Process for producing engineered t cell compositions

Abstract

The present invention provides methods for genetically engineering T cells, such as CD4 + T cells and / or CD8 + T cells, for use in cell therapy. In some aspects, provided methods include one or more steps for combining enriched CD4 + and CD8 + cells in a ratio of 1: 1, and then incubating the cells under stimulated conditions, introducing a recombinant polypeptide into the cells by transduction or transfection, and / or incubating the cells under conditions that promote proliferation and / or expansion. In some aspects, the provided methods are effective and reliable means of highly successfully producing genetically engineered T cells.

Description

[0001] This invention application is a divisional application based on an invention patent application filed on December 7, 2018, with application number 201880088430.X (international application number PCT / US2018 / 064628) and entitled “Process for producing engineered T cell compositions”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 596,774, filed December 8, 2017, entitled “PROCESS FOR PRODUCING A COMPOSITION OF ENGINEERED T CELLS”; U.S. Provisional Application No. 62 / 614,965, filed January 8, 2018, entitled “PROCESS FOR PRODUCING A COMPOSITION OF ENGINEERED T CELLS”; U.S. Provisional Application No. 62 / 716,971, filed August 9, 2018, entitled “PROCESS FOR PRODUCING A COMPOSITION OF ENGINEERED T CELLS”; and U.S. Provisional Application No. 62 / 716,971, filed August 9, 2018, entitled “PROCESS FOR PRODUCING A COMPOSITION OF ENGINEERED T CELLS”. and U.S. Provisional Application No. 62 / 721,604, filed on October 3, 2018, entitled “PROCESS FOR PRODUCING A COMPOSITION OF ENGINEERED TCELLS”; U.S. Provisional Application No. 62 / 740,903, filed on October 3, 2018, entitled “PROCESS FOR PRODUCING A COMPOSITION OF ENGINEERED TCELLS”; U.S. Provisional Application No. 62 / 754,564, filed on November 1, 2018, entitled “PROCESS FOR PRODUCING A COMPOSITION OF ENGINEERED TCELLS”; U.S. Provisional Application No. 62 / 774,165, filed on November 30, 2018, entitled “PROCESS FOR PRODUCING A COMPOSITION OF ENGINEERED TCELLS”; and U.S. Provisional Application No. 62 / 780,903, filed on December 3, 2018, entitled “PROCESS FOR PRODUCING A COMPOSITION OF ENGINEERED TCELLS”. PRODUCING A COMPOSITION OF ENGINEERED T CELLS), the contents of which are incorporated by reference in their entirety for all purposes.

[0004] Incorporation by Reference into the Sequence Listing

[0005] This application is filed with a sequence listing in electronic format. The sequence listing is provided as a 68-kilobyte file named 735042014340SeqList.txt, created on December 7, 2018. The information in the electronic sequence listing is incorporated by reference in its entirety. Technical Field

[0006] The present disclosure provides methods for genetically engineering T cells, such as CD4+T cells and / or CD8+T cells, for use in cell therapy. In some aspects, the methods provided include one or more steps for the following: merging enriched CD4+ and CD8+ cells at a ratio of 1:1, and then incubating the cells under stimulating conditions, introducing recombinant polypeptides into the cells by transduction or transfection, and / or cultivating the cells under conditions that promote proliferation and / or amplification. In some aspects, the methods provided are effective and reliable means for highly successfully producing genetically engineered T cells. Background Art

[0007] A variety of cell therapy approaches are available for treating diseases and conditions. Cell therapy approaches include methods involving immune cells (e.g., T cells) genetically engineered with recombinant receptors (e.g., chimeric antigen receptors). Improved methods for manufacturing and / or engineering such cell therapies are needed, including to provide more efficient processes and / or improved cell composition products. Summary of the Invention

[0008] In some embodiments, provided herein are methods for producing an engineered cell composition, the methods comprising: (a) combining a CD4+ T cell composition and a CD8+ T cell composition at a ratio of CD4+ to CD8+ T cells between 2:1 and 1:2, thereby producing an input composition; (b) incubating the input composition under stimulating conditions, thereby producing a stimulating composition; wherein the stimulating conditions comprise the presence of a stimulating agent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules; and wherein the input composition comprises a concentration of less than 5 x 10 6 At least 100 x 10 cells / ml 6 Total CD4+ and CD8+ T cells.

[0009] In some embodiments, provided herein is a method for producing an engineered cell composition, the method comprising incubating an input composition under stimulating conditions, thereby producing a stimulating composition, wherein: the input composition comprises CD4+ and CD8+ T cells at a ratio between 2:1 and 1:2, and wherein the input composition comprises a concentration of less than 5 x 10 6At least 100 x 10 cells / ml 6 total CD4+ and CD8+ T cells; and the stimulatory conditions include the presence of a stimulatory agent that is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules.

[0010] In some embodiments, provided herein are methods for producing an engineered cell composition comprising introducing a recombinant receptor into cells of a T cell composition comprising at least or at least about 1 x 10 6 The method comprises the step of: providing a concentration of viable cells / mL, wherein at least 80%, at least 85%, at least 90%, or at least 95% of the cells in the T cell composition are CD4+ T cells or CD8+ T cells.

[0011] In certain embodiments, the incubation is performed in serum-free medium. In certain embodiments, the input composition comprises at least 80%, at least 85%, at least 90%, or at least 95% of the cells that are CD4+ T cells or CD8+ T cells. In certain embodiments, the input composition comprises at least 100 x 10 6 500x 10 6 In certain embodiments, the input composition comprises at or about 300 x 10 6 In some embodiments, the total CD4+ and CD8+ T cells are viable cells. In some embodiments, the input composition comprises 1 x 10 6 cells / mL and 5x 10 6 In certain embodiments, the input composition comprises at or about 3 x 10 6 In some embodiments, the input composition comprises CD4+ to CD8+ cells at a ratio of between 1.5:1 and 1:1.5. In some embodiments, the input composition comprises CD4+ to CD8+ cells at a ratio of between 1.2:1 and 0.8:1.

[0012] In certain embodiments, the input composition comprises CD4+ to CD8+ cells at a ratio of at or about 1 : 1. In certain embodiments, the input composition comprises CD4+ and CD8+ cells that are surface positive for CD45RA and CCR7.

[0013] In certain embodiments, the ratio of CD4+ cells that are surface positive for CD45RA and CCR7 to CD8+ cells that are surface positive for CD45RA and CCR7 is at or about 1.1:1.

[0014] In certain embodiments, the input composition comprises CD4+ and CD8+ cells that are surface positive for CD27 and CCR7.

[0015] In certain embodiments, the ratio of CD4+ cells surface positive for CD27 and CCR7 to CD8+ cells surface positive for CD27 and CCR7 is at or about 1.69:1.

[0016] In certain embodiments, the input composition comprises CD4+ and CD8+ cells that are surface positive for CCR7 and surface negative for CD62L, optionally at a ratio of between 2.0:1 and 1.5:1.

[0017] Some embodiments further comprise: introducing the recombinant receptor into cells from the stimulatory composition, thereby producing an engineered cell composition, wherein the introducing comprises contacting the cells of the stimulatory composition with an agent comprising a polynucleotide encoding the recombinant receptor. Some embodiments further comprise: introducing the recombinant receptor into cells from the stimulatory composition, thereby producing an engineered cell composition, wherein the introducing comprises transducing the cells of the stimulatory composition with a viral vector comprising a polynucleotide encoding the recombinant receptor. In some embodiments, the introducing is performed in serum-free medium.

[0018] In certain embodiments, provided herein are methods for producing an engineered cell composition, the methods comprising: (a) combining a CD4+ T cell composition and a CD8+ T cell composition at a ratio of CD4+ to CD8+ T cells between 2:1 and 1:2, thereby producing an input composition; (b) incubating the input composition under stimulating conditions, thereby producing a stimulating composition; wherein the stimulating conditions comprise the presence of a stimulating agent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules; and wherein the input composition comprises a concentration of less than 5 x 10 6 At least 100 x 10 cells / mL 6 Total CD4+ and CD8+ T cells.

[0019] In some embodiments of any of the provided methods, the CD4+ and CD8+ cells in the input composition are enriched or selected from a primary sample from a subject, optionally wherein the CD4+ and CD8+ cells in the input composition are separately enriched or selected from a primary sample from a subject. In certain embodiments of any of the provided methods, the CD4+ T cell composition comprises at least 80%, at least 85%, at least 90%, or at least 95% CD4+ T cells. In specific embodiments of any of the provided methods, the CD8+ T cell composition comprises at least 80%, at least 85%, at least 90%, or at least 95% CD8+ T cells.

[0020] In some embodiments, provided herein is a method for producing an engineered cell composition, the method comprising incubating an input composition under stimulating conditions, thereby producing a stimulating composition, wherein: the input composition comprises CD4+ and CD8+ T cells at a ratio between 2:1 and 1:2, and wherein the input composition comprises a concentration of less than 5 x 10 6 At least 100 x 10 cells / mL 6 total CD4+ and CD8+ T cells; and the stimulatory conditions include the presence of a stimulatory agent that is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules.

[0021] In certain embodiments of any of the provided methods, the incubation is performed in serum-free medium. In specific embodiments of any of the provided methods, the input composition comprises at least 80%, at least 85%, at least 90%, or at least 95% of the cells that are CD4+ T cells or CD8+ T cells. In specific embodiments of any of the provided methods, the input composition comprises at least 100 x 10 6 500x 10 6 In certain embodiments of any of the provided methods, the input composition comprises at or about 300 x 10 6 Total CD4+ and CD8+ T cells.

[0022] In some embodiments of any of the provided methods, the total CD4+ and CD8+ T cells are viable cells. In certain embodiments of any of the provided methods, the input composition comprises 1 x 10 6 cells / mL and 5x 10 6 In certain embodiments of any of the provided methods, the input composition comprises at or about 3 x 10 6In certain embodiments of any of the provided methods, the input composition comprises CD4+ to CD8+ cells at a ratio of between 1.5:1 and 1:1.5. In some embodiments of any of the provided methods, the input composition comprises CD4+ to CD8+ cells at a ratio of between 1.2:1 and 0.8:1.

[0023] In certain embodiments of any of the provided methods, the input composition comprises CD4+ to CD8+ cells at a ratio of at or about 1:1. In specific embodiments of any of the provided methods, the input composition comprises CD4+ and CD8+ cells that are surface positive for CD45RA and CCR7. In some embodiments of any of the provided methods, the ratio of CD4+ cells that are surface positive for CD45RA and CCR7 to CD8+ cells that are surface positive for CD45RA and CCR7 is at or about 1.1:1. In certain embodiments of any of the provided methods, the input composition comprises CD4+ and CD8+ cells that are surface positive for CD27 and CCR7. In specific embodiments of any of the provided methods, the ratio of CD4+ cells that are surface positive for CD27 and CCR7 to CD8+ cells that are surface positive for CD27 and CCR7 is at or about 1.69:1. In some embodiments of any of the provided methods, the input composition comprises CD4+ and CD8+ cells that are surface positive for CCR7 and surface negative for CD62L, optionally at a ratio of between 2.0:1 and 1.5:1.

[0024] In certain embodiments of any of the provided methods, the method further comprises: introducing the recombinant receptor into cells from the stimulatory composition, thereby producing an engineered cell composition, wherein the introducing comprises contacting the cells of the stimulatory composition with an agent comprising a polynucleotide encoding the recombinant receptor.

[0025] In specific embodiments of any of the provided methods: contacting is performed by transfection with a vector, wherein the vector is a transposon, optionally a Sleeping Beauty (SB) transposon or a Piggybac transposon; or contacting is performed by transduction with a viral vector.

[0026] In some embodiments of any of the provided methods, the method further comprises: introducing the recombinant receptor into cells from the stimulatory composition, thereby producing an engineered cell composition, wherein the introducing comprises transducing the cells of the stimulatory composition with a viral vector comprising a polynucleotide encoding the recombinant receptor. In certain embodiments of any of the provided methods, the introducing is performed in serum-free medium.

[0027] In certain embodiments of any of the provided methods, the stimulating composition is introduced to comprise less than 300 x 10 6In some embodiments of any of the provided methods, for introduction, the stimulating composition comprises 50 x 10 6 10 and 200x 6 In certain embodiments of any of the provided methods, for introduction, the stimulating composition comprises at or about 100 x 10 6 In certain embodiments of any of the provided methods, the stimulating composition comprises less than 3 x 10 6 In some embodiments of any of the provided methods, for introduction, the stimulating composition comprises 0.5 x 10 6 cells / mL and 2x 10 6 In certain embodiments of any of the provided methods, for introduction, the stimulating composition comprises at or about 1 x 10 6 The concentration of cells / mL.

[0028] Specific embodiments of any of the provided methods include adjusting the composition of the stimulatory composition prior to introducing the recombinant receptor into the cells of the stimulatory composition and after incubation under stimulatory conditions. In some embodiments of any of the provided methods, the cells of the stimulatory composition are living cells. In certain embodiments, provided herein are methods for producing engineered cell compositions comprising introducing a recombinant receptor into cells of a T cell composition comprising at least or at least about 1 x 10 6 The method comprises the step of: providing a concentration of viable cells / mL, wherein at least 80%, at least 85%, at least 90%, or at least 95% of the cells in the T cell composition are CD4+ T cells or CD8+ T cells.

[0029] In certain embodiments of any of the provided methods, the concentration of the T cell composition is less than 5 x 10 6 In some embodiments of any of the provided methods, the T cell composition comprises at least or at least about or about 100 x 10 6 In certain embodiments of any of the provided methods, the T cell composition comprises fewer than 300 x 10 6 In certain embodiments of any of the provided methods, introducing comprises contacting the T cells by transducing a viral vector comprising a polynucleotide encoding the recombinant receptor.

[0030] In some embodiments of any of the provided methods, introducing is performed in a serum-free medium. In certain embodiments of any of the provided methods, one or more cells in the T cell composition are activated and / or comprise surface expression of an LDL receptor.

[0031] In certain embodiments of any of the provided methods, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% of the cells in the cell composition: (i) express surface markers selected from HLA-DR, CD25, CD69, CD71, CD40L, and 4-1BB; (ii) comprise intracellular expression of a cytokine selected from IL-2, IFN-γ, TNF-α; (iii) are in the G1 phase or later of the cell cycle; and / or (iv) are capable of proliferation.

[0032] In some embodiments of any of the provided methods, prior to introduction, the cells of the composition are produced by a process comprising incubating an input composition comprising CD4+ and CD8+ T cells under stimulatory conditions, wherein the stimulatory conditions include the presence of a stimulatory agent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules. In certain embodiments of any of the provided methods, the incubation is performed in serum-free medium.

[0033] In certain embodiments, provided herein are methods for producing an engineered cell composition, the method comprising: (a) incubating an input composition under stimulating conditions, thereby producing a stimulating composition, wherein: the input composition comprises CD4+ and CD8+ T cells at a ratio between 2:1 and 1:2, and comprises a concentration of less than 5 x 10 6 At least 100x10 cells / mL 6 CD4+ and CD8+ T cells; and the stimulation conditions include the presence of a stimulatory agent that is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules; and (b) introducing the recombinant receptor into less than 300 x 10 6 The engineered cell composition is produced by introducing the stimulatory composition into cells of the engineered cell composition, wherein the introducing comprises contacting the cells of the stimulatory composition with a viral vector comprising a polynucleotide encoding the recombinant receptor.

[0034] In some embodiments of any of the provided methods, incubation and / or introduction is performed in serum-free medium. In certain embodiments of any of the provided methods, the CD4+ and CD8+ T cells are viable cells. In specific embodiments of any of the provided methods, the cells from the stimulating composition are viable cells.

[0035] In some embodiments of any of the provided methods, the introduction is initiated within 2 days after the start of incubation under stimulating conditions and / or within 2 days after the CD4+ T cells and CD8+ T cells of the input composition are combined. In certain embodiments of any of the provided methods, the introduction is initiated within 36 hours after the start of incubation under stimulating conditions and / or within 36 hours after the CD4+ T cells and CD8+ T cells of the input composition are combined. In specific embodiments of any of the provided methods, the introduction is initiated within 30 hours after the start of incubation under stimulating conditions and / or within 30 hours after the CD4+ T cells and CD8+ T cells of the input composition are combined.

[0036] Some embodiments of any of the provided methods further comprise culturing the engineered composition under conditions that promote proliferation and / or expansion of the engineered cells, thereby producing an output composition comprising engineered T cells. In certain embodiments of any of the provided methods, culturing is performed in serum-free medium.

