Methods for Producing Cellular Compositions

Abstract

Continuous counter-flow centrifugation methods for producing cell compositions, including those for the production of T cell therapeutics, including cells expressing recombinant receptors such as chimeric antigen receptors (CARs), are provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 304,502, filed January 28, 2022, and U.S. Provisional Application No. 63 / 438,764, filed January 12, 2023, the contents of each of which are incorporated by reference in their entirety for all purposes.

[0002] Incorporation by reference to sequence listing This application is submitted with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042021040SeqList.xml, created on January 23, 2023, and is 35,102 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] The present disclosure relates, in some aspects, to continuous counterflow centrifugation methods for the processing and manufacturing of cell compositions, including engineered cell compositions. In some aspects, the continuous counterflow centrifugation methods enrich cell compositions for live cells, e.g., viable T cells. Resulting cell compositions, e.g., those comprising T cells genetically engineered to express an antigen receptor, are also provided. In some aspects, transduction of cell compositions, e.g., compositions comprising a population of lymphocytes, is carried out by continuous counterflow centrifugation. In some aspects, the present disclosure provides methods for transduction of cell populations, comprising continuous counterflow centrifugation of lymphocytes and viral vector particles, thereby producing a composition containing transduced cells. In some embodiments, the provided cells and compositions can be used in adoptive cell therapy methods. [Background technology]

[0004] Various strategies are available for enriching and / or transducing T cell populations in vitro, for example, for enriching and transducing antigen-specific T cells in vitro for use in adoptive cellular immunotherapy or cancer therapy. Improved strategies for enriching and / or transducing cell populations in vitro, including those for large-scale research, diagnostic, and therapeutic purposes, are needed. Methods and related compositions that address this need are provided. Summary of the Invention

[0005] Provided herein is a method for producing a composition of genetically engineered T cells, the method comprising: (a) applying a first centrifugal force and a first flow rate to a cell composition comprising T cells in a conical fluid enclosure of a centrifuge system to produce a fluidized bed of cells; (b) generating an input composition comprising the cell composition and viral vector particles by injecting viral vector particles into the conical fluid enclosure; and (c) applying a second centrifugal force and a second flow rate to the input composition, wherein the second centrifugal force and the second flow rate recirculate the viral vector particles in a flow path of the centrifuge system, thereby producing the genetically engineered T cells. In some embodiments, the centrifuge system is a continuous counter-flow centrifuge system.

[0006] In some embodiments, the introduction of viral vector particles is carried out during at least a portion of the application in (a). In some embodiments, the introduction of viral vector particles is carried out during at least a portion of the application in (c).

[0007] Also provided herein is a method for producing a composition of genetically engineered T cells, the method comprising: (a) applying a first centrifugal force and a first flow rate to an input composition comprising (i) viral vector particles and (ii) a cell composition comprising T cells in a conical fluid enclosure of a centrifuge system to produce a fluidized bed of cells; and (b) applying a second centrifugal force and a second flow rate to the input composition in the conical fluid enclosure, wherein the second centrifugal force and the second flow rate recirculate the viral vector particles in a flow path of the centrifuge system, thereby producing the genetically engineered T cells. In some embodiments, the centrifuge system is a continuous counterflow centrifuge system.

[0008] In some embodiments, the method includes generating an input composition by introducing a cellular composition and viral vector particles into a conical fluid enclosure. In some embodiments, the introduction of the cellular composition occurs before, during, and / or after the introduction of the viral vector particles. In some embodiments, the introduction of the cellular composition occurs before the introduction of the viral vector particles. In some embodiments, the introduction of the cellular composition occurs during the introduction of the viral vector particles. In some embodiments, the introduction of the cellular composition occurs after the introduction of the viral vector particles. In some embodiments, the introduction of the cellular composition and / or the introduction of the viral vector particles occurs before and / or during the application in (a). In some embodiments, the introduction of the cellular composition occurs before and / or during the application in (a). In some embodiments, the introduction of the cellular composition occurs before the application in (a). In some embodiments, the introduction of the cellular composition occurs during the application in (a). In some embodiments, the introduction of the cellular composition occurs before and during the application in (a). In some embodiments, the introduction of the cellular composition occurs before and during the application in (a). In some embodiments, the introduction of the cellular composition occurs before and during the application in (a). In some embodiments, the introduction of the viral vector particles occurs before the application in (a). In some embodiments, the introduction of viral vector particles is performed during the application in (a). In some embodiments, the introduction of viral vector particles is performed before and during the application in (a).

[0009] In some embodiments, the method includes applying a third centrifugal force and a third flow rate to the genetically engineered T cells in a conical fluid enclosure of the centrifuge system to produce an output composition comprising the genetically engineered T cells.

[0010] In some embodiments, the percentage of viable T cells in the output composition is greater than the percentage of viable T cells in the input composition. In some embodiments, the percentage of viable T cells in the output composition is at least about 5% greater, at least about 10% greater, at least about 15% greater, at least about 20% greater, or at least about 25% greater than the percentage of viable T cells in the input composition. In some embodiments, the percentage of viable T cells in the output composition is about 5% greater than the percentage of viable T cells in the input composition. In some embodiments, the percentage of viable T cells in the output composition is about 10% greater than the percentage of viable T cells in the input composition. In some embodiments, the percentage of viable T cells in the output composition is about 15% greater than the percentage of viable T cells in the input composition.

[0011] In some embodiments, at least or at least about 5%, at least or at least about 10%, at least or at least about 15%, at least or at least about 20%, at least or at least about 25%, or at least about 30% of the T cells in the output composition are transduced with viral vector particles. In some embodiments, at least about 20% of the T cells in the output composition are transduced with viral vector particles. In some embodiments, at least about 25% of the T cells in the output composition are transduced with viral vector particles. In some embodiments, at least about 30% of the T cells in the output composition are transduced with viral vector particles. In some embodiments, at least about 35% of the T cells in the output composition are transduced with viral vector particles. In some embodiments, at least about 40% of the T cells in the output composition are transduced with viral vector particles.

[0012] In some embodiments, the first centrifugal force is between about 2,000 G and about 4,000 G. In some embodiments, the first flow rate is between about 5 mL / min and about 15 mL / min. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition or input composition for about 15 seconds, about 30 seconds, about 45 seconds, or about 60 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition or input composition for about 30 seconds.

[0013] In some embodiments, the second centrifugal force is between about 500 G and about 1,500 G. In some embodiments, the second flow rate is between about 25 mL / min and about 30 mL / min. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for at least about 15 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, or at least about 90 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for about 30 minutes.

[0014] In some embodiments, the second centrifugal force is between about 500 G and about 1,500 G. In some embodiments, the second flow rate is between about 10 mL / min and about 100 mL / min. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for at least about 15 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, or at least about 90 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for about 30 minutes.

[0015] In some embodiments, the second centrifugal force is between about 100 G and about 2,000 G. In some embodiments, the second flow rate is between about 10 mL / min and about 100 mL / min. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for at least about 15 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, or at least about 90 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for about 30 minutes.

[0016] In some embodiments, the ratio of the first centrifugal force to the first flow rate is between about 200 and about 400. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 300. In some embodiments, the first centrifugal force is about 3,000 G and the first flow rate is about 10 mL / min. In some embodiments, the ratio of the second centrifugal force to the second flow rate is between about 20 and about 100, between about 25 and about 85, or between about 30 and about 65. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 35. In some embodiments, the second centrifugal force is about 1,000 G and the second flow rate is about 28.5 mL / min. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 62.5.

[0017] In some embodiments, the second centrifugal force is about 625 G and the second flow rate is about 10 mL / min.

[0018] In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 30. In some embodiments, the second centrifugal force is about 300 G and the second flow rate is about 10 mL / min.

[0019] In some embodiments, the third centrifugal force is between about 2,000 G and about 3,000 G. In some embodiments, the third flow rate is between about 15 mL / min and about 25 mL / min. In some embodiments, the ratio of the third centrifugal force to the third flow rate is between about 100 and about 150. In some embodiments, the ratio of the third centrifugal force to the third flow rate is about 125. In some embodiments, the third centrifugal force is about 2,500 G and the third flow rate is about 20 mL / min.

[0020] In some embodiments, prior to applying the third centrifugal force and the third flow rate, the method includes subjecting the engineered T cells to one or more washing steps. In some embodiments, the one or more washing steps include a medium change. In some embodiments, the one or more washing steps are performed at a second centrifugal force and a second flow rate.

[0021] In some embodiments, the method includes incubating the T cells of the cell composition under stimulatory conditions prior to application in (a). In some embodiments, the T cells of the cell composition are incubated under stimulatory conditions prior to application in (a). In some embodiments, the cell composition comprises activated cells. In some embodiments, the stimulatory conditions include the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules. In some embodiments, the stimulatory reagent includes (i) a primary agent that specifically binds to a member of the TCR complex, optionally, that specifically binds to CD3; and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule. In some embodiments, the primary agent specifically binds to CD3. In some embodiments, the secondary agent specifically binds to a costimulatory molecule selected from CD28, CD137 (4-1-BB), OX40, and ICOS. In some embodiments, the secondary agent specifically binds to CD28.

[0022] In some embodiments, at least one of the first and second agents comprises an antibody or antigen-binding fragment thereof. In some embodiments, the first agent and the second agent each comprise an antibody or antigen-binding fragment thereof. In some embodiments, the first agent is an anti-CD3 antibody or antigen-binding fragment thereof. In some embodiments, the second agent is an anti-CD28 antibody or antigen-binding fragment thereof. In some embodiments, the first agent is an anti-CD3 antibody or antigen-binding fragment thereof, and the second agent is an anti-CD28 antibody or antigen-binding fragment thereof. In some embodiments, the first agent and the second agent are each present on or attached to the surface of a solid support. In some embodiments, the solid support is or comprises beads. In some embodiments, the solid support is a paramagnetic bead having anti-CD3 and anti-CD28 antibodies attached to its surface. In some embodiments, the first agent and the second agent are reversibly bound to the surface of an oligomeric particle reagent comprising a plurality of streptavidin molecules or streptavidin mutein molecules. In some embodiments, the streptavidin or streptavidin mutein molecule binds to or is capable of binding to biotin, avidin, a biotin analog or mutein, an avidin analog or mutein, and / or a biologically active fragment thereof. In some embodiments, the first agent comprises an anti-CD3 Fab and the second agent comprises an anti-CD28 Fab.

[0023] In some embodiments, the stimulatory conditions include the presence of one or more recombinant cytokines, hi some embodiments, the stimulatory conditions include the presence of one or more of recombinant IL-2, IL-7, and IL-15.

[0024] In some embodiments, the method includes collecting the output composition. In some embodiments, the output composition is collected.

[0025] In some embodiments, the method includes incubating the engineered T cells of the collected output composition. In some embodiments, the engineered T cells of the collected output composition are incubated. In some embodiments, the engineered T cells of the collected output composition are incubated immediately after collection for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, or at least about 20 days.

[0026] In some embodiments, the percentage of viable T cells in the collected output composition about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days after collection is greater than the percentage of viable T cells in the input composition. In some embodiments, the percentage of viable T cells in the collected output composition about 1 day after collection is greater than the percentage of viable T cells in the input composition. In some embodiments, the percentage of viable T cells in the collected output composition about 5 days after collection is greater than the percentage of viable T cells in the input composition.

[0027] In some embodiments, the method includes cryopreserving the collected output composition and / or the collected output composition is cryopreserved to produce a cryopreserved composition. In some embodiments, the method includes cryopreserving the collected output composition to produce a cryopreserved composition. In some embodiments, the collected output composition is cryopreserved to produce a cryopreserved composition. In some embodiments, the cryopreserved composition is thawed to produce a thawed composition, and the percentage of viable T cells in the thawed composition is greater than the percentage of viable T cells in the input composition. In some embodiments, the percentage of viable T cells in the thawed composition is at least about 5% greater, at least about 10% greater, at least about 15% greater, at least about 20% greater, at least 25% greater, or at least about 30% greater than the percentage of viable T cells in the input composition.

[0028] In some embodiments, the input composition comprises T cells with an average diameter of greater than or about 6 micrometers, greater than or about 6 micrometers, greater than or about 6 micrometers, greater than or about 7 micrometers, greater than or about 8 micrometers, greater than or about 9 micrometers, greater than or about 10 micrometers, or greater than or about 11 micrometers.

[0029] In some embodiments, the input composition comprises a total of about 1×10 6 Approximately 2 x 10 T cells in total 9 In some embodiments, the input composition comprises between about 1 x 10 T cells in total. 8 T cells, total number at least approximately 2 x 10 8 T cells, total number at least approximately 3 x 10 8 T cells, total number at least approximately 4 x 108 T cells, total number at least approximately 5 x 10 8 T cells, total number at least approximately 6 x 10 8 T cells, total number at least 7 x 10 8 T cells, total number at least approximately 8 x 10 8 T cells, total number at least approximately 7 x 10 8 T cells, total number at least approximately 8 x 10 8 T cells, total number at least approximately 9 x 10 8 T cells, total number at least approximately 1 x 10 9 T cells, total number at least approximately 1.25 x 10 9 T cells, total number at least approximately 1.50 x 10 9 T cells, or a total number of at least approximately 1.75 x 10 9 In some embodiments, the volume of the input composition is between about 5 ml and about 20,000 ml, between about 10 ml and about 2,000 ml, between about 15 ml and about 1,000 ml, between about 20 ml and about 500 ml, between about 25 ml and about 100 ml, or between about 30 ml and about 60 ml. In some embodiments, the volume of the input composition is between about 30 ml and about 60 ml. In some embodiments, the volume of the input composition is about 30 ml. In some embodiments, the volume of the input composition is about 60 ml.

[0030] In some embodiments, the volume of the output composition is between about 2.5 mL and about 60 mL, between about 5 mL and about 40 mL, or between about 10 mL and about 20 mL. In some embodiments, the volume of the output composition is about 5 mL, about 10 mL, about 15 mL, about 20 mL, about 25 mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL, about 50 mL, about 55 mL, or about 60 mL.

[0031] In some embodiments, one or more steps of the method are automated. In some embodiments, one or more steps of the method are automated by a centrifuge system or a component thereof.

[0032] In some embodiments, the viral vector particle comprises a heterologous nucleic acid encoding a recombinant molecule. In some embodiments, the recombinant molecule is a chemokine, a chemokine receptor, a cytokine, a cytokine receptor, an antigen receptor (e.g., a CAR or TCR), or a combination thereof. In some embodiments, the recombinant molecule is an antibody receptor. In some embodiments, the antigen receptor is a transgenic T cell receptor (TCR). In some embodiments, the antigen receptor is a chimeric antigen receptor (CAR).

[0033] In some embodiments, the chimeric antigen receptor (CAR) comprises an extracellular antigen recognition domain that specifically binds to a target antigen and an intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain comprises the intracellular domain of the CD3-zeta (CD3ζ) chain. In some embodiments, the CAR further comprises a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the transmembrane domain comprises the transmembrane portion of CD28. In some embodiments, the intracellular signaling domain further comprises the intracellular signaling domain of a T cell costimulatory molecule. In some embodiments, the T cell costimulatory molecule is selected from the group consisting of CD28 and 4-1BB. In some embodiments, the CAR is recombinantly expressed. In some embodiments, the CAR is expressed from a vector. In some embodiments, the CAR is expressed from a gamma-retroviral vector or a lentiviral vector. In some embodiments, the CAR is expressed from a lentiviral vector.

[0034] In some embodiments, the viral vector particle is a retroviral vector particle. In some embodiments, the retroviral vector particle is a gamma-retroviral vector. In some embodiments, the retroviral vector particle is a lentiviral vector particle.

[0035] In some embodiments, the antigen receptor specifically binds to an antigen associated with a disease or condition. In some embodiments, the disease or condition is cancer, an autoimmune disease or disorder, and / or an infectious disease. In some embodiments, the disease or condition is cancer. In some embodiments, the T cells are primary T cells, optionally from a human subject.

[0036] Also provided herein is a method for enriching a cell composition for viable cells, the method comprising: (a) applying a first centrifugal force and a first flow rate to a cell composition comprising T cells in a conical fluid enclosure of a centrifuge system to produce a fluidized bed of cells, wherein the cell composition comprises viable and nonviable T cells; and (b) applying a second centrifugal force and a second flow rate to the cell composition, wherein the second centrifugal force and the second flow rate recirculate the cells of the cell composition through a flow path of the centrifuge system, thereby elutriating a waste fraction of the cell composition from the conical fluid enclosure, the waste fraction having a higher percentage of nonviable T cells than the percentage of nonviable T cells in the cell composition, and producing an enriched composition in the conical fluid enclosure, the waste fraction having a higher percentage of viable T cells than the percentage of viable T cells in the cell composition. In some embodiments, the centrifuge system is a continuous counterflow centrifuge system.

[0037] In some embodiments, (i) the first centrifugal force is between about 1,000 G and about 4,000 G, and (ii) the first flow rate is between about 5 mL / min and about 15 mL / min.

[0038] In some embodiments, the ratio of the first centrifugal force (in G) to the first flow rate (in mL / min) is between about 200 and about 500. In some embodiments, the ratio of the first centrifugal force (in G) to the first flow rate (in mL / min) is between about 200 and about 400.

[0039] In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 30 seconds.

[0040] In some embodiments, (ii) the second centrifugal force is between about 350 G and about 4,000 G, and (ii) the second flow rate is between about 5 mL / min and about 100 mL / min.

[0041] In some embodiments, the second centrifugal force is between about 350 G and about 3,000 G. In some embodiments, the second centrifugal force is between about 1,500 G and about 3,000 G. In some embodiments, the second centrifugal force is between about 500 G and about 1,500 G.

[0042] In some embodiments, the second flow rate is between about 65 mL / min and about 100 mL / min.

[0043] In some embodiments, the second flow rate is between about 10 mL / min and about 65 mL / min. In some embodiments, the second flow rate is between about 10 mL / min and about 35 mL / min.

[0044] In some embodiments, the second flow rate is between about 25 mL / min and about 30 mL / min.

[0045] In some embodiments, the ratio of the second centrifugal force (in G) to the second flow rate (in mL / min) is between about 30 and about 70. In some embodiments, the ratio of the second centrifugal force (in G) to the second flow rate (in mL / min) is between about 30 and about 40.

[0046] In some embodiments, the T cells have an average diameter of about 9 μm to about 20 μm. In some embodiments, the T cells have an average diameter of less than 9 μm. In some embodiments, the T cells have an average diameter of about 6 μm to about 9 μm.

[0047] In some embodiments, the T cells have an average diameter of about 9 μm to about 20 μm. In some embodiments, the T cells have an average diameter of about 9 μm to about 20 μm, and the ratio of the second centrifugal force (in G) to the second flow rate (in mL / min) is between about 30 and about 70.

[0048] In some embodiments, the T cells have an average diameter of about 10 μm to about 20 μm. In some embodiments, the T cells have an average diameter of about 12 μm to about 20 μm. In some embodiments, the T cells have an average diameter of about 14 μm to about 20 μm.

[0049] In some embodiments, the T cells have an average diameter of less than about 9 μm and the ratio of the second centrifugal force (in G) to the second flow rate (in mL / min) is between about 30 and about 40.

[0050] 1. A method of enriching a cell composition for viable cells, comprising: (a) applying a first centrifugal force and a first flow rate to a cell composition comprising T cells in a conical fluid enclosure of a centrifuge system to produce a fluidized bed of cells, the cell composition comprising viable and nonviable T cells, (i) the first centrifugal force is between about 2,000 G and about 4,000 G, and (ii) the first flow rate is between about 5 mL / min and about 15 mL / min; and (b) applying a second centrifugal force and a second flow rate to the cell composition, the second centrifugal force and the second flow rate being between about 5 mL / min and about 15 mL / min. Also provided herein is a method comprising recirculating cells of a cell composition through a flow path of a stem, thereby producing an enriched composition having a percentage of viable T cells that is higher than the percentage of viable T cells in the cell composition, wherein (i) the second centrifugal force is between about 1,500 G and about 3,000 G, (ii) the second flow rate is between about 65 mL / min and about 100 mL / min, and (iii) the ratio of the second centrifugal force (in G) to the second flow rate (in mL / min) is between about 30 and about 40, wherein the T cells have an average diameter of less than 9 μm.

[0051] In some embodiments, the method includes collecting the elutriated waste fraction. In some embodiments, the elutriated waste fraction is collected in a container in fluid communication with the wide end of the conical fluid enclosure.

[0052] Also provided herein is a method for enriching a cell composition for viable cells, the method comprising: (a) applying a first centrifugal force and a first flow rate to a cell composition comprising T cells in a conical fluid enclosure of a centrifuge system to produce a fluidized bed of cells, wherein the cell composition comprises viable and nonviable T cells; and (b) applying a second centrifugal force and a second flow rate to the cell composition, wherein the second centrifugal force and the second flow rate recirculate the cells of the cell composition through a flow path of the centrifuge system, thereby elutriating a waste fraction of the cell composition having a higher percentage of nonviable T cells than the percentage of nonviable T cells in the cell composition out of the conical fluid enclosure, and producing an enriched composition in the conical fluid enclosure having a higher percentage of viable T cells than the percentage of viable T cells in the cell composition, wherein the cell composition comprises T cells that were cryopreserved and thawed prior to application of the method. In some embodiments, the centrifuge system is a continuous counter-flow centrifuge system.

[0053] Also provided herein is a method for enriching a cell composition for viable cells, the method comprising: (a) applying a first centrifugal force and a first flow rate to a cell composition comprising T cells in a conical fluid enclosure of a centrifuge system to produce a fluidized bed of cells, wherein the cell composition comprises viable and nonviable T cells; (b) applying a second centrifugal force and a second flow rate to the cell composition, wherein the second centrifugal force and the second flow rate recirculate the cells of the cell composition through a flow path of the centrifuge system, thereby elutriating a waste fraction of the cell composition having a higher percentage of nonviable T cells than the percentage of nonviable T cells in the cell composition out of the conical fluid enclosure, to produce an enriched composition in the conical fluid enclosure having a higher percentage of viable T cells than the percentage of viable T cells in the cell composition; and (c) cryopreserving the cells of the enriched composition after steps (a) and (b), to create a cryopreserved cell composition. In some embodiments, the method includes (d) thawing the cryopreserved cell composition after step (c). In some embodiments, the centrifuge system is a continuous counterflow centrifuge system.

[0054] Also provided herein is a method for enriching a cell composition for viable cells, the method comprising: (a) applying a first centrifugal force and a first flow rate to a cell composition comprising T cells in a conical fluid enclosure of a centrifuge system to produce a fluidized bed of cells, wherein the cell composition comprises viable and nonviable T cells; and (b) applying a second centrifugal force and a second flow rate to the cell composition, wherein the second centrifugal force and the second flow rate recirculate the cells of the cell composition in a flow path of the centrifuge system, thereby producing an enriched composition having a percentage of viable T cells that is higher than the percentage of viable T cells in the cell composition. In some embodiments, the centrifuge system is a continuous counterflow centrifuge system.

[0055] In some embodiments, the method includes collecting the elutriated waste fraction. In some embodiments, the elutriated waste fraction is collected in a container in fluid communication with the wide end of the conical fluid enclosure.

[0056] In some embodiments, the second flow rate is about 30 mL / min or less, hi some embodiments, the second flow rate is between about 25 mL / min and about 30 mL / min.

[0057] 1. A method of enriching a cell composition for viable cells, comprising: (a) applying a first centrifugal force and a first flow rate to a cell composition comprising T cells in a conical fluid enclosure of a centrifuge system to produce a fluidized bed of cells, the cell composition comprising viable and nonviable T cells, (i) the first centrifugal force is between about 2,000 G and about 4,000 G, and (ii) the first flow rate is between about 5 mL / min and about 15 mL / min; and (b) applying a second centrifugal force and a first flow rate to a cell composition comprising viable and nonviable T cells in a conical fluid enclosure of a centrifuge system to produce a fluidized bed of cells, the cell composition comprising viable and nonviable T cells, Also provided herein are methods, comprising applying a second centrifugal force and a second flow rate to the cell composition, wherein the second centrifugal force and the second flow rate recirculate cells of the cell composition in a flow path of a centrifuge system, thereby producing an enriched composition having a percentage of viable T cells that is higher than the percentage of viable T cells in the cell composition, wherein (i) the second centrifugal force is between about 500 G and about 1,500 G, and (ii) the second flow rate is between about 25 mL / min and about 30 mL / min.

[0058] In some embodiments, the cell composition comprises T cells that have been cryopreserved and thawed prior to application of the method, hi some embodiments, the method further comprises thawing the cryopreserved cell composition to produce a cell composition comprising the T cells.

[0059] In some embodiments, the second centrifugal force is between about 700 G and about 1,300 G. In some embodiments, the second centrifugal force is between about 800 G and about 1,200 G. In some embodiments, the second centrifugal force is between about 900 G and about 1,100 G. In some embodiments, the second centrifugal force is about 1,000 G.

[0060] In some embodiments, the ratio of the second centrifugal force (in G) to the second flow rate (in mL / min) is between about 30 and about 40.

[0061] In some embodiments, (i) the first centrifugal force is between about 2,000 G and about 4,000 G, and (ii) the first flow rate is between about 5 mL / min and about 15 mL / min. In some embodiments, (i) the second centrifugal force is between about 500 G and about 1,500 G, and (ii) the second flow rate is between about 25 mL / min and about 30 mL / min. In some embodiments, the method includes introducing the cell composition into a centrifuge system. In some embodiments, the introducing is performed before and / or during at least a portion of the applying in (a). In some embodiments, the introducing is performed before the applying in (a). In some embodiments, the introducing is performed during at least a portion of the applying in (a). In some embodiments, the introducing is performed before and during at least a portion of the applying in (a).

[0062] In some embodiments, prior to applying in (a), the method includes producing engineered T cells by contacting T cells of the cell composition with viral particles. In some embodiments, the engineered T cells have been produced by contacting the T cells of the cell composition with viral vector particles. In some embodiments, the T cells of the cell composition are engineered T cells.

[0063] In some embodiments, the percentage of viable T cells in the enriched composition is at least about 10% greater, at least about 20% greater, at least about 30% greater, at least about 40% greater, at least 50% greater, or at least about 60% greater than the percentage of viable T cells in the cell composition.

[0064] In some embodiments, the method includes (c) applying a third centrifugal force and a third flow rate to the concentrated composition in a conical fluid enclosure of the centrifuge system to collect the concentrated composition. In some embodiments, (i) the third centrifugal force is between about 2,000 G and about 3,000 G, and (ii) the third flow rate is between about 15 mL / min and about 25 mL / min. In some embodiments, before applying the third centrifugal force and the third flow rate, the method includes subjecting the concentrated composition to one or more washing steps. In some embodiments, the one or more washing steps include medium exchange. In some embodiments, the one or more washing steps are performed at a second centrifugal force and a second flow rate.

[0065] In some embodiments, the percentage of viable T cells in the collected enriched composition about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days after collection is greater than the percentage of viable T cells in the cell composition. In some embodiments, the percentage of viable T cells in the collected enriched composition about 1 day after collection is greater than the percentage of viable T cells in the cell composition. In some embodiments, the percentage of viable T cells in the collected enriched composition about 5 days after collection is greater than the percentage of viable T cells in the cell composition.

[0066] In some embodiments, the method includes incubating the T cells of the cell composition under stimulatory conditions prior to application in (a). In some embodiments, the T cells of the cell composition are incubated under stimulatory conditions prior to application in (a). In some embodiments, the cell composition comprises activated cells. In some embodiments, the stimulatory conditions include the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules. In some embodiments, the stimulatory reagent includes (i) a primary agent that specifically binds to a member of the TCR complex, optionally, that specifically binds to CD3; and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule. In some embodiments, the primary agent specifically binds to CD3. In some embodiments, the secondary agent specifically binds to a costimulatory molecule selected from CD28, CD137 (4-1-BB), OX40, and ICOS. In some embodiments, the secondary agent specifically binds to CD28.

