to the walls of the chamber and positioned to establish a zero gradient line within the chamber between the inlet and the outlet.

[0247] In some variations of this module, the variations may include at least two inlets and at least two outlets; at least three magnets adjacent or close to the chamber wall and positioned to establish at least two zero gradient lines between the inlets and the outlets within the chamber; at least four magnets arranged in two rows at opposite ends of the chamber in the z direction; at least four magnets arranged in two rows at opposite ends of the chamber in the z direction and oriented crosswise in the xy plane from near one inlet in the z direction to near one outlet at the opposite end of the chamber; a sub-membrane injection port adjacent to the chamber wall, which wall is also adjacent to the at least two magnets and a membrane adjacent to the sub-membrane; or any combination thereof.

[0248] In another embodiment, the spinning membrane de-beading module comprises a de-beading chamber partially defined by a cylindrical sidewall; a porous spinning membrane having an interior and oriented coaxially with the cylindrical sidewall; a sample inlet; a waste output module connected to the interior of the spinning membrane; a cell output module connected to the de-beading chamber; and at least one magnet adjacent to or proximate to the cylindrical sidewall.

[0249] In some variations of this module, the variation may include a reagent module, have pore sizes larger than the diameter of the particles to be de-beaded and smaller than the diameter of the cells to be de-beaded, or both.

[0250] In yet another embodiment, a flow-through cell processing method includes flowing a cell suspension containing paramagnetic particle-bound cells through a flow-through magnetic separation / de-beading module to generate an unbound cell product. The paramagnetic particle-bound cells continue to move through the flow-through magnetic separation / de-beading module throughout the flow process. The flow-through magnetic separation / de-beading module includes a flow chamber defined by walls through which the cell suspension flows, and at least two magnets positioned adjacent to or near at least one wall.

[0251] In some variations of this method, the cell suspension is layered through a flow-through magnetic separation / de-beading module; the cell suspension further comprises unbound cells and the cell suspension is flowed through a flow-through magnetic separation / de-beading module to separate the paramagnetic particle-bound cells from the unbound cells; the cell suspension further comprises free paramagnetic particles and the cell suspension is flowed through a flow-through magnetic separation / de-beading module to separate the free paramagnetic particles from the unbound cells, or any combination thereof.

[0252] The methods described herein may also include flowing the separated, unbound cells through a flow-through magnetic separation / de-beading module two or more times using a return loop; flowing the separated, paramagnetic particle-bound cells through a flow-through magnetic separation / de-beading module two or more times using a return loop; de-beading the paramagnetic particle-bound cells in the flow-through magnetic separation / de-beading module during the second or subsequent times to produce paramagnetic particles and de-beaded, unbound cells; flowing the resulting paramagnetic particles and de-beaded, unbound cells through the flow-through magnetic separation / de-beading module three or more times to separate the paramagnetic particles and de-beaded, unbound cells; or any combination thereof.

[0253] In another variation, which can be combined with all others, the magnet is oriented to establish a single zero gradient line that intersects the direction of flow, so that paramagnetic particle-bound cells are pulled exclusively in one direction to the zero gradient line but are not affected by the magnetic forces from the magnet in the other two directions.

[0254] In another variation, which can be combined with all others, the chamber further comprises a magnetic inlet through which any paramagnetic particles enter the flow chamber, a non-magnetic inlet, a magnetic outlet opposite the non-magnetic inlet, and a non-magnetic outlet opposite the magnetic inlet, in which case the zero gradient line directs all paramagnetic particles and any paramagnetic particle-bound cells towards the magnetic outlet.

[0255] The cell suspension may further contain unbound cells, and the non-magnetic inlet may be larger than the magnetic inlet, but the non-magnetic outlet is larger than the magnetic outlet, so that fluid flowing from the non-magnetic inlet crosses over to the non-magnetic outlet, thereby preventing any unbound cells from entering the magnetic outlet.

[0256] Alternatively, the cell suspension may further comprise non-bound cells, and the non-magnetic inlet and magnetic inlet may be substantially the same size, or the non-magnetic outlet and magnetic outlet may be substantially the same size, or both, and the respective flow rates of fluids entering said inlets, the respective flow rates of fluids exiting said outlets, or both, may be adjusted so that fluid flowing from the non-magnetic inlet traverses to the non-magnetic outlet, thereby preventing any non-bound cells from entering the magnetic outlet.

[0257] In another variation, which can be combined with all others, the method includes flowing the paramagnetic particle-bound cells through a rotating membrane de-beading module to generate an unbound cell product. The rotating membrane de-beading module can include a cylindrical de-beading chamber through which the paramagnetic particle-bound cells flow, the chamber being defined in part by a cylindrical sidewall and containing a coaxial rotating membrane; and at least one magnet positioned adjacent or proximate to the cylindrical sidewall to establish at least one zero gradient line within the cylindrical de-beading chamber.

[0258] The present disclosure relates to systems and methods for flow-through separation, de-beading, paramagnetic particle separation, or any combination thereof, of paramagnetic particle-bound or unbound cells in a cell suspension in the presence of paramagnetic particles. The systems and methods use a flow-through magnetic separation / de-beading module, a spinning membrane de-beading module, or both. While the systems and methods described herein can be used to remove paramagnetic particles from any cell type, they are particularly well suited for use in removing paramagnetic particles from cells to be used in cell therapy. In addition, because de-beading is typically only performed during positive selection methods, some portions of this description focus on positive selection of paramagnetic particle-bound cells, but the systems and methods can also be used for negative selection. When used for negative selection, any de-beading modules and steps would typically be omitted.

[0259] Separation and Debeading Systems and Modules 1A is a schematic diagram of a cell processing system 2 for flow-through separation, de-beading, paramagnetic particle separation, or any combination thereof, of paramagnetic particle-bound cells in a cell suspension. System 2 includes a cell suspension module 4, a buffer module 6, a flow-through magnetic separation / de-beading module 8, a non-magnetic output module 10, and a magnetic output module 12. System 2 can also optionally include at least one additional module 16, at least one return loop 14, or both.

[0260] System 2 may further include fluid conduits such as tubing or hoses, connectors, valves, switches, clamps, welds, housings, motors, pumps, other mechanical mechanisms, circuitry, monitoring and control devices. System 2 may further include a computer programmed to control System 2 or any of its components to perform a flow-through separation process, a flow-through de-beading process, a flow-through paramagnetic particle separation process, or any combination thereof.

[0261] System 2 may have a static configuration or an adaptable configuration. One adaptable configuration may allow for the replacement of modules or the insertion of additional modules. Another adaptable configuration may have an unchangeable set of modules but may allow for changes in fluid routing for at least one of the modules. Another adaptable configuration may allow for both the replacement and addition of modules and changes in fluid routing. Components of System 2 may facilitate adaptable configuration. For example, a programmed computer in System 2 may detect or use information about which modules are present or control fluid routing. Additionally, System 2 may have housings or fluid conduits with attached connectors, valves, clamps, or switches that allow for the removal or insertion of different modules in the same location. Modules or other components may include identification elements, such as bar codes or radio frequency identification device (RFID) chips, to enable automatic detection of their presence or absence. Modules or other components may also include one or more designation elements that can ensure compliance with good manufacturing practices and other safety regulations. For example, a temperature-sensitive indicator element can indicate that a module or other component has been heat sterilized or has not been exposed to temperatures that could compromise its integrity or effectiveness. The indicator element can also clearly identify the module or other component being used. The indicator element can also be automatically detected by system 2, which helps minimize human error.

[0262] Components of System 2 that are not required for a particular process may be absent, disconnected, or closed. For example, rather than separate non-magnetic output module 10 and magnetic output module 12, there may be a single output module as shown in FIG. 1H. Additionally, as also disclosed in FIG. 1H, components of System 2 may have fluid conduits with different paths and connections than those shown in FIG. 1A, depending on the configuration of valves, clamps, welds, switches, and connectors.

[0263] The cell suspension module 4 contains cells to be separated or de-beaded, suspended in a suspension fluid. When cells are to be separated, the cell suspension typically contains both paramagnetic particle-bound cells and unbound cells. The paramagnetic particle-bound cells may be desired cells, in which case positive selection for the paramagnetic particle-bound cells will be performed in the system 2, or the paramagnetic particle-bound cells may be undesirable, in which case negative selection for the paramagnetic particle-bound cells will be performed.

[0264] When cells are to be de-beaded or paramagnetic particles are to be separated from cells, paramagnetic particle-bound cells are desired cells.These cells can be separated from undesired cells in advance using System 2 or another system.If the presence of undesired cells is not a problem or there are no undesired cells to be separated, the cells to be de-beaded do not need to be subjected to a separation process in advance.

[0265] The cells can be obtained directly from a biological sample such as blood or from cell culture.

[0266] The suspending fluid can be any fluid capable of supporting cell viability throughout the separation, de-beading, or particle removal process. For example, the suspending fluid can be culture medium, a cryogen such as a DMSO-containing fluid, another fluid with a set or controlled pH, or another fluid with nutrients. The suspending fluid can also be a buffer, which can be the same as or different from the buffer in buffer module 6. The suspending fluid can have a different viscosity than the buffer. The suspending fluid can also have a different viscosity than a medium, such as high-density Ficoll, which can be very dense when cells enter system 2.

[0267] The buffer in buffer module 6 can be any fluid that can be combined with the suspending fluid and still allow the suspending fluid to continue to support cell viability. For example, the buffer may have a set or controlled pH. The buffer may also include one or more cytocompatible salts. Although the buffer is provided separately in buffer module 6, once the buffer mixes with the cell suspension, it is considered part of the suspending fluid.

[0268] The suspending fluid or buffer may contain antimicrobial agents, but will typically not contain antimicrobial agents when the cells are later provided to a patient unless system 2 removes these agents, such as by a spinning membrane de-beading module or another module, or unless the antimicrobial agents are later removed by an additional process, module, or system.

[0269] Paramagnetic particles can be formed from any paramagnetic and / or magnetizable material, such as a metal or metal alloy. Typically, the paramagnetic material is not toxic or is coated to avoid toxicity to the cells or to the patient to whom the cells are subsequently administered. Paramagnetic materials have a high magnetic saturation flux (m s) Generally, the suitability of a paramagnetic material will be influenced by the magnets used in system 2 and the configuration of the modules that use the magnets, as these factors will affect the ability of the paramagnetic material to reach magnetic saturation.

[0270] The paramagnetic particles can be coated with a binding agent, such as a growth agent, a receptor or ligand, an antigen, an antibody, or any binding fragment or chimeric variant thereof, such as a chimeric antigen receptor ligand. The binding agent may, in some cases, be reversible, allowing the paramagnetic particles to be detached spontaneously or using specific chemicals. The binding agent may also include a photocleavable linker, in which case the system 2 may include a light source, particularly a high-power light source, as a module or as part of a separate module to enable photocleavage of the linker and separation of the cells and the paramagnetic particles. In some cases, the coating may interact with the cells. In other cases, the coating may interact with at least one undesirable component of the cell suspension to be removed. This undesirable component may be active or inactive and may have previously performed a useful function for the cells or cell suspension fluid. Exemplary undesirable components include antibodies, growth factors, other proteins, and polymers.

[0271] In some cell suspensions, different types of paramagnetic particles may be present, such as particles with different binding agents or particles formed from different magnetic materials, allowing for complex separation or repeated removal of binding agents. Additional paramagnetic particles, which may be coated with any binding agent, may also be bound to the cells.

[0272] Other particles that are not paramagnetic may be present in the cell suspension, and these particles may be coated with whatever is used to coat paramagnetic particles.

[0273] The modules can be formed from or lined with any biologically compatible material, such as cell storage bags, etc. Fluid conduits and any other components of system 2 that come into contact with the cell suspension or buffer can also be formed from or lined with any biologically compatible material.

[0274] Components of System 2 that come into contact with the cell suspension or buffer may be sterilized prior to contact with the cell suspension.

[0275] Components of System 2 may be disposable, especially components that come into contact with the cell suspension, to avoid contamination and sterility concerns.

[0276] 2A is a cross-sectional schematic diagram of a flow-through magnetic separation / de-beading module 8 with an x-oriented magnet configuration, while FIG. 2B is a semi-transparent three-dimensional schematic diagram of the same magnetic separation module 8 with the same configuration. Flow-through magnetic separation / de-beading module 8 includes a flow chamber 50 defined by walls 52. An external dipole magnet 54 generates magnetic field lines 56. Magnet 54 is housed on a movable platform 60. Flow-through magnetic separation / de-beading module 8 further includes an inlet 62 and an outlet 64, through which a cell suspension can flow through module 8 in the y-direction.

[0277] 2A and 2B depict a single row of magnets 54, the flow-through magnetic separation / de-beading module 8 may have two or more rows, as shown in FIG. 4A, or may have more than two magnets per row. Additionally, the magnets 54 may be in a permanent position, in which case the separation / de-beading module 8 may lack a movable platform 60 or may have a movable flow chamber 50. Furthermore, while the magnets 54 are shown in an x-oriented configuration, they can be at any angle in the x-y plane, including a y-oriented or cross-x-y orientation.

[0278] Inlet 62 and outlet 64 can have any configuration sufficient to establish laminar flow of the cell suspension through chamber 50. The inlet geometry, outlet geometry, and flow rate all affect the flow of the cell suspension through chamber 50. In some cases, turbulent flow may be tolerated.

[0279] Wall 52 may be a rigid structure or may be flexible. For example, wall 52 may be the wall of a cell storage bag or other similar component. If wall 52 is flexible, the z-dimension of chamber 50 may vary depending on the flow rate of the cell suspension through chamber 50.

[0280] The z-dimension of chamber 50 can be between 5 μm and 100 μm, between 5 μm and 500 μm, or between 5 μm and 1000 μm, between 5 μm and 1 cm, or generally 100 μm, 500 μm, 1000 μm, or 1 cm or less. Dimensions in both the x, y, and z directions can be constrained to achieve sufficiently high hydrodynamic forces to move cells or paramagnetic particles through chamber 50.

[0281] The magnets 54 may have a high magnetic field strength. For example, the magnets 54 may contain a rare earth metal, such as neodymium or samarium, alloyed with another metal, such as cobalt. The magnets 54 may be dipole magnets as depicted, or other types of magnets, such as quadrupole magnets. The magnets 54 may have an adjustable magnetic field strength. For example, the magnets 54 may be electromagnets. The magnets 54 may be arranged to maximize the magnetic attraction of magnets on the same side of the chamber 50, to maximize the magnetic repulsion of magnets on opposite sides of the chamber 50, or both. While a specific magnet configuration is depicted in FIG. 2A , opposite or matching polarity configurations of the magnets may also be used depending on the effect to be achieved.

[0282] 3 is a schematic side cross-sectional view of a flow-through magnetic separation / de-beading module 8 having a sub-membrane fluid injection port 72 and a membrane 70 positioned above a magnet 54. Fluid from the buffer module 6 or another fluidic module can be introduced via the fluid injection port 72 to facilitate de-beading of cells positioned near the membrane 70.

[0283] The movable platform 60 may be movable in the z-direction, thereby allowing movement of the magnet 54 in the z-direction from a position adjacent to or close to the chamber 50 (not shown), as shown in FIGS. 2A and 2B, to a position away from the chamber 50 (as shown in FIG. 7C). For example, the position away from the chamber 50 may be at least 1 cm from the nearest wall 52. This distance is sufficient to prevent the magnet 54 from substantially affecting any paramagnetic particles in the chamber 50 with their magnetic field. This distance may be substantially shorter if magnetic insulating material is inserted between the magnet 54 and the chamber 50. If the magnet 54 has an adjustable magnetic field strength, rather than moving the magnet 54, it may simply be adjusted to a lower or zero magnetic field strength to avoid any substantial effect on any paramagnetic particles in the chamber 50.

[0284] Specifically, when using the zero-gradient configuration, module 8 can have an upper row of magnets 54 and a lower row of magnets 54, as depicted in Figures 4A and 4B. Movable platform 60 can be rotatable in the xy plane, or magnets 54 can be permanently oriented so that they are in an xy-cross-orientation configuration, such as the configuration depicted in the top-view, longitudinal cross-section schematic of flow-through magnetic separation / de-beading module 8 in Figure 4C. For use in the zero-gradient configuration, flow-through magnetic separation / de-beading module 8 can have two inlets 62, non-magnetic inlet 62a and magnetic inlet 62b, and two outlets 64, magnetic outlet 64a and non-magnetic outlet 64b. In this case, a zero-gradient line 58 in the xy-cross-orientation direction forms a zero-gradient filter when module 8 is in use. As illustrated in FIG. 4D, the inclusion of an additional magnet 54 can result in two zero gradient lines 58a and 58b within the same module 8, thereby allowing separation of different paramagnetic particles into different outlets 64a and 64b or providing a backup filter.

[0285] The zero gradient line 58 can be a zero gradient band having a dimension in the x-direction if the magnets 54 are not adjacent but are spaced far enough apart as depicted in FIGS. 2A and 2B.

[0286] The magnet for use with the flow-through magnetic separation / de-beading module may be located either externally or internally to the chamber through which the cell suspension flows. If the magnet is internal, it may be coated with a biocompatible material. In particular, if the magnet is internal, it is disposable.

[0287] FIG. 1B is a schematic diagram of a cell processing system 2 including multiple flow-through magnetic separation / de-beading modules 8a, 8b, and 8c in parallel. While only three flow-through magnetic separation / de-beading modules 8 are shown, the plurality can be any number greater than two. When flow-through magnetic separation / de-beading modules 8 are present in parallel, the modules will typically be of the same type and configuration, and therefore each module will perform the same function. Parallel flow-through magnetic separation / de-beading modules 8, when combined with additional modules, can be particularly useful for rapid cell suspension processing or fluid volume management. Additionally, since all modules must be used simultaneously, arranging flow-through magnetic separation / de-beading modules 8 in parallel provides flexibility in fluid flow control.