[0037] In certain embodiments, provided herein are methods for producing an engineered cell composition, the method comprising: (a) incubating an input composition under stimulating conditions, thereby producing a stimulating composition; wherein the input composition comprises CD4+ and CD8+ T cells at a ratio of between 2:1 and 1:2, and wherein the input composition comprises CD4+ and CD8+ T cells at a concentration of less than 5 x 10 6 At least 100 x 10 cells / mL 6 total CD4+ and CD8+ T cells; and wherein the stimulation conditions include the presence of a stimulatory agent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules; and (b) introducing the recombinant receptor into less than 300 x 10 6 The invention further comprises introducing into cells of the stimulatory composition, thereby producing an engineered cell composition, wherein the introducing comprises transducing the cells of the stimulatory composition with a viral vector comprising a polynucleotide encoding the recombinant receptor; and (c) culturing the engineered composition under conditions that promote proliferation and / or expansion of the engineered cells, thereby producing an output composition comprising engineered T cells.

[0038] In some embodiments of any of the provided methods, the incubating, introducing, and / or cultivating is performed in a serum-free medium.

[0039] In certain embodiments of any of the provided methods, the input composition comprises CD4+ to CD8+ cells at a ratio of between 1.5:1 and 1:1.5, CD4+ to CD8+ cells at a ratio of between 1.2:1 and 0.8:1, optionally CD4+ to CD8+ T cells at a ratio of at or about 1:1. In specific embodiments of any of the provided methods, wherein the input composition comprises CD4+ and CD8+ that are surface positive for CD45RA and CCR7. In some embodiments of any of the provided methods, the ratio of CD4+ cells that are surface positive for CD45RA and CCR7 to CD8+ cells that are surface positive for CD45RA and CCR7 is at or about 1.1:1. In certain embodiments of any of the provided methods, the input composition comprises CD4+ and CD8+ cells that are surface positive for CD27 and CCR7. In specific embodiments of any of the provided methods, the ratio of CD4+ cells that are surface positive for CD27 and CCR7 to CD8+ cells that are surface positive for CD27 and CCR7 is at or about 1.69:1. In some embodiments of any of the provided methods, the input composition comprises CD4+ and CD8+ cells that are surface positive for CCR7 and surface negative for CD62L.

[0040] In certain embodiments of any of the provided methods, for introduction, the stimulation composition comprises less than 300 x 10 6 In certain embodiments of any of the provided methods, for introduction, the stimulating composition comprises 50 x 10 6 10 and 200x 6 cells, optionally at or about 100 x 10 6 In some embodiments of any of the provided methods, for introduction, the stimulating composition comprises less than 3 x 10 6 In certain embodiments of any of the provided methods, for introduction, the stimulating composition comprises 0.5 x 10 6 cells / mL and 2x 10 6 cells / mL, optionally at or about 1 x 10 6 In certain embodiments of any of the provided methods, the composition of the stimulatory composition is adjusted prior to introducing the recombinant receptor into the cells of the stimulatory composition and after incubation under stimulatory conditions.

[0041] In specific embodiments of any of the provided methods, incubation is carried out in the presence of one or more cytokines, for example, in serum-free medium. In certain embodiments of any of the provided methods, the one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and / or recombinant IL-15. In some embodiments of any of the provided methods, the one or more cytokines comprise: recombinant IL-2 between 10 IU / mL and 200 IU / mL; recombinant IL-7 between 100 IU / mL and 1,000 IU / mL; and / or recombinant IL-15 between 10 IU / mL and 200 IU / mL. In specific embodiments of any of the provided methods, the one or more cytokines comprise: recombinant IL-2 between 10 IU / mL and 200 IU / mL; recombinant IL-7 between 100 IU / mL and 1,000 IU / mL; and recombinant IL-15 between 10 IU / mL and 200 IU / mL.

[0042] In certain embodiments of any of the provided methods, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% of the cells of the stimulatory composition: (i) express surface markers selected from HLA-DR, CD25, CD69, CD71, CD40L, and 4-1BB; (ii) comprise intracellular expression of a cytokine selected from IL-2, IFN-γ, TNF-α; (iii) are in the G1 phase or later of the cell cycle; and / or (iv) are capable of proliferation.

[0043] In some embodiments of any provided method, the stimulating reagent includes a first-level agent specifically bound to a member of the TCR complex (optionally specifically bound to CD3). In a specific embodiment of any provided method, the stimulating reagent also includes a second agent specifically bound to a T cell costimulatory molecule, optionally wherein the costimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40 or ICOS. In certain embodiments of any provided method, the first-level and / or second-level agent includes an antibody, optionally wherein the stimulating reagent includes incubating with anti-CD3 antibodies and anti-CD28 antibodies or their antigen-binding fragments.

[0044] In some embodiments of any of the provided methods, the primary agent and / or secondary agent is present on the surface of a solid support. In specific embodiments of any of the provided methods, the solid support is or comprises beads. In certain embodiments of any of the provided methods, the beads comprise a diameter greater than or greater than about 3.5 μm but no more than about 9 μm or no more than about 8 μm or no more than about 7 μm or no more than about 6 μm or no more than about 5 μm. In some embodiments of any of the provided methods, the beads comprise a diameter of or about 4.5 μm. In specific embodiments of any of the provided methods, the beads are inert. In certain embodiments of any of the provided methods, the beads are or comprise a polystyrene surface. In some embodiments of any of the provided methods, the beads are magnetic or superparamagnetic.

[0045] In specific embodiments of any of the provided methods, the ratio of beads to cells is less than 3: 1. In certain embodiments of any of the provided methods, the ratio of beads to cells is at or about 2: 1 to about 0.5: 1. In some embodiments of any of the provided methods, the ratio of beads to cells is at or about 1: 1.

[0046] In certain embodiments of any of the provided methods, the input composition is incubated under stimulating conditions for less than 48 hours. In certain embodiments of any of the provided methods, the input composition is incubated under stimulating conditions for between 12 hours and 36 hours, inclusive. In some embodiments of any of the provided methods, the input composition is incubated under stimulating conditions for between 18 hours and 30 hours, inclusive. In certain embodiments of any of the provided methods, the input composition is incubated under stimulating conditions for at or about 24 hours.

[0047] In certain embodiments of any of the provided methods, the contacting (optionally transducing) is performed for less than 48 hours. In some embodiments of any of the provided methods, the contacting (optionally transducing) is performed for between 12 hours and 36 hours, inclusive. In specific embodiments of any of the provided methods, the contacting (optionally transducing) is performed for between 18 hours and 30 hours, inclusive. In certain embodiments of any of the provided methods, the contacting (optionally transducing) is performed for at or about 24 hours.

[0048] In some embodiments of any provided method, the viral vector is a retroviral vector. In a specific embodiment of any provided method, the viral vector is a lentiviral vector or a gamma retroviral vector. In certain embodiments of any provided method, the contacting (optionally transduction) is carried out in the absence of a transduction adjuvant. In some embodiments of any provided method, the introduction is carried out in the presence of one or more cytokines, for example, in serum-free medium. In a specific embodiment of any provided method, the one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and / or recombinant IL-15.

[0049] In certain embodiments of any of the provided methods, the one or more cytokines comprise: between 10 IU / mL and 200 IU / mL of recombinant IL-2; between 100 IU / mL and 1,000 IU / mL of recombinant IL-7; and / or between 10 IU / mL and 200 IU / mL of recombinant IL-15. In some embodiments of any of the provided methods, the one or more cytokines comprise: between 10 IU / mL and 200 IU / mL of recombinant IL-2; between 100 IU / mL and 1,000 IU / mL of recombinant IL-7; and between 10 IU / mL and 200 IU / mL of recombinant IL-15.

[0050] In certain embodiments of any of the provided methods, at least a portion of the cultivation is performed with mixing and / or perfusion. In certain embodiments of any of the provided methods, at least a portion of the cultivation is performed with perfusion at a rate of 500 mL / day, about ... In some embodiments of any of the provided methods, at least a first portion of the cultivating is conducted at a perfusion rate of, about, or at least 500 mL / day, 750 mL / day, or 1,000 mL / day, and wherein at least a second portion of the cultivating is conducted at a perfusion rate of, about, or at least 1,200 mL / day, 1,400 mL / day, or 1,500 mL / day.

[0051] In certain embodiments of any of the provided methods, perfusion is initiated and / or increased when the cells reach a specific density. In certain embodiments of any of the provided methods, the specific density is, is about, or is at least 0.4 x 10 6 cells, 0.5 x 10 6 cells, 0.6 x 10 6cells, 0.8 x 10 6 cells, 1.0x 10 6 cells, 1.2 x 10 6 cells, 1.4x10 6 cells, 1.6 x 10 6 cells, 1.8 x 10 6 cells, 2.0 x 10 6 cells, 2.2 x 10 6 cells, or 2.4x10 6 In some embodiments of any of the provided methods, when the cells reach at or about 0.6 x 10 6 When the cells reach a density of at or about 2.0 x 10 cells / mL, perfusion is started and / or increased to a rate of at or about 750 mL / day. In certain embodiments of any of the provided methods, when the cells reach a density of at or about 2.0 x 10 6 When the cell density reaches 1500 cells / mL, perfusion is initiated and / or increased to a rate of at or about 1500 mL / day.

[0052] In certain embodiments of any of the provided methods, the cultivation is carried out in the presence of one or more cytokines, for example, in a serum-free medium. In some embodiments of any of the provided methods, the one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and / or recombinant IL-15. In specific embodiments of any of the provided methods, the one or more cytokines comprise: recombinant IL-2 between 50 IU / mL and 400 IU / mL; recombinant IL-7 between 100 IU / mL and 2,000 IU / mL; and / or recombinant IL-15 between 50 IU / mL and 400 IU / mL. In specific embodiments of any of the provided methods, the cultivation is carried out in the presence of recombinant IL-2 between 50 IU / mL and 400 IU / mL; recombinant IL-7 between 100 IU / mL and 2,000 IU / mL; and recombinant IL-15 between 50 IU / mL and 400 IU / mL, for example, in a serum-free medium. In certain embodiments of any of the provided methods, the one or more cytokines comprise: between 50 IU / mL and 400 IU / mL of recombinant IL-2; between 100 IU / mL and 1,000 IU / mL of recombinant IL-7; and / or between 10 IU / mL and 200 IU / mL of recombinant IL-15.

[0053] In some embodiments of any of the provided methods, the cultivation is initiated within 3 days after the start of incubation under stimulating conditions and / or within 3 days after the CD4+ T cells and CD8+ T cells of the input composition are combined. In specific embodiments of any of the provided methods, the cultivation is initiated within 60 hours after the start of incubation under stimulating conditions and / or within 60 hours after the CD4+ T cells and CD8+ T cells of the input composition are combined. In certain embodiments of any of the provided methods, the cultivation is initiated within 48 hours after the start of incubation under stimulating conditions and / or within 48 hours after the CD4+ T cells and CD8+ T cells of the input composition are combined.

[0054] In some embodiments of any of the provided methods, the culturing is performed at least until the composition comprises a threshold number of T cells. In certain embodiments of any of the provided methods, the threshold number of T cells is, is about, or is at least 2400 x 10 6 In certain embodiments of any of the provided methods, the threshold number of T cells is, is about, or is at least 5500 x 10 6 cells.

[0055] In certain embodiments of any of the methods provided, the culturing is continued for at least one day after a threshold number of T cells is reached. In certain embodiments of any of the methods provided, the threshold number of T cells is, is about, or is at least 900 x 10 6 In certain embodiments of any of the provided methods, the threshold number of T cells is, is about, or is at least 1200 x 10 6 In certain embodiments of any of the provided methods, when the number of T cells is, is about, or is at least 2400 x 10 6 In some embodiments of any of the provided methods, the threshold number of T cells is, is about, or is at least 3500 x 10 6 In certain embodiments of any of the provided methods, when the number of T cells is, is about, or is at least 5500 x 10 6 When the number of cells reached 1, the culture was terminated.

[0056] Certain embodiments of any of the provided methods include collecting the cells that output the composition after said incubation. Some embodiments of any of the provided methods include collecting the cells that output the composition after said incubation, wherein the cells that output the composition are collected at least 9 days after initiation of incubation under stimulating conditions. Certain embodiments of any of the provided methods include collecting the cells that output the composition after said incubation, wherein the cells that output the composition are collected at least 10 days after initiation of incubation under stimulating conditions.

[0057] Specific embodiments of any of the provided methods include a 95% confidence interval for the amount of time between starting incubation and collecting the cells for outputting the composition between 8 days and 25 days. Some embodiments of any of the provided methods include a 95% confidence interval for the amount of time between starting incubation and collecting the cells for outputting the composition between 9 days and 21 days. Some embodiments of any of the provided methods include a 95% confidence interval for the amount of time between starting incubation and collecting the cells for outputting the composition between 9 days and 16 days.

[0058] Specific embodiments of any of the provided methods further comprise formulating the cells of the output composition, optionally in the presence of a pharmaceutically acceptable excipient, for cryopreservation and / or administration to a subject. In certain embodiments of any of the provided methods, the cells of the output composition are formulated in the presence of a cryoprotectant. In some embodiments of any of the provided methods, the cryoprotectant comprises DMSO. In specific embodiments of any of the provided methods, the cells of the output composition are formulated in a container, optionally a vial or bag.

[0059] In some embodiments of any provided method, the separation includes separating CD4+ and / or CD8+ T cells from the biological sample before incubation. In some embodiments of any provided method, the separation includes selecting cells based on the surface expression of CD4 and / or CD8 (optionally by positive or negative selection). In a specific embodiment of any provided method, the separation includes performing a selection based on immunoaffinity. In some embodiments of any provided method, the biological sample includes primary T cells obtained from a subject. In some embodiments of any provided method, the subject is a human subject.

[0060] In certain embodiments of any of the provided methods, the biological sample is or comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, a single blood product, or a leukocyte single blood product. In certain embodiments of any of the provided methods, the recombinant receptor is capable of binding to a target antigen that is associated with, is specific to, and / or is expressed on a cell or tissue of a disease, disorder, or condition. In some embodiments of any of the provided methods, the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer.

[0061] In certain embodiments of any of the methods provided, the target antigen is a tumor antigen. In certain embodiments of any of the methods provided, the target antigen is selected from 5T4, 8H9, avb6 integrin, B7-H6, B cell maturation antigen (BCMA), CA9, cancer-testis antigen, carbonic anhydrase 9 (CAIX), CCL-1, CD19, CD20, CD22, CEA, hepatitis B surface antigen, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD138, CD171, carcinoembryonic antigen (CEA), CE7, cyclin, cyclin A2, c-Met, dual antigen, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, ephrin B2, erb-B2, erb-B3, erb-B4, erbB dimer, EGFR vIII, estrogen receptor, fetal AChR, folate receptor alpha, folate binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, G250 / CAIX, GD2, GD3, gp100, Her2 / neu (receptor tyrosine kinase erbB2), HMW-MAA, IL-22R-alpha, IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domain receptor (KDR), kappa light chain, Lewis Y, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE) -A1, MAGE-A3, MAGE-A6, MART-1, mesothelin, murine CMV, mucin 1 (MUC1), MUC16, NCAM, NKG2D, NKG2D ligand, NY-ESO-1, O-acetylated GD2 (OGD2), carcinoembryonic antigen, preferentially expressed antigen in melanoma (PRAME), PSCA, progesterone receptor, survivin, ROR1, TAG72, VEGF receptor, VEGF-R2, Wilms tumor 1 (WT-1), pathogen-specific antigens, and antigens associated with universal tags.

[0062] In some embodiments of any of the provided methods, the recombinant receptor is or comprises a functional non-TCR antigen receptor or a TCR or antigen-binding fragment thereof.

[0063] In a specific embodiment of any provided method, the recombinant receptor is a chimeric antigen receptor (CAR). In certain embodiments of any provided method, the recombinant receptor is an anti-BCMA CAR. In some embodiments of any provided method, the chimeric antigen receptor comprises an extracellular domain containing an antigen binding domain. In a specific embodiment of any provided method, the antigen binding domain is or comprises an antibody or an antibody fragment thereof, and the antibody fragment is optionally a single-chain fragment. In certain embodiments of any provided method, the fragment comprises an antibody variable region connected by a flexible linker. In some embodiments of any provided method, the fragment comprises scFv. In a specific embodiment of any provided method, the chimeric antigen receptor further comprises a spacer and / or a hinge region.

[0064] In certain embodiments of any of the provided methods, the chimeric antigen receptor comprises an intracellular signaling region. In some embodiments of any of the provided methods, the intracellular signaling region comprises an intracellular signaling domain. In specific embodiments of any of the provided methods, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM). In certain embodiments of any of the provided methods, the intracellular signaling domain is or comprises an intracellular signaling domain of a CD3 chain (optionally a CD3-zeta (CD3ζ) chain), or a signaling portion thereof.

[0065] In some embodiments of any of the provided methods, the chimeric antigen receptor further comprises a transmembrane domain disposed between the extracellular domain and the intracellular signaling region. In specific embodiments of any of the provided methods, the intracellular signaling region further comprises a costimulatory signaling region. In certain embodiments of any of the provided methods, the costimulatory signaling region comprises an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof. In some embodiments of any of the provided methods, the costimulatory signaling region comprises an intracellular signaling domain of CD28, 4-1BB, or ICOS or a signaling portion thereof.