[0067] In some embodiments, at least one of the first and second agents comprises an antibody or antigen-binding fragment thereof. In some embodiments, the first agent and the second agent each comprise an antibody or antigen-binding fragment thereof. In some embodiments, the first agent is an anti-CD3 antibody or antigen-binding fragment thereof. In some embodiments, the second agent is an anti-CD28 antibody or antigen-binding fragment thereof. In some embodiments, the first agent is an anti-CD3 antibody or antigen-binding fragment thereof, and the second agent is an anti-CD28 antibody or antigen-binding fragment thereof. In some embodiments, the first agent and the second agent are each present on or attached to the surface of a solid support. In some embodiments, the solid support is or comprises beads. In some embodiments, the solid support is a paramagnetic bead having anti-CD3 and anti-CD28 antibodies attached to its surface. In some embodiments, the first agent and the second agent are reversibly bound to the surface of an oligomeric particle reagent comprising a plurality of streptavidin molecules or streptavidin mutein molecules. In some embodiments, the streptavidin or streptavidin mutein molecule binds to or is capable of binding to biotin, avidin, a biotin analog or mutein, an avidin analog or mutein, and / or a biologically active fragment thereof. In some embodiments, the first agent comprises an anti-CD3 Fab and the second agent comprises an anti-CD28 Fab.

[0068] In some embodiments, the stimulatory conditions include the presence of one or more recombinant cytokines. In some embodiments, the stimulatory conditions include the presence of one or more of recombinant IL-2, IL-7, and IL-15. In some embodiments, the cell composition comprises activated T cells.

[0069] In some embodiments, the method includes cryopreserving the collected enriched composition to produce a cryopreserved enriched composition. In some embodiments, the collected enriched composition is cryopreserved to produce a cryopreserved enriched composition. In some embodiments, the cryopreserved enriched composition is thawed to produce a thawed enriched composition, and the percentage of viable T cells in the thawed enriched composition is greater than the percentage of viable T cells in the cell composition. In some embodiments, the percentage of viable T cells in the thawed enriched composition is at least about 5% greater, at least about 10% greater, at least about 15% greater, at least about 20% greater, at least about 25% greater, or at least about 30% greater than the percentage of viable T cells in the cell composition.

[0070] In some embodiments, the method includes cryopreserving the cells of the concentrated composition after applying steps (a) and (b) to create a cryopreserved cell composition. In some embodiments, cryopreservation includes suspending the cells in a medium containing a cryoprotectant and freezing the cells. In some embodiments, the freezing is in a controlled-rate freezer.

[0071] In some embodiments, the method further comprises thawing the cryopreserved cell composition, hi some embodiments, the thawing occurs after the cryopreserved cell composition has been frozen for at least 3 days.

[0072] In some embodiments, one or more steps of the method are automated. In some embodiments, one or more steps of the method are automated by a centrifuge system or a component thereof.

[0073] In some embodiments, the viral vector particle comprises a heterologous nucleic acid encoding a recombinant molecule. In some embodiments, the recombinant molecule is a chemokine, a chemokine receptor, a cytokine, a cytokine receptor, an antigen receptor (e.g., a CAR or TCR), or a combination thereof. In some embodiments, the recombinant molecule is an antibody receptor. In some embodiments, the antigen receptor is a transgenic T cell receptor (TCR). In some embodiments, the antigen receptor is a chimeric antigen receptor (CAR).

[0074] In some embodiments, the chimeric antigen receptor (CAR) comprises an extracellular antigen recognition domain that specifically binds to a target antigen and an intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain comprises the intracellular domain of the CD3-zeta (CD3ζ) chain. In some embodiments, the CAR further comprises a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the transmembrane domain comprises the transmembrane portion of CD28. In some embodiments, the intracellular signaling domain further comprises the intracellular signaling domain of a T cell costimulatory molecule. In some embodiments, the T cell costimulatory molecule is selected from the group consisting of CD28 and 4-1BB. In some embodiments, the CAR is recombinantly expressed. In some embodiments, the CAR is expressed from a vector. In some embodiments, the CAR is expressed from a gamma-retroviral vector or a lentiviral vector. In some embodiments, the CAR is expressed from a lentiviral vector.

[0075] In some embodiments, the viral vector particle is a retroviral vector particle. In some embodiments, the retroviral vector particle is a gamma-retroviral vector. In some embodiments, the retroviral vector particle is a lentiviral vector particle.

[0076] In some embodiments, the antigen receptor specifically binds to an antigen associated with a disease or condition. In some embodiments, the disease or condition is cancer, an autoimmune disease or disorder, and / or an infectious disease. In some embodiments, the disease or condition is cancer. In some embodiments, the T cells are primary T cells, optionally from a human subject.

[0077] Provided herein are compositions comprising engineered T cells produced by any of the methods provided herein. In some embodiments, the composition comprises about 1.0 x 10 6 Approximately 2.0 × 10 CAR-expressing T cells 9 In some embodiments, the composition comprises a CAR-expressing T cell between CAR-expressing T cells. In some embodiments, the composition comprises a pharmaceutically acceptable carrier. In some embodiments, the composition comprises a cryoprotectant.

[0078] Provided herein are methods for treating a subject with a disease or disorder, comprising administering any of the compositions provided herein to the subject. Provided herein are uses of any of the compositions provided herein for treating a disease or disorder in a subject. Provided herein are any of the compositions provided herein for use in treating a disease or disorder in a subject. In some embodiments, the genetically engineered cells are engineered to express an antigen receptor that specifically binds to an antigen associated with the disease or disorder. [Brief explanation of the drawings]

[0079] [Figure 1] Figures 1A and 1B show the results of three different experiments [Runs 1-3] in which input cell compositions were enriched for viable cells by continuous counterflow centrifugation. Figure 1A shows the total viable and dead cell counts for the input composition and the product and waste fractions after continuous counterflow centrifugation. Figure 1B shows the increase in the percentage of viable cells after enrichment by continuous counterflow centrifugation. [Figure 2]2A and 2B show the transduction efficiency in the transduction product fraction and the percentage of viable cells in the various product fractions after transduction with the input composition and viral vector, respectively, performed by either the continuous counterflow centrifugation-based method or the scaled-down spinoculation method. As a control, cells were incubated with the viral vector by the scaled-down method but were not subjected to any centrifugation ("no spin"). [Figure 3] Figure 3 shows the percentage of viable cells in various product fractions after transduction with input compositions and viral vectors performed by either the continuous counterflow centrifugation-based method or the scaled-down spinoculation method. The input compositions subjected to the continuous counterflow centrifugation-based method of transduction had a total volume of either 30 mL or 60 mL. [Figure 4]Figure 4A shows the percentage of viable cells in input compositions and washed product fractions generated from 11 different human donors after either a continuous countercurrent elutriation (CCE)-based method of concentration and buffer exchange ("wash") or an alternative method of dead-end centrifugation and buffer exchange. Figure 4B shows the percentage of viable cells in the pre-wash product ("recovered product"; HP), washed product (WP), formulated drug product (FDP), and cryopreserved drug product (CDP) fractions using the CCE-based method or an alternative method. Figure 4C shows the viability of cells in the washed product (WP), formulated drug product (FDP), and cryopreserved drug product (CDP) fractions relative to the viability of the pre-wash product (HP). Figure 4D shows the total number of viable and nonviable cells in the pre-wash product (HP), washed product (WP), and washed waste fractions. Figure 4E shows the viability ratio of thawed cryopreserved drug product (CDP) fractions to pre-wash product (HP) fractions at various centrifugal forces. Figure 4F shows the final product yield using either the modified CCE-based method or the alternative method. Figure 4G shows the average viability of thawed cryopreserved drug product (CDP) fractions using the modified CCE-based method (Mod CCE) or the alternative (Alt) method at low, medium, or high cell inputs. Figures 4H, 4I, and 4J show the predicted improvement in cell viability of CDP produced by the modified CCE-based method compared to the alternative method, including between donors with pre-wash product (HP) fractions exhibiting lower viability (Figure 4H) and donors with lower viability using the alternative method (Figure 4J). Figure 4K shows the theoretical and measured concentrations of impurities in the inflow medium and the theoretical concentration of the same impurities in the effluent medium with increasing wash volume for the modified CCE-based method. [Figure 5]Figure 5A shows the number of total and nonviable cells in the input composition and the product and waste fractions after a continuous counterflow centrifugation method of concentration. The wash step of the concentration method was performed at either a centrifugal force to flow rate ratio (G / FR) of 62.5 or 33.3. Figure 5B shows the percentage of viable cells in the input composition and the washed product fraction after a wash step performed at either a centrifugal force to flow rate ratio (G / FR) of 62.5 or 33.3. Figure 5C shows the size of viable cells (V) and nonviable cells (NV) in the input composition for viability enhancement stimulated for 96 hours (upper panel) or expanded in culture for 15 days after the start of activation (lower panel). [Figure 6] Figure 6A shows the percentage of CD3+CAR+ cells after a continuous counterflow centrifugation-based method of transduction with viral vectors performed under different centrifugal forces and flow rates. For comparison, the percentage of CD3+CAR+ cells was assessed after a scaled-down spinoculation method of transduction (Figure 6B). Figure 6C shows the percentage of CD3+CAR+ cells after a continuous counterflow centrifugation-based method of transduction with viral vectors performed under conditions using a centrifugal force of 3000 G and a flow rate of 30 mL / min. Figure 6D shows the percentage of CD3+CAR+ cells after a continuous counterflow centrifugation-based method of transduction with viral vectors performed under conditions in which the centrifugal force and flow rate conditions were periodically changed throughout the incubation. For comparison, cells were transduced with viral vectors using a scaled-down spinoculation method (693 G). [Figure 7] Figure 7A shows the percentage of CD3+CAR+ cells among viable CD45+ cells after continuous counterflow centrifugation-based transduction of cells with either 11.1 μL or 3.33 μL of vector particles per million cells. Figure 7B shows the percentage of CD3+CAR+ cells after continuous counterflow centrifugation-based transduction of cells with 6 μL of vector particles per million cells. For comparison, cells were transduced with viral vectors using a scaled-down spinoculation method (693G). [Figure 8]Figure 8A shows flow cytometry analysis of CD3+CAR+ cells after a continuous counterflow centrifugation-based method of transduction, where the input composition had a total volume of either 30 mL or 60 mL. Results are quantified in Figure 8B. [Figure 9] Figure 9A shows flow cytometry analysis of CD3+CAR+ cells following a continuous counterflow centrifugation-based method of transduction in which the input composition included either 600 x 106 total cells or 200 x 106 total cells. As a control, a total of 15 x 106 cells were subjected to a scaled-down spinoculation method of transduction. The results are quantified in Figure 9B. [Figure 10] Figure 10A shows the percentage of CAR+ Jurkat cells after transduction with viral vector supernatant collected from an inverted centrifuge system during continuous counterflow centrifugation-based transduction of primary T cells. Figure 10B shows the percentage of CD3+ CAR+ primary T cells after 30 or 90 minutes of continuous counterflow centrifugation-based transduction in an inverted centrifuge system. [Figure 11] FIG. 11 shows viral vector concentration and distribution in a continuous counterflow centrifuge system during a continuous counterflow centrifugation-based method of transduction. DETAILED DESCRIPTION OF THE INVENTION

[0080] Provided herein are methods for transducing cells by centrifugation of target cells, such as those contained in a cell composition, and viral vector particles. In some embodiments, the centrifugation method is based on continuous countercurrent elutriation (CCE). In some embodiments, the method involves CCE-based centrifugation under conditions in which the target cells are repeatedly contacted by viral vector particles in a centrifuge enclosure (e.g., a conical enclosure), thereby producing a composition comprising a plurality of target cells transduced with the viral vector. In some embodiments, the centrifugation-based method of transduction also enriches for viable target cells, removes impurities, or both. Related compositions containing transduced cell populations, such as those produced by the methods according to the provided disclosure, are also provided.

[0081] Also provided are methods for transferring a viral vector into cells (e.g., T cells), including transducing cells, such as immune cells, e.g., T cells, by centrifugation. In some embodiments, the provided methods involve centrifuging cells, such as immune cells (e.g., T cells), and viral vector particles, such as lentiviral vectors. In some embodiments, the centrifugation method is or is based on a continuous countercurrent elutriation (CCE) method of centrifugation, for example, performed in an inverse centrifuge system (e.g., a countercurrent centrifugation system). In some embodiments, the centrifugation system is any of those described in WO2018 / 204992 and WO2019 / 140491, each of which is incorporated herein by reference in its entirety. In some embodiments, the provided methods involve centrifuging cells, such as immune cells (e.g., T cells), and viral vector particles, such as lentiviral vectors, in an inverse centrifugation system (e.g., a countercurrent centrifugation system), in which the viral vector particles are circulated through the system and repeatedly contacted with the cells.

[0082] Also provided are methods for concentrating viable cells in a cell composition, such as a transduced cell composition, by centrifugation. In some embodiments, the cell composition is a T cell composition, e.g., a transduced T cell composition. In some embodiments, the centrifugation method is or is based on a CCE method of centrifugation, e.g., performed in a reverse centrifuge system (e.g., a counterflow centrifugation system). Related compositions containing cell populations enriched in viable cells, such as those produced by methods according to the provided disclosure, are also provided.

[0083] Also provided are methods of removing beads (debeading) from a cell composition, e.g., a T cell composition, by centrifugation. In some embodiments, the centrifugation method is or is based on a CCE method of centrifugation, e.g., performed in a reverse centrifuge system (e.g., a counterflow centrifugation system). Related compositions containing debeaded cell populations, such as those produced by methods according to the provided disclosure, are also provided.

[0084] Countercurrent (also known as "countercurrent") centrifugation is a technique in which the settling velocity of particles in a fluid under centrifugal acceleration is counterbalanced by the flow of a support medium. The particles are thereby suspended as a fluidized bed. Countercurrent centrifugation is gentle enough to allow cells to be cultured and subsequently expanded in a fluidized bed state. Cell aggregation can also be reduced. Furthermore, this technique allows for the separation of dead cells from live cells due to differences in density and morphology. Delivery of a radially inward fluid flow to cells or particles under centrifugal acceleration creates a countercurrent situation. The centrifugal acceleration experienced by each particle is proportional to its radial distance from the center of rotation. To create a fluidized particle bed, the countercurrent flow rate must be adjusted for each radius of rotation. In any of the provided embodiments, the flow rate is provided by a peristaltic pump. This is generally achieved by shaping the chamber as a cone with its apex facing radially outward. A countercurrent fluid flow is input through the apex of the cone. The fluid flow enters the apex of the cone at a relatively high velocity, and the velocity of the fluid flow gradually decreases as it moves radially inward due to the increasing cross-sectional area of the cone. Chamber shapes and specific cone geometries for counterflow centrifugation have long been studied, as documented by RJ Sanderson, K.E. Bird, N.F. Palmer, and J. Brenman in the paper "Design Principles for a Counter Flow Centrifugation Cell Separation Chamber," Analytical Biochemistry 71, 615-622 (1976).

[0085] In some embodiments, the provided methods are used to engineer such cells with heterologous molecules encoding recombinant antigen receptors, such as chimeric antigen receptors (CARs) or transgenic T cell receptors (TCRs). The resulting engineered cells can be used in adoptive immunotherapy. In some such embodiments, the provided methods can be used to prepare immune cells, such as T cells, for adoptive therapy without using spinoculation-based methods of transduction. In some aspects, the provided methods can enrich cell compositions, cell and viral vector particle compositions, and / or transduced cell populations for viable cells. In some aspects, the enrichment of viable cells is more pronounced in compositions with low starting viability. In some aspects, the provided methods enrich for viable cells while maintaining a comparable final cell yield compared to alternative methods (e.g., dead-end centrifugation methods).

[0086] Generally, spinoculation-based methods can be used to transduce cells, such as immune cells (e.g., T cells), with viral vector particles. However, such methods have the drawback of producing a low number of live cells and / or a low number of viable transduced cells. This is because spinoculation-based transduction methods do not inherently enrich cell compositions for live cells. Therefore, existing spinoculation-based methods may not necessarily be able to transduce a large number of cells that remain viable for downstream use, such as for use in cell therapy.

[0087] Additionally, the clearance of impurities in the cell composition is improved compared to alternative methods and systems due to the continuous counter-flow centrifugation system, which dilutes impurities at a continuous exponential rate. In some embodiments, the impurities may include proteins, DNA, cellular debris, reagents used in manufacturing, or any combination thereof.

[0088] Additionally, alternative methods and systems for centrifugation may not necessarily be automated and / or integrated into other systems. In contrast, different steps of the methods provided herein can be automated using a continuous countercurrent centrifugation system (e.g., a CTS Rotea™ countercurrent centrifugation system). Furthermore, such systems are compatible with and can be integrated with other instruments and systems.

[0089] The provided methods are based on the observation that a continuous counter-flow elutriation-based method of centrifugation, such as that carried out in an inverted centrifuge system (e.g., a CTS Rotea™ counter-flow centrifugation system), can achieve efficient transduction of primary cells obtained from a subject and can enrich the cell composition for viable cells.

[0090] In some embodiments, the method is capable of achieving a specific number or percentage of viable cells. For example, in some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the cells in the composition produced by the method are viable. In some embodiments, the method produces an output composition in which the percentage of viable cells is greater than the input composition, e.g., 5%, 10%, 15%, or 20% greater than the input composition.

[0091] In some such embodiments, the number and percentage of live cells can be monitored and / or observed by measuring the expression level of the marker that indicates cell viability or lack thereof.Some well-known methods can be used to evaluate cell viability, such as detecting cell death markers, for example, chromatin condensation, Annexin V, caspase, propidium iodide (PI), and / or phosphatidylserine (PS), for example, for cell surface proteins, by flow cytometry or cell staining (for example, immunohistochemistry).In some examples, expression is measured by detecting the level of molecules produced by cells, for example, lactate dehydrogenase (LDH) or adenosine triphosphate (ATP).

[0092] In some embodiments, the methods can be used to transduce a population of T cells, wherein at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the T cells in the population are viable T cells, e.g., T cells that lack a cell death marker, e.g., a surface marker or secreted molecule or other marker.

[0093] In some embodiments, the methods produce an output composition in which at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the total cells (or of a particular target cell type, such as T cells) in the output composition are viable and / or do not express cell death markers, e.g., surface or secreted or other markers.

[0094] In some embodiments, the provided methods result in relatively high viability of immune cells, e.g., T cells. In some embodiments, at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells, such as T cells, in a cell population, e.g., an output composition, are viable in accordance with the provided methods.

[0095] In some embodiments, no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the T cells in the output composition are nonviable, express a surface marker selected from the group consisting of annexin V, caspase, propidium iodide (PI), and / or phosphatidylserine (PS), and / or secrete relatively high levels of LDH and / or relatively low levels of ATP. For example, in some aspects, the population of cells in the input and / or output compositions are at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% surface negative for annexin V, caspase, propidium iodide (PI), and / or phosphatidylserine (PS).

[0096] Methods and techniques for assessing cell viability are known in the art. Antibodies and reagents for the detection of such markers are well known in the art and readily available.

[0097] In some embodiments, the method is capable of achieving at least a particular transduction efficiency under certain conditions. For example, when the input composition comprises virus and cells in a ratio of 1 or about 1 infectious unit (IU) per cell to 10 IU per cell, e.g., at least 1 or about 1 infectious unit (IU) per cell, or at least 2 or about 2 IU per cell, at least 5 or about 5 IU per cell, or at least 10 or about 10 IU per cell, the method is capable of producing an output composition in which at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% of the cells in the composition produced by the method comprise a viral vector, e.g., are transduced with a viral vector.

[0098] In some such embodiments, the efficiency of cell transduction with a viral vector particle (e.g., a retroviral vector) can be monitored and / or observed by measuring the level of expression of a recombinant molecule or protein, e.g., a heterologous antigen receptor, encoded by a nucleic acid contained in the genome of the viral vector after transduction or other form of transfer of the vector into a cell, e.g., a viable host cell, e.g., a viable T cell, or a population of such cells. Several well-known methods for assessing the expression level of a recombinant molecule can be used, such as affinity-based methods, e.g., immunoaffinity-based methods, e.g., for cell surface proteins, detection by flow cytometry, etc. In some examples, expression is measured by detection of a transduction marker and / or a reporter construct. In some embodiments, a nucleic acid encoding a truncated surface protein is included in the vector and used as a marker of expression and / or its enhancement.

[0099] In some embodiments, the provided methods may include a cryopreservation step before or after incubation of the cells with the viral particles, e.g., transduction of the cells with the viral particles. In some embodiments, the provided methods include a cryopreservation step before or after incubation of the cells with the viral particles, e.g., transduction of the cells with the viral particles. In some embodiments, such a step may also include a process interruption step to allow for shipping of material, sampling of material, or a "pause" of treatment pending the patient's condition. In some embodiments, the improved cell viability achieved by the provided methods is maintained in cryopreserved drug products or in previously cryopreserved, thawed drug products. In some embodiments, the improved cell viability achieved by the provided methods is maintained in cryopreserved drug products. In some embodiments, the improved cell viability achieved by the provided methods is maintained in previously cryopreserved, thawed drug products.

[0100] In some embodiments, provided methods are performed such that one, more, or all steps in preparing cells for clinical use, e.g., in adoptive cell therapy, are performed without exposing the cells to non-sterile conditions and without the need to use a sterile room or cabinet. In some embodiments of such processes, the cells are enhanced for viability and / or transduced, all within a closed system. In some embodiments, the closed system is or includes the conical enclosure of an inverted centrifuge system. In some embodiments, the method, or any portion thereof, is performed in an automated manner. In some embodiments, the entire method is performed in an automated manner.

[0101] In some embodiments, the provided methods provide optimized or improved processes in which cells are transduced in the absence of spinoculation and / or are enhanced for cell viability, thus improving downstream processing and products. In some aspects, the provided methods can also produce transduced cells for administration to a subject that have a better or more desirable phenotype, e.g., more viable cells.

[0102] In some embodiments, such cells produced by the methods, or compositions comprising such cells, are administered to a subject to treat a disease or condition.

[0103] In some embodiments, the provided method comprises administering a suboptimal dose of cells to a subject. In some embodiments, the dose of cells is less than 1.5, 2, 3, 4, 5, or 10 times, or less than about 1.5, 2, 3, 4, 5, or 10 times, of the therapeutically effective dose of cells for treating a disease or condition. In such instances, expansion of cells to provide a therapeutically effective dose of cells can occur in vivo upon administration of the cells to a subject.

[0104] In some embodiments, the provided method comprises in vivo expansion of cells. In some aspects, in vivo expansion of cells can occur in vivo by transgene-specific activation or stimulation of administered cells. In some embodiments, antigen receptors (e.g., CARs) are stimulated, e.g., activated or expanded, upon recognition of antigens. In some embodiments, one or more agents are administered to a subject to boost, increase, or enhance the stimulation, activation, or expansion of cells in vivo in the subject.

[0105] In some embodiments, provided methods produce engineered T cells that exhibit increased persistence and / or expansion when administered to a subject. In some embodiments, engineered cells with increased persistence and / or expansion exhibit better efficacy in the subjects to which they are administered. In some embodiments, provided viral vectors and methods reduce variability in treatment outcomes in adoptive cell therapy methods, for example, by minimizing and / or reducing ex vivo manipulation of T cells prior to administration to a subject. In some embodiments, enriching for viable T cells prior to or in parallel with transduction improves the process of producing or preparing engineered T cells for adoptive cell therapy by reducing the time and reagents required for ex vivo manipulation. In some embodiments, selection of transduced or engineered cells is performed after genetic manipulation.

[0106] Also provided are among the embodiments genetically engineered cells produced by any of the methods provided herein, e.g., cells expressing recombinant antigen receptors (e.g., TCRs or CARs), as well as methods and uses of such genetically engineered cells for adoptive cell therapy.

[0107] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. To the extent that a definition set forth herein contradicts or otherwise conflicts with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0108] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0109] I. Methods for Transducing Cells Methods are provided for producing a composition of genetically engineered T cells in a centrifuge system. In some embodiments, the method includes transducing an input composition comprising a cell composition in the centrifuge system with viral vector particles. In some embodiments, the centrifuge system is a continuous countercurrent elutriation ("CCE") centrifuge system, also known as a reverse centrifuge system. In some embodiments, the cells produced are for use in cell therapy, such as primary cells prepared for autologous or allogeneic transplantation, e.g., in adoptive cell therapy. The method may include additional cell processing steps, e.g., cell washing, isolation, separation, harvesting, formulation, or other steps associated with producing the cell composition.

[0110] In some embodiments, the provided methods are used to introduce viral vector particles, such as retroviral vector particles, into cells, such as T cells. In some embodiments, the viral vector particles contain a heterologous polynucleotide encoding an antigen receptor, such as a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR). Thus, in some embodiments, the provided methods can be used to express a genetically engineered antigen receptor, such as a transgenic TCR or CAR, in cells, such as T cells. Cells transduced by such particles and methods, as well as compositions containing such cells, and methods for using them, are also provided.

[0111] In some embodiments, the methods include features that result in increased viability of transduced cells and / or certain populations or subpopulations thereof, desirable for use in adoptive cell therapy. In some embodiments, the provided methods enrich for viable cells, including viable genetically engineered cells. Thus, in some embodiments, compositions of cells resulting from the provided methods exhibit increased cell viability after transduction compared to before transduction. In some embodiments, the increased viability is observed immediately after transduction and / or is maintained over a period of time (e.g., hours or days) after transduction.

[0112] A. Input Composition In some embodiments, provided methods include genetically engineering T cells by contacting a cell composition comprising T cells with viral vector particles containing a heterologous polynucleotide encoding an antigen receptor (collectively, "input composition") in a centrifuge system. In some embodiments, the method includes applying a first centrifugal force and a first flow rate to the cell composition comprising the T cells in a conical fluid enclosure of the centrifuge system to produce a fluidized bed of cells. In some embodiments, the method includes loading the cell composition into the conical fluid enclosure prior to application of the first centrifugal force and the first flow rate.

[0113] In some embodiments, the centrifuge system includes a cannula within a conical fluid enclosure. In some embodiments, the cannula extends along the length of the conical fluid enclosure. In some embodiments, one end of the cannula is at or near the apex of the conical fluid enclosure. In some embodiments, the other end of the cannula is at or near the wide end of the conical fluid enclosure, for example, at or near the center of the wide end.

[0114] In some embodiments, the cell composition is introduced into the conical fluid enclosure via a cannula. In some embodiments, the cell composition is introduced into the conical fluid enclosure at or near the apex of the conical fluid enclosure. In some embodiments, the cell composition is introduced into the conical fluid enclosure by entering the end of the cannula at or near the wide end of the conical fluid enclosure and exiting the end of the cannula at or near the apex of the conical fluid enclosure.

[0115] In some embodiments, the method includes introducing viral vector particles into a conical fluid enclosure of a centrifuge system, and the resulting composition comprising the cellular composition and the viral particles is an input composition. In some embodiments, the introduction of the viral vector particles is performed simultaneously with or during the application of a first centrifugal force and a first flow rate.

[0116] In some embodiments, the viral vector particles are introduced into the conical fluid enclosure via a cannula. In some embodiments, the viral vector particles are introduced into the conical fluid enclosure at or near the apex of the conical fluid enclosure. In some embodiments, the viral vector particles are introduced into the conical fluid enclosure by entering the end of the cannula at or near the wide end of the conical fluid enclosure and exiting the end of the cannula at or near the apex of the conical fluid enclosure.