[0288] FIG. 1C is a schematic diagram of a cell processing system 2 including multiple flow-through magnetic separation / de-beading modules 8a, 8b, and 8c arranged in series. While only three flow-through magnetic separation / de-beading modules 8 are shown, the plurality can be any number greater than two. When flow-through magnetic separation / de-beading modules 8 are arranged in series, the modules can be of the same type and configuration so that each module performs the same function, but typically will be of different types and configurations so that each module performs a different function. For example, module 8a can separate paramagnetic particle-bound and unbound cells, module 8b can de-bead the paramagnetic particle-bound cells, and module 8c can de-bead the paramagnetic particle-bound cells under a larger magnetic field gradient.

[0289] 1D is a schematic diagram of a cell processing system 2 in which a return loop 14 directs paramagnetic particle-bound cells back through a flow-through magnetic separation / de-beading module 8. Such a system can be used to achieve better separation of paramagnetic particle-bound and unbound cells, or better de-beading or separation of unbound cells and paramagnetic particles, compared to a similar system without a return loop 14. Fluid can be directed into the return loop 14 or to the magnetic output module 12 by a valve or switch.

[0290] 1E is a schematic diagram of a cell processing system 2 in which a cell suspension module 4 and a buffer module 6 are separately connected to a flow-through magnetic separation / de-beading module 8. Such a system may be particularly useful when the flow-through magnetic separation / de-beading module 8 has two inlets 62 and two outlets 64 and a magnet 54 in an xy cross-oriented configuration, as shown in and described with respect to FIG.

[0291] FIG. 1F is a schematic diagram of a cell processing system 2 with a spinning membrane de-beading module 18 located downstream of a flow-through magnetic separation / de-beading module 8. The spinning membrane de-beading module 18 is connected to a waste output module 20 and a cell output module 22. In this system 2, the flow-through magnetic separation / de-beading module 8 separates paramagnetic particle-bound and non-bound cells, while the spinning membrane de-beading module 18 may perform all de-beading or may also perform de-beading. A reagent module 24 may optionally be present if a reagent, such as a chemical, is added to the suspending fluid in the spinning membrane de-beading module 18. While FIG. 1F shows one spinning membrane de-beading module 18, the system 2 can include multiple modules 18 in series or in parallel. If modules 18 are in series, chemicals can be added only to the last module 18 to minimize exposure of cells to the chemicals.

[0292] The reagent in the reagent module 24 can be any chemical that weakens the bond between the particles and the cells. The particles can be paramagnetic or non-paramagnetic particles.

[0293] FIG. 5A is a schematic longitudinal cross-sectional view of the spinning membrane de-beading module 18. The module 18 includes a sample inlet 80 that allows a cell suspension to enter a cylindrical de-beading chamber 82, which is partially defined by a cylindrical sidewall 84 and contains a coaxially oriented cylindrical spinning membrane 86. The wall 84 has magnets 88 aligned on its exterior. The de-beading chamber 18 allows fluid that passes through the spinning membrane 86 to exit via the waste output module 20, while the remaining fluid and cells exit via the cell output module 22. The spinning membrane 86 has an average pore size that is smaller than the average diameter of cells but larger than any paramagnetic particles to be removed by de-beading. This average pore size can also be larger than any paramagnetic particles to be removed, allowing the spinning membrane to remove these paramagnetic particles as a primary particle removal method or as a backup to magnetic removal.

[0294] The magnet 88 may substantially surround the wall 84, as shown in FIG. 5B, or may be spaced apart along the wall 84, as shown in FIG. 5C. The magnet 88 may be mounted on a movable platform to allow movement of the magnet 88 from a position adjacent or close to the wall 88 (not shown), as shown in FIGS. 5A-5C, to a position distant from the wall 84. For example, the distant position may be at least 1 cm from the wall 84. This movement to the distant position prevents the magnet 88 from substantially affecting any paramagnetic particles in the chamber 82 with their magnetic field. If a magnetic insulating material is inserted between the magnet 88 and the chamber 82, this distant position may be shorter than if the magnetic insulating material were not present. If the magnet 88 has an adjustable magnetic field strength, rather than moving the magnet 88, it may simply be adjusted to a lower or zero magnetic field strength to avoid substantially affecting any paramagnetic particles in the chamber 82.

[0295] Although multiple magnets 88 are shown in FIG. 5, it is possible to have only a single magnet 88.

[0296] The magnet 88 may have a high magnetic field strength. For example, the magnet 88 may contain a rare earth metal, such as neodymium or samarium, alloyed with another metal, such as cobalt. The magnet 88 may be a dipole magnet, a quadrupole magnet, or any other type of magnet. The magnet 88 may have an adjustable magnetic field strength and may be, for example, an electromagnet. The magnet 88 may be arranged, for example, in a wound configuration, to maximize magnetic attraction.

[0297] Magnets for use with rotating membrane modules may be located external to the chamber through which the cell suspension flows or may be located internal to the chamber. If the magnet is internal, it may be coated with a biocompatible material. In particular, if the magnet is internal, it may be disposable.

[0298] Exemplary spinning membranes suitable for use in the modules disclosed herein include the 4 μm track-etched polycarbonate spinning membrane used in the LOVO® cell processing system (Fresenius Kabi, Fenwal, Lake Zurich, IL) and the spinning membrane used in the ISOLEX® magnetic cell separation system (Baxter, Deerfield, IL).

[0299] The elements of FIGS. 1A-1F, including the flow-through magnetic separation / de-beading module 8 shown in FIGS. 2-4 or the magnetic rotation membrane de-beading module 18 shown in FIG. 5, can be combined with one another in the cell processing system 2, depending on the specific cell processing to be performed. These elements may be combined as depicted or in other reasonable variations. For example, a reagent module 24 may be included in the system otherwise depicted in FIG. 1A to add chemicals to the suspending fluid in the flow-through magnetic separation / de-beading module 8 when used for de-beading. Modules (including additional buffering or output modules) may be positioned and used to ensure adequate fluid volumes and flow rates, particularly within modules 8 and 18.

[0300] An exemplary cell processing system 2 incorporating multiple modules and loops is shown in FIG. 1G. The system includes a cell suspension module 4 and a buffer module 6a connected to a first flow-through magnetic separation / de-beading module 8a, which has a non-magnetic output module 10a and a magnetic output module 12a. The magnetic output module 12a is connected to a second serial flow-through magnetic separation / de-beading module 8b, which is also connected to a buffer module 6b and has a non-magnetic output module 10b, a magnetic output module 12a, and a return loop 14a. The return loop 14a connects back to module 8b. The magnetic output module 12b is connected to a first spinning membrane de-beading module 18a, which has a waste output module 20a and a cell output module 22a. The cell output module 22a is connected to a second in-line rotating membrane de-beading module 18b, which is also connected to a waste output module 20b, a return loop 14b, and a cell output module 22b, as well as to a reagent module 24. The return loop 14b is connected back to a second in-line flow-through magnetic separation / de-beading module 8b.

[0301] Another exemplary cell processing system 2 having various additional modules with unique fluid conduits is shown in FIG. 1H. A cell suspension module 4 and a satellite module 30, which may be empty or contain a buffer solution, are connected to a flow-through magnetic separation / de-beading module 8. The flow-through magnetic separation / de-beading module 8 is separately connected to a buffer module 6 and a storage module 26. The storage module 26 is further connected to a recovery module 28. Many of the connections are made using spiked tubing 32. The system further includes roller clamps 34, welds 36, slide clamps 38, and pinch clamps 40 along the various fluid conduits.

[0302] The cell processing system 2 can include a variety of additional modules 16, such as magnetic columns, other physical separation modules, cell washing modules, cell concentration modules, and medium exchange modules.

[0303] Cell Separation and Debeading Methods System 2 can be used to separate paramagnetic particle-bound and non-bound cells in a flow-through process, de-bead the magnetic particle-bound cells, separate paramagnetic particle and non-bound cells, or any combination thereof. In a flow-through process, all cells continue to move while in flow-through magnetic separation / de-beading module 8. Paramagnetic particle-bound cells that pass through module 8 and stop along wall 52 are either nonexistent, less than 0.01%, less than 0.05%, or less than 1%.

[0304] Flow-through magnetic separation / de-beading process 1A , flow-through magnetic separation / de-beading module 8 can be primed as needed by flowing buffer from buffer module 6 through module 8 to non-magnetic output module 10 or magnetic output module 12, or to another output or additional module 16. A cell suspension containing paramagnetic particle-bound cells is flowed through module 8.

[0305] When system 2 is configured for separation, the cell suspension is directed to non-magnetic output module 10. Module 8 can be configured to be periodic so that the buffer from buffer 6 does not attract the paramagnetic particles while flowing through module 8 to magnetic output module 12. To ensure better separation of the cells and higher purity of the magnetic or non-magnetic cell product, the cell suspension can be directed through loop 14 for a second or subsequent pass through module 8. The paramagnetic particle-bound cells can be directed to additional component 16, for example, flow-through magnetic separation / de-beading module 8 configured for de-beading or to spinning membrane de-beading module 18.

[0306] If system 2 is configured for de-beading, after flowing through module 8, the cell suspension is directed to non-magnetic output module 10. Alternatively, the cell suspension can be directed by loop 14 for a second or subsequent pass through module 8 before being directed to non-magnetic output module 10.

[0307] A process using the flow-through magnetic separation / de-beading module 8 subjects each paramagnetic particle-bound cell 100, which has at least one paramagnetic particle 102 bound thereto, to a fluid (shear or drag) force 104. Each cell 100 is also subjected to a magnetic force 106, as shown in FIG. 6 . The fluid force 104 and magnetic force 106 can be used in combination to maintain the paramagnetic particles 102 bound to the paramagnetic particle-bound cell 100, thereby allowing the cell 100 to be separated from unbound cells. The fluid force 104 and magnetic force 106 can also be used in combination to cause the paramagnetic particles 102 to detach from the paramagnetic particle-bound cell, thereby allowing the cell 100 to be de-beaded. Secondary forces, such as diffusion and gravity, also act on the paramagnetic particles 102, but their effect on de-beading is typically much smaller than the fluid and magnetic forces and are often ignored when calculating the appropriate flow rate through the module.

[0308] System 2 can generally be configured so that desired cells are no longer subject to passage through the modules as quickly as possible, thereby reducing cell damage. For example, in a de-beading configuration, only magnetic output module 12 can include a return loop to flow-through magnetic separation / de-beading module 8, thereby allowing more easily de-beaded cells to pass through module 8 fewer times than cells with more recalcitrantly bound paramagnetic particles.

[0309] The flow-through module 8 may be of laminar or turbulent flow type. The magnetic force 106 is determined by the saturation magnetic flux (m s ). The magnetic force 106 is also affected by the strength of the magnetic field 56 to which the paramagnetic particles 102 are exposed, which is affected by the strength of the magnet 54 as well as the z-depth of the chamber 50 and the position of the cell 100 within that chamber. The magnetic force 106 is further affected by the magnetic field gradient to which the paramagnetic particles 102 are exposed as they move through the chamber 50, which is affected by the strength and placement of the magnet 54 as well as the position of the paramagnetic particles 102 relative to the magnet 54.

[0310] Additionally, fluid forces are affected by the fluid flow rate, which is typically fastest in the center of the chamber and zero at the walls. This means that cells attached to the walls cannot experience sufficient fluid force to detach from their paramagnetic particles, and cells along the walls may be largely stationary, shielding downstream cells from the fluid force. This results in a loss of desired cells, which is avoided by continuously flowing the cell suspension through the chamber 50. This loss can be avoided by a flow-through approach, in which cells are not held stationary by magnetic forces. This loss can also be avoided by modules in which the velocity at the walls is not zero or near zero, such as a plug flow module, in which the fluid flow rate is uniform throughout the chamber.

[0311] Flow-through separation process The flow-through separation process can be performed using a flow-through magnetic separation / de-beading module 8 configured as shown in Figures 2A and 2B. The flow rate is such that the paramagnetic particles 102 can remain bound to the cells 100 by the hydrodynamic forces 104 and magnetic forces 106. The flow rate is also such that the cells 100 are not lysed by the hydrodynamic forces.

[0312] Figure 7A shows module 8 of Figure 2B when paramagnetic particle-bound cells 100 and unbound cells 110 have just entered chamber 50. In Figure 7B, cells 100 have stopped, while unbound cells 110 continue at their original speed. In Figure 7C, the magnet has moved away, and cells 100 continue to move, while unbound cells 110 have exited chamber 50.

[0313] When magnet 54 is adjacent or close to chamber 50, the suspending fluid exiting chamber 50 enters non-magnetic output module 10. Periodically, the flow of cell suspension from cell suspension module 4 is stopped and buffer solution flows into chamber 50 from buffer module 6 while magnet 54 moves away from chamber 50, causing paramagnetic particle-bound cells 100 to be swept by the buffer solution into magnetic output module 12.

[0314] This flow-through separation process, especially when repeated to allow multiple passages of the cells through module 8, can remove at least 80%, at least 90%, at least 95%, or at least 99% of the paramagnetic particle-bound cells 100 from the cell suspension before it enters non-magnetic output module 10. A single-pass process, in which the cells pass through module 8 only once, is less efficient. For example, in a single-pass process, module 8 can remove at least 25% or at least 50% of the paramagnetic particle-bound cells 100 from the cell suspension before it enters non-magnetic output module 10. Single or multiple passes through the flow-through magnetic separation / de-beading module 8 with either non-magnetic or magnetic output can result in a paramagnetic particle-bound cell product having 1% or less unbound cells 110 and containing at least 99% of the paramagnetic particle-bound cells 100 found in the cell suspension before the flow-through separation process; an unbound cell product having 1% or less paramagnetic particle-bound cells 100 and containing at least 99% of the unbound cells 110 found in the cell suspension before the flow-through process; or both.

[0315] Flow-through de-beading process The flow-through de-beading process can also be performed using a flow-through magnetic separation / de-beading module 8 configured as shown in Figures 2A and 2B. The flow rate is such that the fluidic forces 104 and magnetic forces 106 cause, on average, at least one paramagnetic particle 102 per cell in the cell suspension to detach from the cell 100 as the cell 100 passes through the chamber 50. The flow rate is also such that the cells are not lysed by the fluidic forces.

[0316] FIG. 8A shows module 8 of FIG. 2B when paramagnetic particle-bound cells 100a and 100b have just entered chamber 50. Cell 100a has one paramagnetic particle 102, while cell 100b has two paramagnetic particles 102. In FIG. 8B, one paramagnetic particle 102 has detached from both cell 100a and cell 100b and is stationary at wall 52, while cells 100a and 100b continue to pass through chamber 50. Cell 100a no longer has any paramagnetic particles 102, while cell 100b retains one paramagnetic particle 102. To remove this second paramagnetic particle 102, cell 100b may be passed through module 8 a second time. Alternatively, module 8 may be configured so that cell 100a exits chamber 50 while cell 100b remains within chamber 50, similar to the paramagnetic particle-bound cells in FIG. 7.

[0317] This flow-through debeading process is capable of removing at least 99% of the paramagnetic particles from the cells.

[0318] When CLT0119 T cells were de-beaded using system 2, which includes a recirculation loop 14 from magnetic output module 12, the cells at the magnetic output were resuspended in buffer from buffer module 6 and passed through module 8 once more, followed by a third pass. A comparison of the results of this process with those of a conventional stop-flow process is provided in Figure 9, which presents the end-to-end yield, which is the ratio of the number of cells in the final product to the number of cells that entered the de-beading system.

[0319] The flow-through magnetic separation / de-beading module 8 shown in Figure 3 can be used to de-bead cells similar to the module 8 shown in Figures 2A and 2B. Fluid introduced via port 72 provides sufficient force to cells 100 to detach them from membrane 70. Because the velocity of the suspending fluid approached zero near membrane 70, these cells might not otherwise be separated or de-beaded and could be lost.

[0320] This flow-through de-beading process is also able to remove at least 99% of the paramagnetic particles from the cells.

[0321] Flow-through zero gradient filter process A flow-through separation process can be performed using a flow-through magnetic separation / de-beading module 8 configured as shown in Figures 4A and 4B. The flow rate is such that the paramagnetic particles 102 remain bound to the cells 100 by the hydrodynamic forces 104 and magnetic forces 106. The flow rate is also such that the cells 100 are not arrested within the chamber 50 and are not lysed by the hydrodynamic forces. While various configurations are shown in Figures 10-13, these configurations can be modified for use with different zero gradient filtration processes, e.g., to adjust the number and proportional size of the inlets 62 and outlets 64. For example, the same effect achieved by having different sized inlets 62 and outlets 64 can also be achieved by providing different flow rates, typically controlled by pumps, through the same sized inlets and outlets.

[0322] Figure 10 provides a basic description of how paramagnetic particle-bound cells 100 flow through module 8 in the zero-gradient configuration. As shown in Figure 10A, after entering module 8, cell 100 (left) is pulled in the x-direction toward zero-gradient line 58 until it nearly exits module 8 (right). As shown in Figures 10A and 10B, cell 100 is not subjected to magnetic forces in the y- or z-directions as it enters module 8 (left) or even as it approaches the exit (right).

[0323] Figure 11 shows a zero-gradient module 8 with magnet 54 oriented as shown in Figure 4A. Fluid enters the module via inlet 62. Cells 100 with magnetic particles 102 follow zero-gradient line 58 and are directed toward magnetic outlet 64b. Unbound cells 110 are unaffected by zero-gradient line 58 and flow toward non-magnetic outlets 64a and 64c. In this configuration, some unbound cells 110 will also enter magnetic outlet 64b.