[0066] In certain embodiments of any of the provided methods, the costimulatory signaling region is located between the transmembrane domain and the intracellular signaling region. In certain embodiments of any of the provided methods, an output composition comprising a threshold number or more of cells is generated in greater than or about 85%, greater than or about 90%, or greater than or about 95% of the iterations of the method.

[0067] In some embodiments of any of the provided methods, the serum-free medium comprises: 0.5 mM to 5 mM L-glutamine in dipeptide form; 0.5 mM to 5 mM L-glutamine; and at least one protein in a basal medium, wherein the medium is serum-free. In specific embodiments of any of the provided methods, the dipeptide form of L-glutamine is L-alanyl-L-glutamine.

[0068] In certain embodiments of any of the provided methods, the concentration of L-glutamine in the dipeptide form in the serum-free medium is or is about 2 mM. In some embodiments of any of the provided methods, the concentration of L-glutamine in the serum-free medium is or is about 2 mM. In specific embodiments of any of the provided methods, the at least one protein comprises one or more of albumin, insulin, or transferrin, optionally one or more of human or recombinant albumin, insulin, or transferrin.

[0069] In some any embodiments of the methods provided herein, during at least a portion of cultivation, the cell viability, concentration, density, quantity or a combination thereof of the monitoring cell. In some any such embodiments, monitoring is performed by an optical method (optionally microscopy). In some any such embodiments, monitoring is performed by bright field microscopy, fluorescence microscopy, differential interference contrast microscopy, phase contrast microscopy, digital holographic microscopy (DHM), differential digital holographic microscopy (DDHM) or a combination thereof. In some any such embodiments, monitoring is performed by differential digital holographic microscopy (DDHM). In some any such embodiments, monitoring is performed intermittently or continuously during at least a portion of cultivation, optionally performed once at least every 1 hour, 6 hours, 12 hours, 18 hours, 24 hours or 26 hours during cultivation. In some any such embodiments, monitoring is performed until cell reaches the threshold number of T cells, the threshold number of live T cells, the threshold concentration of T cells or the threshold concentration of live T cells. In some any such embodiments, monitoring and cultivation are performed in a closed system.

[0070] In some any embodiments of the methods provided herein, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% of the cells in the output composition have a memory phenotype; wherein at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% of the cells in the output composition have a central memory phenotype; wherein at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% of the cells in the output composition have a central memory phenotype; In some embodiments, the output composition comprises at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% of the cells are CD27+, CD28+, CCR7+, CD45RA-, CD45RO+, CD62L+, CD3+, CD95+, granzyme B-, and / or CD127+; and / or wherein at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% of the cells in the output composition are CCR7+ / CD45RA- or CCR7+ / CD45RO+.

[0071] In some any embodiments of the methods provided herein, iterations of the methods produce a plurality of output compositions (optionally from a human biological sample), wherein the methods are performed in a plurality of different individual subjects, wherein: the average percentage of cells with a memory phenotype in the plurality of output compositions is between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%; the average percentage of cells with a central ... between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%; and the average percentage of cells that are CD27+, CD28+, CCR7+, CD45RA-, CD45RO+, CD62L+, CD3+, CD95+, granzyme B-, and / or CD127+ in the plurality of output compositions is between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, , between about 55% and about 60%, or between about 60% and about 65%; the average percentage of cells that are CCR7+ / CD45RA- or CCR7+ / CD45RO+ in the plurality of output compositions is between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%; the average percentage of central memory CD4+ T cells in the engineered CD4+ T cells (optionally CAR+CD4+ T cells) of the plurality of output compositions is between about 50% and about 65%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%. between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%; the average percentage of central memory CD8+ T cells in the engineered CD8+ T cells (optionally CAR+CD8+ T cells) of the plurality of output compositions is between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%;and / or the average percentage of central memory T cells (optionally CD4+ central memory T cells and CD8+ central memory T cells) in the engineered T cells (optionally CAR+ T cells) of the plurality of output compositions is between about 40% and about 65%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, or between about 60% and about 65%.;

[0072] In some any embodiments of the methods provided herein, the method produces an output composition exhibiting a predetermined characteristic in at least about 80%, about 90%, about 95%, about 97%, about 99%, about 100% or 100% of a human biological sample, wherein the predetermined characteristic is optionally a threshold value number of cells expressing CAR in the output composition, wherein the method is performed in a plurality of different individual subjects. In some any such embodiments, the plurality of different individual subjects include subjects with a disease or condition. In some any such embodiments, the disease or condition is cancer. In some any such embodiments, cancer is a blood cancer, optionally multiple myeloma. In some any such embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% of the cells in the composition have a memory phenotype; wherein at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% of the cells in the composition have a central memory phenotype; wherein at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% of the cells in the composition have a central memory phenotype; At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% of the cells in the exported composition are CD27+, CD28+, CCR7+, CD45RA-, CD45RO+, CD62L+, CD3+, granzyme B-, and / or CD127+; and / or wherein at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% of the cells in the exported composition are CCR7+ / CD45RA- or CCR7+ / CD45RO+.

[0073] In certain embodiments, provided herein are compositions comprising engineered cells produced by any of the methods provided herein. Some embodiments of any provided compositions comprise a pharmaceutically acceptable carrier. Specific embodiments of any provided compositions comprise a cryoprotectant, optionally DMSO.

[0074] In certain embodiments, provided herein is an article of manufacture comprising any of the compositions provided herein and instructions for administering the output composition to a subject. In certain embodiments of any provided article of manufacture, the subject suffers from a disease or condition, optionally wherein the recombinant receptor specifically recognizes or specifically binds to an antigen associated with the disease or condition or expressed or present on cells of the disease or condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1A Shown is a figure of a bivariate fitting analysis of the ratio of live CD4+ cells to live CD8+ cells (live CD4+ / CD8+ ratio) in apheresis samples compared to the ratio of CAR+CD4+T cells to CAR+CD8+T cells (CAR+CD4+ / CD8+ ratio) in T cell compositions after T cell activation, transduction with chimeric antigen receptor (CAR) constructs and amplification. Curve represents the boundary of a bivariate normal ellipse at p=0.990. Data points represent the average ratio of four samples from each subject, including healthy subjects (circles) and subjects with myeloma (plus signs). Figure 1B Shown is a graph of a bivariate fit analysis of CD45RA+ / CCR7+CD4 / CD8 ratios in the starting mixture of selected CD4 and CD8 cells compared to CAR+CD4+ / CD8+ ratios in T cell compositions after T cell activation, transduction with chimeric antigen receptor (CAR) constructs, and expansion. The curve represents the boundary of a bivariate normal ellipse at p=0.990. The data points represent the average ratios of four samples from each subject, including healthy subjects (circles) and subjects with myeloma (plus signs).

[0076] Figure 2A-2C Graph showing bivariate fit analysis of the ratios of cells of different phenotypes in the starting mixture of selected CD4 and CD8 cells compared to the CAR+CD4+ / CD8+ ratios in the engineered CAR+ T cell composition. Figure 2A Graph showing bivariate fit analysis of the ratio of CD45RA+ / CCR7+ / CD4+ to CD45RA+ / CCR7+ / CD8+ cells in the starting mixture of selected CD4 and CD8 cells compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+ T cell composition. Figure 2B Graph showing bivariate fit analysis of the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells in the starting mixture of selected CD4 and CD8 cells compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+ T cell composition. Figure 2CShown is a graph of a bivariate fit analysis of the ratio of CD27+ / CCR7+ / CD4+T cells to CD27+ / CCR7+ / CD8+T cells in the starting mixture of selected CD4 and CD8 cells compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+T cell composition. Curve represents the boundary of a bivariate normal ellipse at p=0.950. Data points represent the average ratio of multiple compositions from each subject, including healthy donors (circles) and patients with multiple myeloma (plus signs).

[0077] Figure 3A-3C Graph showing bivariate fit analysis of the ratios of cells of different phenotypes in the starting mixture of selected CD4 and CD8 cells from seven patients with multiple myeloma compared to the CAR+CD4+ / CD8+ ratios in the resulting engineered CAR+ T cell compositions. Figure 3A Graph showing bivariate fit analysis of the ratio of CD27+ / CCR7+ / CD4+ to CD27+ / CCR7+ / CD8+ cells in the starting mixture of selected CD4 and CD8 cells compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+ T cell composition. Figure 3B Graph showing bivariate fit analysis of the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells in the starting mixture of selected CD4 and CD8 cells compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+ T cell composition. Figure 3C A graph showing a bivariate fit analysis of the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells in the starting mixture of selected CD4 and CD8 cells compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+ T cell composition. The curve represents the boundary of the bivariate normal ellipse at p=0.950.

[0078] Figure 4A and Figure 4B Depicted in experimental run 1 ( Figure 4A ) and Run 2( Figure 4B ), viable cell counts (VCC; x 10) assessed using continuous monitoring by differential DHM (“Continuous”, lines) or manual sampling (“Manual”, dots). 6 The top graph depicts the measured values ​​of each cell type, and the bottom graph depicts the linear regression analysis and R 2 and slope for comparison between continuous monitoring and manual sampling.

[0079] Figure 5Figure 3 Viable cell count (VCC; x 10) assessed using continuous monitoring by differential DHM during automated amplification compared to manual amplification. 6 cells / mL) and cell viability (%).

[0080] Figures 6A-6D Depicts exemplary phenotypic profiles of 40 engineered CAR+ T cell compositions, each derived from a multiple myeloma patient. Figure 6A ) and CD8+ population ( Figure 6B ), showing the CD45RA×CCR7 expression profile in the CAR+T cell composition. Figure 6C ) and CD8+ population ( Figure 6D ), showing the CD27×CD28 expression profile in the CAR+T cell composition. Each CAR+T cell composition is represented by a dot (●), a cross (×), a diamond (◇), or a triangle (Δ). DETAILED DESCRIPTION

[0081] Provided herein are methods for generating or producing compositions of engineered cells, such as engineered CD4+ and CD8+ T cells, that express recombinant receptors. In certain embodiments, the methods are used in conjunction with a process comprising: incubating cells, such as a composition of cells, under stimulatory conditions; genetically engineering the cells, for example, by transduction or transfection of a polynucleotide encoding the recombinant receptor; and / or cultivating the engineered cells under conditions that promote cell proliferation and / or expansion.

[0082] In some embodiments, provided herein are methods for producing engineered cell compositions comprising combining CD4+ T cells and CD8+ T cells at a ratio between 2:1 and 1:2 to produce an input composition, and incubating the input composition under stimulatory conditions. In certain embodiments, the method comprises incubating an input composition comprising CD4+ T cells and CD8+ T cells at a ratio between 2:1 and 1:2. In certain embodiments, the input composition comprises a concentration of less than 5 x 10 6 At least 100 x 10 cells / mL 6 Total CD4+ and CD8+ T cells.

[0083] In certain embodiments, provided herein are methods for producing engineered cell compositions comprising introducing a recombinant receptor into a set or fixed number of cells in the cell composition, e.g., at least or about 1 x 10 6 10x10 6 100x 10 6 or 1,000x 10 6In some embodiments, the cells are stimulated cells. In certain embodiments, the cells are living cells. In specific embodiments, the introduction comprises transducing T cells with a viral vector comprising a polynucleotide encoding a recombinant receptor. In certain embodiments, the cell composition comprises at least 80%, at least 85%, at least 90%, or at least 95% of the cells being CD4+ T cells or CD8+ T cells.

[0084] In some embodiments, provided herein are methods for producing an engineered cell composition, the method comprising (i) incubating an input composition under stimulating conditions, thereby producing a stimulating composition; wherein the input composition comprises CD4+ and CD8+ T cells at a ratio of between 2:1 and 1:2, and wherein the input composition comprises CD4+ and CD8+ T cells at a concentration of less than 5 x 10 6 At least 100 x 10 cells / mL 6 Total CD4+ and CD8+ T cells; (ii) introducing the recombinant receptor into less than 300 x 10 6 The invention further comprises introducing into cells of the stimulatory composition, thereby producing an engineered cell composition, wherein the introducing comprises transducing the cells of the stimulatory composition with a viral vector comprising a polynucleotide encoding the recombinant receptor; and (iii) culturing the engineered composition under conditions that promote proliferation and / or expansion of the engineered cells, thereby producing an output composition comprising engineered T cells.

[0085] Different processes can be used to produce genetically engineered T cell populations, including engineered T cells that can be used to produce expression of chimeric antigen receptors. However, in some embodiments, some of these processes may require long or relatively long amounts of time to produce engineered cells. In certain embodiments, some of these processes may differ in their ability to successfully produce engineered cells suitable for therapy from different subjects. In certain embodiments, some of these processes can produce genetically engineered T cell compositions with a high degree of variability for parameters such as cell health, vigor, transduction efficiency, and / or cell activity.

[0086] The embodiments provided address one or more of these problems. In particular embodiments, the methods provided produce engineered T cells suitable for therapy (e.g., autologous cell therapy) in a short or relatively short amount of time compared to some existing processes. In addition, in some embodiments, the methods provided result in a more consistent, less variable process in terms of the amount of time required to produce engineered cells from samples collected from different subjects. In particular embodiments, the methods provided are capable of successfully producing engineered T cells suitable for cell therapy from a high proportion of subjects. In certain embodiments, the resulting cell composition contains a high or relatively high portion of healthy cells, such as cells that are alive and / or do not express apoptosis markers; a high or relatively high portion of cells that express recombinant receptors; and / or cells that have a high or relatively high activity (e.g., cytotoxicity, anti-tumor, and / or cytokine production) in response to antigen stimulation. In some embodiments, provided method provides the method for producing engineered cell products, and has a specific success rate in some aspects, such as a high success rate or a success rate greater than a threshold rate, such as those that can produce therapeutic cell compositions, such as for a large amount or high percentage of samples, such as for all or high percentage of samples, compositions with some required or desired characteristics can be produced, each of the samples is derived from different individual subjects or patients, such as subjects or patients to be treated with therapeutic compositions (for example, in the context of autologous cell therapy). In some aspects, subjects or patients suffer from disease or illness, such as cancer, such as blood cancer or blood cancer such as multiple myeloma. In some aspects, the sample (for the sample of high percentage, therapeutic cell compositions can be produced therefrom) is a patient sample, including, for example, those that are variable with respect to cell phenotype or other parameters of sample or its cells.

[0087] In some embodiments, the methods provided produce engineered T cell compositions that have improved or high cell health, for example, compared to cell compositions produced by other processes. In some embodiments, the compositions comprise a high percentage of cells that are negative for apoptosis markers. In some embodiments, the methods provided produce T cell compositions comprising multifunctional cells with robust cytokine production. In some embodiments, the methods provided produce T cell compositions that are enriched for memory phenotypes, enriched for central memory phenotypes, and / or enriched for cells that are CD27+, CD28+, CCR7+, CD45RA-, CD45RO+, CD62L+, CD3+, CD95+, granzyme B-, and / or CD127+. In some embodiments, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% or more of the cells in the composition, the T cells in the composition, or the engineered T cells in the composition (or, for at least half or most samples produced using the method, or on average for samples produced using the method, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% or more of the cells in the composition) are T cells with a central memory phenotype; are CD27+, CD28+; are CCR7+, CD45RA-; and / or are CCR7+, CD45RO+. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75% or 80% or 85% or 90% or 95% or more of the cells in the composition, the T cells in the composition, or the engineered T cells in the composition (or, for at least half or most samples produced using the method, or on average for samples produced using the method, at least 50%, 55%, 60%, 65%, 70%, 75% or 80% or 85% or 90% or 95% or more of the cells in the composition) are T cells with a memory phenotype; are CD45RA-; and / or are CD45RO+.

[0088] In specific embodiments, provided method is used in combination with the process of effectively producing or producing the engineered cells suitable for use in cell therapy.In certain embodiments, the time arrangement, condition and reagent for each step of the process improve the efficiency of each subsequent step and / or the whole process.For example, in some embodiments, can be high enough to achieve the desired effect (for example, the transduction efficiency of the stimulation or improvement of the cell) under the cell concentration, but low enough to avoid growth slowing down or survival reduction in the subsequent treatment step, hatch, transduce and / or cultivate cells.In addition, in some embodiments, the step of the process is timed to start or end at a specific time point, to improve the efficiency of the subsequent process step and / or the whole process.For example, in some embodiments, the step for hatching and engineering approaches (for example, transduction or transfection cell) is completed earlier than alternative methods in the process, and in certain embodiments, this improves the survival and / or health and / or cell proliferation and amplification speed during the subsequent cultivation step.Therefore, in one aspect, the specific time arrangement, condition and reagent of each step affect the cell outside the single step, and in certain embodiments, affect the performance of the whole process.

[0089] In some embodiments, the method is used in combination with the process of producing or producing the genetically engineered cells suitable for cell therapy in a faster and more efficient manner than an alternative process. In certain embodiments, provided herein is a method with a high success rate to produce or produce engineered cell compositions from a wider range of subject populations compared to those that may come from alternative processes. In certain embodiments, the engineered cells produced or produced by the provided method may have more health, vigor, activity, and may have higher recombinant receptor expression compared to the cells produced by alternative methods. Therefore, in some respects, the speed and efficiency of the provided method for producing engineered cells for cell therapy allow for easier planning and coordination of cell therapy such as autologous therapy for a wider range of subject populations compared to those that may be produced by some alternative methods.