[0117] In some embodiments, the method includes applying a second centrifugal force and a second flow rate to the input composition. In some embodiments, the second centrifugal force and the second flow rate recirculate the viral vector particles through the flow path of the centrifuge system. Thus, in some embodiments, the viral vector particles repeatedly contact the fluidized bed of cells while recirculating through the centrifuge system. In some embodiments, the method produces genetically engineered T cells that express an antigen receptor.

[0118] In some embodiments, a waste fraction of the cell composition is elutriated out of the conical fluid enclosure by applying a second centrifugal force and a second flow rate, hi some embodiments, the elutriated waste fraction has a higher percentage of nonviable T cells than the percentage of nonviable T cells in the cell composition.

[0119] In some embodiments, the elutriated cells exit the conical fluid enclosure via an opening at the wide end of the conical fluid enclosure. In some embodiments, the opening at least partially surrounds the end of the cannula at or near the wide end of the conical fluid enclosure. In some embodiments, the opening surrounds the end of the cannula at or near the wide end of the conical fluid enclosure.

[0120] In some embodiments, the method includes collecting the elutriated waste fraction. In some embodiments, the elutriated waste fraction is collected in a container. In some embodiments, the container is in fluid communication with the wide end of the conical fluid enclosure.

[0121] In some embodiments, applying a second centrifugal force and a second flow rate produces an enriched composition having a percentage of viable T cells that is higher than the percentage of viable T cells in the cell composition within the conical fluid enclosure.

[0122] In some embodiments, the introduction of the viral vector particles is performed simultaneously with or during the application of the second centrifugal force and the second flow rate. In some embodiments, the viral vector particles are present in the centrifuge system during at least a portion of the application of the second centrifugal force and the second flow rate.

[0123] In some embodiments, the method includes: (a) applying a first centrifugal force and a first flow rate to a cell composition comprising T cells in a conical fluid enclosure of a centrifuge system to produce a fluidized bed of cells; (b) generating an input composition comprising the cell composition and the viral vector particles by injecting viral vector particles containing a heterologous polynucleotide encoding an antigen receptor into the conical fluid enclosure; and (c) applying a second centrifugal force and a second flow rate to the input composition, wherein the second centrifugal force and the second flow rate recirculate the viral vector particles in the flow path of the centrifuge system, such that the viral vector particles repeatedly contact the fluidized bed of cells, thereby generating the genetically engineered T cells.

[0124] In some embodiments, the method includes applying a first centrifugal force and a first flow rate to an input composition including viral vector particles containing a heterologous polynucleotide encoding an antigen receptor and a cell composition including T cells. In some embodiments, the viral vector particles and the cell composition are both present in a conical fluid enclosure of a centrifuge system when the first centrifugal force and the first flow rate are applied. In some embodiments, the application of the first centrifugal force and the first flow rate produces a fluidized bed of cells. In some embodiments, the method includes loading the cell composition and the viral vector particles into the conical fluid enclosure before application of the first centrifugal force and the first flow rate. In some embodiments, loading of the cell composition occurs before, during, and / or after loading of the viral vector particles. In some embodiments, the method includes loading the input composition into the conical fluid enclosure before application of the first centrifugal force and the first flow rate. In some embodiments, the cell composition and / or the viral vector particles are loaded into the conical fluid enclosure before or during application of the first centrifugal force and the first flow rate.

[0125] In some embodiments, the first centrifugal force is between about 625 G and about 3,000 G. In some embodiments, the first centrifugal force is between about 625 G and about 3,000 G, between about 625 G and about 2,500 G, between about 625 G and about 2,000 G, between about 625 G and about 1,500 G, between about 625 G and about 1,000 G, between about 1,000 G and about 3,000 G, between about 1,000 G and about 2,500 G, between about 1,000 G and about 2,500 G, between about 1,000 G and about 3,0 ... to about 2,000 G, between about 1,000 G and about 1,500 G, between about 1,500 G and about 3,000 G, between about 1,500 G and about 2,500 G, between about 1,500 G and about 2,000 G, between about 2,000 G and about 3,000 G, between about 2,000 G and about 2,500 G, or between about 2,500 G and about 3,000 G. In some embodiments, the first centrifugal force is between about 2,000 G and about 4,000 G.

[0126] In some embodiments, the first centrifugal force is between about 1,000 G and about 5,000 G, between about 1,500 G and about 4,500 G, between about 2,000 G and about 4,000 G, between about 1,500 G and about 3,500 G, or between about 2,000 G and about 3,000 G. In some embodiments, the first centrifugal force is about 1,000 G. In some embodiments, the first centrifugal force is about 1,500 G. In some embodiments, the first centrifugal force is about 2,000 G. In some embodiments, the first centrifugal force is about 2,500 G. In some embodiments, the first centrifugal force is about 3,000 G. In some embodiments, the first centrifugal force is about 3,500 G. In some embodiments, the first centrifugal force is about 4,000 G. In some embodiments, the first centrifugal force is about 4,500 G. In some embodiments, the first centrifugal force is about 5,000 G.

[0127] In some embodiments, the first flow rate is radially inward. In some embodiments, the first flow rate is directed away from the apex of the conical fluid enclosure. In some embodiments, the first centrifugal force is counteracted by the first flow rate. In some embodiments, the first flow rate is a counterflow rate.

[0128] In some embodiments, the first flow rate is influenced by the flow of medium through the cannula. In some embodiments, the flow of medium through the cannula is from the wide end to the tip of the conical fluid enclosure. In some embodiments, the medium exits the cannula and enters the conical fluid enclosure at its tip.

[0129] In some embodiments, the first flow rate is between about 1 mL / min and about 20 mL / min, between about 3 mL / min and about 18 mL / min, between about 5 mL / min and about 15 mL / min, or between about 8 mL / min and about 12 mL / min. In some embodiments, the first flow rate is about 1 mL / min. In some embodiments, the first flow rate is about 3 mL / min. In some embodiments, the first flow rate is about 5 mL / min. In some embodiments, the first flow rate is about 8 mL / min. In some embodiments, the first flow rate is about 9 mL / min. In some embodiments, the first flow rate is about 10 mL / min. In some embodiments, the first flow rate is about 11 mL / min. In some embodiments, the first flow rate is about 12 mL / min. In some embodiments, the first flow rate is about 15 mL / min. In some embodiments, the first flow rate is about 18 mL / min. In some embodiments, the first flow rate is about 20 mL / min.

[0130] In some embodiments, the first flow rate is between about 1 mL / min and about 50 mL / min, between about 1 mL / min and about 40 mL / min, between about 1 mL / min and about 30 mL / min, between about 1 mL / min and about 20 mL / min, between about 1 mL / min and about 10 mL / min, between about 10 mL / min and about 50 mL / min, between about 10 mL / min and about 40 mL / min, between about 10 mL / min and about 30 mL / min, between about 10 mL / min and about 20 mL / min, between about 20 mL / min and about 50 mL / min, between about 20 mL / min and about 40 mL / min, between about 20 mL / min and about 30 mL / min, between about 30 mL / min and about 50 mL / min, between about 30 mL / min and about 40 mL / min, or between about 40 mL / min and about 50 mL / min. In some embodiments, the first flow rate is between about 10 mL / min and about 40 mL / min.

[0131] In some embodiments, the first flow rate is between about 10 mL / min and about 50 mL / min, between about 20 mL / min and about 50 mL / min, between about 30 mL / min and about 50 mL / min, or between about 35 mL / min and about 45 mL / min, hi some embodiments, the first flow rate is about 40 mL / min.

[0132] In some embodiments, the first flow rate is between about 5 mL / min and about 15 mL / min. In some embodiments, (i) the first centrifugal force is between about 2,000 G and about 4,000 G, and (ii) the first flow rate is between about 5 mL / min and about 15 mL / min.

[0133] In some embodiments, the ratio of the first centrifugal force to the first flow rate is between about 200 and about 400. Ratios of centrifugal force to flow rate herein are the ratio of centrifugal force in G to flow rate in mL / min, unless otherwise indicated.

[0134] In some embodiments, the ratio of the first centrifugal force to the first flow rate is between about 200 and about 400, between about 225 and about 375, between about 250 and about 350, or between about 275 and about 325. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 200. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 200. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 225. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 250. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 275. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 300. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 325. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 350. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 375. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 400. In some embodiments, the first centrifugal force is about 3,000 G and the first flow rate is about 10 mL / min.

[0135] In some embodiments, the ratio of the second centrifugal force to the first flow rate is between about 20 and 200, between about 20 and 180, between about 20 and 160, between about 20 and 140, between about 20 and 120, between about 20 and 100, between about 20 and 80, between about 20 and 60, between about 20 and 40, between about 40 and 200, between about 40 and 180, between about 40 and 160, between about 40 and 140, between about 40 and 120, between about 40 and 100, between about 40 and 80, between about 40 and 60, between about 60 and 200, between about 60 and 180, between about 60 and 160, between about 60 and 140, between about 60 and 120, between about 60 and 60 between about 60 and 80, between about 80 and 200, between about 80 and 180, between about 80 and 160, between about 80 and 140, between about 80 and 120, between about 80 and 100, between about 100 and 200, between about 100 and 180, between about 100 and 160, between about 100 and 140, between about 100 and 120, between about 120 and 200, between about 120 and 180, between about 120 and 160, between about 120 and 140, between about 140 and 200, between about 140 and 180, between about 140 and 160, between about 160 and 200, between about 160 and 180, or between about 180 and 200. In some embodiments, the ratio of the first centrifugal force to the first flow rate is between about 40 and 200, between about 40 and 180, between about 40 and 160, between about 40 and 140, between about 40 and 120, between about 40 and 100, between about 40 and 80, or between about 50 and 60. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 62.5.

[0136] In some embodiments, the ratio of the first centrifugal force to the first flow rate is between about 62.5 and about 300, between about 62.5 and about 250, between about 62.5 and about 200, between about 62.5 and about 150, or between about 62.5 and about 100. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 62.5.

[0137] In some embodiments, the ratio of the first centrifugal force to the first flow rate is between about 62.5 and about 300, between about 100 and about 300, between about 150 and about 300, between about 200 and about 300, and between about 250 and about 300.

[0138] In some embodiments, the first centrifugal force is about 2,500 G and the first flow rate is about 40 mL / min.

[0139] In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 15 seconds, about 30 seconds, about 45 seconds, about 60 seconds, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, or about 15 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 15 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 30 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 45 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 60 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 1 minute to about 15 minutes, about 3 minutes to about 12 minutes, or about 5 minutes to about 10 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 1 minute. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 2 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 3 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 4 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 5 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 6 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 7 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 8 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 9 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 10 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 11 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 12 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 13 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 14 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 15 minutes.In some embodiments, a first centrifugal force and a first flow rate are applied to the cell composition at least until a fluidized bed is established.

[0140] In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 15 seconds, at least about 30 seconds, at least about 45 seconds, at least about 60 seconds, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 10 minutes, or at least about 15 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 15 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 20 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 25 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 30 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 45 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 60 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 2 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 3 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 4 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 5 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 10 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 15 minutes.

[0141] In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for between 15 seconds and 60 seconds, or for about 15 seconds and about 60 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for between 25 seconds and 60 seconds, or for about 25 seconds and about 60 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for between 30 seconds and 60 seconds, or for about 30 seconds and about 60 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for between 15 seconds and 2 minutes, or for about 15 seconds and about 2 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for between 25 seconds and 2 minutes, or for about 25 seconds and about 2 minutes.

[0142] In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition until a predetermined number of cells are loaded into the conical fluid enclosure. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition until the predetermined number of cells become part of a fluidized bed of cells. In some embodiments, the predetermined number of cells is a predetermined number of T cells. In some embodiments, the predetermined number of cells, e.g., T cells, is between about 10 million and 100 million, between about 10 million and 90 million, between about 10 million and 80 million, between about 10 million and 70 million, between about 10 million and 60 million, between about 10 million and 50 million, between about 10 million and 40 million, between about 10 million and 30 million, between about 10 million and 20 million, between about 20 million and 100 million, or about 20 million. Between 0 and 90 million, between about 20 and 80 million, between about 20 and 70 million, between about 20 and 60 million, between about 20 and 50 million, between about 20 and 40 million, between about 20 and 30 million, between about 30 and 100 million, between about 30 and 90 million, between about 30 and 80 million, between about 30 and 70 million, between about 30 and 60 million Between about 30 million and 50 million, Between about 30 million and 40 million, Between about 40 million and 100 million, Between about 40 million and 90 million, Between about 40 million and 80 million, Between about 40 million and 70 million, Between about 40 million and 60 million, Between about 40 million and 50 million, Between about 50 million and 100 million, Between about 50 million and 90 million, Between about 50 million and 80 million, Between about 50 million and 70 million In some embodiments, the predetermined number of cells, e.g., T cells, is about 50 million, between about 50 million and 60 million, between about 60 million and 100 million, between about 60 million and 90 million, between about 60 million and 80 million, between about 60 million and 70 million, between about 70 million and 100 million, between about 70 million and 90 million, between about 70 million and 80 million, between about 80 million and 100 million, between about 80 million and 90 million, or between about 90 million and 100 million cells, e.g., T cells. In some embodiments, the predetermined number of cells, e.g., T cells, is about 50 million cells, e.g., T cells.

[0143] In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 15 seconds, about 30 seconds, about 45 seconds, about 60 seconds, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, or about 15 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 15 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 30 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 45 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 60 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 1 minute to about 15 minutes, about 3 minutes to about 12 minutes, or about 5 minutes to about 10 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 1 minute. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 2 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 3 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 4 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 5 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 6 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 7 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 8 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 9 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 10 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 11 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 12 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 13 minutes.In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 14 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for about 15 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition at least until a fluidized bed is established.

[0144] In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for at least about 15 seconds, at least about 30 seconds, at least about 45 seconds, at least about 60 seconds, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 10 minutes, or at least about 15 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for at least about 15 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for at least about 20 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for at least about 25 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for at least about 30 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for at least about 45 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for at least about 60 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for at least about 2 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for at least about 3 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for at least about 4 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for at least about 5 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for at least about 10 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for at least about 15 minutes.

[0145] In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for between 15 seconds and 60 seconds, or for about 15 seconds and about 60 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for between 25 seconds and 60 seconds, or for about 25 seconds and about 60 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for between 30 seconds and 60 seconds, or for about 30 seconds and about 60 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for between 15 seconds and 2 minutes, or for about 15 seconds and about 2 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the input composition for between 25 seconds and 2 minutes, or for about 25 seconds and about 2 minutes.

[0146] In some embodiments, a first centrifugal force and a first flow rate are applied to the input composition until a predetermined number of cells are loaded into the conical fluid enclosure. In some embodiments, the first centrifugal force and a first flow rate are applied to the input composition until the predetermined number of cells become part of the fluidized bed of cells. In some embodiments, the predetermined number of cells is a predetermined number of T cells. In some embodiments, the predetermined number of cells, e.g., T cells, is between about 10 million and 100 million, between about 10 million and 90 million, between about 10 million and 80 million, between about 10 million and 70 million, between about 10 million and 60 million, between about 10 million and 50 million, between about 10 million and 40 million, between about 10 million and 30 million, between about 10 million and 20 million, between about 20 million and 100 million, or about 20 million. Between 0 and 90 million, between about 20 and 80 million, between about 20 and 70 million, between about 20 and 60 million, between about 20 and 50 million, between about 20 and 40 million, between about 20 and 30 million, between about 30 and 100 million, between about 30 and 90 million, between about 30 and 80 million, between about 30 and 70 million, between about 30 and 60 million Between about 30 million and 50 million, Between about 30 million and 40 million, Between about 40 million and 100 million, Between about 40 million and 90 million, Between about 40 million and 80 million, Between about 40 million and 70 million, Between about 40 million and 60 million, Between about 40 million and 50 million, Between about 50 million and 100 million, Between about 50 million and 90 million, Between about 50 million and 80 million, Between about 50 million and 70 million In some embodiments, the predetermined number of cells, e.g., T cells, is about 50 million, between about 50 million and 60 million, between about 60 million and 100 million, between about 60 million and 90 million, between about 60 million and 80 million, between about 60 million and 70 million, between about 70 million and 100 million, between about 70 million and 90 million, between about 70 million and 80 million, between about 80 million and 100 million, between about 80 million and 90 million, or between about 90 million and 100 million cells, e.g., T cells. In some embodiments, the predetermined number of cells, e.g., T cells, is about 50 million cells, e.g., T cells.

[0147] In some embodiments, the second flow rate is radially inward. In some embodiments, the second flow rate is directed away from the apex of the conical fluid enclosure. In some embodiments, the second centrifugal force is counteracted by the second flow rate. In some embodiments, the second flow rate is an opposing flow rate.

[0148] In some embodiments, the second flow rate is influenced by the flow of medium through the cannula. In some embodiments, the flow of medium through the cannula is from the wide end to the tip of the conical fluid enclosure. In some embodiments, the medium exits the cannula and enters the conical fluid enclosure at its tip.

[0149] In other embodiments, the second flow rate is directed radially outward. In some embodiments, the second flow rate is directed toward the apex of the conical fluid enclosure. In some embodiments, the second centrifugal force and the second flow rate are in the same or substantially the same direction.

[0150] In some embodiments, the second flow rate is influenced by the flow of medium through the conical fluid enclosure. In some embodiments, the flow of medium through the conical fluid enclosure is from the wide end to the tip of the conical fluid enclosure. In some embodiments, the medium exits the tip of the conical fluid enclosure and enters the cannula.

[0151] In some embodiments, the second centrifugal force is between about 100 G and about 2,000 G, between about 200 G and about 1,800 G, between about 500 G and about 1,500 G, or between about 750 G and about 1,250 G. In some embodiments, the second centrifugal force is about 250 G. In some embodiments, the second centrifugal force is about 500 G. In some embodiments, the second centrifugal force is about 600 G. In some embodiments, the second centrifugal force is about 700 G. In some embodiments, the second centrifugal force is about 800 G. In some embodiments, the second centrifugal force is about 900 G. In some embodiments, the second centrifugal force is about 1,000 G. In some embodiments, the second centrifugal force is about 1,100 G. In some embodiments, the second centrifugal force is about 1,200 G. In some embodiments, the second centrifugal force is about 1,300 G. In some embodiments, the second centrifugal force is about 1,400 G. In some embodiments, the second centrifugal force is about 1,500 G. In some embodiments, the second centrifugal force is about 1,300 G. In some embodiments, the second centrifugal force is about 1,750 G. In some embodiments, the second centrifugal force is about 2,000 G.

[0152] In some embodiments, the second centrifugal force is between about 100 G and about 4,000 G, between about 100 G and about 3,750 G, between about 100 G and about 3,500 G, between about 100 G and about 3,250 G, between about 100 G and about 3,000 G, between about 100 G and about 2,750 G, between about 100 G and about 2,500 G, between about 100 G and about 2,250 G, between about 100 G and about 2,000 G, between about 100 G and about 1,750 G, between about 100 G and about 1,500 G, between about 100 G and about 1,250 G, between about 100 G and about 1,000 G, between about 100 G and about 7 Between 50G, between about 100G and about 500G, between about 100G and about 250G, between about 250G and about 4,000G, between about 250G and about 3,750G, between about 250G and about 3,500G, between about 250G and about 3,250G, between about 250G and about 3,000G, between about 250G and about 2,750G, between about 250G and about 2,500G, between about 250G and about 2,250G, between about 250G and about 2,000G, between about 250G and about 1,750G, between about 250G and about 1,500G, between about 250G and about 1,250G, between about 250G and about 1 ,000G, between about 250G and about 750G, between about 250G and about 500G, between about 500G and about 4,000G, between about 500G and about 3,750G, between about 500G and about 3,500G, between about 500G and about 3,250G, between about 500G and about 3,000G, between about 500G and about 2,750G, between about 500G and about 2,500G, between about 500G and about 2,250G, between about 500G and about 2,000G, between about 500G and about 1,750G, between about 500G and about 1,500G, between about 500G and about 1,250G, between about 500G and Between about 1,000G, between about 500G and about 750G, between about 750G and about 4,000G, between about 750G and about 3,750G, between about 750G and about 3,500G, between about 750G and about 3,250G, between about 750G and about 3,000G, between about 750G and about 2,750G, between about 750G and about 2,500G, between about 750G and about 2,250G, between about 750G and about 2,000G, between about 750G and about 1,750G, between about 750G and about 1,500G, between about 750G and about 1,250G, between about 750G and about 1,000G, between about 1,Between about 1,000G and about 4,000G, between about 1,000G and about 3,750G, between about 1,000G and about 3,500G, between about 1,000G and about 3,250G, between about 1,000G and about 3,000G, between about 1,000G and about 2,750G, between about 1,000G and about 2,500G, between about 1,000G and about 2,250G, between about 1,000G and about 2,000G, between about 1,000G and about 1,750G, between about 1,000G and about 1,500G, between about 1,000G and about 1,250G, between about 1,250G and about 4,000G, between about 1,25 Between 0G and approximately 3,750G, between approximately 1,250G and approximately 3,500G, between approximately 1,250G and approximately 3,250G, between approximately 1,250G and approximately 3,000G, between approximately 1,250G and approximately 2,750G, between approximately 1,250G and approximately 2,500G, between approximately 1,250G and approximately 2,250G, between approximately 1,250G and approximately 2,000G, between approximately 1,250G and approximately 1,750G, between approximately 1,250G and approximately 1,500G, between approximately 1,500G and approximately 4,000G, between approximately 1,500G and approximately 3,750G, between approximately 1,500G and approximately 3,500G, approximately 1,500G between about 1,500G and about 3,000G, between about 1,500G and about 2,750G, between about 1,500G and about 2,500G, between about 1,500G and about 2,250G, between about 1,500G and about 2,000G, between about 1,500G and about 1,750G, between about 1,750G and about 4,000G, between about 1,750G and about 3,750G, between about 1,750G and about 3,500G, between about 1,750G and about 3,250G, between about 1,750G and about 3,000G, between about 1,750G and about 2,750G, between about 1,750G and Between about 2,500G, between about 1,750G and about 2,250G, between about 1,750G and about 2,000G, between about 2,000G and about 4,000G, between about 2,000G and about 3,750G, between about 2,000G and about 3,500G, between about 2,000G and about 3,250G, between about 2,000G and about 3,000G, between about 2,000G and about 2,750G, between about 2,000G and about 2,500G, between about 2,000G and about 2,250G, between about 2,250G and about 4,000G, between about 2,250G and about 3,750G, between about 2,250G and about 3,Between 500G, between about 2,250G and about 3,250G, between about 2,250G and about 3,000G, between about 2,250G and about 2,750G, between about 2,250G and about 2,500G, between about 2,500G and about 4,000G, between about 2,500G and about 3,750G, between about 2,500G and about 3,500G, between about 2,500G and about 3,250G, between about 2,500G and about 3,000G, between about 2,500G and about 2,750G, between about 2,750G and about 4,000G, between about 2,750G and about 3,750G, Between about 3,500G, between about 2,750G and about 3,250G, between about 2,750G and about 3,000G, between about 3,000G and about 4,000G, between about 3,000G and about 3,750G, between about 3,000G and about 3,500G, between about 3,000G and about 3,250G, between about 3,250G and about 4,000G, between about 3,250G and about 3,750G, between about 3,250G and about 3,500G, between about 3,500G and about 4,000G, between about 3,500G and about 3,750G, or between about 3,750G and about 4,000G.

[0153] In some embodiments, the second centrifugal force is between about 500 G and about 1,500 G. In some embodiments, the second centrifugal force is between about 500 G and about 1,500 G, between about 500 G and about 1,400 G, between about 500 G and about 1,300 G, between about 500 G and about 1,200 G, between about 500 G and about 1,100 G, between about 500 G and about 1,000 G, between about 500 G and about 900 G, between about 500 G and about 800 G, or between about 500 G and about 700 G. In some embodiments, the second centrifugal force is about 625 G.

[0154] In some embodiments, the second centrifugal force is between about 100 G and about 2,000 G. In some embodiments, the second centrifugal force is between about 100 G and about 2,000 G, between about 100 G and about 1,900 G, between about 100 G and about 1,800 G, between about 100 G and about 1,700 G, between about 100 G and about 1,600 G, between about 100 G and about 1,500 G, between about 100 G and about 1,400 G, between about 100 G and about 1,300 G, or between about 100 G and about 2,000 G. G, between about 100 G and about 1,200 G, between about 100 G and about 1,100 G, between about 100 G and about 1,000 G, between about 100 G and about 900 G, between about 100 G and about 800 G, between about 100 G and about 700 G, between about 100 G and about 600 G, between about 100 G and about 500 G, or between about 100 G and about 400 G. In some embodiments, the second centrifugal force is about 300 G.

[0155] In some embodiments, the second flow rate is between about 10 and about 100 mL / min. In some embodiments, (i) the second centrifugal force is between about 100 G and about 2,000 G, and (ii) the second flow rate is between about 10 mL / min and about 100 mL / min. In some embodiments, (i) the second centrifugal force is between about 500 G and about 1,500 G, and (ii) the second flow rate is between about 10 mL / min and about 100 mL / min.

[0156] In some embodiments, the second flow rate is between about 10 and about 100 mL / min, between about 15 and about 90 mL / min, between about 20 and about 80 mL / min, between about 25 and about 70 mL / min, between about 30 and about 60 mL / min, or between about 35 and about 50 mL / min. In some embodiments, the second flow rate is about 20 mL / min, about 21 mL / min, about 22 mL / min, about 23 mL / min, about 24 mL / min, about 25 mL / min, about 25.5 mL / min, about 26 mL / min, about 26.5 mL / min, about 27 mL / min, about 27.5 mL / min, about 28 mL / min, about 28.5 mL / min, about 29 mL / min, about 29.5 mL / min, about 30 mL / min, about 31 mL / min, about 32 mL / min, about 33 mL / min, about 34 mL / min, or about 35 mL / min. In some embodiments, the second flow rate is about 25 mL / min. In some embodiments, the second flow rate is about 25.5 mL / min. In some embodiments, the second flow rate is about 26 mL / min. In some embodiments, the second flow rate is about 26.5 mL / min. In some embodiments, the second flow rate is about 27 mL / min. In some embodiments, the second flow rate is about 27.5 mL / min. In some embodiments, the second flow rate is about 28 mL / min. In some embodiments, the second flow rate is about 28.5 mL / min. In some embodiments, the second flow rate is about 29 mL / min. In some embodiments, the second flow rate is about 29.5 mL / min. In some embodiments, the second flow rate is about 30 mL / min.