[0324] FIG. 12 shows cells migrating through module 8 of FIG. 4C. Cells 100 carrying paramagnetic particles 102 follow zero gradient line 58 and are directed toward magnetic outlet 64b. Unbound cells 110 are unaffected by zero gradient line 58 and flow toward non-magnetic outlet 64b. Thus, zero gradient line 58 acts as a magnetic filter, allowing all cells to continue migrating through chamber 50 without being pushed toward either wall 52. As shown, non-magnetic inlet 62a is larger than magnetic inlet 62b, and non-magnetic outlet 64b is larger than magnetic outlet 64a. If the fluid flow within module 8 is laminar, fluid from non-magnetic inlet 62a crosses to non-magnetic outlet 64b, thereby preventing any unbound cells 110 from entering magnetic outlet 64a. This method can also be used with turbulent flow, but with less efficiency due to the loss of unbound cells to magnetic outlet 64a.

[0325] This flow-through separation process can remove at least 80%, at least 90%, at least 95%, or at least 99% of the paramagnetic particle-bound cells 100 from the cell suspension before it enters the non-magnetic output module 10. Multiple passes through the flow-through magnetic separation / de-beading module 8 with either the non-magnetic or magnetic output can result in a paramagnetic particle-bound cell product having 1% or less unbound cells 11 and containing at least 99% of the paramagnetic particle-bound cells 100 found in the cell suspension before the flow-through separation process; an unbound cell product having 1% or less paramagnetic particle-bound cells 100 and containing at least 99% of the unbound cells 110 found in the cell suspension before the flow-through process; or both.

[0326] Flow-through zero-gradient paramagnetic particle selection process The flow-through paramagnetic particle separation process can also be performed using a flow-through magnetic separation / de-beading module 8 configured as shown in FIG. 4C. FIG. 13 shows de-beaded, unbound cells 110 and paramagnetic particles 102 moving through module 8. The cells 110 may have been previously de-beaded, for example, by a spinning membrane de-beading module 18, a non-spinning membrane de-beading module, or the same or a separate module 8 in a de-beading configuration. Alternatively, the cells 110 may already be in the presence of, but not bound to, paramagnetic particles 102. The paramagnetic particles 102 follow the zero gradient line 58 toward the magnetic outlet 64a. The de-beaded, unbound cells 110 are unaffected by the zero gradient line 58 and flow toward the non-magnetic outlet 64b. Thus, the zero gradient line 58 acts as a magnetic filter while allowing the cells 110 to continue moving through the chamber 50 without being pushed toward either wall 52.

[0327] This flow-through paramagnetic particle separation process can remove at least 80%, at least 90%, at least 95%, or at least 99% of the beads from the cell suspension.

[0328] This process removes the paramagnetic particles and can also remove undesirable components of the cell suspension.

[0329] Although the cell separation process and particle separation process are described separately above, both can be performed simultaneously in the same module or system. For example, a separation module will typically remove both paramagnetic particle-bound cells and free paramagnetic particles from a cell suspension.

[0330] Spinning membrane debeading process In a flow-through process using system 2 of FIG. 1F, separated paramagnetic particle-bound cells 100 are directed via sample inlet 80 to a spinning membrane module 18, as shown in FIG. 14. In de-beading chamber 82, spinning membrane 86 generates a recirculating Taylor-Couette flow in the cell suspension, which generates hydrodynamic forces in addition to those generated by flow through chamber 82 from inlet 80 to outlets 20 and 22. As a result, paramagnetic particles 102 are still subjected to hydrodynamic and magnetic forces, but the relationship between these forces and how they affect de-beading is difficult to model. However, hydrodynamic forces are affected by at least the size and rotation speed of spinning membrane 80, the flow rate of the cell suspension through chamber 82, and the viscosity of the suspending fluid. Magnetic forces are affected by the properties of paramagnetic particles 102, the properties of magnet 88, and the design of module 18, particularly the distance between cells 100 and magnet 88. Hydrodynamic forces are typically not high enough to lyse cells. The Taylor-Couette flow is sufficient to keep the cells away from and out of contact with the wall 84 and the spinning membrane 86 .

[0331] Paramagnetic particles 102 removed from cells 100 migrate toward wall 84, specifically, along wall 84 to the zero gradient line or zone. Any paramagnetic particles 120 are also removed from cells 100 by fluidic forces alone. The paramagnetic particles 120 pass through pores in the rotating membrane 86 and then exit chamber 82 via waste outlet module 20. Any chemicals 122 added from any reagent chambers 24 also pass through pores in the rotating membrane 86 and exit chamber 82 via waste outlet module 20, thereby limiting exposure of cells 100 to chemicals 120. De-beaded, unbound cells 110 exit chamber 82 via cell outlet module 22.

[0332] For example, the paramagnetic particles 102 can be periodically removed from the wall 84 by stopping the flow of cell suspension through the chamber 82, moving the magnet 88 away from the wall 84, and then flowing a buffer solution through the chamber 82.

[0333] Some paramagnetic particles 102 may be removed by passing through the rotating membrane 80. If the magnet 88 is not present or is far enough away from the chamber 82, removal of all paramagnetic particles may be accomplished by the rotating membrane 80.

[0334] This flow-through de-beading process can also remove at least 80%, at least 90%, at least 95% or 99% of the paramagnetic particles from the cells, remove at least 80%, at least 90%, at least 95% or at least 99% of all paramagnetic particles from the cells, or both.

[0335] A rotating membrane can also be used to separate the paramagnetic particles 102 from the unbound cells 102 .

[0336] The spinning membrane 86 can have a pore size small enough to exclude all cells in the cell suspension.

[0337] The spinning membrane module 18 can also be used to remove undesired components from the cell suspension. These components may simply be filtered by the spinning membrane 88 or may interact with the coating of the paramagnetic particles 102, the paramagnetic particles 120, or both, and be removed along with the particles. De-beading and removal of undesired components may be performed separately or simultaneously.

[0338] Other debeading and continuous particle separation processes The modular design of the system 2 also makes it compatible with other de-beading and paramagnetic particle separation processes by simply inserting the appropriate additional module 16. For example, columns and physical separation methods, including magnetic columns, are often used to de-bead cells and can be included as additional modules 16.

[0339] Other Embedded Processes System 2, due to its modular design, is compatible with other integrated processes. These processes can be performed in at least one additional module 16. For example, a module can be used to wash cells. A module can also be used to concentrate cells. A module can be used to change the medium in which the cells are located. One module can be used for more than one of these steps.

[0340] Multi-module flow-through process System 2, as shown in FIG. 1G, can be used in a multi-module flow-through process. Optionally, buffer from buffer module 6a can be flowed through flow-through magnetic separation / de-beading module 8a and, optionally, through one or more of modules 8b, 18a, and 18b. A cell suspension containing desired paramagnetic particle- and non-paramagnetic particle-bound cells and undesired non-bound cells is flowed through module 8a, which is configured for separation as shown in FIG. 4, or alternatively, module 8a can be configured for separation as shown in FIGS. 2A and 2B. The non-bound cells are directed as waste to non-magnetic output module 10a. Paramagnetic particle-bound cells are directed via magnetic output module 12a to flow-through magnetic separation / de-beading module 8b, which is configured as shown in FIGS. 2A and 2B. The paramagnetic particle-bound cells are flowed through return loop 14a at least once before entering magnetic output module 12b as waste. The de-beaded, unbound cells are sent to the spinning membrane de-beading module 18a via the non-magnetic output module 10b. Any remaining paramagnetic and paramagnetic particles are removed, and the paramagnetic particles flow into the waste output module 20a, while the unbound paramagnetic particles remain in module 18. The unbound cells and cells with paramagnetic and / or paramagnetic particles flow into the cell output module 22a. The cell output module 22a is connected to the second spinning membrane module 18b. Chemicals that can aid in the removal of the paramagnetic and / or paramagnetic particles from the cells are added from the reagent module 24. The paramagnetic particles and chemicals flow into the waste module 20b. The paramagnetic particles remain in module 18. The unbound cells and cells with paramagnetic and / or paramagnetic particles flow into the return loop 14b at least once before being sent to the cell output module 22b as the final cell product of the flow-through process.

[0341] Clinical applications All of the processes herein can be performed in accordance with clinical good manufacturing practice (cGMP) standards.

[0342] These processes can be used for cell purification, concentration, recovery, washing, enrichment or cell culture medium exchange, particularly during the collection of starting material (especially cells) at the beginning of the manufacturing process, as well as during the manufacturing process for the selection or expansion of cells for cell therapy.

[0343] The cells can include any number of cells. The cells can be of the same cell type or of mixed cell types. In addition, the cells can be from one donor, such as an autologous donor or a single allogeneic donor, for cell therapy. The cells can be obtained from a patient, for example, by leukapheresis or apheresis. The cells can include T cells, and can include a population with, for example, more than 50% T cells, more than 60% T cells, more than 70% T cells, more than 80% T cells, or more than 90% T cells.

[0344] The selection process can be particularly useful for selecting cells before culturing and expansion. For example, paramagnetic particles coated with anti-CD3 and / or anti-CD28 can be used to select T cells for expansion or for introduction of nucleic acids encoding chimeric antigen receptors (CARs) or other proteins. Such a process is used to generate CTL019 T cells for the treatment of acute lymphoblastic leukemia (ALL).

[0345] The de-beading process and module disclosed herein can be particularly useful in the production of cells for cell therapy, for example, for purifying cells before or after culture and expansion. For example, paramagnetic particles coated with anti-CD3 and / or anti-CD28 antibodies can be used to selectively expand T cells, such as T cells that have been or will be modified by the introduction of a nucleic acid encoding a chimeric antigen receptor (CAR) or other protein so that the CAR is expressed by the T cells. During the production of such T cells, the de-beading process or module can be used to separate the T cells from the paramagnetic particles. Such a de-beading process or module can be used, for example, to produce CTL019 T cells for the treatment of acute lymphoblastic leukemia (ALL).

[0346] In one such process disclosed herein, cells, e.g., T cells, are collected from a donor (e.g., a patient to be treated with an autologous chimeric antigen receptor T cell product) by apheresis (e.g., leukapheresis). The collected cells can then be optionally purified, e.g., by an elutriation step. Paramagnetic particles, e.g., anti-CD3 / anti-CD28 coated paramagnetic particles, can then be added to the cell population to expand the T cells. This process can also include a transduction step in which a nucleic acid encoding one or more desired proteins, e.g., a CAR, e.g., a CAR targeting CD19, is introduced into the cells. The nucleic acid may be introduced by a lentiviral vector. The cells, e.g., lentivirally transduced cells, can then be expanded for several days, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more, e.g., in the presence of a suitable medium. After expansion, the desired T cells can be separated from the paramagnetic particles using the de-beading process / module disclosed herein. This process can include one or more de-beading steps according to the process of the present disclosure.The de-beaded cells can then be formulated for administration to patients.Examples of CAR T cells and their production are further described in, for example, WO2012 / 079000, which is incorporated herein by reference in its entirety.The system and method of the present disclosure can be used for any cell separation / purification / de-beading process described in or related to WO2012 / 079000.

[0347] The systems and methods herein may be useful for other cell therapy products as well by discarding fewer desired cells, causing less cell damage, and more reliably removing magnetic and any non-magnetic particles from cells with less or no exposure to chemicals compared to conventional systems and methods.

[0348] While merely exemplary embodiments of the present disclosure have been specifically described above, it will be understood that modifications and variations of these examples are possible without departing from the spirit and intended scope of the present disclosure. For example, the magnetic modules and systems including them can be arranged and used in various configurations in addition to those described. Additionally, the systems and methods may include additional components and steps not specifically described herein. For example, the methods may include priming, in which a fluid is initially introduced into a component to remove air bubbles and reduce resistance to movement of the cell suspension or buffer. Furthermore, embodiments may include only a portion of the systems described herein for use with the methods described herein. For example, embodiments may relate to disposable modules, hoses, etc. that can be used within non-disposable equipment to form a complete system capable of separating or de-beading cells to produce a cellular product.

[0349] Embodiments of various aspects of the flow-through devices or cell processing systems described herein can be defined in any of the following numbered paragraphs:

[0350] 1. at least one cell suspension module; at least one buffer module; at least one flow-through magnetic separation / de-beading module; at least one non-magnetic output module; At least one magnetic output module and A cell processing system comprising:

[0351] 2. The cell processing system of paragraph 1, further comprising at least one return loop returning upstream of the at least one flow-through magnetic separation / de-beading module.

[0352] 3. A cell processing system according to paragraph 1, comprising at least two flow-through magnetic separation / de-beading modules in parallel.

[0353] 4. A cell processing system according to paragraph 1, comprising at least two flow-through magnetic separation / de-beading modules in series.

[0354] 5. The cell processing system of paragraph 1, further comprising at least one additional module.

[0355] 6. The cell processing system of paragraph 5, wherein the at least one additional module comprises at least one spinning membrane de-beading module.

[0356] 7. A cell processing system according to paragraph 6, comprising at least two rotating membrane debeading modules in parallel.

[0357] 8. A cell processing system according to paragraph 6, comprising at least two spinning membrane debeading modules in series.

[0358] 9. The cell processing system of paragraph 5, wherein the at least one additional module comprises at least one physical separation module.

[0359] 10. The cell processing system of paragraph 9, wherein the at least one additional module comprises at least one magnetic column module.

[0360] 11. The cell processing system of paragraph 5, wherein the at least one additional module comprises at least one medium exchange module.

[0361] 12. The cell processing system of paragraph 5, wherein the at least one additional module comprises at least one cell enrichment module.

[0362] 13. The cell processing system of paragraph 5, wherein the at least one additional module includes at least one cell washing module.

[0363] 14. The flow-through magnetic separation / de-beading module a chamber defined by walls and having an x-direction, a y-direction, and a z-direction; an inlet and an outlet disposed at opposite ends of the chamber in the y direction; at least two magnets adjacent to or proximate to a wall of the chamber and positioned to establish a zero gradient line between the inlet and the outlet within the chamber; 2. The cell processing system according to claim 1, comprising:

[0364] 15. The rotating membrane de-beading module comprises: a de-beading chamber defined in part by a cylindrical sidewall; a porous rotating membrane having an interior and oriented coaxially with said cylindrical side wall; A sample inlet; a waste output module connected to the interior of the rotating membrane; a cell output module connected to the de-beading chamber; 7. The cell processing system according to claim 6, comprising:

[0365] 16. The cell processing system of paragraph 15, wherein the rotating membrane de-beading module further comprises at least one magnet adjacent to or proximate to the cylindrical side wall.

[0366] 17. A chamber defined by walls and having an x-direction, a y-direction, and a z-direction; an inlet and an outlet disposed at opposite ends of the chamber in the y direction; at least two magnets adjacent to or proximate to a wall of the chamber and positioned to establish a zero gradient line between the inlet and the outlet within the chamber; A flow-through magnetic separation / de-beading module including:

[0367] 18. The module of paragraph 17, including at least two inlets and at least two outlets.

[0368] 19. The module of paragraph 17, further comprising at least three magnets adjacent or proximate to a wall of the chamber and positioned to establish at least two zero gradient lines between the inlet and the outlet within the chamber.

[0369] 20. The module described in paragraph 17, further comprising at least four magnets arranged in two rows at opposite ends of the chamber in the z-direction.

[0370] 21. The module described in paragraph 18, further comprising at least four magnets arranged in two rows on either side of the chamber in the z direction and cross-oriented in the xy plane from near one inlet in the z direction of the chamber to near one outlet at the opposite end.

[0371] 22. a sub-membrane injection port adjacent to a wall of said chamber that is also adjacent to at least two magnets; a membrane adjacent to the sub-membrane; 20. The module of claim 17, further comprising:

[0372] 23. A de-beading chamber defined in part by a cylindrical sidewall; a porous rotating membrane having an interior and oriented coaxially with said cylindrical side wall; A sample inlet; a waste output module connected to the interior of the rotating membrane; a cell output module connected to the de-beading chamber; at least one magnet adjacent to or proximate to said cylindrical side wall; a rotating membrane debeading module comprising:

[0373] 24. The spinning membrane de-beading module of claim 23, further comprising a reagent module.

[0374] 25. A spinning membrane de-beading module as described in paragraph 23, wherein the porous spinning membrane has a pore size larger than the diameter of the particles to be de-beaded and smaller than the diameter of the cells to be de-beaded.

[0375] 26. A method of flow-through cell processing, comprising: flowing the cell suspension containing the paramagnetic particle-bound cells through a flow-through magnetic separation / de-beading module to generate an unbound cell product; the paramagnetic particle-bound cells continue to move through a flow-through magnetic separation / de-beading module by a flow process; The flow-through magnetic separation / de-beading module a flow chamber defined by a wall through which the cell suspension flows; at least two magnets positioned adjacent to or in close proximity to at least one wall; A method comprising:

[0376] 27. The method of claim 26, wherein the cell suspension is flowed in layers through the flow-through magnetic separation / de-beading module.

[0377] 28. The method described in paragraph 26, wherein the cell suspension further contains unbound cells, and the paramagnetic particle-bound cells and the unbound cells are separated by flowing the cell suspension through the flow-through magnetic separation / de-beading module.

[0378] 29. The method of claim 28, wherein the cell suspension further contains free paramagnetic particles, and the free paramagnetic particles and the unbound cells are separated by passing the cell suspension through the flow-through magnetic separation / de-beading module.

[0379] 30. The method of claim 28, further comprising passing the separated, unbound cells through the flow-through magnetic separation / de-beading module two or more subsequent times using a return loop.