[0090] All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If definitions set forth herein are contrary to or otherwise inconsistent with definitions set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over those incorporated herein by reference.

[0091] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0092] I. Process for Generating Engineered Cells

[0093] Provided herein are methods for producing output compositions of engineered cells, such as engineered CD4+T and CD8+T cells expressing recombinant proteins (e.g., recombinant receptors such as T cell receptors (TCRs) or chimeric antigen receptors (CARs)). In certain embodiments, the methods provided herein are used in conjunction with the manufacture of cell therapies or production thereof, and may be used in conjunction with additional processing steps (such as separation, separation, selection, activation or stimulation, transduction, washing, suspension, dilution, concentration, and / or formulation of cells). In some embodiments, the method for producing or producing engineered cells (e.g., engineered CD4+ and CD8+T cells) includes isolating cells from a subject; preparing, treating, incubating under stimulating conditions; and / or engineering (e.g., transducing) cells, one or more. In some embodiments, the method includes processing steps performed in the following order: first, isolating (e.g., selecting or separating) input cells, e.g., primary CD4+ and CD8+ cells, from a biological sample; incubating the input cells under stimulating conditions, engineering with vector particles (e.g., viral vector particles) to introduce recombinant polynucleotides into the cells, e.g., by transduction or transfection; cultivating the engineered cells, e.g., transduced cells, e.g., to expand the cells; and collecting, harvesting, and / or filling all or a portion of the cells that will be formulated into the output composition in a container (e.g., a bag or vial). In some embodiments, the cells of the generated output composition are reintroduced into the same subject before or after cryopreservation. In some embodiments, the output composition of the engineered cells is suitable for use in therapy (e.g., autologous cell therapy).

[0094] In certain embodiments, the methods provided are used in conjunction with output compositions of cells expressing recombinant receptors produced from an initial or input composition of cells. In certain embodiments, the input composition is produced, generated and / or prepared by combining, mixing and / or merging cells, the cells including cells from a cell composition containing enriched T cells, enriched CD4+T cells and / or enriched CD8+T cells (hereinafter also referred to as a composition enriched in T cells, a composition enriched in CD4+T cells and a composition enriched in CD8+T cells, respectively). In some embodiments, the input composition of the cells is a composition of merged, mixed and / or merged CD4+ and CD8+T cells. In certain embodiments, the methods provided are used in conjunction with one or more of the following: activating and / or stimulating cells, such as cells of the input composition; and / or genetically engineering the activated and / or stimulated cells, for example, to introduce a polynucleotide encoding a recombinant protein by transduction or transfection; and / or cultivating engineered cells, for example, under conditions that promote proliferation and / or amplification. In certain embodiments, the methods can also be used in conjunction with isolating or selecting cells from a biological sample (e.g., from a biological sample obtained, collected, and / or acquired from a subject) to produce an input composition enriched in T cells. In specific embodiments, the provided methods can be used in conjunction with harvesting, collecting, and / or formulating a composition enriched in T cells after the cells have been incubated, activated, stimulated, engineered, transduced, transfected, and / or cultured.

[0095] In some embodiments, incubating the cells under stimulating conditions is or includes incubating the cells with a stimulating agent (e.g., a stimulating agent as described herein in Section IB-1). In certain embodiments, the cells are incubated at a set or fixed concentration (e.g., less than 5 x 10 6 A set or fixed amount of cells (e.g., greater than at least 100 x 10 cells / mL) is incubated under stimulating conditions. 6 In certain embodiments, the incubation is performed for a set or fixed amount of time (such as an amount of time less than 2 days) or for an amount of time between 18 hours and 30 hours.

[0096] In certain embodiments, the methods provided herein are performed in conjunction with engineered (e.g., transduced or transfected) cells. In some embodiments, a set or fixed amount of cells (e.g., live CD4+ and CD8+ cells) are engineered. In some embodiments, a set or fixed concentration (e.g., less than 3 x 10 6 cells / mL) were incubated under stimulating conditions at a concentration greater than at least 10 x 10 6 In certain embodiments, the engineering is performed for a set or fixed amount of time (such as an amount of time within 2 days) or for an amount of time between 18 hours and 30 hours.

[0097] In certain embodiments, at least a portion of the cultivation step is performed with a bioreactor in a closed system, for example, with constant mixing and / or perfusion. In certain embodiments, mixing and / or perfusion is combined with stabilizing and / or progressively replacing spent or old cell culture medium or solution with fresh culture medium or solution. In some embodiments, cultivation is started within a certain amount of time, for example, within 2, 3, 4, or 5 days after incubation starts or starts under stimulating conditions; within 2, 3, 4, or 5 days after mixing, merging, and / or combining cells (e.g., CD4+ and CD8+ cells) to produce an input composition; within 3, 4, 5, or 6 days after a biological sample is collected; and / or within 3, 4, 5, or 6 days after separating, selecting, and / or enriching a composition of enriched T cells (e.g., CD4+ and / or CD8+ T cells) from a biological sample.

[0098] In some embodiments, one or more processing steps are performed at least in part in a serum-free medium. In some embodiments, the serum-free medium is a defined and / or well-defined cell culture medium. In certain embodiments, the serum-free medium is a controlled medium that has been treated, for example, filtered, to remove inhibitors and / or growth factors. In some embodiments, the serum-free medium contains protein. In certain embodiments, the serum-free medium may contain serum albumin, hydrolysates, growth factors, hormones, carrier proteins, and / or attachment factors.

[0099] In some embodiments, provide method is carried out so that one, multiple or all steps in the preparation of the cell for clinical use (such as adoptive cell therapy) are not exposed to cell and are carried out under non-sterile conditions.In some embodiments of this process, cell is separated, separated or selected, transduced, washed, optionally activated or stimulated and prepared, all carried out in closed system.In some embodiments, described one or more steps are carried out outside closed system or device.In some such embodiments, the composition of enriched cell is aseptically transferred out from closed system or device, such as, by aseptic transfer in a separate closed system.

[0100] In a specific embodiment, before, during or after any stage or step of the process of producing the output composition of enriched T cells expressing recombinant receptors, the composition of enriched T cells can be collected, formulated for cryoprotection, cryogenic freezing, and / or stored at less than 0°C, less than -20°C or stored at or below -70°C or -80°C. In some embodiments, cells can be stored for a period of time within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, or for a period of time within 1, 2, 3, 4, 5, 6, 7, 8 weeks, or for a period of time of at least 1, 2, 3, 4, 5, 6, 7 or 8 weeks, or for more than 8 weeks. After storage, the composition of enriched T cells can be thawed, and the process can be recovered from the same point in the process. In some embodiments, the input composition of enriched T cells is cryogenically frozen and stored before further processing (e.g., incubation under stimulating conditions). In a specific embodiment, the composition of enriched T cells cultivated and / or formulated is cryogenically frozen and stored before being given to the subject, for example, as an autologous cell therapy.

[0101] In certain embodiments, provided herein is a method for producing an engineered cell in combination with the process of the following process, comprising the steps of: incubating cells under stimulation conditions, transducing cells to express recombinant receptors (such as CAR), and cultivating cells under conditions of promoting proliferation or amplification. In a specific embodiment, incubation is continued between 18 and 30 hours, for example, for 24 hours or about 24 hours, and subsequently transduction is continued between 18 and 30 hours, for example, for 24 hours or about 24 hours. In certain embodiments, after cells have been stimulated and transduced, cells are cultivated under conditions of promoting stimulation and / or amplification. In certain embodiments, incubation is started, for example, by contacting cells with stimulating reagents, and transduction is started within 48 hours, 36 hours, or 30 hours after incubation has begun. In some embodiments, cultivation is carried out after incubation and transduction, and cultivation is started within 72 hours, 66 hours, or 60 hours after incubation has begun. In certain embodiments, the incubation is carried out until a threshold amount, density and / or expansion of cells is achieved, until at least one day after a threshold amount, density and / or expansion of cells is achieved, and / or until at least 8, 9, 10, 11 or 12 days after initiation of the incubation.

[0102] In certain embodiments, in any stage or step of the process, a portion of cells can be sampled or collected, for example, as during separation, incubation, engineering, cultivation and / or formulation, cells can be obtained from a composition enriched in T cells, while the composition is retained in a closed system. In certain embodiments, the markers, features or characteristics of such cells can be analyzed, including but not limited to viability, apoptosis, activation, stimulation, growth and / or exhaustion. In some embodiments, cells are sampled or collected by an automated process, while the composition enriched in T cells is retained in a closed system. In some embodiments, the analysis of the cells sampled or collected is automated. In specific embodiments, analysis is performed in a closed system under aseptic conditions.

[0103] In some embodiments, provided herein are processes for producing engineered cells comprising the steps of incubating an input cell composition under stimulatory conditions (e.g., to activate T cells in the composition), engineering (e.g., transducing) the cell composition to express a recombinant receptor (e.g., CAR), culturing the cells under conditions that promote cell proliferation or expansion, and / or harvesting or collecting the cells to produce a cell composition comprising engineered cells (e.g., engineered T cells for cell therapy). In some embodiments, the input cell composition is incubated under stimulating conditions comprising: a stimulating reagent (e.g., a bead reagent described in Section IB-1) to cell ratio of at or about 1:1; the input composition comprises CD4+ T cells and CD8+ T cells at a ratio of at or about 1:1 (e.g., a composition enriched for CD4+ T cells and a composition enriched for CD8+ T cells can be combined, mixed, and / or combined at a ratio of equal to or about 1:1 to produce the input composition); the total duration of incubation under stimulating conditions (e.g., with a stimulating reagent) is between about 12 hours and about 36 hours, e.g., between about 18 hours and about 30 hours; the total duration of incubation under stimulating conditions, e.g., in the presence of a stimulating reagent at a concentration of about 5 x 10 5 cells / mL and approximately 5x 10 7 cells / mL (e.g., equal to or about 3 x 10 6 In some embodiments, the cells are engineered under conditions including: contacting the cells with a nucleic acid molecule encoding a recombinant protein (e.g., a recombinant receptor) at a density of about 5 x 10 cells / mL; and / or stimulating and / or activating the cells (e.g., the cells input into the composition) in a serum-free medium (e.g., a serum-free medium comprising one or more recombinant cytokines (e.g., IL-2, IL-7, and IL-15). In some embodiments, the cells are engineered under conditions including: contacting the cells with a nucleic acid molecule encoding a recombinant protein (e.g., a recombinant receptor) at a density of about 5 x 10 5 cells / mL and approximately 5x 107 cells / mL (e.g., equal to or about 1 x 10 6 cells / mL); approximately 100 x 10 6 In some embodiments, the cells are engineered in a serum-free medium (e.g., a serum-free medium comprising one or more recombinant cytokines (e.g., IL-2, IL-7, and IL-15)). In some embodiments, the cells are cultured under conditions that promote cell proliferation or expansion, comprising: culturing the cells under rocking and / or perfusion conditions; and / or culturing the cells in a serum-free medium (e.g., a serum-free medium comprising one or more recombinant cytokines (e.g., IL-2, IL-7, and IL-15), optionally using a higher concentration of recombinant cytokines than the serum-free medium used for stimulation / activation and / or engineering). In some embodiments, when the cells achieve a threshold amount, concentration, and / or expansion, e.g., at least about 3500x10 6 cells or approximately 5500x10 6 In some embodiments, when cells do not achieve a target or threshold at a given time during the stimulation / activation, engineering, cultivation, and / or harvesting process, the cells can be stimulated / activated, engineered, and / or cultivated until the next time point when the target or threshold is achieved.

[0104] In some embodiments, provided herein are processes for producing engineered cells comprising the steps of incubating an input cell composition under stimulatory conditions (e.g., to activate T cells in the composition), engineering (e.g., transducing) the cell composition to express a recombinant receptor (e.g., CAR), culturing the cells under conditions that promote cell proliferation or expansion, and / or harvesting or collecting the cells to produce a cell composition comprising engineered cells (e.g., engineered T cells for cell therapy). In some embodiments, the input cell composition is incubated under stimulating conditions comprising: a stimulating reagent (e.g., a bead reagent described in Section IB-1) to cell ratio of at or about 1:1; the input composition comprises CD4+ T cells and CD8+ T cells at a ratio of at or about 1:1 (e.g., a composition enriched for CD4+ T cells and a composition enriched for CD8+ T cells can be combined, mixed, and / or combined at a ratio of equal to or about 1:1 to produce the input composition); the total duration of incubation under stimulating conditions (e.g., with a stimulating reagent) is between about 12 hours and about 36 hours, e.g., between about 18 hours and about 30 hours; the total duration of incubation under stimulating conditions, e.g., in the presence of a stimulating reagent at a concentration of about 5 x 10 5 cells / mL and approximately 5x 10 7 cells / mL (e.g., equal to or about 3 x 10 6 In some embodiments, the cells are engineered under conditions including: contacting the cells with a nucleic acid molecule encoding a recombinant protein (e.g., a recombinant receptor) at a density of about 5 x 10 cells / mL; and / or stimulating and / or activating the cells (e.g., the cells input into the composition) in a serum-free medium (e.g., a serum-free medium comprising one or more recombinant cytokines (e.g., IL-2, IL-7, and IL-15). In some embodiments, the cells are engineered under conditions including: contacting the cells with a nucleic acid molecule encoding a recombinant protein (e.g., a recombinant receptor) at a density of about 5 x 10 5 cells / mL and approximately 5 x 10 7 cells / mL (e.g., equal to or about 1 x 10 6 cells / mL); at least about 100 x 10 6 cells and up to approximately 200 x 10 6In some embodiments, the cells are engineered in a serum-free medium (e.g., a serum-free medium comprising one or more recombinant cytokines (e.g., IL-2, IL-7, and IL-15)). In some embodiments, the cells are cultured under conditions that promote cell proliferation or expansion, comprising: culturing the cells under rocking and / or perfusion conditions; and / or culturing the cells in a serum-free medium (e.g., a serum-free medium comprising one or more recombinant cytokines (e.g., IL-2, IL-7, and IL-15), optionally using a higher concentration of recombinant cytokines than the serum-free medium used for stimulation / activation and / or engineering). In some embodiments, when the cells achieve a threshold amount, concentration, and / or expansion, e.g., at least about 2400 x 10 6 In some embodiments, when a cell reaches a threshold cell count (e.g., total nucleated cell count) of cells, and when the cells achieve a threshold viability, such as at least about 75% or at least about 85% of the cells are viable, the cultivation is terminated and the cells are harvested. In some embodiments, when cells do not achieve a target or threshold at a given time during the stimulation / activation, engineering, cultivation, and / or harvesting process, the cells can be stimulated / activated, engineered, and / or cultivated until the next time point when the target or threshold is achieved.

[0105] In some embodiments, the cell or cell composition produced and / or processed by the provided method can be compared with the cell or cell composition processed or produced by exemplary and / or alternative processes. In some embodiments, alternative and / or exemplary processes can be different in one or more specific aspects, but in addition contain the similar or identical features, aspects, steps, stages, reagents and / or conditions of the embodiments or aspects of the provided methods being compared. For example, when the provided method is used in conjunction with hatching cells in the presence of a reagent, such cells can be compared with cells that are not hatched with the reagent in the exemplary and / or alternative processes. In some embodiments, unless otherwise specified, the provided method and exemplary and / or alternative processes are originally similar and / or identical, for example, have similar or identical separation, selection, enrichment, activation, stimulation, engineering approaches, transfection, transduction, cultivation and / or preparation steps. In some embodiments, unless otherwise specified, the provided method and alternative processes separate, select and / or enrich cells from the process cells and / or input cells of the same or similar type of biological sample and / or the same cell type.

[0106] Also provided are cells and compositions (including pharmaceutical compositions and formulations) prepared by the methods, as well as kits, systems, and devices for performing the methods. Also provided are methods of using the cells and compositions prepared by the methods (including therapeutic methods, such as methods for adoptive cell therapy), and pharmaceutical compositions for administration to a subject.

[0107] A. Sample and Cell Preparation

[0108] In certain embodiments, provided method is used in combination with one or more input compositions that separate, select and / or enrich cells from biological sample to produce enriched cells (such as T cells).In some embodiments, provided method includes separating cells or its compositions from biological sample, and described biological sample is for example obtained from or derived from those biological samples of experimenter, and described experimenter is for example suffering from specific disease or disease or needs cell therapy or will be given the experimenter of cell therapy.In some respects, experimenter is people, for example, is the experimenter of the patient who needs specific treatment intervention, and described treatment intervention is for example the adoptive cell therapy for its separation, processing and / or engineered cells.Therefore, in some embodiments, cell is primary cell, for example primary human cell.Sample includes tissue, fluid and other samples directly obtained from experimenter.Biological sample can be the sample directly obtained from biological source or the sample through processing.Biological sample includes but is not limited to body fluid (such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat), tissue and organ sample, including the processed sample derived therefrom.