[0157] In some embodiments, the second flow rate is between about 5 and about 100 mL / min, between about 5 and about 90 mL / min, between about 5 and about 80 mL / min, between about 5 and about 70 mL / min, between about 5 and about 60 mL / min, between about 5 and about 50 mL / min, between about 5 and about 40 mL / min, between about 5 and about 30 mL / min, between about 5 and about 25 mL / min, between about 5 and about 20 mL / min, between about 5 and about 15 mL / min, between about 5 and about 10 mL / min, between about 10 and about 100 mL / min, between about 10 and about 90 mL / min, between about 10 and about 80 mL / min, between about 10 and about 70 mL / min, between about 10 and about 60 mL / min, between about 10 and about 50 mL / min, between about 10 and about 40 mL / min, between about 10 and about 30 mL / min, between about 10 and about 25 mL / min, between about 10 and about 20 mL / min, between about 10 and about 15 mL / min, between about 15 and about 100 mL / min, between about 15 and about 90 mL / min, between about 15 and about 80 mL / min, between about 15 and about 70 mL / min, between about 15 and about 60 mL / min, between about 15 and about 50 mL / min, between about 15 and about 40 mL / min, between about 15 and about 30 mL / min, between about 15 and about 25 mL / min, between about 15 and about Between 20 mL / min, between about 20 and about 100 mL / min, between about 20 and about 90 mL / min, between about 20 and about 80 mL / min, between about 20 and about 70 mL / min, between about 20 and about 60 mL / min, between about 20 and about 50 mL / min, between about 20 and about 40 mL / min, between about 20 and about 30 mL / min, between about 20 and about 25 mL / min, between about 25 and about 100 mL / min, between about 25 and about 90 mL / min, between about 25 and about 80 mL / min, between about 25 and about 70 mL / min, between about 25 and about 60 mL / min, between about 25 and about 50 mL / min, between about 25 and about 40 mL / min minutes, between about 25 and about 30 mL / min, between about 30 and about 100 mL / min, between about 30 and about 90 mL / min, between about 30 and about 80 mL / min, between about 30 and about 70 mL / min, between about 30 and about 60 mL / min, between about 30 and about 50 mL / min, between about 30 and about 40 mL / min, between about 40 and about 100 mL / min, between about 40 and about 90 mL / min, between about 40 and about 80 mL / min, between about 40 and about 70 mL / min, between about 40 and about 60 mL / min, between about 40 and about 50 mL / min, between about 50 and about 100 mL / min, between about 50 and about 90 mL / minThe second flow rate may be between about 50 and about 80 mL / min, between about 50 and about 70 mL / min, between about 50 and about 60 mL / min, between about 60 and about 100 mL / min, between about 60 and about 90 mL / min, between about 60 and about 80 mL / min, between about 60 and about 70 mL / min, between about 70 and about 100 mL / min, between about 70 and about 90 mL / min, between about 70 and about 80 mL / min, between about 80 and about 100 mL / min, between about 80 and about 90 mL / min, or between about 90 and about 100 mL / min. In some embodiments, the second flow rate is between about 10 mL / min and about 30 mL / min. In some embodiments, the second flow rate is between about 25 mL / min and about 30 mL / min.

[0158] In some embodiments, (i) the second centrifugal force is between about 500 G and about 1,500 G, and (ii) the second flow rate is between about 25 mL / min and about 30 mL / min.

[0159] In some embodiments, the second flow rate is between about 5 and about 40 mL / min, between about 5 and about 35 mL / min, between about 5 and about 30 mL / min, between about 5 and about 25 mL / min, between about 5 and about 20 mL / min, or between about 5 and about 15 mL / min, hi some embodiments, the second flow rate is about 10 mL / min.

[0160] In some embodiments, the ratio of the second centrifugal force to the second flow rate is between about 20 and about 100. In some embodiments, the ratio of the second centrifugal force to the second flow rate is between about 25 and about 85. In some embodiments, the ratio of the second centrifugal force to the second flow rate is between about 30 and about 65.

[0161] In some embodiments, the ratio of the second centrifugal force to the second flow rate is between about 20 and about 100, between about 25 and about 80, or between about 30 and about 60. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 20. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 25. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 30. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 35. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 40. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 45. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 50. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 55. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 60. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 65. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 70. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 75. In some embodiments, the second centrifugal force is about 1,000 G and the second flow rate is about 28.5 mL / min.

[0162] In some embodiments, the ratio of the second centrifugal force to the second flow rate is between about 20 and 200, between about 20 and 180, between about 20 and 160, between about 20 and 140, between about 20 and 120, between about 20 and 100, between about 20 and 80, between about 20 and 60, between about 20 and 40, between about 40 and 200, between about 40 and 180, between about 40 and 160, between about 40 and 140, between about 40 and 120, between about 40 and 100, between about 40 and 80, between about 40 and 60, between about 60 and 200, between about 60 and 180, between about 60 and 160, between about 60 and 140, between about 60 and 120, between about 60 and 60 between about 60 and 80, between about 80 and 200, between about 80 and 180, between about 80 and 160, between about 80 and 140, between about 80 and 120, between about 80 and 100, between about 100 and 200, between about 100 and 180, between about 100 and 160, between about 100 and 140, between about 100 and 120, between about 120 and 200, between about 120 and 180, between about 120 and 160, between about 120 and 140, between about 140 and 200, between about 140 and 180, between about 140 and 160, between about 160 and 200, between about 160 and 180, or between about 180 and 200.

[0163] In some embodiments, the ratio of the second centrifugal force to the second flow rate is between about 20 and about 100, between about 20 and about 80, between about 20 and about 60, or between about 20 and about 40. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 30. In some embodiments, the second centrifugal force is about 300 G and the second flow rate is about 10 mL / min.

[0164] In some embodiments, the ratio of the second centrifugal force to the second flow rate is between about 40 and 200, between about 40 and 180, between about 40 and 160, between about 40 and 140, between about 40 and 120, between about 40 and 100, between about 40 and 80, or between about 50 and 60. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 62.5. In some embodiments, the second centrifugal force is about 625 G and the second flow rate is about 10 mL / min.

[0165] In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for about 5 minutes to about 100 minutes, about 10 minutes to about 90 minutes, about 15 minutes to about 80 minutes, about 20 minutes to about 70 minutes, about 25 minutes to about 60 minutes, about 30 minutes to about 50 minutes, or about 35 minutes to about 40 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for about 5 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for about 10 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for about 15 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for about 20 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for about 25 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for about 30 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for about 45 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for about 60 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for about 75 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for about 90 minutes.

[0166] In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for at least about 15 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for at least about 30 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for at least about 45 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for at least about 60 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for at least about 75 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for at least about 90 minutes.

[0167] In some embodiments, the second centrifugal force and the second flow rate are applied sequentially to the input composition.

[0168] In other embodiments, the second centrifugal force and the second flow rate are applied to the input composition discontinuously. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition intermittently. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition at regularly occurring intervals.

[0169] In any of the foregoing embodiments, the amount of time the second centrifugal force and the second flow rate are applied to the input composition can be the total amount of time the second centrifugal force and the second flow rate are discontinuously applied. For example, in some embodiments, the second centrifugal force and the second flow rate are discontinuously applied to the input composition for a total of about 5 minutes to about 25 minutes over a 30-minute period. In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for a total of about 15 minutes over a 30-minute period.

[0170] In some embodiments, the second centrifugal force and the second flow rate are applied to the input composition for a first period of time, followed by a second period of time during which the second centrifugal force and the second flow rate are not applied to the input composition. In some embodiments, a centrifugal force and a flow rate different from the second centrifugal force and the second flow rate are applied to the input composition. In some embodiments, the centrifugal force and the flow rate applied during the second period of time are any of the centrifugal forces and flow rates described herein, for example, any of the second centrifugal forces and second flow rates described herein, for example, any of the centrifugal forces and flow rates at the ratio of the second centrifugal force to the second flow rate described herein.

[0171] In some embodiments, the first period and the second period are approximately equal in length.

[0172] In some embodiments, the first period of time and the second period of time are different in length.

[0173] In some embodiments, the first and second time periods are repeated a predetermined number of times. In some embodiments, the first and second time periods are repeated until a predetermined amount of time has elapsed. In some embodiments, the predetermined amount of time is between about 5 and about 100 minutes, between about 10 and about 90 minutes, between about 15 and about 80 minutes, between about 20 and about 70 minutes, between about 25 and about 60 minutes, between about 30 and about 50 minutes, or between about 35 and about 40 minutes.

[0174] In some embodiments, the first period of time is between about 1 minute and about 10 minutes, between about 1 minute and about 9 minutes, between about 1 minute and about 8 minutes, between about 1 minute and about 7 minutes, between about 1 minute and about 6 minutes, between about 1 minute and about 5 minutes, between about 1 minute and about 4 minutes, between about 1 minute and about 3 minutes, between about 1 minute and about 2 minutes, between about 2 minutes and about 10 minutes, between about 2 minutes and about 9 minutes, between about 2 minutes and about 8 minutes, between about 2 minutes and about 7 minutes, between about 2 minutes and about 6 minutes, between about 2 minutes and about 5 minutes, between about 2 minutes and about 4 minutes, between about 2 minutes and about 3 minutes, between about 3 minutes and about 10 minutes, between about 3 minutes and about 9 minutes, between about 3 minutes and about 8 minutes, between about 3 minutes and about 7 minutes, between about 3 minutes and about 6 minutes, Between 3 minutes and about 5 minutes, between about 3 minutes and about 4 minutes, between about 4 minutes and about 10 minutes, between about 4 minutes and about 9 minutes, between about 4 minutes and about 8 minutes, between about 4 minutes and about 7 minutes, between about 4 minutes and about 6 minutes, between about 4 minutes and about 5 minutes, between about 5 minutes and about 10 minutes, between about 5 minutes and about 9 minutes, between about 5 minutes and about 8 minutes, between about 5 minutes and about 7 minutes, between about 5 minutes and about 6 minutes, between about 6 minutes and about 10 minutes, between about 6 minutes and about 9 minutes, between about 6 minutes and about 8 minutes, between about 6 minutes and about 7 minutes, between about 7 minutes and about 10 minutes, between about 7 minutes and about 9 minutes, between about 7 minutes and about 8 minutes, between about 8 minutes and about 10 minutes, between about 8 minutes and about 9 minutes, or between about 9 minutes and about 10 minutes. In some embodiments, the first period of time is between about 1 minute and about 10 minutes, between about 1 minute and about 9 minutes, between about 1 minute and about 8 minutes, between about 1 minute and about 7 minutes, between about 1 minute and about 6 minutes, between about 1 minute and about 5 minutes, between about 1 minute and about 4 minutes, between about 1 minute and about 3 minutes, or between about 1 minute and about 2 minutes. In some embodiments, the first period of time is about 1 minute.

[0175] In some embodiments, the second period of time is between about 1 minute and about 10 minutes, between about 1 minute and about 9 minutes, between about 1 minute and about 8 minutes, between about 1 minute and about 7 minutes, between about 1 minute and about 6 minutes, between about 1 minute and about 5 minutes, between about 1 minute and about 4 minutes, between about 1 minute and about 3 minutes, between about 1 minute and about 2 minutes, between about 2 minutes and about 10 minutes, between about 2 minutes and about 9 minutes, between about 2 minutes and about 8 minutes, between about 2 minutes and about 7 minutes, between about 2 minutes and about 6 minutes, between about 2 minutes and about 5 minutes, between about 2 minutes and about 4 minutes, between about 2 minutes and about 3 minutes, between about 3 minutes and about 10 minutes, between about 3 minutes and about 9 minutes, between about 3 minutes and about 8 minutes, between about 3 minutes and about 7 minutes, between about 3 minutes and about 6 minutes, Between 3 minutes and about 5 minutes, between about 3 minutes and about 4 minutes, between about 4 minutes and about 10 minutes, between about 4 minutes and about 9 minutes, between about 4 minutes and about 8 minutes, between about 4 minutes and about 7 minutes, between about 4 minutes and about 6 minutes, between about 4 minutes and about 5 minutes, between about 5 minutes and about 10 minutes, between about 5 minutes and about 9 minutes, between about 5 minutes and about 8 minutes, between about 5 minutes and about 7 minutes, between about 5 minutes and about 6 minutes, between about 6 minutes and about 10 minutes, between about 6 minutes and about 9 minutes, between about 6 minutes and about 8 minutes, between about 6 minutes and about 7 minutes, between about 7 minutes and about 10 minutes, between about 7 minutes and about 9 minutes, between about 7 minutes and about 8 minutes, between about 8 minutes and about 10 minutes, between about 8 minutes and about 9 minutes, or between about 9 minutes and about 10 minutes. In some embodiments, the second period of time is between about 1 minute and about 10 minutes, between about 1 minute and about 9 minutes, between about 1 minute and about 8 minutes, between about 1 minute and about 7 minutes, between about 1 minute and about 6 minutes, between about 1 minute and about 5 minutes, between about 1 minute and about 4 minutes, between about 1 minute and about 3 minutes, or between about 1 minute and about 2 minutes. In some embodiments, the second period of time is about 1 minute.

[0176] In some embodiments, the second centrifugal force is about 1,500 G and the second flow rate is about 10 mL / min, and the second centrifugal force and second flow rate are applied to the input composition for a first period of about 1 minute, after which a centrifugal force of about 300 G and a flow rate of about 10 mL / min are applied to the input composition for a second period of about 1 minute. In some embodiments, the first and second periods are repeated until about 30 minutes have elapsed.

[0177] In some embodiments, the volume of the input composition includes volumes between about 5 ml and about 20,000 ml, between about 10 ml and about 2,000 ml, between about 15 ml and about 1,000 ml, between about 20 ml and about 500 ml, between about 25 ml and about 100 ml, or between about 30 ml and about 60 ml. In some embodiments, the volume of the input composition is between about 30 ml and about 60 ml. In some embodiments, the volume of the input composition is about 5 ml. In some embodiments, the volume of the input composition is about 10 ml. In some embodiments, the volume of the input composition is about 15 ml. In some embodiments, the volume of the input composition is about 20 ml. In some embodiments, the volume of the input composition is about 25 ml. In some embodiments, the volume of the input composition is about 30 ml. In some embodiments, the volume of the input composition is about 35 ml. In some embodiments, the volume of the input composition is about 40 ml. In some embodiments, the volume of the input composition is about 45 ml. In some embodiments, the volume of the input composition is about 50 ml. In some embodiments, the volume of the input composition is about 55 ml. In some embodiments, the volume of the input composition is about 60 ml. In some embodiments, the volume of the input composition is about 65 ml. In some embodiments, the volume of the input composition is about 70 ml. In some embodiments, the volume of the input composition is about 75 ml.

[0178] The term "G" or "relative centrifugal force" (RCF) is generally understood to be the effective force exerted on an object or substance (e.g., a cell, sample, or pellet and / or spot in a rotating chamber or other container) relative to the Earth's gravity at a particular location in space relative to the axis of rotation. The value can be determined using well-known formulas taking into account gravity, the speed of rotation, and the radius of rotation (the distance from the axis of rotation to the object, substance, or particle for which the RCF is being measured).

[0179] In some embodiments, cells produced from the provided methods (also referred to hereinafter as "genetically engineered T cells" or "output compositions") comprise cells transduced with a viral vector, such as a viral vector containing a polynucleotide encoding a heterologous protein, such as a recombinant receptor, e.g., a CAR. Heterologous in this context refers to a protein that is not normally expressed from the virus and / or encoded by the viral genome. In some embodiments, integration of the viral vector into the host genome can be assessed by measuring the expression level of a recombinant protein, e.g., a heterologous protein, encoded by a nucleic acid contained in the genome of the viral vector particle after incubation. Several well-known methods for assessing the expression level of a recombinant molecule can be used, such as affinity-based methods, e.g., immunoaffinity-based methods, e.g., for cell surface proteins, detection by flow cytometry, etc. In some examples, expression is measured by detection of a transduction marker and / or reporter construct. In some embodiments, a nucleic acid encoding a truncated surface protein is included in the vector and used as a marker of expression and / or its enhancement.

[0180] i. Cell composition In some embodiments, a cell composition comprising T cells is transduced with a viral vector particle, e.g., according to the methods described. In some embodiments, the concentration of cells in the input composition is 1.0 x 10 5 cells / mL~1.0×10 8 cells / mL or approximately 1.0 x 10 5 cells / mL ~ approx. 1.0×10 8 cells / mL, e.g., at least or about at least or about 1.0 x 10 5 cells / mL, 5×10 5 cells / mL, 1×10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL or 1 x 10 8In some embodiments, the cell composition is about 1 x 10 cells / mL. 6 In some embodiments, the cell composition comprises about 1.25 x 10 cells / mL. 6 In some embodiments, the cell composition comprises about 1.5 x 10 cells / mL. 6 In some embodiments, the cell composition comprises about 1.75 x 10 cells / mL. 6 In some embodiments, the cell composition comprises about 2 x 10 cells / mL. 6 In some embodiments, the cell composition comprises about 2.25 x 10 cells / mL. 6 In some embodiments, the cell composition comprises about 2.5 x 10 cells / mL. 6 In some embodiments, the cell composition comprises about 2.75 x 10 cells / mL. 6 In some embodiments, the cell composition comprises about 3 x 10 cells / mL. 6 Contains cells / mL.

[0181] In some embodiments, the volume of the cellular composition is between about 20 mL and about 300 mL, between about 25 mL and about 250 mL, between about 30 mL and about 200 mL, between about 35 mL, and between about 150 mL, or between about 40 mL and about 100 mL. In some embodiments, the volume of the cellular composition is about 20 mL. In some embodiments, the volume of the cellular composition is about 25 mL. In some embodiments, the volume of the cellular composition is about 30 mL. In some embodiments, the volume of the cellular composition is about 35 mL. In some embodiments, the volume of the cellular composition is about 40 mL. In some embodiments, the volume of the cellular composition is about 45 mL. In some embodiments, the volume of the cellular composition is about 50 mL. In some embodiments, the volume of the cellular composition is about 55 mL. In some embodiments, the volume of the cellular composition is about 60 mL. In some embodiments, the volume of the cellular composition is about 70 mL. In some embodiments, the volume of the cellular composition is about 80 mL. In some embodiments, the volume of the cell composition is about 100 mL. In some embodiments, the volume of the cell composition is about 125 mL. In some embodiments, the volume of the cell composition is about 150 mL. In some embodiments, the volume of the cell composition is about 175 mL. In some embodiments, the volume of the cell composition is about 200 mL.

[0182] In some embodiments, the cell composition comprises a total of about 1 x 10 8 cells, total number approximately 2 × 10 8 cells, total number approximately 3 × 10 8 cells, total number approximately 4 × 10 8 cells, total number approximately 5 × 10 8 cells, total number approximately 6 × 10 8 cells, total number approximately 7 × 10 8 cells, total number approximately 8 × 10 8 cells, total number approximately 9 × 10 8 of cells, or a total of approximately 1 x 10 9 Contains cells of.

[0183] In some embodiments, the T cells have an average diameter of about 5 μm to about 25 μm, about 5 μm to about 20 μm, about 5 μm to about 15 μm, about 5 μm to about 10 μm, about 10 μm to about 25 μm, about 10 μm to about 20 μm, about 10 μm to about 15 μm, about 15 μm to about 25 μm, about 15 μm to about 20 μm, or about 20 μm to about 25 μm. In some embodiments, the T cells have an average diameter of about 9 μm to about 20 μm.

[0184] In some embodiments, the T cells have an average diameter of about 10 μm to about 20 μm. In some embodiments, the T cells have an average diameter of about 12 μm to about 20 μm. In some embodiments, the T cells have an average diameter of about 14 μm to about 20 μm.

[0185] In some embodiments, the T cells have an average diameter of less than 9 μm. In some embodiments, the T cells have an average diameter of about 3 μm to about 9 μm, about 4 μm to about 9 μm, about 5 μm to about 9 μm, about 6 μm to about 9 μm, about 7 μm to about 9 μm, or about 8 μm to about 9 μm. In some embodiments, the T cells have an average diameter of about 6 μm to about 9 μm.

[0186] In some embodiments, the cell composition contains T cells that have been cryopreserved and thawed prior to application of the method, hi some embodiments, the method further comprises thawing the cryopreserved cell composition to produce a cell composition comprising T cells.

[0187] In some embodiments, the cell composition contains T cells that have not been cryopreserved or thawed prior to application of the method.

[0188] In some embodiments, cells are incubated and / or cultured prior to transduction by the provided methods. The incubation step may include culture, cultivation, stimulation, activation, and / or proliferation. In some embodiments, the method includes incubating the T cells of the cell composition under stimulatory conditions prior to application of a first centrifugal force and a first flow rate. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells within the population, to mimic antigen exposure, and / or to stimulate cells for genetic manipulation, e.g., introduction of a recombinant antigen receptor. The conditions may include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells.

[0189] In some embodiments, the T cells of the cell composition are incubated under stimulatory conditions prior to application of the first centrifugal force and the first flow rate. In some embodiments, the method includes incubating the T cells of the cell composition under stimulatory conditions prior to loading the cell composition into a centrifuge system. In some embodiments, the T cells of the cell composition are incubated under stimulatory conditions prior to loading the cell composition into a centrifuge system. In some embodiments, the cell composition comprises activated T cells. In some embodiments, the cell composition comprises T cells that express HLA-DR, CD25, CD69, CD71, CD40L, 4-1BB, or any combination thereof.

[0190] In some embodiments, the stimulatory condition comprises the presence of a stimulatory reagent. In some embodiments, the stimulatory reagent can activate an intracellular signaling domain of a TCR complex. In some aspects, the agent activates or initiates a TCR / CD3 intracellular signaling cascade in T cells. Such agents can include antibodies, e.g., antibodies specific for TCR components and / or costimulatory receptors, e.g., anti-CD3, anti-CD28, and / or one or more cytokines, bound to a solid support, e.g., beads. In some embodiments, the stimulatory reagent can activate one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules. In some embodiments, the stimulatory reagent comprises (i) a primary agent that specifically binds to a member of the TCR complex; and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule. In some embodiments, the primary agent specifically binds to CD3. In some embodiments, the costimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS. In some embodiments, at least one of the primary and secondary agents comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the first agent is or comprises an anti-CD3 antibody or an antigen-binding fragment thereof. In some embodiments, the second agent is or comprises an anti-CD28 antibody or an antigen-binding fragment thereof. Optionally, the expansion method may further comprise adding an anti-CD3 and / or anti-CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml).

[0191] In some embodiments, the stimulating agent comprises IL-2 and / or IL-15, e.g., an IL-2 concentration of at least about 10 units / mL. In some aspects, the incubation is carried out according to techniques such as those described in U.S. Patent No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0192] In some embodiments, the stimulatory conditions include a temperature suitable for the growth of human T lymphocytes, e.g., at least about 25 degrees Celsius, typically at least about 30 degrees Celsius, and typically at or about 37 degrees Celsius. Optionally, the incubation may further include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. The LCL may be irradiated with gamma radiation in the range of about 6000 to 10,000 rads. The LCL feeder cells, in some aspects, are provided in any suitable amount, e.g., at a ratio of at least about 10:1 LCL feeder cells to initial T lymphocytes.

[0193] In embodiments, antigen-specific T cells, e.g., antigen-specific CD4+ and / or CD8+ T cells, are obtained by stimulating naive or antigen-specific T lymphocytes with an antigen. For example, antigen-specific T cell lines or clones against a cytomegalovirus antigen can be generated by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen.

[0194] In some cases, viral vector particles may be used that do not require cells, such as T cells, to be activated. In some such instances, cells may be selected and / or transduced prior to and / or in the absence of activation.

[0195] In some embodiments, at least 40%, 50%, 60%, 70%, 80%, 90% or more of the cells, e.g., T cells, in the cell composition are activated, e.g., in some cases, surface positive for one or more of HLA-DR, CD25, CD69, CD71, CD40L, and / or 4-1BB. In some embodiments, the cells are activated with an activating agent, e.g., in the presence of anti-CD3 / anti-CD28, prior to the initiation of application of the first centrifugal force and the first flow rate, e.g., prior to establishment of the fluidized bed and / or prior to the initiation of transduction. Methods for expanding T cell populations in vitro in the absence of or with low amounts of exogenous growth factors are known in the art (see, e.g., U.S. Pat. No. 6,352,694 B1 and European Patent EP 0 700 430 B1). Generally, such methods utilize a solid surface of greater than 1 μM onto which various binding agents (e.g., anti-CD3 and / or anti-CD28 antibodies) are immobilized. For example, Dynabeads® CD3 / CD28 (Invitrogen) is a commercially available reagent for T cell expansion, which is a uniform, 4.5 μm superparamagnetic, sterile, non-pyrogenic polystyrene bead coated with a mixture of affinity-purified monoclonal antibodies against CD3 and CD28 cell surface molecules on human T cells. In some embodiments, activating agents, such as anti-CD3 and / or anti-CD28, can be immobilized on beads, such as magnetic beads.

[0196] In some embodiments, cell activation is also carried out in the presence of IL-2 (e.g., 50 IU / mL to 200 IU / mL or about 50 IU / mL to about 200 IU / mL, e.g., 100 IU / mL or about 100 IU / mL). In some embodiments, activation is carried out for between 1 hour and 96 hours, 1 hour and 72 hours, 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours, or 12 hours and 24 hours, or for about 1 hour and about 96 hours, about 1 hour and about 72 hours, about 1 hour and about 48 hours, about 4 hours and about 36 hours, about 8 hours and about 30 hours, or about 12 hours and about 24 hours, e.g., at least or about at least 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours. In some embodiments, activation is carried out at a temperature above or above about 25°C, e.g., generally above or above about 32°C, 35°C, or 37°C, e.g., 37°C±2°C or about 37°C±2°C, e.g., at a temperature of 37°C or about 37°C.

[0197] In some embodiments, the cells are not activated with an activating agent prior to the initiation of contact, e.g., prior to the initiation of transduction, e.g., in the presence of anti-CD3 / anti-CD28. In some embodiments, the cell composition comprises a plurality of resting cells. In some embodiments, at least 40%, 50%, 60%, 70%, 80%, 90% or more of the T cells in the population are resting T cells, e.g., T cells lacking T cell activation markers, e.g., surface markers or intracellular cytokines or other markers, and / or T cells in the G0 or G1 phase of the cell cycle.

[0198] In certain aspects, the provided methods allow transduction of T cells without the need for activation prior to contact and / or incubation. In some embodiments, the methods involve transducing a population of T cells containing resting or naive T cells with a viral vector without first activating and / or stimulating the T cells, e.g., prior to transduction. In some such embodiments, the provided methods can be used to prepare cells, e.g., T cells, for adoptive therapy and do not include activating and / or stimulating the T cells.

[0199] In some embodiments, the cells are generally eukaryotic cells, e.g., mammalian cells, typically human cells. In some embodiments, the cells are derived from blood, bone marrow, lymph, or lymphoid organs, or are cells of the immune system, e.g., myeloid or lymphoid cells, including cells of innate or adaptive immunity, e.g., lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells are typically primary cells, e.g., isolated directly from a subject and / or isolated and frozen from a subject. In some embodiments, the cells are primary T cells. In some embodiments, the cells are primary T cells from a human subject. In some embodiments, the cells include one or more subsets of T cells or other cell types, e.g., the total T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof, e.g., those defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence capacity, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. In some embodiments, the cells comprise CD3+ T cells or are enriched for CD3+ T cells. In some embodiments, the cells comprise CD4+ T cells or are enriched for CD4+ T cells. In some embodiments, the cells comprise CD8+ T cells or are enriched for CD8+ T cells. In some embodiments, the cells comprise CD4+ and CD8+ T cells. The cells can be allogeneic and / or autologous relative to the subject to be treated. Methods also include established methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and / or multipotent, e.g., stem cells, e.g., induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from a subject, preparing, treating, culturing, and / or manipulating them as described herein, and reintroducing them into the same patient, before or after lyophilization.

[0200] Among the subtypes and subpopulations of T cells and / or CD4+ T cells and / or CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells.

[0201] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0202] In some embodiments, the preparation of cells comprises one or more culturing and / or preparation steps. Cells can be isolated from a sample, e.g., a biological sample, e.g., obtained from or derived from a subject. In some embodiments, the subject from which the cells are isolated has a disease or condition, or is in need of cell therapy, or is to be administered cell therapy. In some embodiments, the subject is a human being in need of a specific therapeutic intervention, such as adoptive cell therapy, from which cells are isolated, treated, and / or manipulated.