[0380] 31. The method of claim 28, further comprising flowing the separated paramagnetic particle-bound cells through the flow-through magnetic separation / de-beading module two or more subsequent times using a return loop.

[0381] 32. The method of claim 31, further comprising de-beading the paramagnetic particle-bound cells in the flow-through magnetic separation / de-beading module during the second or subsequent run to produce paramagnetic particles and de-beaded unbound cells.

[0382] 33. The method of claim 32, further comprising passing the resulting paramagnetic particles and de-beaded, non-bound cells through the flow-through magnetic separation / de-beading module three or more times to separate the paramagnetic particles from the de-beaded, non-bound cells.

[0383] 34. The method of paragraph 26, wherein the magnets are oriented to establish a single zero gradient line that intersects the direction of flow, so that paramagnetic particle-bound cells are attracted exclusively in one direction to the zero gradient line but are not affected by the magnetic forces of the two magnets in the other two directions.

[0384] 35. The chamber comprises: a magnetic inlet through which any paramagnetic particles enter the flow chamber; a non-magnetic inlet; a magnetic outlet opposite the non-magnetic inlet; a non-magnetic outlet opposite the magnetic inlet; further comprising 27. The method of clause 26, wherein the zero gradient line directs all paramagnetic particles and any bound cells towards the magnetic outlet.

[0385] 36. The method of claim 35, wherein the cell suspension further comprises non-bound cells, the non-magnetic inlet is larger than the magnetic inlet, the non-magnetic outlet is larger than the magnetic outlet, and fluid flowing from the non-magnetic inlet crosses to the non-magnetic outlet, thereby preventing any non-bound cells from entering the magnetic outlet.

[0386] 37. The method of claim 35, wherein the cell suspension further comprises non-bound cells, the non-magnetic inlet and the magnetic inlet are substantially the same size, or the non-magnetic outlet and the magnetic outlet are substantially the same size, or both, and the respective flow rates of fluids entering the inlets, the respective flow rates of fluids exiting the outlets, or both, are adjusted so that fluid flowing from the non-magnetic inlet crosses into the non-magnetic outlet, thereby preventing any non-bound cells from entering the magnetic outlet.

[0387] 38. Further comprising flowing the paramagnetic particle-bound cells through a rotating membrane de-beading module to produce an unbound cell product; The rotating membrane de-beading module a cylindrical de-beading chamber through which the paramagnetic particle-bound cells flow, the chamber being defined in part by a cylindrical sidewall and containing a coaxially rotating membrane; at least one magnet positioned adjacent or proximate to the cylindrical sidewall so as to establish at least one zero gradient line within the cylindrical de-beading chamber; 27. The method of claim 26, comprising:

[0388] 39. The method of claim 26, further comprising flowing the paramagnetic particle-bound cells through a magnetic column module to produce unbound products.

[0389] 40. The method of claim 26, further comprising flowing the paramagnetic particle-bound cells or the unbound cell product through a cell washing module.

[0390] 41. The method of claim 26, further comprising flowing the paramagnetic particle-bound cells or the unbound cell product through a medium exchange module.

[0391] 42. The method of claim 26, further comprising flowing the paramagnetic particle-bound cells or the unbound cell product through a cell enrichment module.

[0392] 43. A method for producing a cell therapy composition, comprising: contacting the cell population with paramagnetic particles coated with one or more agents that support the expansion of one or more cell types within the cell population; introducing a nucleic acid into a cell within said cell population; Expanding cells within the cell population; de-beading the cell population according to the method of any of paragraphs 26 to 42 or using the system of any of paragraphs 1 to 16 or the module of any of paragraphs 17 to 25; formulating said cell population for cell therapy. A method comprising:

[0393] 44. The method of paragraph 43, wherein the one or more agents that support the expansion of one or more cell types comprise an anti-CD3 antibody or antigen-binding fragment thereof, an anti-CD28 antibody or antigen-binding fragment thereof, and combinations thereof.

[0394] 45. The method of any of items 43 to 44, wherein the nucleic acid is introduced by lentivirus or mRNA transduction.

[0395] 46. The method of any one of items 43 to 45, wherein the cell therapy is chimeric antigen receptor T cell therapy.

[0396] 47. The method of paragraph 46, wherein the cell therapy is anti-CD19 chimeric antigen receptor T cell therapy.

[0397] Improved cleaning process The cellular composition of apheresis, e.g., leukapheresis, products varies significantly from patient to patient. Leukapheresis products with a high percentage of granulocytes (e.g., neutrophils) have been correlated with increased instances of cell aggregation during CAR T cell manufacturing using Process B. Without wishing to be bound by theory, it is believed that such irreversible aggregation reduces the number of available cells and adversely affects cell yield by interfering with the enrichment process (e.g., positive selection), resulting in an overall decrease in cell number and purity. Additionally, without wishing to be bound by theory, reduced cell purity and yield directly impacts the performance of subsequent processes (e.g., transduction efficiency and expansion) and the number and quality of the final product cells. The net result is a reduced ability of the manufactured product to meet specifications at the end of the processing cycle. Thus, without wishing to be bound by theory, it is believed that preventing aggregation can reduce cell loss and improve T cell purity, which can produce better quality and quantity of starting material for subsequent processing steps, resulting in an overall improved therapeutic product.

[0398] In current manufacturing processes in the art, such as Process B, patient cell leukapheresis material is thawed in a Plasmatherm (Genesis), washed using a CellSaver 5+ device (Haemonetics), and then resuspended in a cell expansion medium based on X-VIVO15 medium (Lonza), referred to as "modified medium," or in a buffered isotonic saline solution such as phosphate-buffered saline (PBS) for subsequent lymphocyte selection by Ficoll. The modified medium is prepared according to the protocol provided in Example 2. However, as described in Example 2, transferring thawed cells into either the modified medium or PBS solution can cause the cells to clump.

[0399] Therefore, an improved cell washing method for preventing aggregation is also provided herein, which is compatible with subsequent manufacturing steps, such as positive selection by stimulation with anti-CD3 / CD28 CTS Dynabeads (Thermo Fisher). Additionally, the improved washing steps described herein are performed, for example, on thawed cells to remove intracellular debris, free hemoglobin, and cryoprotectants, to achieve volume reduction, and to enable subsequent density gradient separation. In some embodiments, the washing steps are performed using an alternative cell resuspension buffer to modified medium or PBS solution. In some embodiments, the washing steps are performed using a buffer containing dextrose and / or sodium chloride. In some embodiments, the buffer contains between about 5% and about 0.45% sodium chloride, e.g., D5 1 / 2 NS medium. In certain embodiments, the buffer stabilizes the cell suspension and prevents clumping, for example, for at least 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, or 6 hours.

[0400] In one embodiment, the improved washing steps described herein are performed using a device, e.g., a cell separation device, e.g., the same device used for density gradient centrifugation. For example, the improved washing step is performed using a Sepax 2 RM device (Biosafe).

[0401] In certain embodiments, the washing steps disclosed herein can be used on fresh apheresis samples or previously frozen, e.g., thawed, apheresis samples. In embodiments, the washing steps disclosed herein can be used before or after any of the elutriation, density gradient centrifugation, or selection methods described herein. In another embodiment, the washing steps disclosed herein are performed after a density gradient centrifugation step, e.g., density gradient centrifugation using OptiPrep media.

[0402] Improved Manufacturing Process Provided herein are methods that improve the quality and yield of immune effector cells suitable for expressing a CAR more than methods currently used in the art. In one embodiment, the elutriation, density gradient centrifugation, positive and negative selection under flow conditions, and improved washing steps described in the sections above can be used in any combination with each other or with additional methods currently used in the art or described herein to isolate or enrich for desired immune effector cells suitable for expressing a CAR.

[0403] Generally, methods for generating or enriching a population of immune effector cells that can be engineered to express a CAR include providing an input sample, performing an enrichment step, and performing a selection step, thereby generating an output sample containing immune effector cells that are suitable for expression of a CAR. Methods for producing a population of immune effector cells that express a CAR include methods for generating or enriching a population of immune effector cells that can be engineered to express a CAR, and further include a stimulation step, e.g., stimulating the cells to proliferate or persist, and further include introducing a nucleic acid encoding a CAR. Further disclosure regarding stimulation and introduction / expression of a CAR is further described in subsequent sections.

[0404] In one embodiment, the input sample is a fresh sample obtained from a subject, such as a fresh apheresis, leukapheresis, or whole blood sample. In another embodiment, the input sample is a frozen sample. In embodiments where the input sample is a frozen sample, such as a frozen or cryopreserved apheresis, leukapheresis, or whole blood sample, the method includes thawing the frozen sample or preparing a thawed sample. A frozen sample, such as a cryopreserved sample, can be thawed by passive or active means. Thawing by passive means includes thawing the sample, for example, by bringing it to ambient temperature, for example, room temperature, or the temperature of a buffer or solution into which the sample is transferred or mixed. Thawing by active means includes using a device that thaws the sample faster than thawing by passive means, for example, by bringing the sample to ambient temperature.

[0405] In one embodiment, the concentration step comprises performing elutriation or density gradient centrifugation. Elutriation can be performed using elutriation conditions known in the art or using the improved settings described herein for elutriation of frozen or previously frozen samples. Density gradient centrifugation can be performed using Ficoll or a medium containing iodixanol, for example, about 60% iodixanol in water, such as OptiPrep™.

[0406] In any of the methods described herein, the selection step can include performing a positive selection step and / or a negative selection step. The positive selection step can include, for example, using a separation agent, e.g., beads coupled to anti-CD3 and / or anti-CD28 antibodies, under either static or flow conditions, to select CD3+ / CD28+ cells, e.g., using a. The negative selection step can include, for example, using a separation agent, e.g., beads coupled to anti-CD19 antibodies, to negatively select CD19+ B cells or CD19+ lymphoblasts.

[0407] In any of the methods described herein, a wash step can be performed after sample collection, after thawing the sample, before the enrichment step, after the enrichment step, before the selection step, or after the selection step, or any combination thereof.

[0408] Further described herein are exemplary methods of generating or enriching populations of immune effector cells that can be engineered to express a CAR, including one or more of elutriation, density gradient centrifugation, positive or negative selection, e.g., under flow conditions, or improved washing steps.

[0409] In one embodiment, a method of generating or enriching a population of immune effector cells that can be engineered to express a CAR includes providing a frozen input sample comprising immune effector cells; thawing the frozen input sample to generate a thawed sample; performing an enrichment step that includes performing elutriation on the input sample, wherein the input sample can be the thawed input sample; and performing a selection step, wherein the selection is a positive selection, e.g., positive selection of CD3 / CD28+ cells, or a negative selection, e.g., negative selection of CD19+, CD25+, or CD14+ cells.

[0410] In another embodiment, a method of generating or enriching a population of immune effector cells that can be engineered to express a CAR includes providing a fresh or frozen input sample comprising immune effector cells; optionally, if the input sample is a frozen input sample, thawing the frozen input sample to generate a thawed sample; performing an enrichment step that includes performing a density centrifugation step using a medium comprising iodixanol, e.g., a 60% aqueous iodixanol solution, e.g., OptiPrep medium, or a medium having a higher density than Ficoll (e.g., higher than 1.077 g / ml, e.g., about 1.32 g / ml); and performing a selection step, wherein the selection is positive selection, e.g., positive selection of CD3 / CD28+ cells, or negative selection, e.g., negative selection of CD19+, CD25+, or CD14+ cells.

[0411] In another embodiment, a method of generating or enriching a population of immune effector cells that can be engineered to express a CAR includes providing a fresh or frozen input sample containing immune effector cells; performing an enrichment step that includes performing elutriation or density centrifugation (e.g., using Ficoll or Optiprep medium); and performing a positive selection step, e.g., of CD3 / CD28+ cells, under flow conditions.

[0412] In another embodiment, a method of generating or enriching a population of immune effector cells that can be engineered to express a CAR includes providing a fresh or frozen input sample containing immune effector cells; performing an enrichment step that includes performing elutriation or density centrifugation (e.g., using Ficoll or Optiprep medium); and performing a negative selection step, e.g., of CD19+, CD25+, or CD14+ cells, under flow conditions.

[0413] In any of the methods described herein, a wash step can be performed after sample collection, after thawing the sample, before the enrichment step, after the enrichment step, before the selection step, or after the selection step, or any combination thereof.

[0414] To optimize the enrichment of desired immune effector cells and ensure manufacturing success and product quality, control limits can be determined that identify ranges or thresholds for characteristics of the input sample or after one or more steps in the methods described herein and indicate or determine the next step. In embodiments, the control limits can vary depending on the type of cancer from which the input sample is taken. By way of example, control limits for the presence of monocytes in an input sample taken from a subject with ALL or DLBCL are as follows: if monocytes are >20% of the input sample, e.g., leukapheresis total blood cells, the optimal method involves elutriation under flow conditions and / or CD3 / CD28 positive selection; or if monocytes are <20% of the input sample, the input sample is washed, and the optimal method is determined based on the number of blast cells contained. In another example, the control limits for the presence of blast cells in an input sample taken from a subject with ALL or DLBCL are as follows: if blast cells are ≧20% of the incoming leukapheresis WBCs, then elutriation (to remove monocytes, granulocytes, and cellular debris) and / or modified CD19 negative selection (to remove blasts) or other techniques to deplete blasts should be performed; or if blast cells are <20% of the incoming leukapheresis, then the leukapheresis material should be washed and the process determined based on the number of monocytes contained.

[0415] After enrichment of immune effector cells suitable for expressing a CAR, in one embodiment, the immune effector cells are stimulated, e.g., to proliferate, using any of the methods known in the art or described herein, e.g., as described in the section entitled "Activation and Expansion of Immune Effector Cells."

[0416] After enrichment of immune effector cells suitable for expressing a CAR, and optionally after stimulation and / or expansion as described herein, a nucleic acid encoding a CAR, e.g., a nucleic acid encoding a CAR described herein, can be introduced into the immune effector cells. Methods for introducing a nucleic acid, e.g., a nucleic acid encoding a CAR, are well known in the art and are described herein, e.g., as described in the sections entitled "Nucleic Acid Constructs Encoding a CAR," "RNA Transfection," and "Non-Viral Delivery Methods."

[0417] Immune effector cell sources This section provides additional methods or steps for obtaining an input sample containing desired immune effector cells, for isolating and processing the desired immune effector cells, e.g., T cells, and for removing undesired material, e.g., undesired cells. The additional methods or steps described in this section may be used in combination with any of the elutriation, density gradient centrifugation, selection under flow conditions, or improved washing steps described in the previous sections.

[0418] Cell source, for example, T cell or natural killer (NK) cell source can be obtained from subject.Examples of subject include human, monkey, chimpanzee, dog, cat, mouse, rat and their transgenic species.T cell can be obtained from many sources, such as peripheral blood mononuclear cell, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from infected site, ascites, pleural effusion, spleen tissue and tumor.

[0419] In certain embodiments of the present disclosure, immune effector cells, e.g., T cells, can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art and any of the methods described herein, in any combination of those steps. In one embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, such as T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction, and the cells can be placed in an appropriate buffer or medium for subsequent processing steps. In one embodiment, cells are washed with phosphate-buffered saline (PBS). In another embodiment, the wash solution is calcium-free, may be magnesium-free, or may not contain most, if not all, divalent cations. In another embodiment, cells are washed using the improved washing process described herein.

[0420] An initial activation step in the absence of calcium can enhance activation. As those skilled in the art will readily recognize, the washing step may be accomplished by methods known in the art, such as using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processing device, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in various biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or other buffer-containing or buffer-free saline solutions. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0421] In one embodiment, desired immune effector cells, e.g., T cells, are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, e.g., by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation.

[0422] The methods described herein can involve the selection of a specific subpopulation of immune effector cells, e.g., T cells, that are a regulatory T cell-depleted population, a CD25+ cell-depleted population, e.g., using a negative selection technique, e.g., a negative selection technique described herein. In some embodiments, the regulatory T cell-depleted population contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% CD25+ cells.

[0423] In one embodiment, regulatory T cells, e.g., CD25+ T cells, are removed from the population using an anti-CD25 antibody, or a fragment thereof, or a CD25-binding ligand, e.g., IL-2. In one embodiment, the anti-CD25 antibody, or a fragment thereof, or a CD25-binding ligand is conjugated to or otherwise coated on a substrate, e.g., a bead. In one embodiment, the anti-CD25 antibody or a fragment thereof is conjugated to a substrate described herein.

[0424] In one embodiment, regulatory T cells, e.g., CD25+ T cells, are removed from the population using CD25 depletion reagent from Miltenyi™. In one embodiment, the ratio of cells to CD25 depletion reagent is 1 x 10 cells to 20 μL. 7 or 1 x 10 cells per 15 μL 7 or 1 x 10 cells per 10 μL 7 or 1 x 10 cells for 5 μL 7 or 1 x 10 cells for 2.5 μL 7 or 1 x 10 cells per 1.25 μL 7 In one embodiment, for example, for regulatory T cells, e.g., CD25+ depletion, greater than 500 million cells / ml are used. In further embodiments, cell concentrations of 600 million, 700 million, 800 million, or 900 million cells / ml are used.

[0425] In one embodiment, the population of immune effector cells to be depleted is about 6×10 9In other embodiments, the population of immune effector cells to be depleted comprises about 1 x 10 CD25+ T cells. 9 From 1×10 10 In one embodiment, the resulting regulatory T cell depleted population comprises 2 x 10 CD25+ T cells and any integer value therebetween. 9 or fewer regulatory T cells, e.g., CD25+ cells (e.g., 1 x 10 9 , 5×10 8 , 1×10 8 , 5×10 7 , 1×10 7 or fewer CD25+ cells).