[0109] In some aspects, sample is blood or blood-derived sample, or or be derived from apheresis or leukocyte apheresis product.Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, intestinal associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix uteri, testis, ovary, tonsil or other organs and / or cells derived therefrom.In the context of cell therapy (for example, adoptive cell therapy), sample includes the sample from autologous and allogeneic sources.

[0110] In some examples, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. In some aspects, the sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and / or platelets, and in some aspects contains cells other than red blood cells and platelets.

[0111] In some embodiments, the blood cells collected from the experimenter are washed to, for example, remove the plasma fraction, and the cells are placed in appropriate buffer or medium for subsequent processing steps. In some embodiments, cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, the washing step is completed according to the instructions of the manufacturer by a semi-automatic " flow-through " centrifuge (for example, Cobe 2991 cell processor, Baxter). In some aspects, the washing step is completed according to the instructions of the manufacturer by tangential flow filtration (TFF). In some embodiments, after washing, the cells are resuspended in a variety of biocompatible buffers (for example, as a filtration buffer without Ca). 2+ / Mg 2+ In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

[0112] In some embodiments, preparation method comprises the step of freezing (such as cryopreservation) cell before or after separation, selection and / or enrichment and / or for transduction and engineered hatching.In some embodiments, freezing and subsequent thawing step remove the granulocyte in the cell mass, and remove monocyte to a certain extent.In some embodiments, for example, after washing step, cell is suspended in freezing solution to remove plasma and platelet.In some respects, any of multiple known freezing solution and parameter can be used. In some embodiments, the cells are frozen (e.g., cryopreserved or cryopreserved) in a medium and / or solution at a final concentration of at or about 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or 5.0% DMSO, or between at or about 1% and at or about 15%, between at or about 6% and at or about 12%, between at or about 5% and at or about 10%, or between at or about 6% and at or about 8% DMSO. In a specific embodiment, cells are frozen (for example, cryogenic freezing or cryopreservation) in a medium and / or solution at a final concentration of equal to or about 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5% or 0.25% HSA or between 0.1% and -5%, between 0.25% and 4%, between 0.5% and 2% or between 1% and 2%. One example relates to the use of PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing mediums. The cell is then diluted 1:1 with culture medium so that the final concentrations of DMSO and HSA are 10% and 4% respectively. The cell is then typically frozen to a rate equal to or about 1 ° / minute equal to or about -80 °C and stored in the gas phase of a liquid nitrogen storage tank.

[0113] In some embodiments, the separation of cell or colony includes one or more preparations and / or cell separation steps based on non-affinity.In some examples, cell is washed, centrifuged and / or hatched in the presence of one or more reagents, for example, to remove unwanted components, components required for enrichment, cracking or removal of cells sensitive to specific reagents.In some examples, based on one or more characteristics (such as density, adhesion properties, size, sensitivity and / or resistance to specific components) separation cells.In some embodiments, described method includes density-based cell separation method, such as by dissolving red blood cells and centrifuging by Percoll or Ficoll gradient to prepare white blood cells from peripheral blood.

[0114] In some embodiments, at least a portion of the selection step includes hatching the cell with a selection reagent.For example, hatching with one or more selection reagents as a part of a selection method can be carried out as follows: use one or more selection reagents, for expression or presence based on one or more specific molecules (such as surface markers (such as surface proteins), intracellular markers or nucleic acids) in a cell or on a cell to select one or more different cell types. In some embodiments, any known method can be used, using one or more selection reagents, separated based on this type of mark. In some embodiments, one or more selection reagents cause separation, and the separation is based on the separation of affinity or immunoaffinity. For example, in some respects, selection includes hatching with one or more reagents, for separating cells and cell mass based on cellular expression or expression level of one or more marks (typically cell surface markers), for example, by hatching with the antibody or binding partner that are specifically bound to this type of mark, usually washing steps are carried out afterwards and the cell that has been bound to antibody or binding partner is separated from those cells that are not bound to antibody or binding partner.

[0115] In some aspects of this type of process, a certain volume of cells is mixed with a certain amount of required selection reagent based on affinity. Selection based on immunoaffinity can be carried out using any system or method, which results in favorable energy interactions between the cells separated and the molecules specifically binding the label (for example, antibodies or other binding partners on a solid surface (for example, particles)) on the cell. In some embodiments, the method is to use particles (such as beads, for example magnetic beads) coated with a selection agent (for example, an antibody) specific for cell markers. Particles (such as beads) can be incubated or mixed with cells in a container (such as a tube or bag) with a constant cell density and the ratio of particles (such as beads) while shaking or mixing to help promote energy-favorable interactions. In other cases, the method includes selecting cells, wherein all or part of the selection is carried out in the lumen of a centrifugal chamber, for example, under centrifugal rotation. In some embodiments, hatching cells with selection reagent (for example, selection reagent based on immunoaffinity) is carried out in a centrifugal chamber. In certain embodiments, the separation or isolation is performed using a system, device or apparatus as described in International Patent Application Publication No. WO 2009 / 072003 or US20110003380 A1. In one example, the system is a system as described in International Publication No. WO 2016 / 073602.

[0116] In some embodiments, by performing such selection steps or portions thereof (e.g., incubation with antibody-coated particles (e.g., magnetic beads)) in the cavity of a centrifugal chamber, the user is able to control certain parameters, such as the volume of various solutions, the addition of solutions during processing, and their timing, which can provide various advantages compared to other available methods. For example, the ability to reduce the volume of liquid in the cavity during incubation can increase the concentration of particles (e.g., bead reagents) used in the selection, and thereby increase the chemical potential of the solution, without affecting the total number of cells in the cavity. This, in turn, can enhance pairwise interactions between the cells being processed and the particles used for selection. In some embodiments, for example, when associated with the systems, circuits, and controls as described herein, performing the incubation step in the chamber allows the user to agitate the solution at one or more desired times during the incubation, which can also improve interactions.

[0117] In some embodiments, at least a portion of the selection step is carried out in a centrifugal chamber, which includes incubating cells with a selection reagent. In some aspects of this type of process, a certain volume of cells is mixed with a certain amount of the selection reagent required based on affinity, and the volume and amount are significantly less than the volume and amount usually used when similar selection is carried out to select the cells of the same number and / or the cells of the same volume according to the instructions of manufacturers in a tube or container. In some embodiments, the amount of the one or more selection reagents adopted is no more than or no more than about 5%, no more than or no more than about 10%, no more than or no more than about 15%, no more than or no more than about 20%, no more than or no more than about 25%, no more than or no more than about 50%, no more than or no more than about 60%, no more than or no more than about 70% or no more than or no more than about 80% for the amount of the same one or more selection reagents of the cells selected for the same number and / or the cells of the same volume in the incubation based on a tube or container according to the instructions of manufacturers.

[0118] In some embodiments, for selection of cells, e.g., immunoaffinity-based selection, the cells are incubated in a chamber cavity in a composition that further contains a selection buffer with a selection agent, e.g., a molecule, e.g., an antibody, that specifically binds to a surface marker on the cells one wishes to enrich and / or deplete (but not on other cells in the composition), optionally coupled to a scaffold (e.g., a polymer or surface, e.g., beads, e.g., magnetic beads, e.g., magnetic beads coupled to monoclonal antibodies specific for CD4 and CD8). In some embodiments, the selection agent is added to the cells in the chamber cavity as described in an amount that is significantly less than or about less than the amount of selection agent typically used or required to achieve approximately the same or similar selection efficiency for the same number of cells or the same volume of cells when selection is performed in a shaken or rotating tube (e.g., no more than or about no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the amount). In some embodiments, incubate when adding selection buffer to cells and selection reagent, to realize the target volume of hatching for example for or about 10mL to for or about 200mL (for example at least or at least about or about or 10mL, 20mL, 30mL, 40mL, 50mL, 60mL, 70mL, 80mL, 90mL, 100mL, 150mL or 200mL) reagent.In some embodiments, buffer and selection reagent are pre-mixed before being added to cells.In some embodiments, buffer and selection reagent are selected to be added to cells separately.In some embodiments, selection hatching is to carry out under periodic gentle mixing condition, and this can contribute to promoting the interaction that is favourable on energy, thereby allows to use less total selection reagent when realizing high selection efficiency.

[0119] In some embodiments, the total duration of incubation with the selection agent is from 5 minutes or about 5 minutes to 6 hours or about 6 hours, such as 30 minutes to 3 hours, for example at least or at least about 30 minutes, 60 minutes, 120 minutes, or 180 minutes.

[0120] In some embodiments, incubation is typically carried out under mixing conditions, such as in the presence of rotation, typically with relatively low force or speed, such as a speed lower than that used to pellet the cells, such as from or about 600 rpm to or about 1700 rpm (e.g., at or about or at least 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm), such as at an RCF of from or about 80 g to 100 g (e.g., at or about or at least 80 g, 85 g, 90 g, 95 g, or 100 g) at the sample or chamber wall or other container wall. In some embodiments, rotation is performed using repeated intervals of such low-speed rotation followed by a rest period, such as rotation and / or rest for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, such as rotation for about 1 or 2 seconds followed by a rest for about 5, 6, 7, or 8 seconds.

[0121] In some embodiments, this process is performed in a completely closed system with the chamber integrated in. In some embodiments, this process (and in some aspects one or more additional steps, such as a pre-wash step to wash a sample containing cells, such as an apheresis sample) is performed in an automated manner such that cells, reagents, and other components are drawn into and out of the chamber and centrifuged at appropriate times so that the washing and binding steps are accomplished in a single closed system using an automated procedure.

[0122] In some embodiments, after hatching and / or mixing cells and one and / or multiple selection reagents, the hatched cell experiences are separated to select cells based on the presence or absence of one or more specific reagents. In some embodiments, separation is carried out in the same closed system, wherein cells are hatched together with the selection reagent. In some embodiments, after hatching together with the selection reagent, the hatched cells (including cells wherein in conjunction with the selection reagent) are transferred to the system for separating cells based on immunoaffinity. In some embodiments, the system for separation based on immunoaffinity is or contains a magnetic separation column.

[0123] Such separation steps can be based on positive selection (wherein cells that have bound an agent (e.g., an antibody or binding partner) are retained for further use) and / or negative selection (wherein cells that have not bound an agent, such as an antibody or binding partner, are retained. In some instances, both fractions are retained for further use. In some aspects, negative selection can be particularly useful in cases where antibodies that specifically identify cell types in a heterogeneous population are unavailable, making it best to perform separation based on markers expressed by cells other than the desired population.

[0124] In some embodiments, the process steps further include negative and / or positive selection of the incubated cells, for example using a system or device that can perform affinity-based selection. In some embodiments, separation is performed by enriching a specific cell population via positive selection, or depleting a specific cell population via negative selection. In some embodiments, positive or negative selection is accomplished by incubating the cells with one or more antibodies or other binding agents that bind to a marker expressed (marked) on the positively or negatively selected cells, respectively. + ) or expressed at relatively high levels (marker 高 ) specifically binds to one or more surface markers of the .

[0125] Separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell (such as those expressing a marker) refers to increasing the number or percentage of such cells, but need not result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal or depletion of a particular type of cell (such as those expressing a marker) refers to reducing the number or percentage of such cells, but need not result in the complete removal of all such cells.

[0126] In some examples, carry out multiple rounds of separation steps, wherein make the fraction from the positive or negative selection of a step experience another separation step, such as follow-up positive or negative selection.In some examples, single separation step can exhaust the cell expressing multiple marks simultaneously, such as by hatching together with cell and multiple antibodies or binding partners (each kind of antibody or binding partners is specific to the mark tool targeted for negative selection).Equally, by hatching together with multiple antibodies or binding partners expressed on various cell types, can simultaneously positive selection multiple cell types.In certain embodiments, separation step is repeated and / or carried out more than once, wherein make the positive or negative selection fraction from a step experience identical separation step, such as repeated positive or negative selection.In some examples, single separation step is repeated and / or carried out more than once, such as to increase the purity of selected cell and / or to further remove and / or exhaust the cell of negative selection from the fraction of negative selection.In certain embodiments, one or more separation steps are carried out twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more than ten times. In certain embodiments, one or more selection steps are performed and / or repeated between one and ten times, between one and five times, or between three and five times.

[0127] For example, in some aspects, specific subpopulations of T cells, such as cells that are positive for or express high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD95+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells) are isolated by positive or negative selection techniques. In some embodiments, such cells are selected by incubating with one or more antibodies or binding partners that specifically bind to such markers. In some embodiments, the antibodies or binding partners can be, for example, directly or indirectly conjugated to a solid support or matrix (such as magnetic beads or paramagnetic beads) that enables selection. For example, anti-CD3 / anti-CD28 conjugated magnetic beads (e.g., M-450CD3 / CD28 T Cell Expander and / or CD3+, CD28+ T cells were positively selected by ELISA.

[0128] In some embodiments, T cells are separated from PBMC samples by negative selection of a marker (such as CD14) expressed on non-T cells (such as B cells, monocytes or other leukocytes). In some aspects, CD4+ or CD8+ selection steps are used to separate CD4+ helper cells and CD8+ cytotoxic T cells. By expressing or expressing with a relatively high degree of marker on one or more immature, memory and / or effector T cell subsets, such CD4+ and CD8+ colonies can be further classified into subgroups.

[0129] In some embodiments, CD8+ cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the corresponding subpopulations. In some embodiments, central memory T cells (T CM ) cells to increase efficacy, such as to improve long-term survival, expansion and / or engraftment after administration, which in some aspects is particularly robust in such subpopulations. See, for example, Terakura et al. (2012) Blood. 1: 72-82; Wang et al. (2012) J Immunother. 35(9): 689-701. In some embodiments, the combination is enriched for T CM The CD8+T cells and CD4+T cells further enhance the efficacy.

[0130] In some embodiments, memory T cells are present in CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes.PBMCs can be enriched or depleted for CD62L-CD8+ and / or CD62L+CD8+ fractions, for example, using anti-CD8 and anti-CD62L antibodies.

[0131] In some embodiments, the central memory T (T CM In some aspects, the enrichment of T cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD27, CD28, CD95, CD3 and / or CD127; in some aspects, it is based on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. ... CM The CD8+ population of cells is isolated by depletion of cells expressing CD4, CD14, CD45RA and positive selection or enrichment of cells expressing CD62L. In one aspect, central memory T (T CM ) The enrichment of cells is carried out starting from the negative cell fraction selected based on CD4 expression, and the negative cell fraction is subjected to negative selection based on the expression of CD14 and CD45RA and positive selection based on CD62L. In some aspects, such selection is carried out simultaneously, and in other aspects, it is carried out sequentially in any order. In some aspects, the same selection step based on CD4 expression used to prepare CD8+ cell populations or subpopulations is also used to produce CD4+ cell populations or subpopulations, so that the positive and negative fractions from the CD4-based separation are retained and used in the subsequent steps of the method, optionally after one or more other positive or negative selection steps. In some embodiments, the selection of CD4+ cell populations and the selection of CD8+ cell populations are carried out simultaneously. In some embodiments, the selection of CD4+ cell populations and the selection of CD8+ cell populations are carried out sequentially in any order. In some embodiments, the method for selecting cells may include those described in disclosed U.S. application number US20170037369. In some embodiments, the selected CD4+ cell populations and the selected CD8+ cell populations can be combined after selection. In some aspects, a selected population of CD4+ cells and a selected population of CD8+ cells can be combined in a bioreactor bag as described herein.

[0132] In some embodiments, the central memory CD8+ cells are CD27+, CD28+, CD62L+, CCR7+, CD45RA- and / or CD45RO+. In some embodiments, the central memory CD8+ cells are CD62L+ and CD45RO+. In some embodiments, the central memory CD8+ cells are CCR7+ and CD45RO+. In some embodiments, the central memory CD8+ cells are CCR7+ and CD45RA-. In some embodiments, the central memory CD8+ cells are CD62L+ and CCR7+. In some embodiments, the central memory CD8+ cells are CD62L+ / CD45RA-, CCR7+ / CD45RA-, CD62L+ / CCR7+, or CD62L+ / CCR7+ / CD45RA-, and have moderate to high expression of CD44. In some embodiments, central memory CD8+ cells are CD27+ / CD28+ / CD62L+ / CD45RA-, CD27+ / CD28+ / CCR7+ / CD45RA-, CD27+ / CD28+ / CD62L+ / CCR7+, or CD27+ / CD28+ / CD62L+ / CCR7+ / CD45RA-.

[0133] In certain embodiments, a biological sample (e.g., a sample of PBMC or other white blood cells) is subjected to selection of CD4+ T cells, wherein negative and positive fractions are retained. In certain embodiments, CD8+ T cells are selected from the negative fraction. In some embodiments, a biological sample is subjected to selection of CD8+ T cells, wherein negative and positive fractions are retained. In certain embodiments, CD4+ T cells are selected from the negative fraction.