[0203] Thus, in some embodiments, the cells are primary cells, for example, primary human cells. Samples include tissues, fluids, and other samples directly collected from subjects, as well as samples obtained through one or more processing steps, for example, separation, centrifugation, genetic manipulation (e.g., transduction with viral vectors), washing, and / or incubation. Biological samples can be samples obtained directly from biological sources or samples that have been processed. Biological samples include, but are not limited to, bodily fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue, and organ samples, including processed samples derived therefrom.

[0204] In some aspects, the sample from which the cells are derived or isolated is a blood or blood-derived sample, or is an apheresis or leukapheresis product, or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ and / or cells derived therefrom. In some embodiments, the cells are PBMCs. Samples include samples from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0205] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. The cells, in some embodiments, are obtained from a heterologous source, e.g., a mouse, a rat, a non-human primate, or a pig.

[0206] In some embodiments, cell isolation involves one or more preparative and / or affinity-based cell separation steps. In some cases, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove undesired components, to concentrate desired components, or to lyse or remove cells sensitive to a particular reagent. In some cases, cells are separated based on one or more characteristics, such as density, adhesive properties, size, sensitivity and / or resistance to a particular component.

[0207] In some examples, the cells are obtained from the subject's circulating blood, for example, by apheresis or leukapheresis. The sample, in some embodiments, contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and / or platelets, and in some embodiments, contains cells other than red blood cells and platelets.

[0208] In some embodiments, blood cells collected from a subject are washed, for example, to remove the plasma fraction and to place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, the wash step is accomplished by a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 Cell Processor, Baxter) according to the manufacturer's instructions. In some aspects, the wash step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in various biocompatible buffers after washing, such as Ca++ / Mg++-free PBS. In certain embodiments, components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

[0209] In some embodiments, the method comprises a density-based cell separation method, for example, preparation of white blood cells from peripheral blood by lysis of red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0210] In some embodiments, cells do not need to be enriched or selected prior to performing the provided methods.

[0211] In some embodiments, the isolation method involves separating different cell types based on the expression or presence of one or more specific molecules in the cells, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method for separating based on such markers can be used. The separation method can include any of those disclosed herein, including methods using reversible reagent systems, such as agents (e.g., receptor binding agents or selection agents) and reagents as described herein.

[0212] In some embodiments, the separation is affinity-based or immunoaffinity-based. For example, isolation in some aspects involves the separation of cells and cell populations based on the cellular expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such marker, generally followed by a washing step and separation of cells that are bound to the antibody or binding partner from cells that are not bound to the antibody or binding partner.

[0213] Such separation steps can be based on positive selection, in which cells that bind to the reagent are retained for further use, and / or negative selection, in which cells that do not bind to the antibody or binding partner are retained. In some cases, both fractions are retained for further use. In some embodiments, negative selection can be particularly useful when antibodies that specifically distinguish cell types in a heterogeneous population are not available, as separation is best performed based on markers expressed by cells other than the desired population.

[0214] Separation does not necessarily result in 100% enrichment or removal of a specific cell population or the cells that express a specific marker.For example, positive selection or enrichment of a specific type of cell, such as that that expresses a marker, refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express a marker.Similarly, negative selection, removal or depletion of a specific type of cell, such as that that expresses a marker, refers to reducing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0215] In some embodiments, the cell composition is enriched for CD3+ T cells. In some embodiments, at least 70%, at least 75%, at least 80%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the cell composition are CD3+ T cells. In some embodiments, the cell composition is enriched for CD4+ T cells. In some embodiments, at least 70%, at least 75%, at least 80%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the cell composition are CD4+ T cells. In some embodiments, the cell composition is enriched for CD8+ T cells. In some embodiments, at least 70%, at least 75%, at least 80%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the cell composition are CD8+ T cells. In some embodiments, the cell composition is enriched for CD4+ and CD8+ T cells. In some embodiments, at least 70%, at least 75%, at least 80%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the cell composition are CD4+ or CD8+ T cells.

[0216] In some cases, multiple rounds of separation steps are performed, in which the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some cases, a single separation step can simultaneously deplete cells expressing multiple markers, for example, by incubating cells with multiple antibodies or binding partners, each specific for a marker targeted by negative selection. Similarly, multiple cell types can be simultaneously positively selected by incubating cells with multiple antibodies or binding partners expressed on various cell types.

[0217] For example, in some embodiments, specific subpopulations of T cells, such as cells positive for one or more surface markers or cells expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques.

[0218] For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28-conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0219] In some embodiments, isolation is carried out by enriching for a particular cell population by positive selection, or by depleting a particular cell population by negative selection. In some embodiments, positive or negative selection is achieved by incubating cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers that are expressed or expressed at relatively high levels (marker high) on the positively or negatively selected cells, respectively.

[0220] In some embodiments, T cells are separated from PBMC samples by negative selection for markers expressed on non-T cells, e.g., B cells, monocytes, or other leukocytes, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed on, or relatively more highly expressed in, one or more naive, memory, and / or effector T cell subpopulations.

[0221] In some embodiments, CD8+ cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, for example, by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is performed to increase efficacy, for example, to improve long-term survival, expansion, and / or engraftment after administration, which in some aspects is particularly robust in such subpopulations. See Terakura et al. (2012) Blood.1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, efficacy is further enhanced by combining TCM-enriched CD8+ T cells and CD4+ T cells.

[0222] In some embodiments, memory T cells are present in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted for the CD62L-CD8+ and / or CD62L+CD8+ fractions, for example, using anti-CD8 and anti-CD62L antibodies.

[0223] In some embodiments, enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127, and in some aspects on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment of central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, and this fraction is subjected to negative selection based on expression of CD14 and CD45RA and positive selection based on CD62L. Such selections are carried out simultaneously in some aspects and sequentially in either order in other aspects. In some embodiments, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation is also used to generate the CD4+ cell population or subpopulation; thus, both the positive and negative fractions obtained from the CD4-based separation are retained and used in subsequent steps of the method, as appropriate, after one or more further positive or negative selection steps.

[0224] In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, and both the negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on markers characteristic of central memory T cells, such as CD62L or CCR7, with the positive and negative selections being performed in either order.

[0225] CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO-.

[0226] In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically contains antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic or paramagnetic bead, to allow for cell separation for positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: SA Brooks and U. Schumacher (c) Humana Press Inc., Totowa, NJ).

[0227] In some embodiments, a sample or composition of cells to be separated is incubated with a small, magnetizable, or magnetically responsive material, e.g., a magnetically responsive particle or microparticle, e.g., a paramagnetic bead (e.g., Dynalbeads or MACS beads, etc.). The magnetically responsive material, e.g., a particle, is generally attached, directly or indirectly, to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., a surface marker, present on a cell, a plurality of cells, or a population of cells that one wishes to separate, e.g., negatively or positively select.

[0228] In some embodiments, the magnetic particles or beads comprise a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials that can be used in magnetic separation methods. Suitable magnetic particles include those described in U.S. Patent No. 4,452,773 to Molday and European Patent Specification EP 452342 B, which are hereby incorporated by reference. Other examples include colloidal-sized particles, such as those described in U.S. Patent No. 4,795,698 to Owen and U.S. Patent No. 5,200,084 to Liberti et al.

[0229] Incubation is generally carried out under conditions in which the antibody or binding partner attached to the magnetic particle or bead, or a molecule that specifically binds to such an antibody or binding partner, e.g., a secondary antibody or other reagent, specifically binds to the cell surface molecule if present on cells in the sample.

[0230] In some embodiments, the sample is placed in a magnetic field, and cells with magnetically responsive or magnetizable particles attached are attracted to the magnet and separated from unlabeled cells. For positive selection, cells that are attracted to the magnet are retained, and for negative selection, cells that are not attracted (unlabeled cells) are retained. In some embodiments, a combination of positive and negative selection is performed during the same selection step, and positive and negative fractions are retained and further processed or subjected to additional separation steps.

[0231] Methods for removing magnetizable particles from cells are known and include, for example, the use of competing unlabeled antibodies, magnetizable particles, or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.

[0232] In some embodiments, affinity-based selection is by magnetically activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). The MACS system allows for high-purity selection of cells with attached magnetized particles. In certain embodiments, MACS operates in a manner in which non-target and target species are sequentially eluted after application of an external magnetic field. That is, cells attached to the magnetized particles are retained in place, while unattached species are eluted. Then, after this first elution step is complete, the species trapped in the magnetic field and prevented from elution are released in some manner so that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from a heterogeneous population of cells.

[0233] In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that performs one or more of the isolation, cell preparation, separation, processing, incubation, culture, and / or formulation steps of the method. In some aspects, the system is used to perform each of these steps in a closed or sterile environment, e.g., to minimize error, user handling, and / or contamination. In one example, the system is a system such as those described in International Patent Application Publication No. WO2009 / 072003 or U.S. Patent Application Publication No. 20110003380A1.

[0234] In some embodiments, the system or device performs one or more, e.g., all, of the isolation, processing, and / or formulation steps in an integrated or self-contained system and / or in an automated or programmable manner. In some aspects, the system or device includes a computer and / or computer program in communication with the system or device that allows a user to program, control, evaluate the results of, and / or adjust various aspects of the processing, isolation, manipulation, and formulation steps.

[0235] In some aspects, the separation and / or other steps are performed using, for example, a CliniMACS system (Miltenyi Biotic) for automated separation of cells at a clinical scale in a closed and sterile system. In certain embodiments, the separation and / or other steps are performed using a CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system is equipped with a cell processing unit that, in some aspects, allows for automated washing and fractionation of cells by centrifugation.

[0236] In some embodiments, the cell populations described herein are collected and enriched (or depleted) by flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluid stream. In some embodiments, the cell populations described herein are collected and enriched (or depleted) by preparative-scale (FACS) sorting. In certain embodiments, the cell populations described herein are collected and enriched (or depleted) by using a microelectromechanical system (MEMS) chip in combination with a FACS-based detection system (see, for example, WO2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376). In both cases, cells can be labeled with multiple markers, allowing for the isolation of clearly defined T cell subsets with high purity.

[0237] In some embodiments, the preparation method includes a step for freezing, e.g., cryopreserving, the cells either before or after isolation, incubation, and / or genetic manipulation. In some embodiments, the freezing and subsequent thawing steps remove granulocytes and, to some extent, monocytes from the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., after a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters can be used in some aspects. One example involves the use of PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing medium. This is then diluted 1:1 with medium to achieve final DMSO and HSA concentrations of 10% and 4%, respectively. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0238] ii. Viral vector particles In some embodiments, the cell composition comprising T cells is transduced with a viral vector, such as any of those described in Section IV, by any of the methods provided herein.

[0239] In some embodiments, the lentiviral vector is introduced into the composition in the centrifuge. In some embodiments, the volume of the composition containing the lentiviral vector particles is about 1 mL, about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, or about 20 mL. In some embodiments, the volume of the composition containing the lentiviral vector is about 5 mL. In some embodiments, the volume of the composition containing the lentiviral vector is about 10 mL. In some embodiments, the volume of the composition containing the lentiviral vector is about 15 mL. In some embodiments, the volume of the composition containing the lentiviral vector is about 20 mL.

[0240] In some embodiments, the viral vector particles are provided at a certain ratio of copy number of viral vector particles or their infectious units (IU) per total number of cells in the input composition or total number of cells to be transduced (IU / cell). For example, in some embodiments, the viral particles are present at 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or 60 IU, or about 0.5, about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 30, about 40, about 50, or about 60 IU, or at least 0.5, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, or at least 60 IU, or at least about 0.5, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about, or at least about 60 IU of viral vector particles per cell during contacting.

[0241] In some embodiments, transduction can be achieved at a multiplicity of infection (MOI) of less than 100, for example, generally less than 60, less than 50, less than 40, less than 30, less than 20, less than 10, less than 5 or even lower.

[0242] B. Output Composition In some embodiments, the method includes applying a third centrifugal force and a third flow rate to the genetically engineered T cells to produce an output composition comprising the genetically engineered T cells. In some embodiments, the output composition is collected or recovered, such as for downstream use in cell therapy. In some embodiments, the third centrifugal force and the third flow rate are applied to the genetically engineered T cells in a conical fluid enclosure of a centrifuge system. In some embodiments, applying the third centrifugal force and the third flow rate to the genetically engineered cells allows for collection or recovery of the output composition.

[0243] In some embodiments, the output composition is collected via a cannula, hi some embodiments, the output composition enters the end of the cannula at or near the tip of the conical fluid enclosure and exits the end of the cannula at or near the wide end of the conical fluid enclosure.

[0244] In some embodiments, the method comprises cryopreserving the output composition. In some embodiments, the level of viable cells in the output composition is maintained in the cryopreserved output composition. In some embodiments, the method comprises thawing the cryopreserved output composition. In some embodiments, the level of viable cells in the output composition is maintained in the thawed output composition.

[0245] In some embodiments, the third centrifugal force is between about 2,000G and about 3,000G.

[0246] In some embodiments, the third centrifugal force is between about 2,000 G and about 3,000 G, between about 2,200 G and about 2,800 G, or between about 2,400 G and about 2,600 G. In some embodiments, the third centrifugal force is about 2,000 G. In some embodiments, the third centrifugal force is about 2,100 G. In some embodiments, the third centrifugal force is about 2,200 G. In some embodiments, the third centrifugal force is about 2,300 G. In some embodiments, the third centrifugal force is about 2,400 G. In some embodiments, the third centrifugal force is about 2,500 G. In some embodiments, the third centrifugal force is about 2,600 G. In some embodiments, the third centrifugal force is about 2,700 G. In some embodiments, the third centrifugal force is about 2,800 G. In some embodiments, the third centrifugal force is about 2,900 G. In some embodiments, the third centrifugal force is about 3,000 G.

[0247] In some embodiments, the third flow rate is directed radially outward. In some embodiments, the third flow rate is directed toward the apex of the conical fluid enclosure. In some embodiments, the third centrifugal force and the third flow rate are in the same or substantially the same direction.

[0248] In some embodiments, the third flow rate is influenced by the flow of medium through the conical fluid enclosure. In some embodiments, the flow of medium through the conical fluid enclosure is from the wide end to the tip of the conical fluid enclosure. In some embodiments, the medium exits the tip of the conical fluid enclosure and enters the cannula.

[0249] In some embodiments, the third flow rate is between about 10 mL / min and about 30 mL / min, between about 12 mL / min and about 28 mL / min, between about 15 mL / min and about 25 mL / min, or between about 18 mL / min and about 22 mL / min. In some embodiments, the third flow rate is about 15 mL / min. In some embodiments, the third flow rate is about 16 mL / min. In some embodiments, the third flow rate is about 17 mL / min. In some embodiments, the third flow rate is about 18 mL / min. In some embodiments, the third flow rate is about 19 mL / min. In some embodiments, the third flow rate is about 20 mL / min. In some embodiments, the third flow rate is about 21 mL / min. In some embodiments, the third flow rate is about 22 mL / min. In some embodiments, the third flow rate is about 23 mL / min. In some embodiments, the third flow rate is about 24 mL / min. In some embodiments, the third flow rate is about 15 mL / min. In some embodiments, the third flow rate is about 25 mL / min.

[0250] In some embodiments, the third flow rate is between about 15 mL / min and about 25 mL / min. In some embodiments, (i) the third centrifugal force is between about 2,000 G and about 3,000 G, and (ii) the third flow rate is between about 15 mL / min and about 25 mL / min.

[0251] In some embodiments, the ratio of the third centrifugal force to the third flow rate is between about 100 and about 150, between about 110 and 140, or between about 120 and 130. In some embodiments, the ratio of the third centrifugal force to the third flow rate is about 100. In some embodiments, the ratio of the third centrifugal force to the third flow rate is about 105. In some embodiments, the ratio of the third centrifugal force to the third flow rate is about 110. In some embodiments, the ratio of the third centrifugal force to the third flow rate is about 115. In some embodiments, the ratio of the third centrifugal force to the third flow rate is about 120. In some embodiments, the ratio of the third centrifugal force to the third flow rate is about 125. In some embodiments, the ratio of the third centrifugal force to the third flow rate is about 130. In some embodiments, the ratio of the third centrifugal force to the third flow rate is about 135. In some embodiments, the ratio of the third centrifugal force to the third flow rate is about 140. In some embodiments, the ratio of the third centrifugal force to the third flow rate is about 145. In some embodiments, the ratio of the third centrifugal force to the third flow rate is about 150. In some embodiments, the third centrifugal force is about 2,500 G and the third flow rate is about 20 mL / min.

[0252] In some embodiments, the ratio of the third centrifugal force to the third flow rate is between about 100 and about 150.

[0253] In some embodiments, the method includes cryopreserving the cells of the output composition to create a cryopreserved composition. In some embodiments, cryopreserving includes suspending the cells in a medium containing a cryoprotectant and freezing the cells. In some embodiments, the freezing is in a controlled-rate freezer. In some embodiments, the method includes thawing the cryopreserved cell composition. In some embodiments, the method includes formulating the thawed cells to create a cell composition for administration as a drug product. In some embodiments, the thawing occurs after the cryopreserved cell composition has been frozen for at least one day. In some embodiments, the thawing occurs after the cryopreserved cell composition has been frozen for at least two days. In some embodiments, the thawing occurs after the cryopreserved cell composition has been frozen for at least three days. In some embodiments, the thawing occurs after the cryopreserved cell composition has been frozen for at least one week, ten days, two weeks, or one month. In some embodiments, the thawing occurs after the cryopreserved cell composition has been frozen for up to ten days, two weeks, one month, two months, three months, or six months.

[0254] C. Other Processing Steps In some embodiments, processing steps for transduction, such as in connection with cell manipulation, may further include washing, culture, cultivation, stimulation, activation, expansion, and / or formulation of the cells. In some embodiments, the genetically engineered cells are subjected to one or more washing steps before being collected as an output composition. In some embodiments, the collected output composition is incubated under stimulatory conditions or in the presence of a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population and / or to mimic antigen exposure. Stimulation can be performed ex vivo or in vivo after administration to a subject.

[0255] i. Cleaning In some embodiments, prior to applying the third centrifugal force and the third flow rate to the genetically engineered cells, the method includes subjecting the genetically engineered cells to one or more washing steps. In some embodiments, the one or more washing steps remove non-viable cells. In some embodiments, the one or more washing steps remove impurities (e.g., proteins, DNA, cellular debris, reagents).

[0256] In some embodiments, the one or more washing steps comprise applying a centrifugal force between about 500 G and about 3,000 G. In some embodiments, the centrifugal force is about 500 G. In some embodiments, the centrifugal force is about 1,000 G. In some embodiments, the centrifugal force is about 1,500 G. In some embodiments, the centrifugal force is about 2,000 G. In some embodiments, the centrifugal force is about 2,500 G. In some embodiments, the centrifugal force is about 3,000 G. In some embodiments, the one or more washing steps comprise applying a flow rate of about 20 mL / min to about 100 mL / min. In some embodiments, the flow rate is about 20 mL / min, about 30 mL / min, about 40 mL / min, about 50 mL / min, about 60 mL / min, about 70 mL / min, about 80 mL / min, about 90 mL / min, or about 100 mL / min. In some embodiments, the flow rate is about 30 mL / min. In some embodiments, the flow rate is about 35 mL / min. In some embodiments, the flow rate is about 40 mL / min. In some embodiments, the flow rate is about 45 mL / min. In some embodiments, the flow rate is about 50 mL / min. In some embodiments, the flow rate is about 55 mL / min. In some embodiments, the flow rate is about 60 mL / min. In some embodiments, the flow rate is about 65 mL / min. In some embodiments, the flow rate is about 70 mL / min. In some embodiments, the flow rate is about 75 mL / min. In some embodiments, the flow rate is about 80 mL / min. In some embodiments, the flow rate is about 85 mL / min. In some embodiments, the flow rate is about 90 mL / min.

[0257] In some embodiments, the one or more washing steps comprise applying a centrifugal force (G) and flow rate (FR) at a ratio between about 20 G / FR and about 80 G / FR. In some embodiments, the one or more washing steps comprise applying a G / F ratio of about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, or about 75. In some embodiments, the G / FR ratio is about 62.5 G / FR. In some embodiments, the one or more washing steps comprise applying a centrifugal force of about 2,500 G and a flow rate of about 40 mL / min. In some embodiments, the G / FR ratio is about 33.3 G / FR. In some embodiments, the one or more washing steps comprise applying a centrifugal force of about 2,500 G and a flow rate of about 75 mL / min. In some embodiments, the G / FR ratio is about 35 G / FR. In some embodiments, the one or more washing steps comprise applying a centrifugal force of about 1,000 G and a flow rate of about 28.5 mL / min.

[0258] In some embodiments, the genetically engineered cells are suspended in medium in a centrifuge before one or more washing steps. In some embodiments, the one or more washing steps include a medium change. Thus, in some aspects, the medium is changed to a different solution during one or more washing steps. In some aspects, nonviable cells are elutriated during one or more washing steps. In some aspects, nonviable cells are collected as a waste fraction during one or more washing steps. In some aspects, the cells of the cell composition are transduced during one or more washing steps. Thus, in some embodiments, the cells of the cell composition are transduced and washed simultaneously.

[0259] ii. Post-transduction activation and / or expansion of cells In some embodiments, the cells, e.g., the collected output composition, are further incubated and / or cultured in connection with the genetic manipulation. The incubation step may include culture, cultivation, stimulation, activation, and / or propagation. In some such embodiments, the further incubation is performed under conditions that result in integration of the viral vector into the host genome of one or more cells. The incubation and / or manipulation may be performed in a culture vessel, such as a culture or cell culture unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container. In some embodiments, the composition or cells are incubated under stimulatory conditions or in the presence of a stimulatory agent. Such conditions include conditions designed to induce proliferation, expansion, activation, and / or survival of cells in a population, mimic antigen exposure, and / or prime for genetic manipulation, such as the introduction of a recombinant antigen receptor.

[0260] In some embodiments, the further incubation is carried out at a temperature above room temperature, for example above or above about 25° C., for example, generally above or above about 32° C., 35° C., or 37° C. In some embodiments, the further incubation is carried out at a temperature of 37° C.±2° C. or about 37° C.±2° C., for example, at a temperature of 37° C. or about 37° C.

[0261] In some embodiments, further incubation is carried out under conditions for stimulation and / or activation of the cells, which may include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agent designed to activate cells.

[0262] In some embodiments, the stimulatory conditions or agents include one or more agents (e.g., stimulatory agents and / or auxiliary agents), such as ligands, capable of activating the intracellular signaling domain of the TCR complex. In some aspects, an agent suitable for delivering a primary signal, e.g., initiating activation of an ITAM-induced signal, e.g., specific for a TCR component, and / or an agent promoting a costimulatory signal, e.g., specific for a T cell costimulatory receptor, e.g., anti-CD3, anti-CD28, or anti-41-BB, appropriately coupled to a solid support such as beads, and / or one or more cytokines, activates or initiates the TCR / CD3 intracellular signaling cascade in T cells. Anti-CD3 / anti-CD28 beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander and / or ExpACT® beads) are among the stimulatory agents. Optionally, the expansion method may further include adding anti-CD3 and / or anti-CD28 antibodies to the culture medium. In some embodiments, the stimulating agent comprises IL-2 and / or IL-15, eg, an IL-2 concentration of at least about 10 units / mL.

[0263] In some embodiments, the stimulatory conditions or agents include one or more agents, e.g., ligands, capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agents activate or initiate the TCR / CD3 intracellular signaling cascade in T cells. Such agents can include antibodies, e.g., antibodies specific for TCR components and / or costimulatory receptors, e.g., anti-CD3, anti-CD28, and / or one or more cytokines, bound to a solid support, e.g., beads. Optionally, the expansion method can further include adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulatory agents include IL-2 and / or IL-15, e.g., an IL-2 concentration of at least about 10 units / mL, at least about 50 units / mL, at least about 100 units / mL, or at least about 200 units / mL.

[0264] Conditions can include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agent designed to activate cells.

[0265] In some embodiments, the incubation is carried out according to techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and / or Wang et al. (2012) J Immunother. 35(9): 689-701.

[0266] In some embodiments, the further incubation is performed in a centrifuge. In some embodiments, the further incubation is performed without spinning or centrifugation, and generally occurs after collection of the output composition. In some embodiments, the further incubation is performed outside the stationary phase, such as outside the chromatography matrix, e.g., in solution.

[0267] In some embodiments, the further incubation is carried out in a different container or device than that in which the contacting took place, such as by transfer, e.g., automated transfer, of the cells, e.g., the collected output composition, to a different container or device after centrifugation.

[0268] In some embodiments, the genetically engineered T cells are expanded by adding feeder cells, e.g., non-dividing peripheral blood mononuclear cells (PBMCs), to the culture starter composition (e.g., so that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells may comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000-3600 rads to prevent cell division. In some aspects, the feeder cells are added to the culture medium prior to the addition of the population of genetically engineered T cells.

[0269] In some embodiments, the stimulatory conditions include a temperature suitable for the growth of human T lymphocytes, e.g., at least about 25°C, typically at least about 30°C, and typically at or about 37°C. Optionally, the incubation may further include adding non-dividing virus-transformed lymphoblastoid cells (LCL) as feeder cells. For example, the virus may be EBV, CMV, or influenza, and the transformed LCLs present antigens from the virus on their surface, optionally in the context of MHC. In some embodiments, the T cells express a TCR that recognizes the viral antigen. In some embodiments, the viral antigen may be from EBV, CMV, or influenza. The LCLs may be irradiated with gamma rays in the range of about 6,000 to 10,000 rads. The LCL feeder cells, in some aspects, are provided in any suitable amount, e.g., at a ratio of about 10:1 LCL feeder cells to initial T lymphocytes.

[0270] In some embodiments, further culturing or incubation, e.g., to promote ex vivo expansion, is carried out for more than 24 hours, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 8 days, more than 9 days, more than 10 days, more than 11 days, more than 12 days, more than 13 days, more than 14 days, or more than 15 days, or for more than about 24 hours, more than about 2 days, more than about 3 days, more than about 4 days, more than about 5 days, more than about 6 days, more than about 7 days, more than about 8 days, more than about 9 days, more than about 10 days, more than about 11 days, more than about 12 days, more than about 13 days, more than about 14 days, or more than about 15 days. In some embodiments, further culturing or incubation is carried out for 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 24 hours or less.

[0271] In some embodiments, for example, the total duration of incubation with the stimulant is between 1 hour and 96 hours, between 1 hour and 72 hours, between 1 hour and 48 hours, between 4 hours and 36 hours, between 8 hours and 30 hours, or between 12 hours and 24 hours, or between about 1 hour and about 96 hours, between about 1 hour and about 72 hours, between about 1 hour and about 48 hours, between about 4 hours and about 36 hours, between about 8 hours and about 30 hours, or between about 12 hours and about 24 hours, e.g., at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, or at least 72 hours, or between about 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, or at least 72 hours. In some embodiments, the further incubation is for a period of 1 to 48 hours, 4 to 36 hours, 8 to 30 hours, or 12 to 24 hours, or for a period of about 1 to 48 hours, 4 to 36 hours, 8 to 30 hours, or 12 to 24 hours.

[0272] In some embodiments, the methods provided herein do not include further culturing or incubation, e.g., do not include an ex vivo expansion step, or include a substantially shorter ex vivo expansion step.

[0273] In some embodiments, the entire process of manipulating the cells, e.g., selection and / or enrichment, incubation associated with transduction, and / or further culturing or cultivation, is carried out within a period of more than 9 days, 8 days or less, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day or less after obtaining the cells from the control, it being understood that such timing does not include any period during which the cells are subjected to cryopreservation.