[0426] In one embodiment, regulatory T cells, e.g., CD25+ cells, are removed from the population using the CliniMAC system in conjunction with a depletion tubing set, e.g., tubing 162-01. In one embodiment, the CliniMAC system is run in a depletion setting, e.g., DEPLETION2.1.

[0427] Without wishing to be bound by a particular theory, it is believed that reducing the levels of negative regulators of immune cells (e.g., unwanted immune cells, e.g., T cells) in a subject prior to apheresis or during the manufacture of a CAR-expressing cell product may contribute to the production of CAR-expressing cells. REG Reducing the number of T cells can significantly reduce a subject's risk of recurrence. REG Methods for depleting cells are known in the art. REG Methods for reducing cells include, but are not limited to, cyclophosphamide, anti-GITR antibodies (such as the anti-GITR antibodies described herein), CD25 depletion, mTOR inhibitors, and combinations thereof.

[0428] In some embodiments, the manufacturing method includes, prior to manufacturing of the cells expressing the CAR, REG For example, the manufacturing method can include reducing (e.g., depleting) the number of cells (e.g., T cells, NK cells) expressing a CAR prior to manufacturing the product. REGThis involves contacting the sample, eg, an apheresis sample, with an anti-GITR antibody and / or an anti-CD25 antibody (or a fragment thereof, or a CD25-binding ligand) to deplete the cells.

[0429] Without wishing to be bound by a particular theory, it is believed that reducing the levels of negative regulators of immune cells (e.g., unwanted immune cells, e.g., T) in a subject prior to apheresis or during the manufacture of a CAR-expressing cell product may increase the risk of CAR-mediated immune cell death. REG In one embodiment, the subject is treated with T cells prior to collection of cells for manufacturing of a CAR-expressing cell product. REG In one embodiment, the subject is pre-treated with one or more therapies that reduce the risk of the subject returning to treatment with CAR-expressing cells. REG Methods for reducing T cells include, but are not limited to, administering to the subject one or more of cyclophosphamide, anti-GITR antibody, CD25 depletion, or a combination thereof. REG Methods for reducing cells include, but are not limited to, administering one or more of cyclophosphamide, anti-GITR antibody, CD25 depletion, mTOR inhibitor, or a combination thereof to the subject.Cyclophosphamide, anti-GITR antibody, CD25-depletion, or a combination thereof can be administered before, during, or after the infusion of a cell product expressing a CAR.Cyclophosphamide, anti-GITR antibody, CD25 depletion, or a combination thereof can be administered before, during, or after the infusion of a cell product expressing a CAR.

[0430] In some embodiments, the manufacturing method includes reducing the number of (e.g., depleting) TREG cells prior to manufacturing of the CAR-expressing cells. For example, the manufacturing method includes contacting a sample, e.g., an apheresis sample, with an anti-GITR antibody and / or an anti-CD25 antibody (or fragment thereof, or a CD25-binding ligand), e.g., to deplete TREG cells prior to manufacturing of the CAR-expressing cell (e.g., T cell, NK cell) product.

[0431] In one embodiment, the subject is pretreated with cyclophosphamide prior to collection of cells for manufacturing of a CAR-expressing cell product, thereby reducing the risk of the subject returning to CAR-expressing cell treatment (e.g., CTL019 treatment). In one embodiment, the subject is pretreated with an anti-GITR antibody prior to collection of cells for manufacturing of a CAR-expressing cell (e.g., T cell or NK cell) product, thereby reducing the risk of the subject returning to CAR-expressing cell treatment.

[0432] In one embodiment, the CAR-expressing cell (e.g., T cell, NK cell) manufacturing process is modified to deplete TREG cells prior to manufacturing of the CAR-expressing cell (e.g., T cell, NK cell) product (e.g., CTL019 product). In one embodiment, CD25 depletion is used to deplete TREG cells prior to manufacturing of the CAR-expressing cell (e.g., T cell, NK cell) product (e.g., CTL019 product).

[0433] In one embodiment, the population of cells removed are not regulatory T cells or tumor cells, but cells that would otherwise negatively affect the expansion and / or function of CART cells, e.g., cells expressing CD14, CD11b, CD33, CD15, or other markers that may be expressed by immunosuppressive cells. In one embodiment, it is envisioned that such cells are removed simultaneously with the regulatory T cells and / or tumor cells, or after the depletion, or in a different order.

[0434] The methods described herein may include more than one selection step, for example, more than one depletion step. Enrichment of a T cell population by negative selection can be achieved, for example, by using a combination of antibodies directed against surface markers unique to the negatively selected cells. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail may include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0435] The methods described herein may further include removing cells expressing a tumor antigen, e.g., a tumor antigen that does not contain CD25, such as CD19, CD30, CD38, CD123, CD20, CD14, or CD11b, from the population to provide a population of regulatory T cell-depleted, e.g., CD25+ depleted, and tumor antigen-depleted cells suitable for expression of a CAR, e.g., a CAR described herein. In one embodiment, cells expressing a tumor antigen are removed simultaneously with regulatory T cells, e.g., CD25+ cells. For example, anti-CD25 antibodies or fragments thereof and anti-tumor antigen antibodies or fragments thereof can be attached to the same substrate, e.g., beads that can be used to remove cells, or anti-CD25 antibodies or fragments thereof or anti-tumor antigen antibodies or fragments thereof can be attached to separate beads, a mixture of which can be used to remove cells. In other embodiments, removal of regulatory T cells, e.g., CD25+ cells, and removal of cells expressing a tumor antigen are sequential, e.g., can be performed in either order.

[0436] Also provided are methods that include providing a population of regulatory T cell-depleted, e.g., CD25+-depleted cells and checkpoint inhibitor-depleted cells, e.g., PD1+, LAG3+ and / or TIM3+-depleted cells, by removing from the population one or more cells that express a checkpoint inhibitor, e.g., a checkpoint inhibitor described herein, e.g., PD1+ cells, LAG3+ cells and TIM3+ cells. Exemplary checkpoint inhibitors include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF (e.g., TGF beta), e.g., as described herein. In one embodiment, cells expressing a checkpoint inhibitor are simultaneously deleted with regulatory T cells, e.g., CD25+ cells. For example, an anti-CD25 antibody or fragment thereof and an anti-check point inhibitor antibody or fragment thereof can be attached to the same bead, which can be used to remove cells, or an anti-CD25 antibody or fragment thereof and an anti-check point inhibitor antibody or fragment thereof can be attached to separate beads, the mixture of which can be used to remove cells. In other embodiments, the removal of regulatory T cells, e.g., CD25+ cells, and the removal of cells expressing a check point inhibitor are sequential, e.g., can occur in either order.

[0437] The methods described herein may include a positive selection step. For example, T cells can be isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3 / CD28T, for a period sufficient for positive selection of the desired T cells. In one embodiment, the period is approximately 30 minutes. In a further aspect, the period ranges from 30 minutes to 36 hours or longer, and all integer values therebetween. In a further embodiment, the period is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours. In yet another embodiment, the period is 10 to 24 hours, e.g., 24 hours. Longer incubation times may be used to isolate T cells in any situation where T cells are scarce compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Furthermore, the use of longer incubation times can increase the efficiency of CD8+ T cell capture. Thus, by simply shortening or lengthening the time that T cells can be bound to the CD3 / CD28 beads and / or increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be selectively selected for at the beginning of the culture or at other times during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies to beads or other surfaces, subpopulations of T cells can be selectively selected for at the beginning of the culture or at other desired times.

[0438] In one embodiment, T cell populations may be selected that express one or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other suitable molecules, e.g., other cytokines. Methods of screening for cell expression can be determined, for example, by the methods described in PCT Publication WO2013 / 126712.

[0439] To isolate a desired population of cells by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) may be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (e.g., increase the cell concentration) to maximize contact between the cells and beads. For example, in one embodiment, a concentration of 10 billion cells / ml, 9 billion cells / ml, 8 billion cells / ml, 7 billion cells / ml, 6 billion cells / ml, or 5 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In further embodiments, a cell concentration of 75 million or more, 80 million or more, 85 million or more, 90 million or more, 95 million or more, or 100 million cells per ml is used. In further embodiments, a concentration of 125 million or 150 million cells per ml can be used.

[0440] The use of high cell concentrations can result in increased cell yield, cell activation, and cell expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express the target antigen of interest, such as CD28-negative T cells, or cells from samples containing many tumor cells (e.g., leukemia blood, tumor tissue, etc.). Such cell populations may have therapeutic value and are desirable to obtain. For example, the use of high cell concentrations allows for more efficient selection of CD8+ T cells, which typically have relatively weak CD28 expression.

[0441] In a related embodiment, it may be desirable to use a lower cell concentration. By significantly diluting the mixture of T cells and a surface (e.g., particles such as beads), the interaction between the particles and the cells is minimized. This selects cells that express a large amount of the desired antigen for binding to the particles. For example, CD4+ T cells express higher levels of CD28, but are captured more efficiently than CD8+ T cells at low concentrations. In one embodiment, the cell concentration used is 5×10 6 In other embodiments, the concentration used is about 1 x 10 5 / ml to 1 × 106 / ml and any integer value therebetween.

[0442] In other embodiments, the cells can be incubated on a rotator for various lengths of time, at various speeds, at either 2-10° C. or room temperature.

[0443] In one embodiment, a plurality of immune effector cells of the population do not express diacylglycerol kinase (DGK), e.g., are DGK-deficient. In one embodiment, a plurality of immune effector cells of the population do not express Ikaros, e.g., are Ikaros-deficient. In one embodiment, a plurality of immune effector cells of the population do not express DGK and Ikaros, e.g., are both DGK- and Ikaros-deficient.

[0444] T cells for stimulation may be frozen after a washing step. Without wishing to be bound by theory, the freezing step followed by thawing results in a more homogenous product by removing granulocytes and, to some extent, monocytes from the cell population. After a washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and are expected to be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or culture medium containing 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A, followed by freezing the cells to -80°C at a rate of 1 degree per minute and storing them in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods may be used, as well as uncontrolled freezing at -20°C immediately or in liquid nitrogen.

[0445] In certain embodiments, cryopreserved cells are thawed, washed as described herein, and allowed to sit at room temperature for 1 hour before activation using the methods of the present invention.

[0446] It is also contemplated in the context of the present invention to collect a blood sample or apheresis product from a subject at a time prior to when cells expanded as described herein may be needed. As such, a source of cells to be expanded can be collected at any time necessary, and desired cells, such as T cells, may be isolated and frozen for later use in immune effector cell therapy for numerous diseases or conditions anticipated to benefit from immune effector cell therapy, such as those described herein. In one embodiment, a blood sample or apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or apheresis is taken from a generally healthy subject who is at risk for developing a disease but has not yet developed the disease, and the subject's cells may be isolated and frozen for later use. In certain embodiments, T cells may be expanded, frozen, and used at a later time. In certain embodiments, a sample is collected from a patient shortly after diagnosis of a particular disease, such as those described herein, but prior to any treatment. In further embodiments, cells are isolated from a blood sample or apheresis from a subject prior to a number of relevant treatment modalities, including, but not limited to, treatment with drugs such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228 and radiation.

[0447] In further embodiments of the present invention, T cells are obtained directly from a patient after a treatment that leaves the subject with functional T cells. In this regard, it has been observed that following certain cancer treatments, particularly treatment with drugs that damage the immune system, the quality of the obtained T cells may be optimal or improved in terms of their ability to expand ex vivo immediately after treatment, during the period when the patient is typically expected to recover from the treatment. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo expansion. Therefore, within the context of the present invention, it is contemplated to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic cell lineage, during this collection period. Furthermore, in certain embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create conditions in the subject that are favorable for the repopulation, recirculation, regeneration, and / or expansion of specific cell types, particularly during defined periods following therapy. Exemplary cell types include T cells, B cells, dendritic cells, and other immune system cells.

[0448] In one embodiment, immune effector cells expressing a CAR molecule, e.g., a CAR molecule described herein, are obtained from a subject that has received a low immune-enhancing dose of an mTOR inhibitor. In one embodiment, the population of immune effector cells, e.g., T cells, engineered to express a CAR is harvested after a sufficient time or after administration of a sufficient low immune-enhancing dose of an mTOR inhibitor such that the level of PD1-negative immune effector cells, e.g., T cells, or the ratio of PD1-negative immune effector cells, e.g., T cells / PD1-positive immune effector cells, e.g., T cells, in or harvested from the subject is at least transiently increased.

[0449] In other embodiments, a population of immune effector cells, e.g., T cells, that have been engineered or are expected to be engineered to express a CAR can be treated ex vivo by contact with an amount of an mTOR inhibitor that increases the number of PD1-negative immune effector cells, e.g., T cells, or increases the ratio of PD1-negative immune effector cells, e.g., T cells / PD1-positive immune effector cells, e.g., T cells.

[0450] It is recognized that the application method can utilize culture medium conditions containing 5% or less, for example, 2%, human AB serum, and can employ known culture medium conditions and compositions, such as those described in Smith et al., "Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement" Clinical & Translational Immunology (2015) 4, e31; doi:10.1038 / cti.2014.31.

[0451] In one embodiment, the method of the present application can employ culture medium conditions that include serum-free medium. In one embodiment, the serum-free medium is OpTmizer CTS (LifeTech), Immunocult XF (Stemcell technologies), CellGro (CellGenix), TexMacs (Miltenyi), Stemline (Sigma), Xvivo15 (Lonza), PrimeXV (Irvine Scientific), or StemXVivo (RandD systems). The serum-free medium can be supplemented with a serum substitute, such as ICSR (Immune Cell Serum Replacement) from LifeTech. The level of serum substitute (e.g., ICSR) can be, for example, 5% or less, e.g., about 1%, 2%, 3%, 4%, or 5%.

[0452] In one embodiment, the T cell population is diacylglycerol kinase (DGK) deficient. DGK-deficient cells include cells that do not express DGK RNA or protein, or have reduced or inhibited DGK activity. DGK-deficient cells can be generated by genetic approaches, for example, by administering RNA interference agents, such as siRNA, shRNA, miRNA, to reduce or prevent DGK expression. Alternatively, DGK-deficient cells can be generated by treatment with DGK inhibitors as described herein.

[0453] In one embodiment, the T cell population is Ikaros-deficient. Ikaros-deficient cells include cells that do not express Ikaros RNA or protein, or have reduced or inhibited Ikaros activity. Ikaros-deficient cells can be generated by genetic approaches, for example, by administering RNA interference agents, such as siRNA, shRNA, or miRNA, to reduce or prevent Ikaros expression. Alternatively, Ikaros-deficient cells can be generated by treatment with an Ikaros inhibitor, such as lenalidomide.

[0454] In embodiments, the T cell population is DGK-deficient and Ikaros-deficient, e.g., does not express DGK and Ikaros or has reduced or inhibited DGK and Ikaros activity. Such DGK- and Ikaros-deficient cells can be generated by any of the methods described herein.

[0455] In one embodiment, the NK cells are obtained from a subject. In another embodiment, the NK cells are an NK cell line, such as the NK-92 cell line (Conkwest).

[0456] Cells expressing allogeneic CAR In the embodiments described herein, the immune effector cells may be allogeneic immune effector cells, e.g., T cells or NK cells. For example, the cells may be allogeneic T cells, e.g., allogeneic T cells that lack expression of a functional T cell receptor (TCR) and / or human leukocyte antigen (HLA), e.g., HLA class I and / or HLA class II.

[0457] T cells lacking a functional TCR may, for example, be engineered so that they do not express any functional TCR on their surface, or so that they do not express one or more subunits that comprise a functional TCR (e.g., engineered to not express (or exhibit reduced expression of) TCR alpha, TCR beta, TCR gamma, TCR delta, TCR epsilon, and / or TCR zeta), or so that they produce very little functional TCR on their surface. Alternatively, the T cell can express a TCR that is substantially impaired in function, e.g., by expression of a mutated or truncated form of one or more of the TCR subunits. The term "substantially impaired in function" means that the TCR is not expected to elicit a deleterious immune response in a host.

[0458] The T cells described herein can be engineered, for example, to not express functional HLA on their surface. For example, the T cells described herein can be engineered to downregulate cell surface expression of HLA, e.g., HLA class I and / or HLA class II. In some embodiments, downregulation of HLA can be achieved by reducing or eliminating expression of beta-2 microglobulin (B2M).

[0459] In some embodiments, the T cells may lack a functional TCR and a functional HLA, eg, HLA class I and / or HLA class II.

[0460] Modified T cells lacking expression of a functional TCR and / or HLA can be obtained by any suitable means, such as knocking out or knocking down one or more subunits of the TCR or HLA. For example, T cells may include knockdown of the TCR and / or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nuclease (TALEN), or zinc finger endonuclease (ZFN).

[0461] In some embodiments, the allogeneic cells may be cells that do not express or express at low levels of an inhibitory molecule, e.g., by any of the methods described herein. For example, the cells may be cells that can reduce the ability of cells that do not express or express at low levels of an inhibitory molecule, e.g., cells that express a CAR, to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF (e.g., TGF beta). Inhibition of inhibitory molecules, e.g., by inhibition at the DNA, RNA, or protein level, can optimize the performance of cells expressing a CAR. In embodiments, inhibitory nucleic acids, e.g., inhibitory nucleic acids described herein, e.g., dsRNA, e.g., siRNA or shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nucleases (TALENs), or zinc finger endonucleases (ZFNs), can be used.

[0462] siRNA and shRNA that inhibit TCR or HLA In some embodiments, TCR expression and / or HLA expression can be inhibited in a cell, e.g., a T cell, using an siRNA or shRNA targeting a nucleic acid encoding a TCR and / or HLA and / or an inhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF-beta).