[0134] In a specific example, a PBMC sample or other leukocyte sample is subjected to selection for CD4+ cells, wherein 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 specific to central memory T cells (such as CD62L or CCR7), wherein the positive and negative selections are performed in either order.

[0135] In some embodiments, CD4+ T helper cells are classified 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+, or CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, central memory CD4+ cells are CD27+, CD28+, CD62L+, CCR7+, CD45RA- and / or CD45RO+. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, central memory CD4+ cells are CCR7+ and CD45RO+. In some embodiments, central memory CD4+ cells are CCR7+ and CD45RA-. In some embodiments, central memory CD4+ cells are CD62L+ and CCR7+. In some embodiments, central memory CD4+ cells are CD62L+ / CD45RA-, CCR 7+ / CD45RA-, CD62L+ / CCR7+, or CD62L+ / CCR7+ / CD45RA-, and have moderate to high expression of CD44. In some embodiments, central memory CD4+ cells are CD27+ / CD28+ / CD62L+ / CD45RA-, CD27+ / CD28+ / CCR7+ / CD45RA-, CD27+ / CD28+ / CD 62L+ / CCR7+, or CD27+ / CD28+ / CD62L+ / CCR7+ / CD45RA-. In some embodiments, effector CD4+ cells are CD62L- and CD45RO-.

[0136] In one example, in order to enrich CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes 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 magnetic beads or paramagnetic beads) to allow separation of cells for positive and / or negative selection. For example, in some embodiments, immunomagnetic (or affinity magnetic) separation techniques are used to separate or isolate cells and cell populations (reviewed in Methods in Molecular Medicine, Vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, pp. 17-25, S.A. Brooks and U. Schumacher, eds. Humana Press Inc., Totowa, New Jersey).

[0137] In some aspects, the incubated cell sample or composition to be separated is mixed with a medium containing small magnetizable or magnetically responsive materials (such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., Dynabeads or The magnetically responsive material (e.g., particle) is typically incubated with a selection agent (e.g., beads). The magnetically responsive material (e.g., particle) is typically attached directly or indirectly to a binding partner (e.g., antibody) that specifically binds to a molecule (e.g., surface marker) present on a cell, a plurality of cells, or a cell population that requires isolation (e.g., negative or positive selection).

[0138] In some embodiments, magnetic particles or magnetic beads include magnetic response materials that are bound to specific binding members (such as antibodies or other binding partners). There are many well-known magnetic response materials for magnetic separation methods. Suitable magnetic particles are included in those described in Molday, U.S. Patent No. 4,452,773 and European Patent Specification EP 452342B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Patent No. 4,795,698 and Liberti et al., U.S. Patent No. 5,200,084 are other examples.

[0139] The incubation is typically performed under conditions such that the antibody or binding partner, or a molecule that specifically binds to such an antibody or binding partner attached to the magnetic particles or beads (such as a secondary antibody or other reagent), specifically binds to the cell surface molecule (if present on cells in the sample).

[0140] In certain embodiments, magnetic responsive particles are coated in a primary antibody or other binding partners, secondary antibodies, lectins, enzymes or streptavidin. In certain embodiments, magnetic particles are attached to cells via the coating of a primary antibody specific for one or more markers. In certain embodiments, cells are labeled with a primary antibody or a binding partner instead of beads, and then the coated magnetic particles of a cell type specific secondary antibody or other binding partner (e.g., streptavidin) are added. In certain embodiments, the streptavidin-coated magnetic particles are used in combination with a biotinylated primary antibody or secondary antibody.

[0141] In some aspects, separation is achieved in the process whereby the sample is placed in a magnetic field, and those cells with magnetic response or magnetizable particles attached thereto will be attracted to the magnet and separated from unlabeled cells. For positive selection, cells attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is carried out during the same selection step, wherein positive and negative fractions are retained and further processed or are subjected to a further separation step.

[0142] In some embodiments, the selection based on affinity is carried out via magnetically activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetic activated cell sorting (MACS) such as CliniMACS system can select the cell with the magnetized particles attached thereto in high purity. In certain embodiments, MACS operates in such a mode, wherein non-target species and target species are sequentially eluted after applying an external magnetic field. That is to say, the cell attached to the magnetized particles is maintained in place, and unattached species are eluted. Then, after completing this first elution step, in some way discharge is captured in the magnetic field and is prevented from eluting species so that they can be eluted and recovered. In certain embodiments, non-target cells are marked and exhausted from heterogeneous cell populations.

[0143] In some embodiments, the magnetically responsive particles remain attached to the cells, which are subsequently incubated, cultured, and / or engineered; in some aspects, the particles remain attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. 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. In some embodiments, the magnetizable particles are biodegradable.

[0144] In some respects, separation and / or other steps are to use CliniMACS system (Miltenyi Biotec) to carry out, for example, for separating cells with clinical scale level automation in closed and aseptic system. Components can include integrated microcomputer, magnetic separation unit, peristaltic pump and various pinch valves. In some respects, all components of integrated computer control instrument also indicate that described system performs repetitive program with standardized sequence. In some respects, magnetic separation unit includes removable permanent magnet and the support for selecting post. Peristaltic pump controls the flow velocity of whole tube group, and together with pinch valve, guarantees the controlled flow of the buffer solution through described system and the continuous suspension of cell.

[0145] In some respects, the CliniMACS system uses the magnetizable particles of antibody coupling, which are provided in a sterile, pyrogen-free solution. In some embodiments, after using magnetic particle labeled cells, cells are washed to remove excessive particles. The cell preparation bag is then connected to the tube set, which is in turn connected to the bag and cell collection bag containing buffer. The tube set is composed of pre-assembled sterile tubes (comprising pre-column and separation column) and is only for disposable use. After starting the separation procedure, the system automatically applies the cell sample to the separation column. The labeled cells are retained in the column, and unlabeled cells are removed by a series of washing steps. In some embodiments, the cell mass used for use with the methods described herein is not labeled and is not retained in the column. In some embodiments, the cell mass used for use with the methods described herein is labeled and retained in the column. In some embodiments, the cell mass used for use with the methods described herein is eluted from the column after removing the magnetic field, and is collected in the cell collection bag.

[0146] In certain embodiments, use CliniMACS Prodigy system (Miltenyi Biotec) to separate and / or other steps. In some aspects, the CliniMACS Prodigy system is equipped with a cell treatment consortium, which allows washing and classification of cells by centrifugal automation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software, which determines the optimal cell classification terminal by identifying the macroscopic layer of the source cell product. For example, peripheral blood is automatically separated into red blood cells, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation room, which realizes cell culture protocols, such as cell differentiation and amplification, antigen loading and long-term cell culture. The input port can allow aseptic removal and supplementary culture medium, and integrated microscope monitoring cells can be used. See, eg, Klebanoff et al. (2012) J Immunother. 35(9):651–660, Terakura et al. (2012) Blood. 1:72–82, and Wang et al. (2012) J Immunother. 35(9):689-701.

[0147] In some embodiments, the cell populations described herein are collected and enriched (or depleted) via flow cytometry, wherein cells stained for a variety of cell surface markers are carried in a fluid stream. In some embodiments, the cell populations described herein are collected and enriched (or depleted) via preparation 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, WO 2010 / 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 a variety of markers, thereby allowing the separation of clearly defined T cell subsets with high purity.

[0148] In some embodiments, antibodies or binding partners are labeled with one or more detectable labels to facilitate separation for positive and / or negative selection. For example, separation can be based on the combination with fluorescently labeled antibodies. In some examples, separation cells are carried in a fluid stream based on the combination of antibodies or other binding partners specific for one or more cell surface markers, such as by fluorescence activated cell sorting (FACS) (including preparation scale (FACS)) and / or microelectromechanical systems (MEMS) chips, for example, in combination with flow cytometry systems. Such methods allow positive and negative selection to be performed simultaneously based on multiple markers.

[0149] In some embodiments, separation and / or selection result in one or more compositions enriched for T cells (e.g., CD3+ T cells, CD4+ T cells, and / or CD8+ T cells). In some embodiments, two or more separate compositions enriched for T cells are separated, selected, enriched, or obtained from a single biological sample. In some embodiments, separate compositions are separated, selected, enriched, and / or obtained from separate biological samples collected, obtained, and / or obtained from the same subject.

[0150] In certain embodiments, the isolation and / or selection results in one or more compositions enriched for T cells comprising 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 99%, at least or about 99.5%, at least or about 99.9%, or about 100% CD3+ T cells. In certain embodiments, the composition enriched for T cells consists essentially of CD3+ T cells.

[0151] In certain embodiments, the isolation and / or enrichment results in a composition enriched for CD4+ T cells comprising at least or at least about 60%, at least or at least about 65%, at least or at least about 70%, at least or at least about 75%, at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 98%, at least or at least about 99%, at least or at least about 99.5%, at least or at least about 99.9%, or at or about 100% CD4+ T cells. In certain embodiments, the infusion composition of CD4+ T cells comprises less than or about 40%, less than or about 35%, less than or about 30%, less than or about 25%, less than or about 20%, less than or about 15%, less than or about 10%, less than or about 5%, less than or about 1%, less than or about 0.1%, or less than or about 0.01% CD8+ T cells, and / or contains no CD8+ T cells, and / or is free of or essentially free of CD8+ T cells. In some embodiments, the composition enriched for T cells consists essentially of CD4+ T cells.

[0152] In certain embodiments, the isolation and / or enrichment results in a composition enriched for CD8+ T cells comprising at least or at least about 60%, at least or at least about 65%, at least or at least about 70%, at least or at least about 75%, at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 98%, at least or at least about 99%, at least or at least about 99.5%, at least or at least about 99.9%, or at or about 100% CD8+ T cells. In certain embodiments, the composition of CD8+ T cells contains less than or about 40%, less than or about 35%, less than or about 30%, less than or about 25%, less than or about 20%, less than or about 15%, less than or about 10%, less than or about 5%, less than or about 1%, less than or about 0.1%, or less than or about 0.01% CD4+ T cells, and / or contains no CD4+ T cells, and / or is free of or essentially free of CD4+ T cells. In some embodiments, the composition enriched for T cells consists essentially of CD8+ T cells.

[0153] In some embodiments, after separation, selection and / or enrichment, one or more compositions of enriched T cells are frozen, such as cryopreserved and / or cryopreserved. In specific embodiments, after separation, selection and / or enrichment, the compositions of enriched CD4+T cells are frozen, such as cryopreserved and / or cryopreserved. In certain embodiments, after separation, selection and / or enrichment, the compositions of enriched CD8+T cells are frozen, such as cryopreserved and / or cryopreserved. In some embodiments, before hatching, activating, stimulating, engineering, transduction, transfection, cultivating, amplifying, harvesting and / or preparing any step of cell composition, one or more compositions of enriched T cells are frozen, such as cryopreserved and / or cryopreserved. In specific embodiments, before hatching, activating, stimulating, engineering, transduction, transfection, cultivating, amplifying, harvesting and / or preparing any step of cell composition, the compositions of enriched CD4+T cells are frozen, such as cryopreserved and / or cryopreserved. In some embodiments, before incubation, activation, stimulation, engineering, transduction, transfection, cultivation, amplification, harvesting and / or preparing any step of cell composition, the composition enriched in CD8+ T cells is frozen, for example, cryopreserved and / or cryopreserved. In a specific embodiment, one or more cryogenically frozen input compositions are stored for example at or at about -80°C for 12 hours and 7 days, between 24 hours and 120 hours, or between 2 days and 5 days. In a specific embodiment, one or more cryogenically frozen input compositions are stored at or at about -80°C for less than 10 days, 9 days, 8 days, 7 days, 6 days, or 5 days, 4 days, 3 days, 2 days or 1 day. In some embodiments, one or more cryogenically frozen input compositions are stored at or at about -80°C or stored for about 1 day, 2 days, 3 days, 4 days, 5 days or 6 days.

[0154] In some embodiments, a sample containing cells (eg, an apheresis product or a leukapheresis product) is washed to remove one or more anticoagulants, such as heparin, added during the apheresis or leukapheresis.

[0155] In some embodiments, a sample containing cells (e.g., a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, a leukapheresis product) is cryopreserved and / or cryoprotected (e.g., frozen) and then thawed prior to any step of isolating, selecting, activating, stimulating, engineering, transducing, transfecting, incubating, culturing, harvesting, formulating a cell population, and / or administering the formulated cell population to a subject.

[0156] In certain embodiments, the apheresis product or leukapheresis product is cryopreserved and / or cryoprotected (e.g., frozen) and then thawed before performing cell selection or separation steps (e.g., T cell selection or separation steps) as described below. In some embodiments, after T cell selection or separation steps are performed to apheresis products or leukapheresis products cryopreserved and / or cryoprotected, during or between any subsequent steps (activation, stimulation, engineering, transduction, transfection, incubation, cultivation, harvesting, preparation of cell populations and / or the steps of administering the prepared cell populations to the subject), no additional cryopreservation and / or cryoprotection steps are performed. For example, T cells selected from apheresis products or leukapheresis products cryopreserved and / or cryoprotected for thawing are not cryopreserved and / or cryoprotected again before being thawed for downstream processes (e.g., T cell activation / stimulation or transduction).

[0157] In certain embodiments, the cryopreserved and / or cryoprotected apheresis product or leukapheresis product is stored (e.g., no T cell selection is performed before freezing the sample), which in some aspects can provide greater flexibility for subsequent manufacturing steps. In one aspect, storing cells before selection increases the cell yield of downstream processes, and storing cells earlier may mean that they are healthier and may be more likely to meet manufacturing success criteria. In another aspect, once thawed, the cryopreserved and / or cryoprotected apheresis product or leukapheresis product can be subjected to one or more different selection methods. The advantages of this method are, among other things, enhancing the availability, efficacy, and / or other aspects of cells for cell therapy for a disease or condition in a subject (e.g., in a sample donor and / or another recipient).

[0158] In some embodiments, the time after the donor is diagnosed with a disease or illness, before cell selection or when not carrying out previous cell selection (for example, without previous T cell selection, such as by chromatography selection) collect and cryopreserve and / or cryoprotection sample (for example apheresis or leukocyte apheresis sample). In some aspects, the time of cryopreservation is also before the donor has accepted one or more of the following: any initial treatment for the disease or illness, any targeted therapy or any treatment marked for the treatment of the disease or illness, or any treatment except radiation and / or chemotherapy. In some embodiments, after the first recurrence of the disease after the initial treatment of the disease and before the donor or subject receives the subsequent treatment of the disease, sample is collected. Initial and / or subsequent treatment can be a therapy except cell therapy. In some embodiments, the collected cells can be used for use in cell therapy after initial and / or subsequent treatment. In one aspect, the cryopreservation and / or cryoprotection sample without previous cell selection can help reduce upfront costs, such as the cost relevant to the untreated patient who may cross and need later treatment in a randomized clinical trial.

[0159] In some embodiments, after the second-line treatment of the disease, after the disease relapses for the second time, and before the donor or subject receives the subsequent treatment of the disease, before cell selection or without previous cell selection (for example, without previous T cell selection, such as by chromatography selection) collect and cryopreserve and / or cryoprotect sample (for example apheresis or leukocyte apheresis sample). In some embodiments, for example, by assessing certain risk factors, the patient is identified as possibly recurring after the second-line treatment. In some embodiments, risk factors are based on disease type and / or genetics, such as double hit lymphoma, primary refractory cancer or activated B cell lymphoma. In some embodiments, risk factors are based on clinical manifestations, such as other poor prognostic indicators (for example, IPI (International Prognostic Index)>2) after early recurrence or treatment after first-line treatment.

[0160] In some embodiments, the time before the donor or subject is diagnosed with the disease, before cell selection or when not carrying out previous cell selection (for example, without previous T cell selection, such as by chromatography selection) collect and cryopreserve and / or cryoprotect sample (such as apheresis or leukocyte apheresis sample). In some aspects, it can be determined that the donor or subject has the risk of suffering from the disease. In some aspects, the donor or subject can be a healthy subject. In some cases, if cell therapy is needed in the later stage of life, the donor or subject can choose to deposit or store cells and will not be considered as having the risk of suffering from the disease or being diagnosed with the disease. In some embodiments, based on factors such as genetic mutation, genetic abnormality, genetic damage, family medical history, protein abnormality (such as protein production and / or processing defects) and the lifestyle selection that may increase the risk of suffering from the disease, it can be thought that the donor or subject has the risk of suffering from the disease. In some embodiments, cells are collected as prevention.

[0161] In some embodiments, a cryopreserved and / or cryoprotected cell sample (e.g., an apheresis or leukapheresis sample), such as a cell sample that has not undergone prior cell selection (e.g., without prior T cell selection, such as selection by chromatography), is stored or stored for a period of greater than or equal to or about 12 hours, 24 hours, 36 hours, or 48 hours. In some embodiments, the sample is stored or stored for a period of greater than or equal to 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the sample is placed in long-term storage or long-term storage. In some aspects, the samples are stored for a period of greater than or equal to or about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, or more.