[0274] In some embodiments of the methods provided herein, the engineered cells, e.g., output compositions or formulated compositions, are administered to a subject immediately after transduction or shortly thereafter without significant ex vivo expansion. In some embodiments, the engineered cells may be administered shortly after the transduction step. In some embodiments, the engineered cells may be administered shortly after the transduction step, e.g., without significant ex vivo expansion or with ex vivo expansion that is substantially shorter than ex vivo expansion in conventional methods that may require significant in vitro activation, expansion, and / or enrichment. For example, in some embodiments of the methods provided herein, the engineered cells may be administered within 3, 2, or 1 day of transduction. In some embodiments, the engineered cells may be administered within 48, 36, 24, 20, 16, 12, 8, 4, 2, 1, or fewer hours of the transduction step. In some embodiments, the engineered cells are subjected to a substantially shorter in vitro expansion than conventional methods, for example, 48, 36, 24, 20, 16, 12, 8, 4, 2, 1 or fewer hours of in vitro expansion.

[0275] In any of such embodiments, expansion and / or activation of the cells, e.g., expansion of the engineered cells within the subject's body following administration of the cells, may occur in vivo following exposure to an antigen. In some embodiments, the extent, degree, or magnitude of in vivo expansion may be further increased, boosted, or enhanced by various methods that may modulate, e.g., increase, the expansion, proliferation, survival, and / or effectiveness of the administered cells, e.g., recombinant receptor-expressing cells.

[0276] In some embodiments, such methods include methods that involve administering engineered cells that are further modified with an agent, e.g., a nucleic acid, to alter (e.g., increase or decrease) the expression or activity of a molecule, where such altered expression or activity increases, boosts, or enhances the expansion, proliferation, survival, and / or effectiveness of the administered cells. In some embodiments, expression of the agent, e.g., nucleic acid, is inducible, repressible, regulatable, and / or user-controlled, such as by administration of an inducer or other modulating molecule.

[0277] In some embodiments, such methods include methods that involve co-administration, e.g., simultaneous or sequential administration, with a drug or agent that can increase, boost, or enhance the expansion, proliferation, survival, and / or effectiveness of the administered cells, e.g., recombinant receptor-expressing cells.

[0278] iii. Formulation In some embodiments, after further incubation, the process for preparing the cells may further include formulating the cells. Thus, the processing step may also include formulating the above-described compositions.

[0279] Also provided are pharmaceutical compositions or formulations for use in the above methods, which in some embodiments are formulated in an automated or partially automated manner, e.g., in a closed system in which other processing steps are performed in conjunction with the provided processing methods.

[0280] In some embodiments, the cells and compositions are administered to a subject in the form of a pharmaceutical composition or formulation, such as a composition comprising a cell or cell population and a pharmaceutically acceptable carrier or excipient.

[0281] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that would be unacceptably toxic to the subject to which the formulation is administered.

[0282] In some embodiments, the pharmaceutical composition further comprises another pharmaceutically active agent or drug, e.g., a chemotherapeutic agent such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the agent is administered in the form of a salt, e.g., a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid, and organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and arylsulfonic acids, e.g., p-toluenesulfonic acid.

[0283] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0284] In some embodiments, the choice of carrier is determined in part by the particular cells and / or the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition may include a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers can be prepared, for example, as described in Remington's Pharmaceutical Sciences 16 th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0285] In some embodiments, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffering agents is used. The buffering agent or mixture thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21 st This is described in more detail in ed. (May 1, 2005).

[0286] The formulation may include an aqueous solution. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably active ingredients with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active agents or drugs, such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine.

[0287] In some embodiments, the pharmaceutical composition contains cells in an amount effective to treat or prevent a disease or condition, e.g., a therapeutically or prophylactically effective amount. Therapeutic or prophylactic effectiveness is, in some embodiments, monitored by periodic evaluation of the treated subject. The desired dosage can be delivered by a single bolus of cells, by multiple boluses of cells, or by continuous infusion of cells.

[0288] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, intrapulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to a subject using peripheral systemic delivery via intravenous, intraperitoneal, or subcutaneous injection.

[0289] In some embodiments, the compositions are provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH in some aspects. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat more convenient to administer, especially by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range that allows for longer contact periods with specific tissues. Liquid or viscous compositions can contain a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0290] Sterile injectable solutions can be prepared by incorporating the cells in a vehicle such as a mixture with a suitable carrier, diluent, or excipient, such as sterile water, saline, glucose, dextrose, etc. The composition can contain auxiliary substances such as wetting agents, dispersing agents, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-enhancing additives, preservatives, flavoring agents, and / or coloring agents, depending on the desired route of administration and preparation. In some embodiments, standard texts may be consulted to prepare appropriate preparations.

[0291] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents and buffers.Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol and sorbic acid.Prolonged absorption of injectable pharmaceutical forms can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0292] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0293] D. Exemplary Characteristics of Output Compositions In some embodiments, the volume of the output composition (e.g., the collected output composition) is between about 1 ml and about 20,000 ml, between about 5 ml and about 2,000 ml, between about 10 ml and about 1,000 ml, between about 15 ml and about 500 ml, or between about 20 ml and about 100 ml. In some embodiments, the volume of the output composition is about 1 ml, about 5 ml, about 10 ml, about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml, about 50 ml, about 55 ml, about 60 ml, about 65 ml, about 70 ml, about 75 ml, about 80 ml, about 85 ml, about 90 ml, about 95 ml, or about 100 ml. In some embodiments, the volume of the output composition is about 1 ml. In some embodiments, the volume of the output composition is about 5 ml. In some embodiments, the volume of the output composition is about 10 ml. In some embodiments, the volume of the output composition is about 15 mL. In some embodiments, the volume of the output composition is about 20 mL. In some embodiments, the volume of the output composition is about 25 mL. In some embodiments, the volume of the output composition is about 30 mL. In some embodiments, the volume of the output composition is about 35 mL. In some embodiments, the volume of the output composition is about 40 mL. In some embodiments, the volume of the output composition is about 45 mL. In some embodiments, the volume of the output composition is about 50 mL.

[0294] In some embodiments, the output composition comprises a greater percentage of viable T cells than the cell composition or the input composition. In some embodiments, the output composition comprises a greater percentage of viable T cells than the cell composition. In some embodiments, the percentage of viable T cells in the output composition is at least about 5% greater, at least about 10% greater, at least about 15% greater, at least about 20% greater, or at least about 25% greater than the percentage of viable T cells in the cell composition. In some embodiments, the percentage of viable T cells in the output composition is about 5% greater than the percentage of viable T cells in the cell composition. In some embodiments, the percentage of viable T cells in the output composition is about 10% greater than the percentage of viable T cells in the cell composition. In some embodiments, the percentage of viable T cells in the output composition is about 15% greater than the percentage of viable T cells in the cell composition. In some embodiments, the percentage of viable T cells in the output composition is about 20% greater than the percentage of viable T cells in the cell composition. In some embodiments, the percentage of viable T cells in the output composition is about 25% greater than the percentage of viable T cells in the cell composition. In some embodiments, the percentage of viable T cells in the output composition is about 30% greater than the percentage of viable T cells in the cell composition.

[0295] In some embodiments, the output composition comprises a greater percentage of viable T cells than the input composition. In some embodiments, the percentage of viable T cells in the output composition is at least about 5% greater, at least about 10% greater, at least about 15% greater, at least about 20% greater, or at least about 25% greater than the percentage of viable T cells in the input composition. In some embodiments, the percentage of viable T input in the output composition is about 5% greater than the percentage of viable T input in the input composition. In some embodiments, the percentage of viable T input in the output composition is about 10% greater than the percentage of viable T input in the input composition. In some embodiments, the percentage of viable T input in the output composition is about 15% greater than the percentage of viable T input in the input composition. In some embodiments, the percentage of viable T inputs in the output composition is about 20% greater than the percentage of viable T inputs in the input composition. In some embodiments, the percentage of viable T inputs in the output composition is about 25% greater than the percentage of viable T inputs in the input composition. In some embodiments, the percentage of viable T inputs in the output composition is about 30% greater than the percentage of viable T inputs in the input composition.

[0296] In certain embodiments, the output composition contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% viable cells. In some embodiments, the output composition contains at least 75% or at least 75% viable cells. In certain embodiments, the output composition contains at least 85%, at least 90%, or at least 90%, or at least 95% or at least 95% viable cells. In some embodiments, the output composition contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% viable CD3+ T cells. In certain embodiments, the output composition contains at least 75% or at least about 75% viable CD3+ T cells. In certain embodiments, the output composition contains at least 85%, at least 90%, or at least 90%, or at least 95% or at least about 95% viable CD3+ T cells.In some embodiments, the output composition contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% viable CD4+ T cells. In certain embodiments, the output composition contains at least 75% or at least about 75% viable CD4+ T cells. In certain embodiments, the output composition contains at least 85%, at least 90%, or at least 90%, or at least 95% or at least about 95% viable CD4+ T cells. In certain embodiments, the output composition contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% viable CD8+ T cells. In some embodiments, the output composition contains at least 75% or at least 75% viable CD8+ T cells. In certain embodiments, the output composition contains at least 85%, at least 90%, or at least 90%, or at least 95% or at least 95% viable CD8+ T cells.

[0297] In certain embodiments, the output cells have a low rate and / or frequency of cells undergoing apoptosis and / or cells prepared, stimulated, and / or entering apoptosis. In certain embodiments, the output cells have a low rate and / or frequency of cells positive for apoptotic markers. In some embodiments, less than 40% or about 40%, less than 35% or about 35%, less than 30% or about 30%, less than 25% or about 25%, less than 20% or about 20%, less than 15% or about 15%, less than 10% or about 10%, less than 5% or about 5%, or less than 1% or about 1% of the cells in the output composition express, contain, and / or are positive for apoptotic markers. In certain embodiments, less than 25% or about 25% of the cells in the output composition express, contain, and / or are positive for apoptotic markers. In certain embodiments, less than 10% or less than about 10% of the cells in the output composition express, contain, and / or are positive for apoptotic markers. In certain embodiments, less than 5% or less than about 5% of the cells in the output composition express, contain, and / or are positive for apoptotic markers. In certain embodiments, less than 1% or less than about 1% of the cells in the output composition express, contain, and / or are positive for apoptotic markers.

[0298] In certain embodiments, the output composition is a composition of cells enriched for CD3+ T cells. In some embodiments, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 98%, at least or about 98.5%, at least or about 99%, at least or about 99.5%, at least or about 99.9%, 100%, or about 100% of the total cells in the output composition are CD3+ T cells. In some embodiments, at least or about 86%, at least or about 86.5%, at least or about 87%, at least or about 87.5%, at least or about 88%, at least or about 88.5%, at least or about 89%, at least or about 89.5%, at least or about 90%, at least or about 90.5%, at least or about 91%, at least or about 91.5%, at least or about 92%, at least or about 92.5%, at least or about 93%, at least or about 93.5%, at least or about 94%, at least or about 94.5%, at least or about 95%, at least or about 95.5%, at least or about 96%, at least or about 96.5%, at least or about 97%, at least or about 97.5%, at least or about 98%, or at least or about 98.5% of all cells in the output composition are CD3+ T cells. In some embodiments, between about 80% and about 100%, between about 85% and about 98%, between about 88% and about 96%, or between about 90% and about 94% of the total cells in the output composition are CD3+ T cells. In some embodiments, the output composition consists essentially of CD3+ T cells. In some embodiments, at least or about 90% of the total cells in the output composition are CD3+ T cells, and at least or about 40% of the total cells in the output composition express a recombinant receptor (e.g., a CAR).

[0299] In certain embodiments, the output composition is a composition of cells enriched for CD4+ T cells and CD8+ T cells. In certain embodiments, CD4+ T cells and CD8+ T cells account for at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 98%, at least or about 98.5%, at least or about 99%, at least or about 99.5%, at least or about 99.9%, 100%, or about 100% of the total cells in the output composition. In some embodiments, CD4+ T cells and CD8+ T cells account for at least or about 86%, at least or about 86.5%, at least or about 87%, at least or about 87.5%, at least or about 88%, at least or about 88.5%, at least or about 89%, at least or about 89.5%, at least or about 90%, at least or about 90.5%, at least or about 91%, at least or about 91.5%, at least or about 92%, at least or about 92.5%, at least or about 93%, at least or about 93.5%, at least or about 94%, at least or about 94.5%, at least or about 95%, at least or about 95.5%, at least or about 96%, at least or about 96.5%, at least or about 97%, at least or about 97.5%, at least or about 98%, or at least or about 98.5% of the total cells in the output composition. In some embodiments, CD4+ T cells and CD8+ T cells comprise between about 80% and about 100%, between about 85% and about 98%, between about 88% and about 96%, or between about 90% and about 94% of the total cells in the output composition. In some embodiments, the output composition consists essentially of CD4+ T cells and CD8+ T cells.

[0300] In certain embodiments, the output composition has a C of 10% to 90% or between about 10% and about 90%, 20% to 80% or between about 20% and about 80%, 25% to 75% or between about 25% and about 75%, 30% to 70% or between about 30% and about 70%, 35% to 65% or between about 35% and about 65%, 40% to 60% or between about 40% and about 60%, 55% to 45% or between about 55% and about 45%, or about 50% or 50%. D4+ T cells and between 10% and 90% or about 10% and about 90%, 20% and 80% or about 20% and about 80%, 25% and 75% or about 25% and about 75%, 30% and 70% or about 30% and about 70%, 35% and 65% or about 35% and about 65%, 40% and 60% or about 40% and about 60%, 55% and 45% or about 55% and about 45%, or about 50% or about 50% CD8+ T cells. In certain embodiments, the output composition contains between 35% and 65%, or about 35% and about 65%, 40% and 60%, or about 40% and about 60%, 55% and 45%, or about 55% and about 45%, or about 50% or 50% CD4+ T cells and 35% and 65%, or about 35% and about 65%, 40% and 60%, or about 40% and about 60%, 55% and 45%, or about 55% and about 45%, or about 50% or 50% CD8+ T cells. In certain embodiments, the output contains between 35% and 65%, or about 35% and about 65% CD4+ T cells and 35% and 65%, or about 35% and about 65% CD8+ T cells. In certain embodiments, the output composition contains a ratio of CD4+ T cells to CD8+ T cells of between 3:1 and 1:3, between 2.5:1 and 1:2.5, between 2:1 and 1:2, between 1.5:1 and 1:1.5, between 1.4:1 and 1:1.4, between 1.3:1 and 1:1.3, between 1.2:1 and 1:1.2, or between 1.1:1 and 1:1.1.In some embodiments, the composition of cells is at or about 3:1, 2.8:1 or about 2.8:1, 2.5:1 or about 2.5:1, 2.25:1 or about 2.25:1, 2:1 or about 2:1, 1.8:1 or about 1.8:1, 1.7:1 or about 1.7:1, 1.6:1 or about 1.6:1, 1.5:1 or about 1.5:1, 1.4:1 or about 1.4:1, 1.3:1 or about 1.3:1, 1.2:1 or about 1.2:1, 1.1:1 or about 1.1:1, 1:1 or about 1:1 or about 1:2.5, 1:2.5 or about 1:2.5, 1:2.8 or about 1:2.8, or 1:3 or about 1:3.

[0301] In some embodiments, the output composition contains a ratio of CD4+ T cells expressing the recombinant receptor, e.g., a CAR, to CD8+ T cells expressing the recombinant receptor, e.g., a CAR, of between 3:1 and 1:3, between 2.5:1 and 1:2.5, between 2:1 and 1:2, between 1.5:1 and 1:1.5, between 1.4:1 and 1:1.4, between 1.3:1 and 1:1.3, between 1.2:1 and 1:1.2, or between 1.1:1 and 1:1.1. In some embodiments, the ratio of recombinant receptor (e.g., CAR)-expressing CD4+ T cells to recombinant receptor (e.g., CAR)-expressing CD8+ T cells in the output composition is at or about 3:1, 2.8:1 or about 2.8:1, 2.5:1 or about 2.5:1, 2.25:1 or about 2.25:1, 2:1 or about 2:1, 1.8:1 or about 1.8:1, 1.7:1 or about 1.7:1, 1.6:1 or about 1.6:1, 1.5:1 or about 1.5:1, 1.4:1 or about 1.4:1, 1.3:1 or about 1.3:1, 1.2:1 or about 1.2:1, 1.4:1 or about 1.4:1, 1.5:1 or about 1.5:1, 1.6:1 or about 1.6:1, 1.5:1 or about 1.5:1, 1.4:1 or about 1.4:1, 1.3:1 or about 1.3:1, 1.2:1 or about 1.2:1, 1.8:1 or about 1.8:1, 1.7:1 or about 1.7 ... 1:1 or about 1.2:1, 1.1:1 or about 1.1:1, 1:1 or about 1:1, 1:1.1 or about 1:1.1, 1:1.2 or about 1:1.2, 1:1.3 or about 1:1.3, 1:1.4 or about 1:1.4, 1:1.5 or about 1:1.5, 1:1.6 or about 1:1.6, 1:1.7 or about 1:1.7, 1:1.8 or about 1:1.8, 1:2 or about 1:2, 1:2.25 or about 1:2.25, 1:2.5 or about 1:2.5, 1:2.8 or about 1:2.8, or 1:3 or about 1:3.

[0302] In some embodiments, output compositions generated or produced in association with the provided methods contain cells expressing a recombinant receptor, e.g., a TCR or a CAR. In some embodiments, expressing a recombinant receptor can include, but is not limited to, having one or more recombinant receptor proteins localized to the cell membrane and / or cell surface, having a detectable amount of recombinant receptor protein, having a detectable amount of mRNA encoding the recombinant receptor, having or containing a recombinant polynucleotide encoding the recombinant receptor, and / or having or containing mRNA or protein that is a surrogate marker for recombinant receptor expression.

[0303] In some embodiments, at least or about 5%, at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 97%, at least or about 99%, or more than about 99% of the cells of the output composition express the recombinant receptor. In certain embodiments, at least or about 50% of the cells of the output composition express the recombinant receptor. In certain embodiments, at least or about 5%, at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 97%, at least or about 99%, or more than about 99% of the CD3+ T cells of the output composition express the recombinant receptor. In some embodiments, at least or about 50% of the CD3+ T cells of the output composition express the recombinant receptor.In certain embodiments, at least or about 5%, at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 97%, at least or about 99%, or more than about 99% of the cells of the output composition are CD3+ T cells expressing the recombinant receptor. In some embodiments, at least or about 50% of the cells of the output composition are CD3+ T cells expressing the recombinant receptor.

[0304] In certain embodiments, at least or about 30%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 97%, at least or about 99%, or more than about 99% of the CD4+ T cells of the output composition express the recombinant receptor. In certain embodiments, at least or about 50% of the CD4+ T cells of the output composition express the recombinant receptor. In some embodiments, at least or about 30%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 97%, at least or about 99%, or more than about 99% of the CD8+ T cells of the output composition express the recombinant receptor. In certain embodiments, at least or about 50% of the CD8+ T cells of the output composition express the recombinant receptor.

[0305] In certain embodiments, at least 50%, or at least about 50%, at least 60%, or at least about 60%, at least 70%, or at least about 70%, at least 75%, or at least about 75%, at least 80%, or at least about 80%, at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, at least 99%, or at least about 99.9%, or at least about 99.9%, of the recombinant receptor-expressing (e.g., CAR+) cells of the output composition are viable cells, e.g., cells negative for apoptotic markers such as caspases (e.g., activated caspase-3). In certain embodiments, at least 85%, or at least about 85%, at least 90%, or at least about 90%, or at least 95%, or at least about 95% of the recombinant receptor-expressing (e.g., CAR+) cells of the output composition are viable cells, e.g., cells negative for apoptotic markers such as caspases (e.g., activated caspase-3). In some embodiments, at least 90% or at least about 90% of the recombinant receptor-expressing (e.g., CAR+) cells of the output composition are viable cells, e.g., cells negative for an apoptotic marker such as a caspase (e.g., activated caspase-3). In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least about 99%, or at least 99.9% or at least about 99.9% of the CD3+ T cells of the output composition are viable cells, e.g., cells negative for an apoptotic marker such as a caspase (e.g., activated caspase-3).In certain embodiments, at least 85%, or at least about 85%, at least 90%, or at least about 90%, or at least 95%, or at least about 95% of the CD3+ T cells of the output composition are viable cells, e.g., cells negative for an apoptotic marker such as a caspase (e.g., activated caspase-3). In certain embodiments, at least 90%, or at least about 90% of the CD3+ T cells of the output composition are viable cells, e.g., cells negative for an apoptotic marker such as a caspase (e.g., activated caspase-3). In some embodiments, at least 50%, or at least about 50%, at least 60%, or at least about 60%, at least 70%, or at least about 70%, at least 75%, or at least about 75%, at least 80%, or at least about 80%, at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, at least 99%, or at least about 99.9%, or at least about 99.9%, of the recombinant receptor-expressing (e.g., CAR) CD3+ T cells of the output composition are viable cells, e.g., cells negative for apoptotic markers such as caspases (e.g., activated caspase-3). In certain embodiments, at least 85%, or at least about 85%, at least 90%, or at least about 90%, or at least 95%, or at least about 95% of the recombinant receptor-expressing (e.g., CAR) CD3+ T cells of the output composition are viable cells, e.g., cells negative for apoptotic markers such as caspases (e.g., activated caspase-3). In certain embodiments, at least 90% or at least about 90% of the recombinant receptor-expressing (e.g., CAR+) CD3+ T cells of the output composition are viable cells, e.g., cells negative for apoptotic markers such as caspases (e.g., activated caspase-3).

[0306] In certain embodiments, on average, at least or at least about 50%, at least or at least about 60%, at least 70%, at least or at least about 70%, at least 75%, at least or at least about 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least about 99%, or at least about 99.9% of the recombinant receptor-expressing (e.g., CAR+) cells of the plurality of output compositions produced by the methods disclosed herein are viable cells, e.g., cells negative for apoptotic markers such as caspases (e.g., activated caspase-3). In certain embodiments, on average, at least 85%, or at least about 85%, at least 90%, or at least about 90%, or at least 95%, or at least about 95% of the recombinant receptor-expressing (e.g., CAR+) cells of the plurality of output compositions produced by the methods disclosed herein are viable cells, e.g., cells negative for apoptotic markers such as caspases (e.g., activated caspase-3). In some embodiments, on average, at least 90%, or at least about 90% of the recombinant receptor-expressing (e.g., CAR+) cells of the plurality of output compositions produced by the methods disclosed herein are viable cells, e.g., cells negative for apoptotic markers such as caspases (e.g., activated caspase-3).In some embodiments, on average, at least or at least about 50%, at least or at least about 60%, at least 70%, at least or at least about 70%, at least 75%, at least or at least about 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least about 99%, or at least about 99.9% of the CD3+ T cells in a plurality of output compositions produced by the methods disclosed herein are viable cells, e.g., cells negative for apoptotic markers such as caspases (e.g., activated caspase-3). In certain embodiments, on average, at least or at least about 85%, at least or at least about 90%, or at least 95% or at least about 95% of the CD3+ T cells in a plurality of output compositions produced by the methods disclosed herein are viable cells, e.g., cells negative for apoptotic markers such as caspases (e.g., activated caspase-3). In certain embodiments, on average, at least 90% or at least about 90% of the CD3+ T cells in a plurality of output compositions produced by the methods disclosed herein are viable cells, e.g., cells negative for apoptotic markers such as caspases (e.g., activated caspase-3).In some embodiments, on average, at least or at least about 50%, at least or at least about 60%, at least 70%, at least or at least about 70%, at least 75%, at least or at least about 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least about 99%, or at least about 99.9% of the recombinant receptor-expressing (e.g., CAR+) CD3+ T cells of the plurality of output compositions produced by the methods disclosed herein are viable cells, e.g., cells negative for apoptotic markers such as caspases (e.g., activated caspase-3). In certain embodiments, on average, at least 85%, at least 90%, or at least 95% of the recombinant receptor-expressing (e.g., CAR+) CD3+ T cells of a plurality of output compositions produced by the methods disclosed herein are viable cells, e.g., cells negative for apoptotic markers such as caspases (e.g., activated caspase-3). In certain embodiments, on average, at least 90% of the recombinant receptor-expressing (e.g., CAR+) CD3+ T cells of a plurality of output compositions produced by the methods disclosed herein are viable cells, e.g., cells negative for apoptotic markers such as caspases (e.g., activated caspase-3).

[0307] In any of the foregoing embodiments, the multiple output compositions produced by the methods disclosed herein can be derived from the same or different donors. In some aspects, at least two of the multiple output compositions are derived from different donors. In some aspects, each of the multiple output compositions is derived from one of several different donors, e.g., from about 2, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, or more than about 60 different donors, e.g., patients in need of cell therapy, such as CAR-T cell therapy.

[0308] In certain embodiments, the majority of the cells in the output composition are naive or naive-like cells, central memory cells, and / or effector memory cells. In certain embodiments, the majority of the cells in the output composition are naive-like cells or central memory cells. In some embodiments, the majority of the cells in the output composition are central memory cells. In some aspects, less differentiated cells, e.g., central memory cells, are longer-lived and less rapidly exhausted, thereby increasing persistence and durability. In some aspects, responders to cell therapy, such as CAR-T cell therapy, have increased expression of central memory genes. See, e.g., Fraietta et al. (2018) Nat Med. 24(5):563-571.

[0309] In certain embodiments, the cells of the output composition have a higher proportion and / or frequency of naive-like T cells or T cells that are surface positive for markers expressed on naive-like T cells. In certain embodiments, the cells of the output composition have a higher proportion and / or frequency of naive-like cells than an output composition generated from an alternative process, e.g., a process involving expansion (e.g., a process involving operation of an expansion unit and / or including steps aimed at causing cell expansion). In certain embodiments, naive-like T cells may include cells in various differentiation states and may be characterized by positive or high expression (e.g., surface or intracellular expression) of certain cell markers and / or negative or low expression (e.g., surface or intracellular expression) of other cell markers. In some aspects, naive-like T cells are characterized by positive or high expression of CCR7, CD45RA, CD28, and / or CD27. In some aspects, naive-like T cells are characterized by negative expression of CD25, CD45RO, CD56, CD62L, and / or KLRG1. In some aspects, naive-like T cells are characterized by low expression of CD95. In certain embodiments, naive-like T cells, or T cells that are surface-positive for markers expressed on naive-like T cells, are CCR7+CD45RA+, and these cells are CD27+ or CD27-. In certain embodiments, naive-like T cells, or T cells that are surface-positive for markers expressed on naive-like T cells, are CD27+CCR7+, and these cells are CD45RA+ or CD45RA-. In certain embodiments, naive-like T cells, or T cells that are surface-positive for markers expressed on naive-like T cells, are CD62L-CCR7+.

[0310] II. Methods for Enriching Viable Cells Methods are provided for enriching a cell composition (e.g., an engineered cell composition) for viable cells in a centrifuge system. In some embodiments, the centrifuge system is a continuous countercurrent elutriation ("CCE") centrifuge system, also known as a reverse centrifuge system. In some embodiments, the cells produced are cells for use in cell therapy, such as primary cells prepared for autologous or allogeneic transplantation, e.g., in adoptive cell therapy. The method may include additional cell processing steps, e.g., cell washing, isolation, separation, collection, formulation, or other steps associated with the production of the cell composition.

[0311] Also provided herein are methods for enriching a cell composition for viable cells, the methods comprising: (a) applying a first centrifugal force and a first flow rate to a cell composition comprising T cells in a conical fluid enclosure of a centrifuge system to produce a fluidized bed of cells, wherein the cell composition comprises viable and nonviable T cells; and (b) applying a second centrifugal force and a second flow rate to the cell composition, wherein the second centrifugal force and the second flow rate recirculate the cells of the input composition in a flow path of the centrifuge system, thereby producing an enriched composition having a percentage of viable T cells that is higher than the percentage of viable T cells in the cell composition. In some embodiments, the method comprises loading the cell composition (e.g., an engineered cell composition) into the centrifuge system, wherein the loading is performed before and / or during at least a portion of the application in (a).