[0463] Expression systems for siRNAs and shRNAs, as well as exemplary shRNAs, are described, for example, in paragraphs 649 and 650 of International Application WO2015 / 142675, filed March 13, 2015, which is incorporated by reference in its entirety.

[0464] CRISPR that inhibits TCR or HLA As used herein, "CRISPR" or "CRISPR for TCR and / or HLA" or "CRISPR inhibiting TCR and / or HLA" refers to a set of clustered regularly interspaced short palindromic repeats or a system comprising such a set of repeats. As used herein, "Cas" refers to a CRISPR-associated protein. A "CRISPR / Cas" system refers to a system derived from CRISPR and Cas that can be used to silence or mutate TCR genes and / or HLA genes in a cell, e.g., a T cell, and / or an inhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF beta).

[0465] The CRISPR / Cas system and its uses are described, for example, in paragraphs 651-658 of International Application WO2015 / 142675, filed March 13, 2015, which is incorporated by reference in its entirety.

[0466] TALENs that inhibit TCR and / or HLA "TALEN" or "TALEN against HLA and / or TCR" or "TALEN that inhibits HLA and / or TCR" refers to a transcription activator-like effector nuclease, an artificial nuclease that can be used to edit HLA genes and / or TCR genes in a cell, e.g., a T cell, and / or an inhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF beta).

[0467] TALENs and their uses are described, for example, in paragraphs 659-665 of International Application WO2015 / 142675, filed March 13, 2015, which is incorporated by reference in its entirety.

[0468] Zinc finger nucleases that inhibit HLA and / or TCR "ZFN" or "zinc finger nuclease" or "ZFN against HLA and / or TCR" or "ZFN inhibiting HLA and / or TCR" refers to a ZFN that inhibits HLA and / or TCR by targeting HLA genes and / or TCR genes in a cell, e.g., a T cell, and / or an inhibitory molecule described herein [e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or TCR-1)]. or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF beta.

[0469] ZFNs and their uses are described, for example, in paragraphs 666-671 of International Application WO2015 / 142675, filed March 13, 2015, which is incorporated by reference in its entirety.

[0470] Telomerase expression Telomeres play a crucial role in somatic cell persistence, and telomere length is maintained by telomerase (TERT). Telomere length in CLL cells can be very short (Roth et al., "Significantly shorter telomeres in T-cells of patients with ZAP-70+ / CD38 chronic lymphocytic leukaemia," British Journal of Haematology, 143, 383-386., August 28, 2008), and may be even shorter in manufactured CAR-expressing cells, such as CART19 cells, thereby limiting the possibility of telomere elongation after adoptive transfer into patients. Telomerase expression can rescue CAR-expressing cells from replicative exhaustion.

[0471] Without intending to be bound by any particular theory, in some embodiments, the persistence of therapeutic T cells in patients is short-lived due to shortening of telomeres in T cells; therefore, transfection of a telomerase gene may lengthen the telomeres of T cells and improve the persistence of T cells in patients. See Carl June, "Adoptive T cell therapy for cancer in the clinic," Journal of Clinical Investigation, 117:1466-1476 (2007). Thus, in certain embodiments, immune effector cells, e.g., T cells, ectopically express a telomerase subunit, e.g., the catalytic subunit of telomerase, e.g., TERT, e.g., hTERT. In some embodiments, the present disclosure provides a method of producing a CAR-expressing cell, the method comprising contacting a cell with a nucleic acid encoding a telomerase subunit, e.g., the catalytic subunit of telomerase, e.g., TERT, e.g., hTERT. The cells can be contacted with the nucleic acid before, in parallel with, or after contact with the construct encoding the CAR.

[0472] Telomerase expression can be stable (e.g., the nucleic acid can be integrated into the cell's genome) or transient (e.g., the nucleic acid is not integrated and expression declines after a period of time, e.g., several days). Stable expression can be achieved by transfecting or transducing cells with DNA encoding the telomerase subunits and a selectable marker and selecting for stable integrants. Alternatively or in combination, stable expression can be achieved by site-specific recombination, e.g., using the Cre / Lox or FLP / FRT systems.

[0473] Transient expression can include transfection or transduction using nucleic acids, such as DNA or RNA, such as mRNA. In some embodiments, transient mRNA transfection avoids the genetic instability that can be associated with stable transfection using TERT. Transient expression of exogenous telomerase activity is described, for example, in International Application WO2014 / 130909, the entire contents of which are incorporated herein by reference. In embodiments, mRNA-based transfection of telomerase subunits is carried out according to the messenger RNA Therapeutics™ platform commercialized by Moderna Therapeutics. For example, the method may be the method described in U.S. Patent Nos. 8,710,200, 8,822,663, 8,680,069, 8,754,062, 8,664,194, or 8,680,069.

[0474] In one embodiment, hTERT has the amino acid sequence of GenBank Protein ID AAC51724.1 [Meyerson et al., "hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization" Cell Volume 90, Issue 4, 22 August 1997, Pages 785-795]: MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDP AAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFA LLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFV LVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPA PGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPA RPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYS SGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPL FLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQ LLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQEL TWKMSVRGCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTET TFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFI PKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLG LDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYC VRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSL NEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAG IRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEAL GGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRR KLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPT FFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVT YVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD (SEQ ID NO: 108) It has the amino acid sequence:

[0475] In some embodiments, the hTERT has a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 108. In some embodiments, the hTERT has the sequence of SEQ ID NO: 108. In some embodiments, the hTERT comprises a deletion (e.g., not more than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, C-terminus, or both. In some embodiments, the hTERT comprises a transgenic amino acid sequence (e.g., not more than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, C-terminus, or both.

[0476] In one embodiment, hTERT is the nucleic acid sequence of GenBank Accession No. AF018167 (Meyerson et al., "hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization" Cell Volume 90, Issue 4, 22 August 1997, Pages 785-795): 1 caggcagcgt ggtcctgctg cgcacgtggg aagccctggc cccggccacc cccgcgatgc 61 cgcgcgctcc ccgctgccga gccgtgcgct ccctgctgcg cagccactac cgcgaggtgc 121 tgccgctggc cacgttcgtg cggcgcctgg ggccccaggg ctggcggctg gtgcagcgcg 181 gggacccggc ggctttccgc gcgctggtgg cccagtgcct ggtgtgcgtg ccctgggacg 241 cacggccgcc ccccgccgcc ccctccttcc gccaggtgtc ctgcctgaag gagctggtgg 301 cccgagtgct gcagaggctg tgcgagcgcg gcgcgaagaa cgtgctggcc ttcggcttcg 361 cgctgctgga cggggcccgc ggggggcccccc ccgaggcctt caccaccagc gtgcgcagct 421 acctgcccaa cacggtgacc gacgcactgc gggggagcgg ggcgtgggg ctgctgttgc 481 gccgcgtggg cgacgacgtg ctggttcacc tgctggcacg ctgcgcgctc tttgtgctgg 541 tggctcccag ctgcgcctac caggtgtgcg ggccgccgct gtaccagctc ggcgctgcca 601 ctcaggcccg gccccccgcca cacgctagtg gaccccgaag gcgtctggga tgcgaacggg 661 cctggaacca tagcgtcagg gaggccgggg tccccctggg cctgccagcc ccgggtgcga 721 ggaggcgcgg gggcagtgcc agccgaagtc tgccgttgcc caagaggccc aggcgtggcg 781 ctgcccctga gccggagcgg acgcccgttg ggcaggggtc ctgggcccac ccgggcagga 841 cgcgtggacc gagtgaccgt ggtttctgtg tggtgtcacc tgccagaccc gccgaagaag 901 ccacctcttt ggagggtgcg ctctctggca cgcgccactc ccacccatcc gtgggccgcc 961 agcaccacgc gggcccccca tccacatcgc ggccaccacg tccctgggac acgccttgtc 1021 ccccggtgta cgccgagacc aagcacttcc tctactcctc aggcgacaag gagcagctgc 1081 ggccctcctt cctactcagc tctctgaggc ccagcctgac tggcgctcgg aggctcgtgg 1141 agaccatctt tctgggttcc aggccctgga tgccagggac tccccgcagg ttgccccgcc 1201 tgccccagcg ctactggcaa atgcggcccc tgtttctgga gctgcttggg aaccacgcgc 1261 agtgccccta cggggtgctc ctcaagacgc actgcccgct gcgagctgcg gtcaccccag 1321 cagccggtgt ctgtgcccgg gagaagcccc agggctctgt ggcggccccc gaggaggagg 1381 acacagaccc ccgtcgcctg gtgcagctgc tccgccagca cagcagcccc tggcaggtgt 1441 acggcttcgt gcgggcctgc ctgcgccggc tggtgccccc aggcctctgg ggctccaggc 1501 acaacgaacg ccgcttcctc aggaacacca agaagttcat ctccctgggg aagcatgcca 1561 agctctcgct gcaggagctg acgtggaaga tgagcgtgcg gggctgcgct tggctgcgca 1621 ggagcccagg ggttggctgt gttccggccg cagagcaccg tctgcgtgag gagatcctgg 1681 ccaagttcct gcactggctg atgagtgtgt acgtcgtcga gctgctcagg tctttctttt 1741 1801 1861 1921 gcttcatccc caagcctgac gggctgcggc cgattgtgaa catggactac gtcgtgggag 1981 ccagaacgtt ccgcagagaa aagagggccg agcgtctcac ctcgagggtg aaggcactgt 2041 tcagcgtgct caactacgag cgggcgcggc gccccggcct cctgggcgcc tctgtgctgg 2101 gcctggacga tatccacagg gcctggcgca ccttcgtgct gcgtgtgcgg gcccaggacc 2161 cgccgcctga gctgtacttt gtcaaggtgg atgtgacggg cgcgtacgac accatcccc 2221 aggacaggct cagggaggtc atcgccagca tcatcaaacc ccagaacacg tactgcctgc 2281 gtcggtatgc cgtggtccag aaggccgccc atgggcacgt ccgcaaggcc ttcaagagcc 2341 acgtctctac cttgacagac ctccagccgt acatgcgaca gttcgtggct cacctgcagg 2401 agaccagccc gctgagggat gccgtcgtca tcgagcagag ctcctccctg aatgaggcca 2461 gcagtggcct cttcgacgtc ttcctacgct tcatgtgcca ccacgccgtg cgcatcaggg 2521 gcaagtccta cgtccagtgc caggggatcc cgcagggctc catcctctcc acgctgctct 2581 gcagcctgtg ctacggcgac atggagaaca agctgtttgc ggggattcgg cgggacgggc 2641 tgctcctgcg tttggtggat gatttcttgt tggtgacacc tcacctcacc cacgcgaaaa 2701 ccttctccag gaccctggtc cgaggtgtcc ctgagtatgg ctgcgtggtg aacttgcgga 2761 agacagtggt gaacttccct gtagaaagacg aggccctggg tggcacggct tttgttcaga 2821 tgccggccca cggcctattc ccctggtgcg gcctgctgct ggatacccgg accctggagg 2881 tgcagagcga ctactccagc tatgcccgga cctccatcag agccagtctc accttcaacc 2941 gcggcttcaa ggctgggagg aacatgcgtc gcaaactctt tggggtcttg cggctgaagt 3001 gtcacagcct gtttctggat ttgcaggtga acagcctcca gacggtgtgc accaacatct 3061 acaagatcct cctgctgcag gcgtacaggt ttcacgcatg tgtgctgcag ctcccatttc 3121 atcagcaagt ttggaagaac cccacatttt tcctgcgcgt catctctgac acggcctccc 3181 tctgctactc catcctgaaa gccaagaacg cagggatgtc gctgggggcc aagggcgccg 3241 ccggccctct gccctccgag gccgtgcagt ggctgtgcca ccaagcattc ctgctcaagc 3301 tgactcgaca ccgtgtcacc tacgtgccac tcctggggtc actcaggaca gcccagacgc 3361 agctgagtcg gaagctcccg gggacgacgc tgactgccct ggaggccgca gccaacccgg 3421 cactgccctc agacttcaag accatcctgg actgatggcc acccgcccac agccaggccg 3481 agagcagaca ccagcagccc tgtcacgccg ggctctacgt cccagggagg gaggggcggc 3541 ccacacccag gcccgcaccg ctgggagtct gaggcctgag tgagtgtttg gccgaggcct 3601 gcatgtccgg ctgaaggctg agtgtccggc tgaggcctga gcgagtgtcc agccaagggc 3661 tgagtgtcca gcacacctgc cgtcttcact tccccacagg ctggcgctcg gctccacccc 3721 agggccagct tttcctcacc aggagcccgg cttccactcc ccacatagga atagtccatc 3781 cccagattcg ccattgttca cccctcgccc tgccctcctt tgccttccac ccccaccatc 3841 caggtggaga ccctgagaag gaccctggga gctctgggaa tttggagtga ccaaaggtgt 3901 gccctgtaca caggcgagga ccctgcacct ggatgggggt ccctgtgggt caaattgggg 3961 ggaggtgctg tgggagtaaa atactgaata tatgagtttt tcagttttga aaaaaaaaaa 4021 aaaaaaa (SEQ ID NO: 23) is coded by

[0477] In some embodiments, hTERT is encoded by a nucleic acid having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 23. In some embodiments, hTERT is encoded by the nucleic acid of SEQ ID NO: 23.

[0478] Chimeric antigen receptor (CAR) The present invention provides immune effector cells (e.g., T cells, NK cells) engineered to contain one or more CARs that direct the immune effector cells to cancer. This targeting is achieved by an antigen-binding domain on the CAR that is specific for a cancer-associated antigen. There are two classes of cancer-associated antigens (tumor antigens) that can be targeted by the CARs described herein: (1) cancer-associated antigens that are expressed on the surface of cancer cells; and (2) cancer-associated antigens that are themselves intracellular, but where fragments (peptides) of such antigens are presented on the surface of cancer cells by MHC (major histocompatibility complex).

[0479] Thus, immune effector cells, for example, immune effector cells obtained by the methods described herein, can be engineered to contain a CAR that targets one of the following cancer-associated antigens (tumor antigens): CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, VEGFR2, Lewis Y, CD24, PDGFR-beta, PRSS21, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-a bl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, legumain, HPV E6, E7, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, and mutated hsp70-2.

[0480] Bispecific CAR In one embodiment, a multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies have specificity for no more than two antigens. Bispecific antibody molecules are characterized by a variable domain sequence of a first immunoglobulin that has binding specificity for a first epitope and a variable domain sequence of a second immunoglobulin that has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or subunits of different multimeric proteins). In one embodiment, a bispecific antibody molecule comprises heavy and light chain variable domain sequences that have binding specificity for a first epitope, and heavy and light chain variable domain sequences that have binding specificity for a second epitope. In one embodiment, a bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In one embodiment, a bispecific antibody molecule comprises a half antibody or fragment thereof having binding specificity for a first epitope and a half antibody or fragment thereof having binding specificity for a second epitope. In one embodiment, a bispecific antibody molecule comprises an scFv or fragment thereof having binding specificity for a first epitope and an scFv or fragment thereof having binding specificity for a second epitope.

[0481] In certain embodiments, the antibody molecule is a multispecific (e.g., bispecific or trispecific) antibody molecule. Protocols for generating bispecific or heterodimeric antibody molecules and various configurations of bispecific antibody molecules are described, for example, in paragraphs 455-458 of WO2015 / 142675, filed March 13, 2015, the entire contents of which are incorporated herein by reference.

[0482] In one embodiment, the bispecific antibody molecule features a first immunoglobulin variable domain sequence, e.g., an scFv, that has binding specificity for CD19, e.g., comprising an scFv described herein or comprising light chain CDRs and / or heavy chain CDRs from an scFv described herein, and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope on a different antigen.

[0483] Chimeric TCR In one embodiment, antibodies and antibody fragments of the invention (e.g., CD19 antibodies and fragments) can be grafted onto one or more constant domains of a T cell receptor ("TCR") chain, e.g., a TCR alpha or TCR beta chain, to create a chimeric TCR. Without being bound by theory, it is believed that the chimeric TCR signals through the TCR complex immediately upon antigen binding. For example, an scFv disclosed herein may be grafted onto a constant domain, e.g., at least a portion of the extracellular constant domain, transmembrane domain, and cytoplasmic domain, of a TCR chain, e.g., a TCR alpha chain and / or a TCR beta chain. As another example, an antibody fragment, e.g., a VL domain described herein, may be grafted onto a constant domain of a TCR alpha chain, and an antibody fragment, e.g., a VH domain described herein, may be grafted onto a constant domain of a TCR beta chain (or alternatively, a VL domain may be grafted onto a constant domain of a TCR beta chain and a VH domain may be grafted onto a TCR alpha chain). As another example, the CDRs of an antibody or antibody fragment may be grafted onto the TCR alpha and / or beta chain to create a chimeric TCR. For example, the LCDR disclosed herein may be grafted onto the variable domain of the TCR alpha chain, and the HCDR disclosed herein may be grafted onto the variable domain of the TCR beta chain, or vice versa. Such chimeric TCRs can be produced, for example, by methods known in the art (e.g., Willemsen RA et al., Gene Therapy 2000; 7: 1369-1377; Zhang T et al., Cancer Gene Ther 2004; 11: 487-496; Aggen et al., Gene Ther. 2012 Apr; 19(4): 365-74).

[0484] Non-antibody scaffolds In embodiments, the antigen-binding domain comprises a non-antibody scaffold, such as fibronectin, ankyrin, domain antibody, lipocalin, small modular immunopharmaceutical, maxybody, protein A, or affilin. The non-antibody scaffold has the ability to bind to a target antigen on a cell. In embodiments, the antigen-binding domain is a polypeptide or fragment thereof of a naturally occurring protein expressed on a cell. In some embodiments, the antigen-binding domain comprises a non-antibody scaffold. A wide variety of non-antibody scaffolds can be utilized, provided that the resulting polypeptide comprises at least one binding region that specifically binds to a target antigen on a target cell.