[0162] In some embodiments, the apheresis or leukapheresis sample obtained from the donor is transported to a storage or processing facility in a cooled environment, and / or stored at low temperatures in a storage facility or processed in a processing facility. In some embodiments, before transport, the sample is processed, for example, by selecting T cells (such as CD4+ and / or CD8+T cells). In some embodiments, this treatment is performed after transport and before the sample is stored at low temperatures. In some embodiments, the sample is processed after thawing after low temperature storage.

[0163] By allowing a donor to store their cells at a stage when the donor, and therefore their cells, have not yet received extensive treatment for the disease and / or before the disease or condition or its diagnosis is narrowed, such cells may have certain advantages for use in cell therapy compared to cells collected after one or more rounds of treatment. For example, cells harvested before one or more rounds of treatment may be healthier, may exhibit higher levels of certain cellular activities, may grow faster, and / or may be more susceptible to genetic manipulation than cells that have already undergone several rounds of treatment. Another example of an advantage according to the embodiments described herein may include convenience. For example, by collecting (optionally processing) and storing donor cells before they are needed for cell therapy, the cells will be readily available if and when the recipient needs them later. This can increase apheresis laboratory capacity, providing technicians with greater flexibility to schedule apheresis collection processes.

[0164] Exemplary methods and systems for cryogenic storage and processing of cells from samples such as apheresis samples may include those described in International Publication No. WO 2018170188. In some embodiments, the methods and systems are directed to collecting apheresis before the patient needs cell therapy, and then cryopreserving the apheresis sample for later use in engineering cells (such as T cells) with recombinant receptors (such as CAR). In some cases, such processes may include those described herein. In some embodiments, apheresis samples are collected from the subject, and are cryopreserved before subsequent T cell selection, activation, stimulation, engineering, transduction, transfection, incubation, cultivation, harvesting, preparing cell mass and / or the prepared cell mass is given to the subject. In such examples, before making the sample undergo one or more selection steps (such as any steps described herein), the cryopreserved apheresis sample is thawed.

[0165] In some embodiments, a cryopreserved and / or cryoprotected cell sample (e.g., apheresis or leukapheresis sample) (e.g., a cell sample that has not undergone previous cell selection (e.g., without previous T cell selection, such as by chromatography)) is thawed prior to its downstream processing for the manufacture of a cell population (e.g., a T cell population containing CAR+T cells) for cell therapy. In some embodiments, such cryopreserved and / or cryoprotected cell sample (e.g., apheresis or leukapheresis sample) is used in combination with a process provided herein for engineering T cell therapy (e.g., CAR+T cell therapy). In a particular example, no further cryopreservation step is performed before or during the harvesting / forming step.

[0166] 1. Input composition

[0167] In certain embodiments, the methods provided are used in conjunction with an input composition for producing and / or preparing cells. In certain embodiments, the input cell composition is a composition for use in genetically engineered cells (e.g., cells that will be genetically engineered and / or will undergo a process for producing genetically engineered cells). In certain embodiments, the cells will be treated, contacted, and / or incubated with nucleic acids encoding recombinant receptors. In certain embodiments, the input cell composition contains CD4+ T cells and CD8+ T cells. In specific embodiments, the input cell composition contains CD4+ T cells and CD8+ T cells that are naive and / or naive-like T cells.

[0168] In some embodiments, a desired ratio, fixed ratio, and / or controlled ratio is a ratio or number of cells at which two types of cells or separate cell populations are included in an input cell composition that is designed to produce an output cell composition having a desired, defined, and / or controlled ratio of engineered CD4+ to CD8+ T cells, or within a tolerance rate or variance thereof, upon completion of the incubation and / or engineering step or other processing step and / or upon thawing and / or immediately prior to administration to a subject.

[0169] In certain embodiments, the input composition is a composition enriched for CD3+ T cells. In some embodiments, the input composition is or comprises 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 99%, at least or about 99.5%, at least or about 99.9%, or about 100% CD3+ T cells. In some embodiments, the input composition consists essentially of CD3+ T cells. In certain embodiments, the input composition is a composition of cells enriched for CD4+ T cells and CD8+ T cells. In certain embodiments, the input composition is or comprises 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 99%, at least or about 99.5%, at least or about 99.9%, or about 100% of cells that are CD4+ or CD8+ T cells. In some embodiments, the input composition consists essentially of CD4+ and CD8+ T cells.

[0170] In certain embodiments, the input composition contains between at or about 30% and at or about 70%, between at or about 35% and at or about 65%, between at or about 40% and at or about 60%, between at or about 45% and at or about 55%, or about 50% or 50% CD4+ T cells, and between at or about 30% and at or about 70%, between at or about 35% and at or about 65%, between at or about 40% and at or about 60%, between at or about 45% and at or about 55%, or about 50% or 50% CD8+ T cells. In certain embodiments, the input composition contains between at or about 45% and at or about 55%, about 50%, or 50% CD4+ T cells, and between at or about 45% and at or about 55%, about 50%, or 50% CD8+ T cells.

[0171] In some embodiments, at least one separate composition of enriched CD4+T cells and at least one separate composition of enriched CD8+T cells are separated, selected, enriched or obtained from a single biological sample (e.g., PBMC or other leukocyte samples from the same donor (e.g., patient or healthy individual). In some embodiments, initially from the same biological sample (e.g., obtained, collected, and / or obtained from a single biological sample of a single subject), a separate composition of (e.g., separated, selected and / or enriched) enriched CD4+T cells and a separate composition of enriched CD8+T cells are derived. In some embodiments, the biological sample is first subjected to the selection of CD4+T cells, wherein negative and positive fractions are retained, and the negative fraction is further subjected to the selection of CD8+T cells. In other embodiments, the biological sample is first subjected to the selection of CD8+T cells, wherein negative and positive fractions are retained, and the negative fraction is further subjected to the selection of CD4+T cells. In some embodiments, selection method is performed as described in International PCT Publication No. WO2015 / 164675. In some aspects, first to biological sample positive selection CD8+T cell to produce at least one composition of enrichment CD8+T cell, and then to negative fraction positive selection CD4+T cell to produce at least one composition of enrichment CD4+T cell, make at least one composition of enrichment CD8+T cell and at least one composition of enrichment CD4+T cell be from same biological sample (for example, from same donor patient or healthy individual) separate composition.In some aspects, by two or more compositions separated of enrichment T cell (for example, at least one is the composition of enrichment CD4+T cell from same donor, and at least one is the independent composition of enrichment CD8+T cell) separately frozen (for example cryogenic freezing or cryopreservation) in cryopreservation medium.In some aspects, the cell composition of independent cryopreservation is stored and / or transported in independent container with one or more transport modes.In some aspects, the cell composition of independent cryopreservation is thawed and optionally washed.

[0172] In some aspects, two or more separate compositions of enriched T cells (for example, at least one is a composition of enriched CD4+T cells from the same biological sample, and at least one is a separate composition of enriched CD8+T cells from the same biological sample) are thawed and mixed, combined and / or merged, and the composition can be optionally washed before or after mixing, combining and / or merging. In some aspects, the composition of enriched T cells, through mixing, combining and / or merging and optionally washed, forms an input composition. In some aspects, by contacting with a stimulating reagent (for example, by incubating with magnetic beads conjugated with CD3 / CD28 for T cell activation), activate and / or stimulate input compositions (for example, comprising a ratio of or about 1:1 CD4+T cells and CD8+T cells), and optionally adjust (for example, reduce) the volume of the cell composition from activation / stimulation to achieve a target volume. In some aspects, for example, a retroviral vector encoding a recombinant protein (such as CAR) is used to engineer, transduce and / or transfect the cell composition activated / stimulated to express the same recombinant protein in the CD4+T cells and CD8+T cells of the cell composition. In some aspects, the volume of the cell composition from the engineering is optionally adjusted (such as reduced) to achieve the target volume. In some aspects, the method includes removing stimulating agents, such as magnetic beads, from the cell composition. In some aspects, the cell composition containing engineered CD4+T cells and engineered CD8+T cells is cultivated, for example, for the expansion of CD4+T cells and / or CD8+T cell colonies therein. In certain embodiments, the cell composition from cultivation is harvested and / or collected and / or prepared by washing the cell composition in a formulation buffer. In certain embodiments, the prepared cell composition comprising CD4+T cells and CD8+T cells is frozen (such as cryogenic freezing or cryopreservation) in a cryopreservation medium. In some aspects, the preparation can be stored and / or transported cryopreserved in one or more containers. In some aspects, the engineered CD4+ T cells and CD8+ T cells in the formulation are derived from the same donor or biological sample and express the same recombinant protein (e.g., CAR), and the formulation is administered to a subject in need thereof (e.g., from the same donor).

[0173] In specific embodiments, the input composition contains a ratio of CD4+ T cells to CD8+ T cells of between 3:1 and 1:3, between 2:1 and 1:2, between 1.5 and 0.75, between 1.25 and 0.75, or between 1.2 and 0.8. In certain embodiments, the input composition contains a ratio of CD4+ T cells to CD8+ T cells of at or about 1:1.

[0174] In some embodiments, the cells from the enriched CD4+T cell compositions and the cells from the enriched CD8+T cell compositions are mixed, combined and / or merged to produce an input composition containing CD4+T cells and CD8+T cells. In certain embodiments, the compositions of enriched CD4+T cells and CD8+T cells are merged, mixed and / or combined under stimulating conditions before incubating cells. In certain embodiments, after separation, enrichment and / or selection of CD4+ and CD8+T cells from a biological sample, the compositions of enriched CD4+ and CD8+T cells are merged, mixed and / or combined. In specific embodiments, after freezing (e.g., cryogenic freezing) and thawing the compositions of enriched CD4+ and CD8+T cells, the compositions of enriched CD4+ and CD8+T cells are merged, mixed and / or combined.

[0175] In certain embodiments, the input composition is produced, generated or prepared by mixing, merging and / or combining cells from a CD4+ cell enriched composition with cells from a CD8+ cell enriched composition. In certain embodiments, the composition enriched for CD4+ T cells contains at least or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 99.9% CD4+ T cells. In specific embodiments, the composition enriched for CD4+ T cells contains 100% CD4+ T cells or contains about 100% CD4+ T cells. In certain embodiments, the composition enriched for T cells comprises or contains less than or less than about 20%, less than or less than about 10%, less than or less than about 5%, less than or less than about 1%, less than or less than about 0.1%, or less than or less than about 0.01% CD8+ T cells, and / or does not contain CD8+ T cells, and / or does not contain or is substantially free of CD8+ T cells. In some embodiments, the cell population is essentially composed of CD4+T cells. In certain embodiments, the composition enriched for CD8+T cells contains at least or at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% or 99.9% CD8+T cells, or contains or contains about 100% CD8+T cells. In certain embodiments, the composition enriched for CD8+T cells includes or contains less than or less than about 20%, less than or less than about 10%, less than or less than about 5%, less than or less than about 1%, less than or less than about 0.1%, or less than or less than about 0.01% CD4+T cells, and / or does not contain CD4+T cells, and / or does not contain or substantially do not contain CD4+T cells. In some embodiments, the cell population is essentially composed of CD8+T cells.

[0176] In certain embodiments, the CD4+ T cells and CD8+ T cells are pooled, mixed and / or combined at a ratio of CD4+ T cells to CD8+ T cells of between 1:10 and 10:1, between 1:5 and 5:1, between 4:1 and 1:4, between 3:1, between 1:3 and 3:1, between 2:1 and 1:2, between 1.5:1 and 1:1.5, between 1.25:1 and 1:1.25, between 1.2:1 and 1:1.2, between 1.1:1 and 1:1.1, or about 1:1 or 1:1. In specific embodiments, CD4+ T cells and CD8+ T cells are pooled, mixed, and / or combined at a ratio of CD4+ T cells to CD8+ T cells of between 2: 1 and 1:2, between 1.5: 1 and 1:1.5, between 1.25: 1 and 1:1.25, between 1.2: 1 and 1:1.2, between 1.1: 1 and 1:1.1, or about 1: 1 or 1: 1. In some embodiments, CD4+ T cells and CD8+ T cells are pooled, mixed, and / or combined at a ratio of CD4+ T cells to CD8+ T cells of at or about 1:1.

[0177] In some embodiments, the CD4+ T cells and CD8+ T cells are pooled, mixed and / or combined at a ratio of between 1:10 and 10:1, between 1:5 and 5:1, between 4:1 and 1:4, between 3:1, between 1:3 and 3:1, between 2:1 and 1:2, between 1.5:1 and 1:1.5, between 1.25:1 and 1:1.25, between 1.2:1 and 1:1.2, between 1.1:1 and 1:1.1, or about 1:1 or 1:1. In specific embodiments, cells from a composition enriched for CD4+ and CD8+ T cells are pooled, mixed, and / or combined at a ratio of CD4+ T cells to CD8+ T cells of between 2: 1 and 1:2, between 1.5: 1 and 1:1.5, between 1.25: 1 and 1:1.25, between 1.2: 1 and 1:1.2, between 1.1: 1 and 1:1.1, or about 1: 1 or 1: 1. In some embodiments, cells from a composition enriched for CD4+ and CD8+ T cells are pooled, mixed, and / or combined at a ratio of CD4+ T cells to CD8+ T cells of at or about 1:1.

[0178] In certain embodiments, the input composition contains a certain ratio (e.g., a defined, controlled, and / or fixed ratio) of CD4+ naive-like T cells to CD8+ naive-like T cells. In specific embodiments, the ratio of CD4+ naive-like T cells to CD8+ naive-like T cells is between 10:1 and 0.05:1, between 8:1 and 0.1:1, between 5:1 and 0.2:1, between 2.5:1 and 0.25:1, between 2.2:1 and 0.8:1, between 2:1 and 0.5:1, or between 1.5:1 and 1:1, inclusive. In certain embodiments, the ratio of CD4+ naive-like T cells to CD8+ naive-like T cells is between 2:1 and 0.8:1, between 1.6:1 and 0.8:1, between 1.4:1 and 0.8:1, between 1.2:1 and 0.8:1, or between 1.2:1 and 0.8:1, inclusive. In some embodiments, the ratio is between 2.2:1 and 0.8:1, inclusive. In certain embodiments, the ratio of CD4+ naive-like T cells to CD8+ naive-like T cells is or is about 2.2:1, 2.1:1, 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, or 0.8:1. In certain embodiments, the ratio is or is about 1.1:1.

[0179] In certain embodiments, the input composition has a mass of at or about 1 x 10 6 , 5x 10 6 , 1x 10 7 , 5x 10 7 , 1.0x10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x 10 8 , 1.5x 10 8 , 1.6x 10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x10 8 , 2.0x 10 8 , 2.1x 10 8 , 2.2x 10 8 , 2.3x 10 8 , 2.4x 10 8 , 2.5x 10 8 , 2.6x 10 8 , 2.7x 10 8 , 2.8x108 , 2.9x 10 8 , 3.0x 10 8 , 3.5x 10 8 , 4.0x 10 8 , 4.5x 10 8 , 5x 10 8 , 5x 10 8 or 1x 10 9 In certain embodiments, the input composition has a count of at or about 1 x 10 6 , 5x 10 6 , 1x 10 7 , 5x10 7 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x 10 8 , 1.5x 10 8 , 1.6x 10 8 , 1.7x 10 8 , 1.8x10 8 , 1.9x 10 8 , 2.0x 10 8 , 2.1x 10 8 , 2.2x 10 8 , 2.3x 10 8 , 2.4x 10 8 , 2.5x 10 8 , 2.6x 10 8 , 2.7x10 8 , 2.8x 10 8 , 2.9x 10 8 , 3.0x 10 8 , 3.5x 10 8 , 4.0x 10 8 , 4.5x 10 8 , 5x 10 8 , 5x 10 8 or 1x 10 9 In some embodiments, the input cell composition has a count of at or about 1 x 10 6 , 5x10 6 , 1x 10 7 , 5x 10 7 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 108 , 1.3x 10 8 , 1.4x 10 8 , 1.5x 10 8 , 1.6x10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x 10 8 , 2.0x 10 8 , 2.1x 10 8 , 2.2x 10 8 , 2.3x 10 8 , 2.4x 10 8 , 2.5x10 8 , 2.6x 10 8 , 2.7x 10 8 , 2.8x 10 8 , 2.9x 10 8 , 3.0x 10 8 , 3.5x 10 8 , 4.0x 10 8 , 4.5x 10 8 , 5x10 8 , 5x 10 8 or 1x 10 9 The amount of naive-like CD4+ and naive-like CD8+ T cells.

[0180] In certain embodiments, the input composition has a concentration of at or about 1 x 10 6 or about 1x 10 10 Between or about 1x 10 7 or about 1x 10 9 between or about v and or about 5x10 8 between or about 1x 10 8 or about 3x 10 8 In certain embodiments, the input composition has cells expressing CD4 or CD8 in an amount of at or about 1 x 10 6 or about 1x 10 10 Between or about 1x 10 7 or about 1x 10 9 Between or about v and or about 5x 10 8 Between, or about 1x10 8 or about 3x 10 8In some embodiments, the input composition has naive-like CD4+ and naive-like CD8+ T cells in an amount of at or about 1 x 10 6 With or about 1x 10 10 Between, between or about 1x 10 7 With or about 1x 10 9 Between, between or about 5x 10 7 or about 5x 10 8 between, or between or about 1x 10 8 or about 3x 10 8 between.