[0312] In some embodiments, the centrifuge system includes a cannula within a conical fluid enclosure. In some embodiments, the cannula extends along the length of the conical fluid enclosure. In some embodiments, one end of the cannula is at or near the apex of the conical fluid enclosure. In some embodiments, the other end of the cannula is at or near the wide end of the conical fluid enclosure, for example, at or near the center of the wide end.

[0313] In some embodiments, the cell composition is introduced into the conical fluid enclosure via a cannula. In some embodiments, the cell composition is introduced into the conical fluid enclosure at or near the apex of the conical fluid enclosure. In some embodiments, the cell composition is introduced into the conical fluid enclosure by entering the end of the cannula at or near the wide end of the conical fluid enclosure and exiting the end of the cannula at or near the apex of the conical fluid enclosure.

[0314] In some embodiments, a waste fraction of the cell composition is elutriated out of the conical fluid enclosure by applying a second centrifugal force and a second flow rate, hi some embodiments, the elutriated waste fraction has a higher percentage of nonviable T cells than the percentage of nonviable T cells in the cell composition.

[0315] In some embodiments, the elutriated cells exit the conical fluid enclosure via an opening at the wide end of the conical fluid enclosure. In some embodiments, the opening at least partially surrounds the end of the cannula at or near the wide end of the conical fluid enclosure. In some embodiments, the opening surrounds the end of the cannula at or near the wide end of the conical fluid enclosure.

[0316] In some embodiments, the method includes collecting the elutriated waste fraction. In some embodiments, the elutriated waste fraction is collected in a container. In some embodiments, the container is in fluid communication with the wide end of the conical fluid enclosure.

[0317] In some embodiments, applying a second centrifugal force and a second flow rate produces an enriched composition having a percentage of viable T cells that is higher than the percentage of viable T cells in the cell composition within the conical fluid enclosure.

[0318] In some embodiments, the provided methods are used to enrich for viable cells, such as T cells (e.g., engineered T cells). In some embodiments, the cells have been previously introduced with a heterologous polynucleotide encoding an antigen receptor, such as a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR). In some embodiments, the cells have been previously introduced with a heterologous nucleic acid via a viral vector particle. In some embodiments, the cells have been previously transduced with a viral vector containing a heterologous polynucleotide encoding an antigen receptor. In some embodiments, the genetic engineering comprises contacting T cells of the cell composition with a viral vector particle containing a heterologous polynucleotide encoding an antigen receptor. In some embodiments, the cells express the antigen receptor. In some embodiments, prior to applying in (a), the method comprises genetically engineering the T cells of the cell composition to express an antigen receptor, and / or the T cells of the cell composition have been genetically engineered to express an antigen receptor. Thus, in some embodiments, the provided methods can be used to enrich for viable T cells that have been previously engineered to express an antigen receptor, such as a transgenic TCR or CAR.

[0319] In some embodiments, the cell composition contains T cells that have been cryopreserved and thawed prior to application of the method, hi some embodiments, the method comprises thawing the cryopreserved cell composition to produce a cell composition comprising T cells.

[0320] In some embodiments, the method includes cryopreserving the cells of the concentrated composition to create a cryopreserved composition. In some embodiments, cryopreserving includes suspending the cells in a medium containing a cryoprotectant and freezing the cells. In some embodiments, the freezing is in a controlled-rate freezer. In some embodiments, the method includes thawing the cryopreserved cell composition. In some embodiments, the method includes formulating the thawed cells to create a cell composition for administration as a drug product. In some embodiments, the thawing occurs after the cryopreserved cell composition has been frozen for at least one day. In some embodiments, the thawing occurs after the cryopreserved cell composition has been frozen for at least two days. In some embodiments, the thawing occurs after the cryopreserved cell composition has been frozen for at least three days. In some embodiments, the thawing occurs after the cryopreserved cell composition has been frozen for at least one week, ten days, two weeks, or one month. In some embodiments, the thawing occurs after the cryopreserved cell composition has been frozen for up to ten days, two weeks, one month, two months, three months, or six months.

[0321] In some embodiments, the cell composition contains T cells that have not been cryopreserved or thawed prior to application of the method.

[0322] Populations of cells produced by such methods, and methods for using the same, are also provided.

[0323] Thus, in some embodiments, the compositions of cells obtained from the provided methods exhibit increased viability after centrifugation compared to before centrifugation, hi some embodiments, the increased viability is observed immediately after centrifugation and / or is maintained for a period of time (e.g., hours or days) after centrifugation.

[0324] In some embodiments, the first centrifugal force is between about 500 G and about 5,000 G, between about 500 G and about 4,500 G, between about 500 G and about 4,000 G, between about 500 G and about 3,500 G, between about 500 G and about 3,000 G, between about 500 G and about 2,500 G, between about 500 G and about 2,000 G, between about 500 G and about 1,500 G, between about 500 G and about 1,000 G, between about 1,000 G and about 5,000 G, between about 1,000 G and about 4,500 G, between about 1,000 G and about Between about 1,000G and about 3,500G, between about 1,000G and about 3,000G, between about 1,000G and about 2,500G, between about 1,000G and about 2,000G, between about 1,000G and about 1,500G, between about 1,500G and about 5,000G, between about 1,500G and about 4,500G, between about 1,500G and about 4,000G, between about 1,500G and about 3,500G, between about 1,500G and about 3,000G, between about 1,500G and about 2,500G, Between 1,500G and approximately 2,000G, between approximately 2,000G and approximately 5,000G, between approximately 2,000G and approximately 4,500G, between approximately 2,000G and approximately 4,000G, between approximately 2,000G and approximately 3,500G, between approximately 2,000G and approximately 3,000G, between approximately 2,000G and approximately 2,500G, between approximately 2,500G and approximately 5,000G, between approximately 2,500G and approximately 4,500G, between approximately 2,500G and approximately 4,000G, between approximately 2,500G and approximately 3,500G, between approximately 2,500G and approximately 3 In some embodiments, the first centrifugal force is between about 1,000 G and about 4,000 G, between about 3,000 G and about 5,000 G, between about 3,000 G and about 4,500 G, between about 3,000 G and about 4,000 G, between about 3,000 G and about 3,500 G, between about 3,500 G and about 5,000 G, between about 3,500 G and about 4,500 G, between about 3,500 G and about 4,000 G, between about 4,000 G and about 5,000 G, or between 4,500 G and 5,000 G or between about 4,500 G and about 5,000 G. In some embodiments, the first centrifugal force is between about 1,000 G and about 4,000 G. In some embodiments, the first centrifugal force is between about 2,000 G and about 4,000 G.

[0325] In some embodiments, the first centrifugal force is between about 1,000 G and about 5,000 G, between about 1,500 G and about 4,500 G, between about 2,000 G and about 4,000 G, between about 1,500 G and about 3,500 G, or between about 2,000 G and about 3,000 G. In some embodiments, the first centrifugal force is about 1,000 G. In some embodiments, the first centrifugal force is about 1,500 G. In some embodiments, the first centrifugal force is about 2,000 G. In some embodiments, the first centrifugal force is about 2,500 G. In some embodiments, the first centrifugal force is about 3,000 G. In some embodiments, the first centrifugal force is about 3,500 G. In some embodiments, the first centrifugal force is about 4,000 G. In some embodiments, the first centrifugal force is about 4,500 G. In some embodiments, the first centrifugal force is about 5,000 G.

[0326] In some embodiments, the first flow rate is radially inward. In some embodiments, the first flow rate is directed away from the apex of the conical fluid enclosure. In some embodiments, the first centrifugal force is counteracted by the first flow rate. In some embodiments, the first flow rate is a counter flow rate.

[0327] In some embodiments, the first flow rate is influenced by the flow of medium through the cannula. In some embodiments, the flow of medium through the cannula is from the wide end to the tip of the conical fluid enclosure. In some embodiments, the medium exits the cannula and enters the conical fluid enclosure at its tip.

[0328] In some embodiments, the first flow rate is between about 1 mL / min and about 20 mL / min, between about 3 mL / min and about 18 mL / min, between about 5 mL / min and about 15 mL / min, or between about 8 mL / min and about 12 mL / min. In some embodiments, the first flow rate is about 1 mL / min. In some embodiments, the first flow rate is about 3 mL / min. In some embodiments, the first flow rate is about 5 mL / min. In some embodiments, the first flow rate is about 8 mL / min. In some embodiments, the first flow rate is about 9 mL / min. In some embodiments, the first flow rate is about 10 mL / min. In some embodiments, the first flow rate is about 11 mL / min. In some embodiments, the first flow rate is about 12 mL / min. In some embodiments, the first flow rate is about 15 mL / min. In some embodiments, the first flow rate is about 18 mL / min. In some embodiments, the first flow rate is about 20 mL / min.

[0329] In some embodiments, the first flow rate is between about 5 mL / min and about 15 mL / min. In some embodiments, (i) the first centrifugal force is between about 1,000 G and about 4,000 G, and (ii) the first flow rate is between about 5 mL / min and about 15 mL / min. In some embodiments, (i) the first centrifugal force is between about 2,000 G and about 4,000 G, and (ii) the first flow rate is between about 5 mL / min and about 15 mL / min.

[0330] In some embodiments, the ratio of the first centrifugal force to the first flow rate is between about 100 and about 600, between about 100 and about 500, between about 100 and about 400, between about 100 and about 300, between about 100 and about 200, between about 200 and about 600, between about 200 and about 500, between about 200 and about 400, between about 200 and about 300, between about 300 and about 600, between about 300 and about 500, between about 300 and about 400, between about 400 and about 600, between about 400 and about 500, or between about 500 and about 600. In some embodiments, the ratio of the first centrifugal force to the first flow rate is between about 200 and about 500. In some embodiments, the ratio of the first centrifugal force to the first flow rate is between about 200 and about 400. Ratios of centrifugal force to flow rate herein are the ratio of centrifugal force in G to flow rate in mL / min unless otherwise indicated.

[0331] In some embodiments, the ratio of the first centrifugal force to the first flow rate is between about 200 and about 400, between about 225 and about 375, between about 250 and about 350, or between about 275 and about 325. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 200. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 200. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 225. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 250. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 275. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 300. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 325. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 350. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 375. In some embodiments, the ratio of the first centrifugal force to the first flow rate is about 400.

[0332] In some embodiments, the first centrifugal force is between about 2,000 G and about 4,000 G and the first flow rate is between about 5 mL / min and about 15 mL / min, hi some embodiments, the first centrifugal force is about 3,000 G and the first flow rate is about 10 mL / min.

[0333] In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition (e.g., the engineered cell composition) for about 15 seconds, about 30 seconds, about 45 seconds, about 60 seconds, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, or about 15 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 15 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 30 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 45 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for about 60 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition at least until a fluidized bed of cells is established.

[0334] In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 15 seconds, at least about 30 seconds, at least about 45 seconds, at least about 60 seconds, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 10 minutes, or at least about 15 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 15 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 20 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 25 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 30 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 45 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 60 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 2 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 3 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 4 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 5 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 10 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for at least about 15 minutes.

[0335] In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for between 15 seconds and 60 seconds, or for about 15 seconds and about 60 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for between 25 seconds and 60 seconds, or for about 25 seconds and about 60 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for between 30 seconds and 60 seconds, or for about 30 seconds and about 60 seconds. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for between 15 seconds and 2 minutes, or for about 15 seconds and about 2 minutes. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition for between 25 seconds and 2 minutes, or for about 25 seconds and about 2 minutes.

[0336] In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition until a predetermined number of cells are loaded into the conical fluid enclosure. In some embodiments, the first centrifugal force and the first flow rate are applied to the cell composition until the predetermined number of cells become part of a fluidized bed of cells. In some embodiments, the predetermined number of cells is a predetermined number of T cells. In some embodiments, the predetermined number of cells, e.g., T cells, is between about 10 million and 100 million, between about 10 million and 90 million, between about 10 million and 80 million, between about 10 million and 70 million, between about 10 million and 60 million, between about 10 million and 50 million, between about 10 million and 40 million, between about 10 million and 30 million, between about 10 million and 20 million, between about 20 million and 100 million, or about 20 million. Between 0 and 90 million, between about 20 and 80 million, between about 20 and 70 million, between about 20 and 60 million, between about 20 and 50 million, between about 20 and 40 million, between about 20 and 30 million, between about 30 and 100 million, between about 30 and 90 million, between about 30 and 80 million, between about 30 and 70 million, between about 30 and 60 million Between about 30 million and 50 million, Between about 30 million and 40 million, Between about 40 million and 100 million, Between about 40 million and 90 million, Between about 40 million and 80 million, Between about 40 million and 70 million, Between about 40 million and 60 million, Between about 40 million and 50 million, Between about 50 million and 100 million, Between about 50 million and 90 million, Between about 50 million and 80 million, Between about 50 million and 700 million 0 million, between about 50 and 60 million, between about 60 and 100 million, between about 60 and 90 million, between about 60 and 80 million, between about 60 and 70 million, between about 70 and 100 million, between about 70 and 90 million, between about 70 and 80 million, between about 80 and 100 million, between about 80 and 90 million, or between about 90 and 100 million cells, e.g., T cells. In some embodiments, the predetermined number of cells, e.g., T cells, is about 50 million cells, e.g., T cells.

[0337] In some embodiments, the second flow rate is radially inward. In some embodiments, the second flow rate is directed away from the apex of the conical fluid enclosure. In some embodiments, the second centrifugal force is counteracted by the second flow rate. In some embodiments, the second flow rate is an opposing flow rate.

[0338] In some embodiments, the second flow rate is influenced by the flow of medium through the cannula. In some embodiments, the flow of medium through the cannula is from the wide end to the tip of the conical fluid enclosure. In some embodiments, the medium exits the cannula and enters the conical fluid enclosure at its tip.

[0339] In other embodiments, the second flow rate is directed radially outward. In some embodiments, the second flow rate is directed toward the apex of the conical fluid enclosure. In some embodiments, the second centrifugal force and the second flow rate are in the same or substantially the same direction.

[0340] In some embodiments, the second flow rate is influenced by the flow of medium through the conical fluid enclosure. In some embodiments, the flow of medium through the conical fluid enclosure is from the wide end to the tip of the conical fluid enclosure. In some embodiments, the medium exits the tip of the conical fluid enclosure and enters the cannula.

[0341] In some embodiments, the second centrifugal force is between about 100 G and about 4,000 G, between about 100 G and about 3,500 G, between about 100 G and about 3,000 G, between about 100 G and about 2,500 G, between about 100 G and about 2,000 G, between about 100 G and about 1,500 G, between about 100 G and about 1,000 G, between about 100 G and about 500 G, between about 100 G and about 350 G, between about 350 G and about 4,000 G, between about 350 G and about 3,500 G, or about 350 G. between about 3,000G, between about 350G and about 2,500G, between about 350G and about 2,000G, between about 350G and about 1,500G, between about 350G and about 1,000G, between about 350G and about 500G, between about 500G and about 4,000G, between about 500G and about 3,500G, between about 500G and about 3,000G, between about 500G and about 2,500G, between about 500G and about 2,000G, between about 500G and about 1,500G, between about 500G and about 1 ,000G, between about 1,000G and about 4,000G, between about 1,000G and about 3,500G, between about 1,000G and about 3,000G, between about 1,000G and about 2,500G, between about 1,000G and about 2,000G, between about 1,000G and about 1,500G, between about 1,500G and about 4,000G, between about 1,500G and about 3,500G, between about 1,500G and about 3,000G, between about 1,500G and about 2,500G, between about 1,500G and between about 2,000G, between about 2,000G and about 4,000G, between about 2,000G and about 3,500G, between about 2,000G and about 3,000G, between about 2,000G and about 2,500G, between about 2,500G and about 4,000G, between about 2,500G and about 3,500G, between about 2,500G and about 3,000G, between about 3,000G and about 4,000G, between about 3,000G and about 3,500G, or between about 3,500G and about 4,000G.

[0342] In some embodiments, the second centrifugal force is between about 350 G and about 4,000 G. In some embodiments, the second centrifugal force is between about 350 G and about 3,000 G. In some embodiments, the second centrifugal force is between about 1,500 G and about 3,000 G. In some embodiments, the T cells are activated T cells. In some embodiments, the T cells, e.g., activated T cells, have an average diameter of about 5 μm to about 25 μm, about 5 μm to about 20 μm, about 5 μm to about 15 μm, about 5 μm to about 10 μm, about 10 μm to about 25 μm, about 10 μm to about 20 μm, about 10 μm to about 15 μm, about 15 μm to about 25 μm, about 15 μm to about 20 μm, or about 20 μm to about 25 μm. In some embodiments, the T cells have an average diameter of about 9 μm to about 20 μm.

[0343] In some embodiments, the T cells have an average diameter of about 10 μm to about 20 μm. In some embodiments, the T cells have an average diameter of about 12 μm to about 20 μm. In some embodiments, the T cells have an average diameter of about 14 μm to about 20 μm.

[0344] In some embodiments, the second centrifugal force is between about 350 G and about 4,000 G. In some embodiments, the second centrifugal force is between about 350 G and about 3,000 G. In some embodiments, the second centrifugal force is between about 500 G and about 1,500 G. In some embodiments, the second centrifugal force is between about 700 G and about 1,300 G. In some embodiments, the second centrifugal force is between about 800 G and about 1,200 G. In some embodiments, the second centrifugal force is between about 900 G and about 1,100 G. In some embodiments, the second centrifugal force is about 1,000 G. In some embodiments, the T cells are non-activated T cells or less-activated T cells. In some embodiments, the T cells are cryopreserved and thawed prior to application of the method. In some embodiments, the T cells have an average diameter of less than 9 μm. In some embodiments, the T cells have an average diameter of about 3 μm to about 9 μm, about 4 μm to about 9 μm, about 5 μm to about 9 μm, about 6 μm to about 9 μm, about 7 μm to about 9 μm, or about 8 μm to about 9 μm. In some embodiments, the T cells have an average diameter of about 6 μm to about 9 μm.

[0345] In some embodiments, the second centrifugal force is between about 100 G and about 2,000 G, between about 200 G and about 1,800 G, between about 500 G and about 1,500 G, or between about 750 G and about 1,250 G. In some embodiments, the second centrifugal force is about 250 G. In some embodiments, the second centrifugal force is about 500 G. In some embodiments, the second centrifugal force is about 600 G. In some embodiments, the second centrifugal force is about 700 G. In some embodiments, the second centrifugal force is about 800 G. In some embodiments, the second centrifugal force is about 900 G. In some embodiments, the second centrifugal force is about 1,000 G. In some embodiments, the second centrifugal force is about 1,100 G. In some embodiments, the second centrifugal force is about 1,200 G. In some embodiments, the second centrifugal force is about 1,300 G. In some embodiments, the second centrifugal force is about 1,400 G. In some embodiments, the second centrifugal force is about 1,500 G. In some embodiments, the second centrifugal force is about 1,300 G. In some embodiments, the second centrifugal force is about 1,750 G. In some embodiments, the second centrifugal force is about 2,000 G.

[0346] In some embodiments, the second flow rate is between about 5 mL / min and about 100 mL / min, between about 5 mL / min and about 90 mL / min, between about 5 mL / min and about 80 mL / min, between about 5 mL / min and about 70 mL / min, between about 5 mL / min and about 60 mL / min, between about 5 mL / min and about 50 mL / min, between about 5 mL / min and about 40 mL / min, between about 5 mL / min and about 30 mL / min, between about 5 mL / min and about 20 mL / min, between about 5 mL / min and about 10 mL / min, between about 10 mL / min and about 100 mL / min, between about 10 mL / min and about 90 mL / min, between about 10 mL / min and about Between about 80 mL / min, between about 10 mL / min and about 70 mL / min, between about 10 mL / min and about 60 mL / min, between about 10 mL / min and about 50 mL / min, between about 10 mL / min and about 40 mL / min, between about 10 mL / min and about 30 mL / min, between about 10 mL / min and about 20 mL / min, between about 20 mL / min and about 100 mL / min, between about 20 mL / min and about 90 mL / min, between about 20 mL / min and about 80 mL / min, between about 20 mL / min and about 70 mL / min, between about 20 mL / min and about 60 mL / min, between about 20 mL / min and about 50 mL / min, about 20 mL / min between about 20 mL / min and about 30 mL / min, between about 30 mL / min and about 100 mL / min, between about 30 mL / min and about 90 mL / min, between about 30 mL / min and about 80 mL / min, between about 30 mL / min and about 70 mL / min, between about 30 mL / min and about 60 mL / min, between about 30 mL / min and about 50 mL / min, between about 30 mL / min and about 40 mL / min, between about 40 mL / min and about 100 mL / min, between about 40 mL / min and about 90 mL / min, between about 40 mL / min and about 80 mL / min, between about 40 mL / min and about 70 mL / min, between about 40 mL / min and about 50 mL / min, between about 50 mL / min and about 100 mL / min, between about 50 mL / min and about 90 mL / min, between about 50 mL / min and about 80 mL / min, between about 50 mL / min and about 70 mL / min, between about 50 mL / min and about 60 mL / min, between about 60 mL / min and about 100 mL / min, between about 60 mL / min and about 90 mL / min, between about 60 mL / min and about 80 mL / min, between about 60 mL / min and about 70 mL / min, between about 70 mL / min and about 100 mL / min, between about 70 mL / min and about 90 mL / min,Between about 70 mL / min and about 80 mL / min, between about 80 mL / min and about 100 mL / min, between about 80 mL / min and about 90 mL / min, or between about 90 mL / min and about 100 mL / min.

[0347] In some embodiments, the second flow rate is between about 5 mL / min and about 100 mL / min. In some embodiments, (i) the second centrifugal force is between about 350 G and about 4,000 G, and (ii) the second flow rate is between about 5 mL / min and about 100 mL / min. In some embodiments, the second flow rate is between about 10 mL / min and about 65 mL / min. In some embodiments, the second flow rate is between about 10 mL / min and about 35 mL / min. In some embodiments, the T cells are activated T cells. In some embodiments, T cells, e.g., activated T cells, have an average diameter of about 5 μm to about 25 μm, about 5 μm to about 20 μm, about 5 μm to about 15 μm, about 5 μm to about 10 μm, about 10 μm to about 25 μm, about 10 μm to about 20 μm, about 10 μm to about 15 μm, about 15 μm to about 25 μm, about 15 μm to about 20 μm, or about 20 μm to about 25 μm. In some embodiments, T cells have an average diameter of about 9 μm to about 20 μm.

[0348] In some embodiments, the T cells have an average diameter of about 10 μm to about 20 μm. In some embodiments, the T cells have an average diameter of about 12 μm to about 20 μm. In some embodiments, the T cells have an average diameter of about 14 μm to about 20 μm.

[0349] In some embodiments, the second flow rate is 30 mL / min or less. In some embodiments, the second flow rate is between about 25 mL / min and about 30 mL / min. In some embodiments, (i) the second centrifugal force is between about 500 G and about 1,500 G, and (ii) the second flow rate is between about 25 mL / min and about 30 mL / min. In some embodiments, the T cells are non-activated T cells or less-activated T cells. In some embodiments, the T cells are cryopreserved and thawed prior to application of the method. In some embodiments, the T cells have an average diameter of less than 9 μm. In some cases, the T cells have an average diameter of about 3 μm to about 9 μm, about 4 μm to about 9 μm, about 5 μm to about 9 μm, about 6 μm to about 9 μm, about 7 μm to about 9 μm, or about 8 μm to about 9 μm. In some embodiments, the T cells have an average diameter of about 6 μm to about 9 μm.

[0350] In some embodiments, the second flow rate is 30 mL / min or less. In some embodiments, the second flow rate is between about 25 mL / min and about 30 mL / min. In some embodiments, (i) the second centrifugal force is between about 500 G and about 1,500 G, and (ii) the second flow rate is between about 25 mL / min and about 30 mL / min. In some embodiments, the T cells are non-activated T cells or less-activated T cells. In some embodiments, the method includes activating and culturing the cells of the cell composition (e.g., culturing the cells for at least 24, 48, 72, or 96 hours) before applying the first centrifugal force and the first flow rate to the cell composition. In some embodiments, the method includes cryopreserving the T cells of the concentrated composition and, if appropriate, thawing them. In some embodiments, the T cells have an average diameter of less than 9 μm. In some embodiments, the T cells have an average diameter of about 3 μm to about 9 μm, about 4 μm to about 9 μm, about 5 μm to about 9 μm, about 6 μm to about 9 μm, about 7 μm to about 9 μm, or about 8 μm to about 9 μm. In some embodiments, the T cells have an average diameter of about 6 μm to about 9 μm.

[0351] In some embodiments, the second flow rate is between about 65 mL / min and about 100 mL / min. In some embodiments, (i) the second centrifugal force is between about 1,500 G and about 3,000 G, and (ii) the second flow rate is between about 65 mL / min and about 100 mL / min. In some embodiments, the second flow rate is between about 65 mL / min and about 90 mL / min. In some embodiments, the second flow rate is between about 65 mL / min and about 80 mL / min. In some embodiments, the T cells are non-activated T cells or less-activated T cells. In some embodiments, the T cells are cryopreserved and thawed prior to application of the method. In some embodiments, the T cells have an average diameter of less than 9 μm. In some embodiments, the T cells have an average diameter of about 3 μm to about 9 μm, about 4 μm to about 9 μm, about 5 μm to about 9 μm, about 6 μm to about 9 μm, about 7 μm to about 9 μm, or about 8 μm to about 9 μm. In some embodiments, the T cells have an average diameter of about 6 μm to about 9 μm.

[0352] In some embodiments, the second flow rate is between about 65 mL / min and about 100 mL / min. In some embodiments, (i) the second centrifugal force is between about 1,500 G and about 3,000 G, and (ii) the second flow rate is between about 65 mL / min and about 100 mL / min. In some embodiments, the second flow rate is between about 65 mL / min and about 90 mL / min. In some embodiments, the second flow rate is between about 65 mL / min and about 80 mL / min. In some embodiments, the T cells are non-activated T cells or less-activated T cells. In some embodiments, the method includes cryopreserving the cells of the concentrated composition and, if appropriate, thawing. In some embodiments, the T cells have an average diameter of less than 9 μm. In some embodiments, the T cells have an average diameter of about 3 μm to about 9 μm, about 4 μm to about 9 μm, about 5 μm to about 9 μm, about 6 μm to about 9 μm, about 7 μm to about 9 μm, or about 8 μm to about 9 μm. In some embodiments, the T cells have an average diameter of about 6 μm to about 9 μm.

[0353] In some embodiments, the second flow rate is between about 10 and about 100 mL / min, between about 15 and about 90 mL / min, between about 20 and about 80 mL / min, between about 25 and about 70 mL / min, between about 30 and about 60 mL / min, or between about 35 and about 50 mL / min. In some embodiments, the second flow rate is about 20 mL / min, about 21 mL / min, about 22 mL / min, about 23 mL / min, about 24 mL / min, about 25 mL / min, about 25.5 mL / min, about 26 mL / min, about 26.5 mL / min, about 27 mL / min, about 27.5 mL / min, about 28 mL / min, about 28.5 mL / min, about 29 mL / min, about 29.5 mL / min, about 30 mL / min, about 31 mL / min, about 32 mL / min, about 33 mL / min, about 34 mL / min, or about 35 mL / min. In some embodiments, the second flow rate is about 25 mL / min. In some embodiments, the second flow rate is about 25.5 mL / min. In some embodiments, the second flow rate is about 26 mL / min. In some embodiments, the second flow rate is about 26.5 mL / min. In some embodiments, the second flow rate is about 27 mL / min. In some embodiments, the second flow rate is about 27.5 mL / min. In some embodiments, the second flow rate is about 28 mL / min. In some embodiments, the second flow rate is about 28.5 mL / min. In some embodiments, the second flow rate is about 29 mL / min. In some embodiments, the second flow rate is about 29.5 mL / min. In some embodiments, the second flow rate is about 30 mL / min.