[0485] Non-antibody scaffolds include fibronectin (Novartis, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA and Ablynx nv, Zwijnaarde, Belgium), lipocalins (Pieris Proteolab AG, Freising, Germany), small modular immunopharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), maxybodies (Avidia, Inc., Mountain View, CA), protein A (Affibody AG, Sweden), and affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

[0486] In certain embodiments, the antigen-binding domain comprises the extracellular domain of a molecule that binds to a counterligand on the surface of a target cell, or a counterligand-binding fragment thereof.

[0487] The immune effector cells can comprise a recombinant DNA construct comprising a sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain (e.g., an antibody or antibody fragment, a TCR or TCR fragment) that specifically binds to a tumor antigen, e.g., a tumor antigen described herein, and an intracellular signaling domain. The intracellular signaling domain can comprise a costimulatory signaling domain and / or a primary signaling domain, e.g., a zeta chain. As described elsewhere, the methods described herein can include transducing cells, e.g., cells from a regulatory T cell-depleted population, with a nucleic acid encoding a CAR, e.g., a CAR described herein.

[0488] In certain embodiments, the CAR comprises an scFv domain, where the scFv may be preceded by an optional leader sequence, such as that provided in SEQ ID NO: 1, followed by an optional hinge sequence, such as that provided in SEQ ID NO: 2 or SEQ ID NO: 36 or SEQ ID NO: 38, a transmembrane region, such as that provided in SEQ ID NO: 6, an intracellular signaling domain comprising SEQ ID NO: 7 or SEQ ID NO: 16, and a CD3 zeta sequence comprising SEQ ID NO: 9 or SEQ ID NO: 10, e.g., said domains are contiguous and in the same reading frame to form a single fusion protein.

[0489] In one embodiment, an exemplary CAR construct comprises an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen binding domain (e.g., an antigen binding domain described herein), a hinge (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular stimulatory domain (e.g., an intracellular stimulatory domain described herein). In one embodiment, an exemplary CAR construct comprises an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen binding domain (e.g., an antigen binding domain described herein), a hinge (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein), an intracellular costimulatory signaling domain (e.g., a costimulatory signaling domain described herein), and / or an intracellular primary signaling domain (e.g., a primary signaling domain described herein).

[0490] An exemplary leader sequence is provided as SEQ ID NO: 1. An exemplary hinge / spacer sequence is provided as SEQ ID NO: 2 or SEQ ID NO: 36 or SEQ ID NO: 38. An exemplary transmembrane domain sequence is provided as SEQ ID NO: 6. An exemplary sequence of the intracellular signaling domain of 4-1BB protein is provided as SEQ ID NO: 7. An exemplary sequence of the intracellular signaling domain of CD27 is provided as SEQ ID NO: 16. An exemplary CD3 zeta domain sequence is provided as SEQ ID NO: 9 or SEQ ID NO: 10.

[0491] In one embodiment, the immune effector cell comprises a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an antigen binding domain, and the sequence is contiguous with and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. Typical intracellular signaling domains that can be used in CAR include, but are not limited to, one or more intracellular signaling domains such as CD3-zeta, CD28, CD27, 4-1BB, etc. In some cases, the CAR may comprise any combination of CD3-zeta, CD28, 4-1BB, etc.

[0492] Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as by screening libraries from cells which express the nucleic acid molecule, by extracting the nucleic acid molecule from a vector known to contain it, or by isolating it directly from cells and tissues containing the gene using standard techniques, etc. Alternatively, the nucleic acid of interest can be produced synthetically rather than cloned.

[0493] For example, a retroviral or lentiviral vector construct can be used to introduce a nucleic acid encoding a CAR into immune effector cells.

[0494] For example, a nucleic acid encoding a CAR can be introduced into immune effector cells using an RNA construct that can be directly transfected into cells. Methods for generating mRNA for use in transfection include in vitro transcription (IVT) of a template using specially designed primers, followed by poly(A) addition, to produce a construct typically 50-2000 bases in length (e.g., those described in the Examples, e.g., SEQ ID NO: 35) containing 3' and 5' untranslated sequences ("UTRs") (e.g., 3' and / or 5' UTRs described herein), a 5' cap (e.g., 5' caps described herein), and / or an internal ribosome entry site (IRES) (e.g., IRES described herein), the nucleic acid to be expressed, and a poly(A) tail. The RNA produced in this manner can be efficiently transfected into different types of cells. In one embodiment, the template includes sequences for the CAR. In one embodiment, the RNA CAR vector is transduced into cells, e.g., T cells, by electroporation.

[0495] antigen-binding domain In one embodiment, a plurality of immune effector cells, for example, regulatory T cell depleted population, comprises nucleic acid encoding CAR, which comprises target-specific binding element, otherwise referred to as antigen binding domain.The selection of binding element depends on the type and number of ligands that characterize the surface of target cell.For example, antigen binding domain can be selected to recognize the ligand that acts as a cell surface marker on target cell related to specific pathology.Therefore, examples of cell surface markers that can act as ligands for antigen binding domain in CAR described herein include those related to virus, bacteria and parasite infection, autoimmune disease and cancer cell.

[0496] In one embodiment, the portion of the CAR comprising the antigen binding domain comprises an antigen binding domain that targets a tumor antigen, e.g., a tumor antigen described herein.

[0497] The antigen-binding domain may be any domain that is bound to an alternative scaffold known in the art to function as an antigen-binding domain, such as, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and functional fragments thereof (including, but not limited to, single-domain antibodies, such as camelid-derived nanobodies, including heavy chain variable domains (VH), light chain variable domains (VL), and variable domains (VHH)), as well as recombinant fibronectin domains, T cell receptors (TCR) or fragments thereof, such as single-chain TCRs, etc. In some cases, it is beneficial for the antigen-binding domain to be derived from the same species as the CAR is expected to ultimately be used. For example, when used in humans, it may be beneficial for the antigen-binding domain of the CAR to contain human or humanized residues in the antigen-binding domain of an antibody or antibody fragment.

[0498] In one embodiment, the antigen-binding domain comprises an anti-CD19 antibody or fragment thereof, such as an scFv. For example, the antigen-binding domain comprises a variable heavy chain and a variable light chain listed in Table 1. The linker sequence joining the variable heavy and light chains may be, for example, any of the linker sequences described herein or alternatively, GSTSGSGKPGSGEGSTKG (SEQ ID NO: 104).

[0499] [Table 3]

[0500] [Table 4]

[0501] [Table 5]

[0502] Any CD19 CAR, for example, the CD19 antigen-binding domain of any known CAR, can be used in accordance with the present disclosure. For example, LG-740; U.S. Patent No. 8,399,645; U.S. Patent No. 7,446,190; Xu et al., Leuk Lymphoma.2013 54(2):255-260(2012); Cruz et al., Blood 122(17):2965-2973 (2013); Brentjens et al., Blood, 118(18):4817-4828 (2011); Kochenderfer et al., Blood 116(20):4099-102 (2010); Kochenderfer et al., Blood 122 (25):4129-39(2013); and the CD19 CAR described in 16th Annu Meet Am Soc Gen Cell Ther (ASGCT) (May 15-18, Salt Lake City) 2013, Abst 10.

[0503] Exemplary target antigens that can be targeted using CAR-expressing cells include, but are not limited to, CD19, CD123, EGFRvIII, mesothelin, among others, as described, for example, in WO2014 / 130635, WO2014 / 130657, and WO2015 / 090230, which are incorporated by reference herein in their entireties.

[0504] In one embodiment, CAR T cells that specifically bind CD19 have the USAN name TISAGENLECLEUCEL-T. CTL019 is generated by genetic modification of T cells, mediated by stable insertion by transduction with a self-inactivating, replication-deficient lentiviral (LV) vector containing the CTL019 transgene under the control of the EF-1 alpha promoter. CTL019 can also be a mixture of transgene-positive and transgene-negative T cells delivered to a subject based on percent transgene-positive T cells.

[0505] In other embodiments, the CAR-expressing cells can specifically bind to human CD19 and can include a CAR molecule or antigen-binding domain (e.g., a humanized antigen-binding domain), for example, according to Table 3 of WO2014 / 153270, which is incorporated herein by reference.

[0506] In other embodiments, the CAR-expressing cells can specifically bind to CD123 and can include a CAR molecule (e.g., any of CAR1-CAR8) or antigen-binding domain, for example, according to Tables 1-2 of WO2014 / 130635, which is incorporated herein by reference.

[0507] In one embodiment, the CAR molecule comprises a CD123 CAR described herein, e.g., the CD123 CAR described in U.S. Patent Application Publication Nos. 2014 / 0322212 A1 or 2016 / 0068601 A1, both of which are incorporated herein by reference. In an embodiment, the CD123 CAR comprises the amino acid or has the nucleotide sequence set forth in U.S. Patent Application Publication Nos. 2014 / 0322212 A1 or 2016 / 0068601 A1, both of which are incorporated herein by reference.

[0508] In other embodiments, the CAR-expressing cells can specifically bind to EGFRvIII and can include a CAR molecule or antigen-binding domain, for example, according to Table 2 or SEQ ID NO: 11 of WO2014 / 130657, which is incorporated herein by reference.

[0509] In one embodiment, the CAR molecule comprises an EGFRvIII CAR molecule described herein, such as the EGFRvIII CAR described in U.S. Patent Application Publication No. 2014 / 0322275 A1, which is incorporated herein by reference. In an embodiment, the EGFRvIII CAR comprises the amino acid or has the nucleotide sequence set forth in U.S. Patent Application Publication No. 2014 / 0322275 A1, which is incorporated herein by reference.

[0510] In other embodiments, the CAR-expressing cells can specifically bind mesothelin and can include a CAR molecule or antigen-binding domain, for example, according to Tables 2-3 of WO2015 / 090230, which is incorporated herein by reference.

[0511] In one embodiment, the CAR molecule comprises a mesothelin CAR described herein, such as the mesothelin CAR described in WO2015 / 090230, which is incorporated herein by reference. In an embodiment, the mesothelin CAR comprises the amino acid or nucleotide sequence set forth in WO2015 / 090230, which is incorporated herein by reference.

[0512] In one embodiment, the CAR molecule comprises a BCMA CAR molecule described herein, such as the BCMA CAR described in U.S. Patent Application Publication No. 2016 / 0046724 A1. In an embodiment, the BCMA CAR comprises the amino acid or nucleotide sequence set forth in U.S. Patent Application Publication No. 2016 / 0046724 A1, which is incorporated herein by reference.

[0513] In one embodiment, the CAR molecule comprises a CLL1 CAR described herein, for example, the CLL1 CAR described in U.S. Patent Application Publication No. 2016 / 0051651 A1, which is incorporated herein by reference. In an embodiment, the CLL1 CAR comprises the amino acid or has the nucleotide sequence set forth in U.S. Patent Application Publication No. 2016 / 0051651 A1, which is incorporated herein by reference.

[0514] In one embodiment, the CAR molecule comprises a CD33 CAR described herein, such as the CD33 CAR described in U.S. Patent Application Publication No. 2016 / 0096892 A1, which is incorporated herein by reference. In an embodiment, the CD33 CAR comprises the amino acid or nucleotide sequence set forth in U.S. Patent Application Publication No. 2016 / 0096892 A1, which is incorporated herein by reference.

[0515] According to any method or composition described herein, in embodiments, the CAR molecule comprises a CD123 CAR described herein, e.g., the CD123 CAR described in U.S. Patent Application Publication No. 2014 / 0322212 A1 or 2016 / 0068601 A1, both of which are incorporated herein by reference. In embodiments, the CD123 CAR comprises the amino acid or has the nucleotide sequence set forth in U.S. Patent Application Publication No. 2014 / 0322212 A1 or 2016 / 0068601 A1, both of which are incorporated herein by reference. In other embodiments, the CAR molecule comprises a CD19 CAR molecule described herein, e.g., the CD19 CAR molecule described in U.S. Patent Application Publication No. 2015 / 0283178 A1, e.g., CTL019. In embodiments, the CD19 CAR has the amino acid or nucleotide sequence set forth in U.S. Patent Application Publication No. 2015 / 0283178 A1, which is incorporated herein by reference. In one embodiment, the CAR molecule comprises a BCMA CAR molecule described herein, for example, the BCMA CAR described in U.S. Patent Application Publication No. 2016 / 0046724 A1. In embodiments, the BCMA CAR has the amino acid or nucleotide sequence set forth in U.S. Patent Application Publication No. 2016 / 0046724 A1, which is incorporated herein by reference. In an embodiment, the CAR molecule comprises a CLL1 CAR described herein, for example, the CLL1 CAR described in U.S. Patent Application Publication No. 2016 / 0051651 A1, which is incorporated herein by reference. In embodiments, the CLL1 CAR has the amino acid or nucleotide sequence set forth in U.S. Patent Application Publication No. 2016 / 0051651 A1, which is incorporated herein by reference. In one embodiment, the CAR molecule comprises a CD33 CAR described herein, for example, the CD33 CAR described in U.S. Patent Application Publication No. 2016 / 0096892 A1, which is incorporated herein by reference.In embodiments, the CD33 CAR has the amino acid or nucleotide sequence set forth in U.S. Patent Application Publication No. 2016 / 0096892 A1, which is incorporated herein by reference. In some embodiments, the CAR molecule comprises an EGFRvIII CAR molecule described herein, such as the EGFRvIII CAR described in U.S. Patent Application Publication No. 2014 / 0322275 A1, which is incorporated herein by reference. In some embodiments, the EGFRvIII CAR has the amino acid or nucleotide sequence set forth in U.S. Patent Application Publication No. 2014 / 0322275 A1, which is incorporated herein by reference. In some embodiments, the CAR molecule comprises a mesothelin CAR described herein, such as the mesothelin CAR described in WO 2015 / 090230, which is incorporated herein by reference. In some embodiments, the mesothelin CAR has the amino acid or nucleotide sequence set forth in WO 2015 / 090230, which is incorporated herein by reference.

[0516] Exemplary CD19 CARs include those described herein, for example, in one or more tables herein, or those described in Xu et al. Blood 123.24(2014):3750-9, Kochenderfer et al. Blood 122.25(2013):4129-39, Cruz et al. Blood 122.17(2013):2965-73, NCT00586391, NCT01087294, NCT02456350, NCT00840853, NCT02659943, NCT02650999, NCT02640209, NCT01747486, NC T02546739, NCT02656147, NCT02772198, NCT00709033, NCT02081937, NCT00924326, NCT02735083, NCT02794246, NCT02746952, NCT015936 96, NCT02134262, NCT01853631, NCT02443831, NCT02277522, NCT02348216, NCT02614066, NCT02030834, NCT02624258, NCT02625480, NCT0 2030847, NCT02644655, NCT02349698, NCT02813837, NCT02050347, NCT01683279, NCT02529813, NCT02537977, NCT02799550, NCT02672501, NCT02819583, NCT02028455, NCT01840566, NCT01318317, NCT01864889, NCT02706405, NCT01475058, NCT01430390, NCT02146924, NCT0205 1257, NCT02431988, NCT01815749, NCT02153580, NCT01865617, NCT02208362, NCT02685670, NCT02535364, NCT02631044, NCT02728882, NC and anti-CD19 CARs described in NCT02735291, NCT01860937, NCT02822326, NCT02737085, NCT02465983, NCT02132624, NCT02782351, NCT01493453, NCT02652910, NCT02247609, NCT01029366, NCT01626495, NCT02721407, NCT01044069, NCT00422383, NCT01680991, NCT02794961, or NCT02456207, said references being incorporated herein by reference in their entireties.

[0517] In one embodiment, the antigen-binding domain comprises one, two, three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2, and HC CDR3, from an antibody described herein (e.g., an antibody described in WO2015 / 142675, US Patent Application Publication No. 2015 / 0283178 A1, US Patent Application Publication No. 2016 / 0046724 A1, US Patent Application Publication No. 2014 / 0322212 A1, US Patent Application Publication No. 2016 / 0068601 A1, US Patent Application Publication No. 2016 / 0051651 A1, US Patent Application Publication No. 2016 / 0096892 A1, US Patent Application Publication No. 2014 / 0322275 A1, or WO2015 / 090230, which are incorporated herein by reference). CDR3 and / or one, two, three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2, and LC CDR3, from an antibody described herein (e.g., an antibody described in WO2015 / 142675, U.S. Patent Application Publication No. 2015 / 0283178 A1, U.S. Patent Application Publication No. 2016 / 0046724 A1, U.S. Patent Application Publication No. 2014 / 0322212 A1, U.S. Patent Application Publication No. 2016 / 0068601 A1, U.S. Patent Application Publication No. 2016 / 0051651 A1, U.S. Patent Application Publication No. 2016 / 0096892 A1, U.S. Patent Application Publication No. 2014 / 0322275 A1, or WO2015 / 090230, which are incorporated herein by reference). In one embodiment, the antigen binding domain comprises the heavy chain variable region and / or the light chain variable region of an antibody listed above.

[0518] In embodiments, the antigen-binding domain is an antigen-binding domain described in WO2015 / 142675, US Patent Application Publication No. 2015 / 0283178 A1, US Patent Application Publication No. 2016 / 0046724 A1, US Patent Application Publication No. 2014 / 0322212 A1, US Patent Application Publication No. 2016 / 0068601 A1, US Patent Application Publication No. 2016 / 0051651 A1, US Patent Application Publication No. 2016 / 0096892 A1, US Patent Application Publication No. 2014 / 0322275 A1 or WO2015 / 090230, which are incorporated herein by reference.