[0354] In some embodiments, the ratio of the second centrifugal force to the second flow rate is between about 20 and about 100, between about 20 and about 90, between about 20 and about 80, between about 20 and about 70, between about 20 and about 60, between about 20 and about 50, between about 20 and about 40, between about 20 and about 30, between about 30 and about 100, between about 30 and about 90, between about 30 and about 80, between about 30 and about 70, between about 30 and about 60, between about 30 and about 50, between about 30 and about 40, between about 40 and about 100, between about 40 and about 90, Between about 40 and about 80, between about 40 and about 70, between about 40 and about 60, between about 40 and about 50, between about 50 and about 100, between about 50 and about 90, between about 50 and about 80, between about 50 and about 70, between about 50 and about 60, between about 60 and about 100, between about 60 and about 90, between about 60 and about 80, between about 60 and about 70, between about 70 and about 100, between about 70 and about 90, between about 70 and about 80, between about 80 and about 100, between about 80 and about 90, or between about 90 and about 100.

[0355] In some embodiments, the ratio of the second centrifugal force to the second flow rate is between about 30 and about 70. In some embodiments, the ratio of the second centrifugal force to the second flow rate is between about 30 and about 40. In some embodiments, the T cells are activated T cells. In some embodiments, the T cells, e.g., activated T cells, have an average diameter of about 5 μm to about 25 μm, about 5 μm to about 20 μm, about 5 μm to about 15 μm, about 5 μm to about 10 μm, about 10 μm to about 25 μm, about 10 μm to about 20 μm, about 10 μm to about 15 μm, about 15 μm to about 25 μm, about 15 μm to about 20 μm, or about 20 μm to about 25 μm. In some embodiments, the T cells have an average diameter of about 9 μm to about 20 μm.

[0356] In some embodiments, the T cells have an average diameter of about 10 μm to about 20 μm. In some embodiments, the T cells have an average diameter of about 12 μm to about 20 μm. In some embodiments, the T cells have an average diameter of about 14 μm to about 20 μm.

[0357] In some embodiments, the T cells have an average diameter of about 9 μm to about 20 μm, and the ratio of the second centrifugal force to the second flow rate is between about 30 and about 70. In some embodiments, the T cells have an average diameter of 10 μm to 20 μm (e.g., 12 μm to 20 μm or 14 μm to 20 μm), and the ratio of the second centrifugal force to the second flow rate is between about 30 and about 70. In some embodiments, the T cells have an average diameter of 12 μm to 20 μm, and the ratio of the second centrifugal force to the second flow rate is between about 30 and about 70. In some embodiments, the T cells have an average diameter of 14 μm to 20 μm, and the ratio of the second centrifugal force to the second flow rate is between about 30 and about 70.

[0358] In some embodiments, the T cells have an average diameter of less than about 9 μm and the ratio of the second centrifugal force to the second flow rate is between about 30 and about 40.

[0359] In some embodiments, the ratio of the second centrifugal force to the second flow rate is between about 30 and about 40. In some embodiments, the T cells are non-activated T cells or less-activated T cells. In some embodiments, the T cells are cryopreserved and thawed prior to application of the method. In some embodiments, the T cells have an average diameter of less than 9 μm. In some embodiments, the T cells have an average diameter of about 3 μm to about 9 μm, about 4 μm to about 9 μm, about 5 μm to about 9 μm, about 6 μm to about 9 μm, about 7 μm to about 9 μm, or about 8 μm to about 9 μm. In some embodiments, the T cells have an average diameter of about 6 μm to about 9 μm.

[0360] In some embodiments, the ratio of the second centrifugal force to the second flow rate is between about 20 and about 100, between about 25 and about 80, or between about 30 and about 60. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 20. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 25. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 30. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 35. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 40. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 45. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 50. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 55. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 60. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 65. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 70. In some embodiments, the ratio of the second centrifugal force to the second flow rate is about 75.

[0361] In some embodiments, the second centrifugal force is between about 500 G and about 1,500 G and the second flow rate is between about 25 mL / min and about 30 mL / min, hi some embodiments, the second centrifugal force is about 1,000 G and the flow rate is about 28.5 mL / min.

[0362] In some embodiments, the second centrifugal force and the second flow rate are applied to the cell composition for about 5 minutes to about 100 minutes, about 10 minutes to about 90 minutes, about 15 minutes to about 80 minutes, about 20 minutes to about 70 minutes, about 25 minutes to about 60 minutes, about 30 minutes to about 50 minutes, or about 35 minutes to about 40 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the cell composition for about 5 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the cell composition for about 10 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the cell composition for about 15 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the cell composition for about 20 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the cell composition for about 25 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the cell composition for about 30 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the cell composition for about 45 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the cell composition for about 60 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the cell composition for about 75 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the cell composition for about 90 minutes.

[0363] In some embodiments, the second centrifugal force and the second flow rate are applied to the cell composition for at least about 15 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the cell composition for at least about 30 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the cell composition for at least about 45 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the cell composition for at least about 60 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the cell composition for at least about 75 minutes. In some embodiments, the second centrifugal force and the second flow rate are applied to the cell composition for at least about 90 minutes.

[0364] A. Cellular Compositions (e.g., Engineered Cellular Compositions) In some embodiments, a cell composition comprising T cells (e.g., T cells previously engineered to express an antigen receptor) is enriched for viable cells in a continuous counterflow centrifuge system. In some embodiments, the concentration of cells in the engineered cell composition is greater than or equal to 1.0 x 10 5 cells / mL~1.0×10 8 cells / mL or approximately 1.0 x 10 5 cells / mL ~ approx. 1.0×10 8 cells / mL, e.g., at least or about at least or about 1.0 x 10 5 cells / mL, 5×10 5 cells / mL, 1×10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL or 1 x 10 8 In some embodiments, the engineered cell composition comprises about 1 x 10 cells / mL. 6 In some embodiments, the engineered cell composition comprises about 1.25 x 10 cells / mL. 6 In some embodiments, the cell composition comprises about 1.5 x 10 cells / mL. 6 In some embodiments, the engineered cell composition comprises about 1.75 x 10 cells / mL. 6 In some embodiments, the engineered cell composition comprises about 2 x 10 cells / mL. 6 In some embodiments, the engineered cell composition comprises about 2.25 x 10 cells / mL. 6 In some embodiments, the engineered cell composition comprises about 2.5 x 10 cells / mL. 6 In some embodiments, the engineered cell composition comprises about 2.75 x 10 cells / mL. 6 In some embodiments, the engineered cell composition comprises about 3 x 10 cells / mL. 6 Contains cells / mL.

[0365] In some embodiments, the volume of the engineered cell composition is between about 20 mL and about 300 mL, between about 25 mL and about 250 mL, between about 30 mL and about 200 mL, between about 35 mL, and about 150 mL, or between about 40 mL and about 100 mL. In some embodiments, the volume of the engineered cell composition is about 20 mL. In some embodiments, the volume of the engineered cell composition is about 25 mL. In some embodiments, the volume of the engineered cell composition is about 30 mL. In some embodiments, the volume of the engineered cell composition is about 35 mL. In some embodiments, the volume of the engineered cell composition is about 40 mL. In some embodiments, the volume of the engineered cell composition is about 45 mL. In some embodiments, the volume of the engineered cell composition is about 50 mL. In some embodiments, the volume of the engineered cell composition is about 55 mL. In some embodiments, the volume of the engineered cell composition is about 60 mL. In some embodiments, the volume of the engineered cell composition is about 70 mL. In some embodiments, the volume of the engineered cell composition is about 80 mL. In some embodiments, the volume of the engineered cell composition is about 100 mL. In some embodiments, the volume of the engineered cell composition is about 125 mL. In some embodiments, the volume of the engineered cell composition is about 150 mL. In some embodiments, the volume of the engineered cell composition is about 175 mL. In some embodiments, the volume of the engineered cell composition is about 200 mL.

[0366] In some embodiments, the engineered cell composition comprises a total of about 1 x 10 8 of cells, total number approximately 2 × 10 8 cells, total number approximately 3 × 10 8 cells, total number approximately 4 × 10 8 cells, total number approximately 5 × 10 8 cells, total number approximately 6 × 10 8 cells, total number approximately 7 × 10 8 cells, total number approximately 8 × 10 8 cells, total number approximately 9 × 10 8 of cells, or a total of approximately 1 x 10 9 Contains cells of.

[0367] In some embodiments, the T cells have an average diameter of about 5 μm to about 25 μm, about 5 μm to about 20 μm, about 5 μm to about 15 μm, about 5 μm to about 10 μm, about 10 μm to about 25 μm, about 10 μm to about 20 μm, about 10 μm to about 15 μm, about 15 μm to about 25 μm, about 15 μm to about 20 μm, or about 20 μm to about 25 μm. In some embodiments, the T cells have an average diameter of about 9 μm to about 20 μm. In some embodiments, the T cells are activated T cells.

[0368] In some embodiments, the T cells have an average diameter of about 10 μm to about 20 μm. In some embodiments, the T cells have an average diameter of about 12 μm to about 20 μm. In some embodiments, the T cells have an average diameter of about 14 μm to about 20 μm. In some embodiments, the T cells are activated T cells.

[0369] In some embodiments, the T cells have an average diameter of less than 9 μm. In some embodiments, the T cells have an average diameter of about 3 μm to about 9 μm, about 4 μm to about 9 μm, about 5 μm to about 9 μm, about 6 μm to about 9 μm, about 7 μm to about 9 μm, or about 8 μm to about 9 μm. In some embodiments, the T cells have an average diameter of about 6 μm to about 9 μm.

[0370] In some embodiments, cells are incubated and / or cultured prior to genetic manipulation by the provided methods. Incubation steps may include culture, cultivation, stimulation, activation, and / or expansion. In some embodiments, the methods include incubating T cells of the cell composition under stimulatory conditions prior to genetic manipulation. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells within the population, to mimic antigen exposure, and / or to stimulate cells for genetic manipulation, e.g., introduction of a recombinant antigen receptor. Conditions may include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells.

[0371] In some embodiments, the T cells of the cell composition are incubated under stimulatory conditions prior to application of the first centrifugal force and the first flow rate. In some embodiments, the method includes incubating the T cells of the cell composition under stimulatory conditions prior to loading the cell composition into a centrifuge system. In some embodiments, the T cells of the cell composition are incubated under stimulatory conditions prior to loading the cell composition into a centrifuge system. In some embodiments, the cell composition comprises activated T cells. In some embodiments, the cell composition comprises T cells that express HLA-DR, CD25, CD69, CD71, CD40L, 4-1BB, or a combination thereof.

[0372] In some embodiments, the stimulatory condition comprises the presence of a stimulatory reagent. In some embodiments, the stimulatory reagent can activate an intracellular signaling domain of a TCR complex. In some aspects, the agent activates or initiates a TCR / CD3 intracellular signaling cascade in T cells. Such agents can include antibodies, e.g., antibodies specific for TCR components and / or costimulatory receptors, e.g., anti-CD3, anti-CD28, and / or one or more cytokines, bound to a solid support, e.g., beads. In some embodiments, the stimulatory reagent can activate one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules. In some embodiments, the stimulatory reagent comprises (i) a primary agent that specifically binds to a member of the TCR complex; and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule. In some embodiments, the primary agent specifically binds to CD3. In some embodiments, the costimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS. In some embodiments, at least one of the primary and secondary agents comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the first agent is or comprises an anti-CD3 antibody or an antigen-binding fragment thereof. In some embodiments, the second agent is or comprises an anti-CD28 antibody or an antigen-binding fragment thereof. Optionally, the expansion method may further comprise adding an anti-CD3 and / or anti-CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml).

[0373] In some embodiments, the stimulating agent comprises IL-2 and / or IL-15, e.g., an IL-2 concentration of at least about 10 units / mL. In some aspects, the incubation is carried out according to techniques such as those described in U.S. Patent No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0374] In some embodiments, the stimulatory conditions include a temperature suitable for the growth of human T lymphocytes, e.g., at least about 25 degrees Celsius, typically at least about 30 degrees Celsius, and typically at or about 37 degrees Celsius. Optionally, the incubation may further include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. The LCL may be irradiated with gamma radiation in the range of about 6000 to 10,000 rads. The LCL feeder cells, in some aspects, are provided in any suitable amount, e.g., at a ratio of at least about 10:1 LCL feeder cells to initial T lymphocytes.

[0375] In embodiments, antigen-specific T cells, e.g., antigen-specific CD4+ and / or CD8+ T cells, are obtained by stimulating naive or antigen-specific T lymphocytes with an antigen. For example, antigen-specific T cell lines or clones against a cytomegalovirus antigen can be generated by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen.

[0376] In some cases, viral vector particles may be used that do not require cells, such as T cells, to be activated. In some such instances, cells may be selected and / or transduced prior to and / or in the absence of activation.

[0377] In some embodiments, at least 40%, 50%, 60%, 70%, 80%, 90% or more of the cells, e.g., T cells, in the cell composition are activated, e.g., in some cases, surface positive for one or more of HLA-DR, CD25, CD69, CD71, CD40L, and / or 4-1BB. In some embodiments, the cells are activated with an activating agent, e.g., in the presence of anti-CD3 / anti-CD28, prior to the initiation of application of the first centrifugal force and the first flow rate, e.g., prior to establishment of the fluidized bed and / or prior to the initiation of transduction. Methods for expanding T cell populations in vitro in the absence of or with low amounts of exogenous growth factors are known in the art (see, e.g., U.S. Pat. No. 6,352,694 B1 and European Patent EP 0 700 430 B1). Generally, such methods utilize a solid surface of greater than 1 μM onto which various binding agents (e.g., anti-CD3 and / or anti-CD28 antibodies) are immobilized. For example, Dynabeads® CD3 / CD28 (Invitrogen) is a commercially available reagent for T cell expansion, which is a uniform, 4.5 μm superparamagnetic, sterile, non-pyrogenic polystyrene bead coated with a mixture of affinity-purified monoclonal antibodies against CD3 and CD28 cell surface molecules on human T cells. In some embodiments, activating agents, such as anti-CD3 and / or anti-CD28, can be immobilized on beads, such as magnetic beads.

[0378] In some embodiments, cell activation is also carried out in the presence of IL-2 (e.g., 50 IU / mL to 200 IU / mL or about 50 IU / mL to about 200 IU / mL, e.g., 100 IU / mL or about 100 IU / mL). In some embodiments, activation is carried out for between 1 hour and 96 hours, 1 hour and 72 hours, 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours, or 12 hours and 24 hours, or for about 1 hour and about 96 hours, about 1 hour and about 72 hours, about 1 hour and about 48 hours, about 4 hours and about 36 hours, about 8 hours and about 30 hours, or about 12 hours and about 24 hours, e.g., at least or about at least 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours. In some embodiments, activation is carried out at a temperature above or above about 25°C, e.g., generally above or above about 32°C, 35°C, or 37°C, e.g., 37°C±2°C or about 37°C±2°C, e.g., at a temperature of 37°C or about 37°C.

[0379] In some embodiments, the cells are not activated with an activating agent, e.g., in the presence of anti-CD3 / anti-CD28, prior to the initiation of contact, e.g., prior to the initiatio...

Claims

1. 1. A method for producing a composition of genetically engineered T cells, comprising: (a) applying a first centrifugal force and a first flow rate to a cell composition comprising T cells in a conical fluid enclosure of a centrifuge system to produce a fluidized bed of cells, wherein the centrifuge system is a continuous counterflow centrifuge system; (b) generating an input composition comprising the cell composition and the viral vector particles by introducing the viral vector particles into the conical fluid enclosure; and (c) applying a second centrifugal force and a second flow rate to the input composition, wherein the second centrifugal force and the second flow rate recirculate the viral vector particles in a flow path of the centrifuge system, thereby producing genetically engineered T cells. A method comprising:

2. 2. The method of claim 1, wherein (i) the second centrifugal force is between about 500 G and about 1,500 G, and (ii) the second flow rate is between about 10 mL / min and about 100 mL / min.

3. 1. A method for producing a composition of genetically engineered T cells, comprising: (a) applying a first centrifugal force and a first flow rate to an input composition comprising (i) viral vector particles and (ii) a cell composition comprising T cells in a conical fluid enclosure of a centrifuge system to produce a fluidized bed of cells, wherein the centrifuge system is a continuous counterflow centrifuge system; and (b) applying a second centrifugal force and a second flow rate to the input composition in the conical fluid enclosure, wherein the second centrifugal force and the second flow rate recirculate the viral vector particles in a flow path of the centrifuge system, thereby producing genetically engineered T cells. A method comprising:

4. 4. The method of any one of claims 1 to 3, further comprising generating the input composition by introducing the cellular composition and the viral vector particles into the conical fluid enclosure, wherein the introduction of the cellular composition occurs before, during, and / or after the introduction of the viral vector particles.

5. 4. The method of any one of claims 1 to 3, further comprising applying a third centrifugal force and a third flow rate to the genetically engineered T cells in a conical fluid enclosure of the centrifuge system to produce an output composition comprising the genetically engineered T cells, and optionally, applying a third centrifugal force and a third flow rate to the genetically engineered cells allows for collection or recovery of the output composition.

6. 6. The method of claim 5, wherein the percentage of viable T cells in the output composition is greater than the percentage of viable T cells in the input composition, optionally by at least about 5%.

7. 6. The method of claim 5, wherein at least 5% or at least about 5% of the T cells in the output composition are transduced with the viral vector particles.

8. (i)(a) the first centrifugal force is between about 625 G and about 3,000 G or between about 2,000 G and about 4,000 G; or (b) the first flow rate is between about 40 mL / min and about 50 mL / min; and / or (ii) the ratio of the first centrifugal force (G) to the first flow rate (mL / min) is between about 40 and 200; 4. The method according to any one of claims 1 to 3. (i) the ratio of the second centrifugal force (in G) to the second flow rate (in mL / min) is between about 20 and about 100; and / or (ii) the second centrifugal force and the second flow rate are applied to the input composition for at least about 15 minutes; 4. The method according to any one of claims 1 to 3.

10. 4. The method of any one of claims 1 to 3, wherein the method comprises incubating the T cells of the cell composition under stimulatory conditions prior to said applying in (a) and / or wherein the T cells of the cell composition are incubated under stimulatory conditions prior to said applying in (a).

11. 11. The method of claim 10, wherein the stimulatory conditions comprise the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules.

12. 12. The method of claim 11, wherein the stimulatory reagent comprises: (i) a first agent that specifically binds to a member of the TCR complex, optionally, that specifically binds to CD3; and (ii) a second agent that specifically binds to a T cell costimulatory molecule, optionally wherein the costimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, and ICOS. (i) at least one of the first and second agents comprises an antibody or an antigen-binding fragment thereof; and / or (ii) the first agent is an anti-CD3 antibody or an antigen-binding fragment thereof, and the second agent is an anti-CD28 antibody or an antigen-binding fragment thereof; The method of claim 12.

14. 13. The method of claim 12, wherein the first agent and the second agent are each present on the surface of a solid support, optionally wherein the solid support is a bead. (i) the stimulatory conditions include the presence of one or more recombinant cytokines; and / or (ii) the stimulatory conditions include the presence of one or more of recombinant IL-2, IL-7, and IL-15; The method of claim 10.

16. The method comprises collecting the output composition, and / or the output composition is collected and, optionally, (i) the method comprises incubating the genetically engineered T cells of the collected output composition, and / or the genetically engineered T cells of the collected output composition are incubated, and / or (ii) the percentage of viable T cells in the collected output composition about 1 day or about 5 days after collection is greater than the percentage of viable T cells in the input composition; The method of claim 5.

17. The input composition has a total number of about 1×10 ６ A total of approximately 2 × 10 T cells ９ T cells among T cells of The input composition comprises a total of at least about 1 x 10 ８ and / or The volume of the input composition is between about 5 ml and about 20,000 ml, optionally between 30 ml and 60 ml; 4. The method according to any one of claims 1 to 3.

18. 6. The method of claim 5, wherein the volume of the output composition is between about 2.5 mL and about 60 mL.

19. 1. A method for enriching a cellular composition for viable cells, comprising: (a) applying a first centrifugal force and a first flow rate to a cell composition comprising T cells in a conical fluid enclosure of a centrifuge system to produce a fluidized bed of cells, wherein the cell composition comprises viable and nonviable T cells, and the centrifuge system is a continuous counterflow centrifuge system; and (b) applying a second centrifugal force and a second flow rate to the cell composition, wherein the second centrifugal force and the second flow rate recirculate cells of the cell composition through a flow path of the centrifuge system, thereby elutriating a waste fraction of the cell composition having a percentage of nonviable T cells that is higher than the percentage of nonviable T cells in the cell composition and out of the conical fluid enclosure, and producing an enriched composition within the conical fluid enclosure having a percentage of viable T cells that is higher than the percentage of viable T cells in the cell composition. A method comprising:

20. 20. The method of claim 19, wherein the ratio of the second centrifugal force (in G) to the second flow rate (in mL / min) is between about 30 and about 40. (i) the first centrifugal force is between about 1,000 G and about 4,000 G, and the first flow rate is between about 5 mL / min and about 15 mL / min; (ii) the second centrifugal force is between about 350 G and about 4,000 G, and the second flow rate is between about 5 mL / min and about 100 mL / min; (iii) the second centrifugal force is between about 350 G and 3,000 G; (iv) the second flow rate is between about 10 mL / min and about 65 mL / min; and / or (v) introducing the cell composition into the centrifuge system, said introducing occurring before and / or during at least a portion of said applying in (a); 21. The method of claim 19 or 20.

22. 1. A method for enriching a cellular composition for viable cells, comprising: (a) applying a first centrifugal force and a first flow rate to a cell composition comprising T cells in a conical fluid enclosure of a centrifuge system to produce a fluidized bed of cells, wherein the cell composition comprises viable and nonviable T cells, (i) the first centrifugal force is between about 2,000 G and about 4,000 G, and (ii) the first flow rate is between about 5 mL / min and about 15 mL / min; and (b) applying a second centrifugal force and a second flow rate to the cell composition, wherein the second centrifugal force and the second flow rate recirculate cells of the cell composition through a flow path of the centrifuge system, thereby producing an enriched composition having a percentage of viable T cells that is higher than the percentage of viable T cells in the cell composition, wherein (i) the second centrifugal force is between about 1,500 G and about 3,000 G; (ii) the second flow rate is between about 65 mL / min and about 100 mL / min; and (iii) the ratio of the second centrifugal force (in G) to the second flow rate (in mL / min) is between about 30 and about 40. Including, The method, wherein the T cells have an average diameter of less than 9 μm.

23. 1. A method for enriching a cellular composition for viable cells, comprising: (a) applying a first centrifugal force and a first flow rate to a cell composition comprising T cells in a conical fluid enclosure of a centrifuge system to produce a fluidized bed of cells, wherein said cell composition comprises viable and non-viable T cells; (b) applying a second centrifugal force and a second flow rate to the cell composition, wherein the second centrifugal force and the second flow rate recirculate cells of the cell composition through a flow path of the centrifuge system, thereby elutriating a waste fraction of the cell composition having a percentage of nonviable T cells higher than the percentage of nonviable T cells in the cell composition out of the conical fluid enclosure, and producing a concentrated composition within the conical fluid enclosure having a percentage of viable T cells higher than the percentage of viable T cells in the cell composition; and (c) cryopreserving the cells of the enriched composition after steps (a) and (b) to produce a cryopreserved cell composition. A method comprising:

24. 1. A method for enriching a cellular composition for viable cells, comprising: (a) applying a first centrifugal force and a first flow rate to a cell composition comprising T cells in a conical fluid enclosure of a centrifuge system to produce a fluidized bed of cells, wherein the cell composition comprises viable and nonviable T cells, and the centrifuge system is a continuous counterflow centrifuge system, (i) the first centrifugal force is between about 2,000 G and about 4,000 G, and (ii) the first flow rate is between about 5 mL / min and about 15 mL / min; and (b) applying a second centrifugal force and a second flow rate to the cell composition, wherein the second centrifugal force and the second flow rate recirculate cells of the cell composition through a flow path of the centrifuge system, thereby producing an enriched composition having a percentage of viable T cells that is higher than the percentage of viable T cells in the cell composition, wherein (i) the second centrifugal force is between about 500 G and about 1,500 G, and (ii) the second flow rate is between about 25 mL / min and about 30 mL / min. A method comprising:

25. 21. The method of claim 19 or 20, wherein prior to the applying in (a), the method comprises contacting the T cells of the cell composition with viral vector particles to produce genetically engineered T cells, and / or the T cells of the cell composition have been contacted with viral vector particles to produce genetically engineered T cells.

26. (i) the percentage of viable T cells in the concentrated composition is at least about 10% greater than the percentage of viable T cells in the cell composition; and / or (ii) the method includes applying a third centrifugal force and a third flow rate to the genetically engineered T cells to produce an output composition comprising the genetically engineered T cells, and optionally, applying the third centrifugal force and the third flow rate to the genetically engineered cells allows for collection or recovery of the output composition.

21. The method of claim 19 or 20.

27. The method of claim 1 , wherein the cell composition comprises activated T cells.

28. 4. The method according to any one of claims 1 to 3, wherein one or more steps of the method are automated, optionally one or more steps of the method being automated by a centrifuge system or a component thereof.

29. 4. The method of any one of claims 1 to 3, wherein the viral vector particle comprises a heterologous nucleic acid encoding a recombinant molecule, optionally wherein the recombinant molecule is a chemokine, a chemokine receptor, a cytokine, a cytokine receptor, an antigen receptor, or a combination thereof, and further optionally wherein the recombinant molecule is a transgenic T cell receptor (TCR) or a chimeric antigen receptor (CAR). (i) the chimeric antigen receptor (CAR) comprises an extracellular antigen recognition domain that specifically binds to a target antigen and an intracellular signaling domain that comprises an immunoreceptor tyrosine-based activation motif (ITAM), and optionally the intracellular signaling domain comprises the intracellular domain of the CD3-zeta (CD3ζ) chain and / or a T-cell costimulatory molecule, optionally CD28 or 4-1BB; and / or (ii) the chimeric antigen receptor (CAR) comprises a transmembrane domain linking an extracellular domain and an intracellular signaling domain, and optionally the transmembrane domain comprises the transmembrane portion of CD28; 30. The method of claim 29.

31. The method of any one of claims 1 to 3, wherein the viral vector particle is a retroviral vector particle, optionally a gamma-retroviral vector or a lentiviral vector particle.

32. 30. The method of claim 29, wherein the antigen receptor specifically binds to an antigen associated with a disease or condition, and optionally the disease or condition is cancer, an autoimmune disease or disorder, and / or an infectious disease.

33. 4. The method of any one of claims 1 to 3, wherein the T cells are primary T cells, suitably from a human subject.

34. 25. A composition comprising genetically engineered T cells produced by the method of any one of claims 1 to 3, 19, 20, and 22 to 24, optionally comprising: (i) about 1.0 × 10 ６ Approximately 2.0 x 10 CAR-expressing T cells ９ CAR-expressing T cells among CAR-expressing T cells; (ii) a pharmaceutically acceptable carrier, and / or (iii) cryoprotectants A composition comprising:

35. 35. The composition of claim 34, for use in treating a disease or disorder, optionally wherein the engineered T cells express an antigen receptor that specifically binds to an antigen associated with the disease or disorder.

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