[0519] In embodiments, the antigen binding domain targets BCMA and is described in U.S. Patent Application Publication No. 2016 / 0046724 A1.

[0520] In embodiments, the antigen binding domain targets CD19 and is described in US Patent Application Publication No. 2015 / 0283178 A1.

[0521] In embodiments, the antigen binding domain targets CD123 and is described in US Patent Application Publication No. 2014 / 0322212 A1, US Patent Application Publication N...

Claims

1. 1. A method of generating a population of immune effector cells (e.g., T cells) that can be engineered to express a chimeric antigen receptor (CAR), comprising: a) providing a frozen input sample containing immune effector cells; b) thawing the frozen input sample to generate a thawed sample; c) performing elutriation on the thawed sample to collect immune effector cells, thereby generating an output sample comprising immune effector cells suitable for expression of a CAR. A method comprising:

2. 10. The method of claim 1, wherein the frozen input sample is a plasma apheresis sample.

3. d) depleting CD19+ cells under flow conditions; e) performing density centrifugation using a medium containing iodixanol, e.g., a 60% iodixanol solution in water, e.g., an Optiprep medium, or a medium having a higher density than Ficoll (e.g., higher than 1.077 g / ml, e.g., about 1.32 g / ml); f) performing (e.g., on the lysed sample) a washing step with a buffer containing dextrose and / or sodium chloride, e.g., D5 medium (5% dextrose and 0.45% sodium chloride), e.g., a washing step performed using a CS5 (CellSaver 5+) device; and g) Performing positive selection of CD3 / CD28+ cells under flow conditions 3. The method of claim 1 or 2, further comprising one, two, three or all of:

4. The method of any one of claims 1 to 3, comprising adjusting the viscosity of the thawed sample, for example by adding an isotonic liquid, for example PBS, to the thawed sample.

5. wherein the elutriation is carried out using a flow rate of about 30-82 mL / min or 50-80 mL / min and / or the collection volume is about 250-1250 mL or 300-1000 mL for each fraction, e.g. the elutriation is carried out using a flow rate of about 30, 40, 50, 60, 70, 72, or 82 mL / min, e.g., about 70 or 72 mL / min; the elutriation is carried out using a harvest volume of about 250, 400, 500, 900, or 975 mL, e.g., about 400 or 975 mL; and / or The elutriation is carried out at about 2400 rpm. The method according to any one of claims 1 to 4.

6. The input sample is at least 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 35% or 40% monocytes; less than 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% T cells and / or At least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% B cells The method according to any one of claims 1 to 5, comprising:

7. The output sample is less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2% or 0.1% monocytes; at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% T cells; less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2% or 0.1% B cells; at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% CD4+CD25+ cells and / or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% CD8+CD25+ cells The method according to any one of claims 1 to 6, comprising:

8. 8. The method of any one of claims 1 to 7, having a T cell yield recovery of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% T cells.

9. The method of any one of claims 1 to 8, wherein the output sample is contacted with a nucleic acid encoding a CAR.

10. After contacting the output sample with a nucleic acid encoding a CAR, the output sample is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% CAR+CD4+ central memory cells; at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CAR+ cells and / or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% CAR+CD8+ central memory cells 10. The method of claim 9, comprising:

11. After contacting the output sample with a nucleic acid encoding a CAR, the output sample is produce less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1 pg IFN-gamma per transduced cell; and / or a cytotoxicity level (e.g., EC ５０ 11. The method of claim 9 or 10, wherein the rec.

12. 1. A method of generating a population of immune effector cells (e.g., T cells) that can be engineered to express a CAR, comprising: a) providing an input sample containing immune effector cells; b) performing density centrifugation using a medium comprising iodixanol, e.g., a 60% aqueous solution of iodixanol, e.g., an Optiprep medium, or a medium having a higher density than Ficoll (e.g., higher than 1.077 g / ml, e.g., about 1.32 g / ml), to generate an output sample comprising immune effector cells suitable for expression of the CAR. A method comprising:

13. c) depleting CD19+ cells under flow conditions; d) elutriation of said input sample, which may be a thawed input sample; e) performing a washing step (e.g., prior to density centrifugation) with a buffer containing dextrose and / or sodium chloride, e.g., D5 medium (5% dextrose and 0.45% sodium chloride), e.g., a washing step performed using a CS5 (CellSaver 5+) device; and f) Performing positive selection of CD3 / CD28+ cells under flow conditions 13. The method of claim 12, further comprising performing one, two, three or all of:

14. The method of claim 12 or 13, which does not include one or more of: using a solution containing glycol, such as a Ficoll solution; or performing a washing step with a buffer containing dextrose and / or sodium chloride, such as a D5 medium, for example, a washing step performed using a CS5 device; or performing a positive selection step.

15. The method of any one of claims 12 to 14, wherein the density centrifugation is performed using a cell separation device, such as a Sepax2 device.

16. The input sample is less than 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16% or 15% T cells; at least 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% monocytes and / or at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% B cells The method according to any one of claims 12 to 15, comprising:

17. The output sample is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% T cells; Less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, or 0.1% monocytes and / or Less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.01% B cells The method according to any one of claims 12 to 16, comprising:

18. 18. The method of any one of claims 12 to 17, having a T cell yield recovery of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% T cells.

19. 1. A method of generating a population of immune effector cells (e.g., T cells) that can be engineered to express a CAR, comprising: a) providing an input sample containing immune effector cells; b) generating an output sample comprising immune effector cells suitable for expression of a CAR by removing CD19+ cells from said input sample under flow conditions, e.g., using a flow-through device. A method comprising:

20. c) elutriation of said input sample, which may be a thawed input sample; d) a density centrifugation step using a medium containing iodixanol, e.g., a 60% aqueous solution of iodixanol, e.g., an Optiprep medium, or a medium having a higher density than Ficoll (e.g., higher than 1.077 g / ml, e.g., about 1.32 g / ml); e) performing a washing step (e.g., before removing CD19+ cells and / or after thawing the input sample) with a buffered dextrose and / or sodium chloride, e.g., D5 medium (5% dextrose and 0.45% sodium chloride), e.g., a washing step performed using a CS5 (CellSaver 5+) device; and f) Positive selection of CD3 / CD28+ cells under flow conditions 20. The method of claim 19, further comprising performing one, two, three or all of:

21. 21. The method of claim 19 or 20, which does not involve performing elutriation or density centrifugation.

22. The method of any one of claims 19 to 21, wherein the CD19+ cells comprise B cells, such as lymphoblasts.

23. The input sample is at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45% or 50% CD19+ cells; at least 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 35%, or 40% monocytes and / or Less than 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% T cells The method of any one of claims 19 to 37, comprising:

24. The output sample is less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.01% CD19+ cells; a percentage of CD19+ cells of less than 50%, 45%, 40%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 2%, 2% or 1% compared to the input sample; less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2% or 0.1% monocytes; at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% T cells and / or Less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.01% B cells The method of any one of claims 19 to 38, comprising:

25. The method of any one of claims 19 to 24, wherein CD19+ cells are removed by magnetic separation.

26. The magnetic separation contacting the cells with a separation reagent comprising a magnetic or paramagnetic member and a CD19 binding member; Flow cytometry and / or Use of a magnetic cell separation device, e.g., a CliniMACs device 26. The method of claim 25, comprising:

27. 26. The method of claim 25, wherein CD19+ cells are removed by FACS.

28. 28. The method of any one of claims 19 to 27, having a T cell yield recovery of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% T cells.

29. 29. The method of any one of claims 19 to 28, wherein the input sample (e.g. the input sample after washing) comprises at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% B cells.

30. 1. A method of generating a population of immune effector cells (e.g., T cells) that can be engineered to express a CAR, comprising: a) providing an input sample containing immune effector cells; b) generating an output sample comprising immune effector cells suitable for expression of a CAR by positively selecting CD3+ / CD28+ cells from said input sample under flow conditions. A method comprising:

31. c) depleting CD19+ cells, e.g., under flow conditions; d) elutriation of said input sample, which may be a thawed input sample; e) a density centrifugation step using a medium containing iodixanol, e.g., a 60% iodixanol solution in water, e.g., an Optiprep medium, or a medium having a higher density than Ficoll (e.g., higher than 1.077 g / ml, e.g., about 1.32 g / ml); f) performing a washing step (e.g., before removing CD19+ cells and / or after thawing the input sample) with a buffered dextrose and / or sodium chloride, e.g., D5 medium (5% dextrose and 0.45% sodium chloride), e.g., a washing step performed using a CS5 (CellSaver 5+) device; 31. The method of claim 30, further comprising performing one, two, three or all of:

32. performing elutriation, washing steps (if necessary) and density centrifugation (e.g., using Ficoll or OptiPrepO media) prior to positive selection; and / or Washing steps (if necessary) and density centrifugation (e.g., using Ficoll or OptiPrep media) should be performed before positive selection.

31. The method of claim 30, further comprising:

33. The method of any one of claims 30 to 32, which does not include performing elutriation.

34. 34. The method of any one of claims 30 to 33, comprising carrying out washing with a buffer comprising dextrose and / or sodium chloride, e.g. D5 1 / 2 NS, e.g. using a CS5+ device.

35. 35. The method of any one of claims 30 to 34, wherein said positive selection of CD3+ / CD28+ cells comprises contacting said input sample with a separation reagent comprising a magnetic or paramagnetic member and a CD3 and / or CD28 binding member.

36. 36. The method of any one of claims 30 to 35, wherein said positive selection of CD3+ / CD28+ cells comprises incubating said input sample with a separation reagent for about 10 to 90 minutes, about 10 to 60 minutes, about 10 to 45 minutes, about 12 to 90 minutes, about 12 to 60 minutes, about 12 to 45 minutes, about 15 to 90 minutes, about 15 to 60 minutes, about 15 to 45 minutes, such as about 30 minutes or about 20 minutes.

37. 37. The method of claim 36, wherein the separation reagent comprises beads coupled (e.g., covalently or non-covalently coupled) to anti-CD3 and / or anti-CD28 antibodies.

38. The output sample is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 15% or 20% T cells; less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, or 0.1% monocytes; at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% T cells, e.g., CD3+CD45+ T cells The method of any one of claims 30 to 37, comprising:

39. 39. The method of any one of claims 30 to 38, performed using a magnetic device, such as a Dynamag CTS, or other apparatus of magnetic elements, and / or using a ratio of magnetic separation members (e.g. beads) to T cells of about 3:

1.

40. 40. The method of any one of claims 30 to 39, wherein said positive selection of CD3+ / CD28+ cells comprises a separation or residence time of less than about 6, 5, 6, 3, 2 or 1 minute, or less than about 50, 40, 30, 20, 10, 5, 4, 3, 2 or 1 second.

41. 41. The method of any one of claims 30 to 40, comprising flowing a fluid comprising said immune effector cells and magnetic separation members in a closed system, e.g. a chamber or bag, in which magnetic separation occurs, optionally said flowing being carried out at a speed such that magnetic separation of said members (which may have bound immune effector cells) occurs.

42. at least one cell suspension module; at least one flow-through magnetic separation / de-beading module; at least one non-magnetic output module; at least one magnetic output module; Optionally, at least one magnetic component external to said magnetic separation / de-beading module, said magnetic component generating a magnetic force and / or a magnetic field gradient; Optionally, at least one buffer module; The method of any one of claims 30 to 41, carried out using a device comprising:

43. The magnetic separation / de-beading module a chamber defined by walls and having an x-direction, a y-direction, and a z-direction; an inlet and an outlet arranged at both ends of the chamber, for example, in the x-direction, the y-direction, or the z-direction; at least two magnets adjacent or proximate to a wall of the chamber and positioned to establish a zero gradient line between the inlet and the outlet within the chamber; 43. The method of claim 42, comprising:

44. The input sample is About 1×10 cells ７ pcs / ml, at least about 5%, 10%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% B cells, e.g., CD45+CD19+ B cells, and / or at least about 5%, 10%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% B cells, e.g., CD45-CD19+ B cells The method of any one of claims 30 to 43, comprising:

45. The output sample is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% B cells, e.g., CD45+CD19+ B cells; Less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% B cells, e.g., CD45-CD19+ B cells The method of any one of claims 30 to 44, comprising:

46. The input sample is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% monocytes; at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% tumor cells, e.g., lymphoblasts and / or Less than 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% immune effector cells, e.g., T cells 46. The method of any one of claims 1 to 45, comprising:

47. The output sample is less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2% or 0.1% monocytes; less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2% or 0.1% tumor cells; at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9% immune effector cells, e.g., T cells; a percentage of monocytes less than 50%, 45%, 40%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 2%, 2% or 1% compared to the input sample; a percentage of tumor cells less than 50%, 45%, 40%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 2%, 2% or 1% compared to the input sample; and / or a percentage of immune effector cells, e.g., T cells, of at least 50%, 45%, 40%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 2%, 2% or 1% compared to the input sample; 47. The method of any one of claims 1 to 46, comprising:

48. 48. The method of any one of claims 1 to 47, further comprising introducing, e.g., by transduction, a nucleic acid encoding a CAR into one or more of said immune effector cells in said output sample, wherein optionally the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, e.g., a signaling domain comprising a primary signaling domain and / or a costimulatory signaling domain, and optionally wherein said transduction results in a transduction efficiency of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

49. 49. The method of claim 48, further comprising a step of assaying the transduction efficiency and / or performing a washing step on the input sample with a buffer containing dextrose and / or sodium chloride, such as D5 medium, e.g., using a CS5+ instrument.

50. 50. The method of any one of claims 1 to 49, wherein the immune effector cells are human immune effector cells.

51. 51. The method of any one of claims 1 to 50, wherein the output sample comprises CD8+ T cells and / or CD4+ T cells.

52. The input sample may be selected from the group consisting of one or more acute leukemias, including but not limited to B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL); one or more chronic leukemias, including but not limited to chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL); one or more chronic leukemias, including but not limited to B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, 52. The method of any one of claims 1-51, wherein the tumor is from a patient having a cancer selected from the group consisting of an additional blood cancer or hematological condition, including a malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, preleukemia, atypical and / or atypical cancer, malignancy, precancerous condition or proliferative disorder, and any combination thereof.

53. assaying one or more cell surface markers on cells in the output sample, such as CD45, CD19, CD3, CD28, CD25, or CD14; and / or Stimulating the immune effector cells, e.g., prior to transduction, by contacting them with, e.g., a reagent that binds CD3 and / or CD28, e.g., a substrate (e.g., beads) coupled to CD3 and / or CD28 antibodies.

53. The method of any one of claims 1 to 52, further comprising:

54. 1. A method of generating a population of immune effector cells (e.g., T cells) that can be engineered to express a chimeric antigen receptor (CAR), comprising: i) providing an input sample containing immune effector cells, e.g., a frozen input sample or a fresh input sample; ii) optionally, if the input sample is a frozen input sample, thawing the frozen input sample to produce a thawed sample; iii) 1. performing elutriation on said input sample, which may be a thawed input sample; or 2. Performing a density centrifugation step using a medium containing iodixanol, e.g., a 60% iodixanol solution in water, e.g., an Optiprep medium, or a medium having a higher density than Ficoll (e.g., higher than 1.077 g / ml, e.g., about 1.32 g / ml). carrying out a concentration step comprising: iv) performing a selection step, wherein the selection is a positive selection, e.g., a positive selection of CD3 / CD28+ cells, or a negative selection, e.g., a negative selection of CD19+, CD25+, or CD14+ cells. thereby generating an output sample comprising immune effector cells that are suitable for expression of a CAR.

55. 1. A method of generating a population of immune effector cells (e.g., T cells) that can be engineered to express a chimeric antigen receptor (CAR), comprising: a) providing an input sample containing immune effector cells, e.g., a frozen input sample or a fresh input sample; b) performing a concentration step, which comprises performing elutriation or density centrifugation (e.g., using Ficoll or Optiprep media); c) carrying out the selection step under flow conditions, for example by using a flow-through device, wherein the selection is a positive selection, e.g. a positive selection of CD3 / CD28+ cells, or a negative selection, e.g. a negative selection of CD19+, CD25+ or CD14+ cells. thereby generating an output sample comprising immune effector cells that are suitable for expression of a CAR.

56. performing a washing step with a buffer containing dextrose and / or sodium chloride, e.g., D5 medium (5% dextrose and 0.45% sodium chloride), e.g., a washing step performed using a CS5 (CellSaver 5+) device; stimulating the output sample with an agent that stimulates the proliferation of immune effector cells, e.g., an agent that stimulates CD3 / TCR complex-associated signals, and / or a ligand that stimulates costimulatory molecules on the surface of T cells, e.g., an anti-CD3 antibody and an anti-CD28 antibody; and / or introducing a nucleic acid encoding a CAR, e.g., by transduction, transfection, or electroporation; 56. The method of claim 54 or 55, further comprising:

57. A reaction mixture obtainable using the method according to any one of claims 1 to 56.

58. 1. A reaction mixture comprising at least 80%, 85%, 90%, or 95% T cells and less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% monocytes, wherein the total number of cells in said reaction mixture totals 100%, and optionally, one or more of said T cells express a CAR.

59. A total of at least 1 x 10 ６ , 2 × 10 ６ , 5 x 10 ６ , 1 x 10 ７ , 2 × 10 ７ , 5 x 10 ７ , 1×10 ８ , 2 × 10 ８ , or 5 x 10 ８ individual cells, less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% B cells and / or Less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% cancer cells, e.g., lymphoblasts 59. The reaction mixture of claim 58, comprising:

60. 60. The reaction mixture of any one of claims 57 to 59, further comprising a nucleic acid encoding the CAR, e.g., a nucleic acid present within or outside a T cell.

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