[0181] In some embodiments, the input composition has or contains at least or about 1%, 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 50%, at least or about 60%, 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 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 99.5%, at least or about 99.9%, or 100% or about 100% naive-like cells. In certain embodiments, the input composition contains or comprises no more than or about no more than 100%, no more than or about no more than 99%, no more than or about no more than 98%, no more than or about no more than 97%, no more than or about no more than 96%, no more than or about no more than 95%, no more than or about no more than 90%, or no more than or about no more than 85% naive-like cells.

[0182] In certain embodiments, the methods provided herein include one or more steps of producing, generating, and / or preparing an input composition. In certain embodiments, producing, generating, and / or preparing an input composition includes one or more steps of mixing or combining cells of a CD4+ T cell composition with cells of a CD8+ T cell composition.

[0183] In some embodiments, the cells (e.g., CD4+T cells CD8+T cells) of the input composition have been separated and / or selected from a sample (e.g., a biological sample). In certain embodiments, the cell source of the input composition is a cell composition that has been separated and / or selected from a sample, such as a composition of CD4+ and CD8+T cells. In specific embodiments, the CD4+T cell composition and the CD8+T cell composition are separated and / or selected from a sample (e.g., a biological sample). In certain embodiments, the composition of CD4+T cells and the composition of CD8+T cells are separated and / or selected from the same sample. In certain embodiments, the composition of CD4+T cells and the composition of CD8+T cells are separated and / or selected from a sample taken from or obtained from the same subject.

[0184] In certain embodiments, the CD4+ T cell composition contains or comprises at least or about 60%, 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 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 99.5%, at least or about 99.9%, or 100% or about 100% CD4+ T cells. In some embodiments, the CD4+ T cell composition contains or comprises no more than or about 100%, no more than or about 99%, no more than or about 98%, no more than or about 97%, no more than or about 96%, no more than or about 95%, no more than or about 90%, or no more than or about 85% CD4+ T cells.

[0185] In certain embodiments, the CD8+ T cell composition contains or comprises at least or about 60%, 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 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 99.5%, at least or about 99.9%, or 100% or about 100% CD8+ T cells. In specific embodiments, the CD8+ T cell composition contains or comprises no more than or about 100%, no more than or about 99%, no more than or about 98%, no more than or about 97%, no more than or about 96%, no more than or about 95%, no more than or about 90%, or no more than or about 85% CD8+ T cells.

[0186] In certain embodiments, the production, generation and / or preparation of the input composition includes one or more steps of measuring, determining and / or quantifying the amount, portion, number, number per volume, number per weight, and / or percentage of live CD4+ T cells and / or live CD8+ T cells present in the CD4+ T cell composition and / or CD8+ T cell composition, for example, prior to combining or mixing the cells of the cell composition. In specific embodiments, the production, generation and / or preparation of the input composition includes one or more steps of measuring, determining and / or quantifying the amount, portion, number, number per volume, number per weight, and / or percentage of naive-like CD4+ T cells and / or naive-like CD8+ T cells present in the CD4+ T cell composition and / or CD8+ T cell composition. In some embodiments, the naive-like CD4+ and / or naive-like CD8+ T cells are live naive-like cells.

[0187] In certain embodiments, producing, generating and / or preparing an input composition comprises one or more steps of measuring, determining and / or quantifying the amount, portion, number, number per volume, number per weight, and / or percentage of live CD4+ T cells and / or live CD8+ T cells present in a sample (e.g., a biological sample). In specific embodiments, producing, generating and / or preparing an input composition comprises one or more steps of measuring, determining and / or quantifying the amount, portion, number, number per volume, number per weight, and / or percentage of naive-like CD4+ T cells and / or naive-like CD8+ T cells present in a sample. In some embodiments, naive-like CD4+ and / or naive-like CD8+ T cells are live naive-like cells.

[0188] In some embodiments, the cells of the input composition are separated and / or selected from a sample (e.g., a biological sample). In specific embodiments, the share of immature-like cells in the sample (e.g., the share of immature-like CD4+ and CD8+ T cells) is known or has been determined, measured or evaluated. In some embodiments, cells from the sample are separated and / or selected to directly produce a cell composition of immature-like CD4+ T cells and immature-like CD8+ T cells having a defined, fixed or controlled ratio, such as an input composition. In certain embodiments, cells are separated and / or selected using immunoaffinity beads. In some embodiments, cells are separated and / or selected using affinity columns. In specific embodiments, cells from the sample are separated or selected according to any of the methods described in WO 2015 / 164675 to produce a cell composition of immature-like CD4+ cells and immature-like CD8+ cells having a defined ratio, a controlled ratio and / or a fixed ratio.

[0189] In certain embodiments, the input composition comprises cells that are directly isolated and / or selected from a sample by a first and a second separation or selection. In certain embodiments, the input composition is produced by performing a first and a second selection to isolate an amount, number, or concentration of CD4+ T cells and CD8+ T cells sufficient to produce a defined, fixed, and / or controlled ratio of naive-like CD4+ T cells to naive-like CD8+ T cells.

[0190] In some embodiments, cells from a sample are directly separated, selected, and / or enriched to produce an input composition enriched in CD4+ cells and CD8+ cells. In some embodiments, the amount, quantity, percentage, number per volume, and / or number per weight of naive-like CD4+ and naive-like CD8+ cells in a sample have been measured, assessed, and / or determined, and the amount of CD4+ and CD8+ cells sufficient to achieve an input composition having a defined, fixed, or controlled ratio of naive-like CD4+ to naive-like CD8+ T cells is separated, selected, and / or enriched. In some embodiments, cells directly separated, selected, and / or enriched from a sample are input compositions and used in subsequent processing steps, such as subsequent processing steps involving incubation, stimulation, activation, engineering, and / or formulation of the enriched cells.

[0191] In some embodiments, the isolated, selected and / or enriched cells from a sample (e.g., an input composition) contain a ratio of CD4+ cells to CD8+ cells at a defined, fixed or controlled ratio of naive-like CD4+ cells to naive-like CD8+ cells. In certain embodiments, generating, producing and / or preparing an input composition comprises one or more of the following steps: measuring, determining and / or quantifying the amount, fraction, number, number per volume, number per weight and / or percentage of naive-like CD4+ T cells and / or naive-like CD8+ T cells present in the composition of CD4+ T cells and / or the composition of CD8+ T cells.

[0192] In some embodiments, before the first and / or second selection is carried out from sample, the ratio of CD4+ and CD8+T cells in sample (for example, biological sample) is determined.In certain embodiments, before the first and / or second selection is carried out, the ratio of immature sample CD4+T cells and immature sample CD8+T cells in sample is determined.Based on the specific ratio of CD4+ and CD8+T cells and / or the specific ratio (they can change between samples) of immature sample CD4+ and CD8+T cells in sample, specific selection mode can be personalized for sample, such as by sizing chromatographic column or selecting the amount or concentration of immunoaffinity reagent, to achieve required ratio, fixed ratio or controlled ratio.The relative level of various cell populations in subject or frequency can be determined based on the surface expression of one or more marks present in such colony or subpopulation.A variety of well-known methods for assessing the expression level of surface markers or protein can be used, such as by the detection carried out by the method for example based on immunoaffinity based on affinity, such as in the case of cell surface proteins, such as by flow cytometry detection.

[0193] In some situations, the appropriate ratio of naive-like CD4+ to CD8+ T cells can vary depending on, for example, the specific disease, condition, or previous treatment of the subject from which the cells were derived, and / or the specific antigenic specificity of the cells, the relative representation of specific cell types (e.g., CD4+ cells) of the various subpopulations (e.g., effector cells versus memory cells versus naive cells), and / or one or more conditions used to incubate the cells, such as culture medium, stimulators, incubation time, buffer, oxygen content, carbon dioxide content, antigens, cytokines, antibodies, and other components. Thus, a cell type that is typically or generally known to proliferate or expand faster than another cell type may not always have this property in every situation. Thus, in some aspects, the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells is determined based on the known capacity of the cell type under normal or typical circumstances, combined with an assessment of the phenotype or state of the cell or the subject from which the cell was derived, and / or empirical evidence.

[0194] In some embodiments, the separation and / or steps are performed using immunomagnetic beads. In some embodiments, a cell sample containing CD4+ and CD8+ cells is contacted with magnetic beads containing a first immunoaffinity reagent that binds to CD4 or CD8 and a second immunoaffinity reagent that binds to the other of CD4 or CD8. The separation and / or steps can be performed simultaneously and / or sequentially.

[0195] In some embodiments, the first and / or second immunoaffinity reagent is present in the incubation composition at a suboptimal yield concentration, whereby the enriched composition contains less than all (e.g., 70%) of the total CD4+ cells in the incubation composition and / or less than all (e.g., 70%) of the CD8+ cells in the incubation composition, thereby producing a composition enriched for CD4+ and CD8+ T cells.

[0196] In some embodiments, a suboptimal yield concentration of an affinity agent is a concentration that is lower than the concentration used or required to achieve an optimal or maximum yield of bound cells in a given selection or enrichment involving incubating cells with the agent and recovering or isolating cells that have bound to the agent (e.g., "yield" is the number of cells so recovered or selected compared to the total number of cells in the incubation that are targeted by the agent, or for which the agent is specific, or have a marker for which the agent is specific and capable of binding). A suboptimal yield concentration is typically a concentration or amount of an agent that achieves less than a full (e.g., no more than 70%) yield of bound cells (e.g., CD4+ and / or CD8+ T cells) after recovering cells that have bound to the agent in this process or step. In some embodiments, no more than or no more than about 50%, 45%, 40%, 30%, or 25% yield is achieved with a suboptimal concentration of an affinity agent. The concentration can be expressed as the number or mass of particles or surfaces per cell and / or the mass or molecules of an agent (e.g., an antibody, such as an antibody fragment) per cell. In certain embodiments, the suboptimal yield concentration is sufficient to drive or achieve a fixed, controlled, and / or defined ratio of naive-like CD4+ T cells to naive-like CD8+ T cells.

[0197] In some embodiments, for example, when operating at suboptimal yield concentrations of each or one or more of two or more selection agents having affinity for CD4+ and / or CD8+ T cells, one or more of such agents is used at a higher concentration than one or more of the other such agents to bias the ratio of cell types recognized by the agent as compared to the one or more cell types recognized by the other agent(s). For example, an agent that specifically binds to a marker for which it is desired to bias the ratio can be included at a concentration (e.g., dose or mass per cell) that is increased by half, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to the other agent(s), depending on how much it is desired to increase the ratio.

[0198] In some embodiments, when operating within a suboptimal range and / or with cells sufficient to achieve saturation of the reagent, the amount of immunoaffinity reagent is proportional to the approximate yield of enriched cells. In certain embodiments, the appropriate amount or concentration of the immunoaffinity reagent can be routinely determined and depends on the desired ratio of the resulting composition containing enriched or selected CD4+ and CD8+ T cells.

[0199] In some embodiments, the separation and / or isolation step is performed using magnetic beads to which the immunoaffinity reagent is reversibly bound, such as by interacting with a peptide ligand of a streptavidin mutein, as described in WO 2015 / 164675. Examples of such magnetic beads are In some embodiments, the separation and / or separation steps are performed using magnetic beads, such as those commercially available from Miltenyi Biotec.

[0200] In some embodiments, the first selection or enrichment of CD4+ and CD8+ cells from the sample is performed using immunoaffinity-based reagents comprising at least a first and a second affinity chromatography matrix, respectively, on which antibodies are immobilized. In some embodiments, one or both of the first and / or second selections can employ a plurality of affinity chromatography matrices and / or antibodies, whereby the plurality of matrices and / or antibodies used for the same selection (i.e., the first selection or the second selection) are linked in series. In some embodiments, the one or more affinity chromatography matrices employed in the first and / or second selections adsorb or are capable of selecting or enriching at least or at least about 50 x 10 6 cells / mL, 100x 10 6 cells / mL, 200x10 6 cells / mL or 400 x 10 6 In some embodiments, the adsorption capacity can be adjusted based on the diameter and / or length of the column. In some embodiments, the culture initiation ratio of the selected or enriched composition is achieved by selecting an amount of matrix sufficient to achieve the culture initiation ratio and / or a relative amount sufficient to achieve the culture initiation ratio, based on, for example, the adsorption capacity of the one or more columns used to select the cells.

[0201] In an exemplary embodiment, the CD4+ T cells and CD8+ T cells have equal or similar proportions of naive-like cells, and the adsorption capacity of the one or more matrices is the same between the first selection and the second selection, for example, at or about 1 x 10 8cells / mL, whereby the enrichment or selection of cells in the first selection and the second selection produces a composition containing CD4+ cells and CD8+ cells, said composition having a naive-like CD4+ to CD8+ T cell ratio of or about 1: 1. In certain embodiments, the appropriate volume, diameter or number of affinity matrix chromatography columns used for the first and / or second selection can be selected or determined in a routine manner, depending on the fraction of naive-like cells and on the desired ratio of the input composition to be produced.

[0202] In some embodiments, the adsorptive capacity of one or more column matrices is regulated to compensate for the frequency of the cell of the corresponding CD4+ or CD8+ parental colony from the starting sample of the experimenter, compared with the frequency difference of immature sample cells (e.g., immature sample CD4+ or CD8+ cell). The relative level or frequency of various cell populations in the experimenter can be determined based on the surface expression of one or more marks present on such colonies or subpopulations of the experimenter. A variety of well-known methods for evaluating the expression level of surface markers or proteins can be used, such as the detection performed by the method based on affinity, such as immunoaffinity, in the case of cell surface proteins, such as by flow cytometry.

[0203] In some embodiments, naive-like cells (e.g., naive-like CD4+ and / or CD8+ T cells) are assessed, measured, and / or detected in a cell composition (e.g., a CD4+ and / or CD8+ T cell composition) or in a sample (e.g., a biological sample). In some embodiments, naive-like T cells are T cells that are positive for expression of one or more markers, and the marker indicates that the cell is a naive cell and / or a naive-like cell. In certain embodiments, naive-like T cells are cells that are positive for expression of a marker, and the marker is associated with a naive state or a naive-like state in a T cell. In specific embodiments, naive-like T cells are T cells that are negative for expression of one or more markers, and the marker indicates that the cell is not a naive cell and / or is not a naive-like cell. In certain embodiments, naive-like T cells are cells that are negative for expression of a marker, and the marker is associated with a non-naive state or a non-naive-like state in a T cell. In certain embodiments, the non-naive state or non-naive-like state in a T cell includes, for example, but is not limited to, effector T (T EFF ) cells, memory T cells, central memory T cells (T CM ), effect memory T(T EM ) cells and combinations thereof.

[0204] In some embodiments, the naive-like T cells are positive for the expression of at least one, two, three, four, five, six, seven, eight, nine, ten or more markers, which indicate that the cell is a naive cell and / or a naive-like cell, and / or the markers are associated with a naive state or a naive-like state in the T cell. In some embodiments, the markers are expressed on the surface of the cell. In certain embodiments, the naive-like T cells are negative for the expression of at least one, two, three, four, five, six, seven, eight, nine, ten or more markers, which indicate that the cell is a non-naive cell and / or a non-naive-like cell, and / or the markers are associated with a non-naive state or a non-naive-like state in the T cell.

[0205] Indication that T cells are naive cells and / or naive-like T cells, and / or markers associated with the naive state or naive-like state in T cells include, but are not limited to, CD27, CD28, CD45RA, CD62L, and / or CCR7. In some embodiments, naive-like T cells (e.g., naive-like CD4+ and / or CD8+ T cells) are positive for expression of CD27, CD28, CD45RA, CD62L, and / or CCR7. In certain embodiments, naive-like T cells are positive for surface expression of one or more of CD27, CD28, CD45RA, CD62L, and / or CCR7.

[0206] Indicator cells are non-immature cells and / or non-immature-like T cells and / or markers associated with a non-immature state or a non-immature-like state in T cells include, but are not limited to, CD25, CD45RO, CD56, KLRG1, and / or CD95. In some embodiments, immature-like T cells (e.g., immature-like CD4+ and / or CD8+ T cells) are negative for expression of CD25, CD45RO, CD56, and / or KLRG1. In specific embodiments, immature-like T cells (e.g., immature-like CD4+ and / or CD8+ T cells) have low expression of markers associated with non-immature cells or non-immature-like cells. In specific embodiments, immature-like T cells have low expression of CD95. In certain embodiments, immature-like T cells are negative for surface expression of one or more of CD25, CD45RO, CD56, and / or KLRG1.

[0207] In some embodiments, low expression of a marker associated with non-naive cells or non-naive-like cells is or includes at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% less expression of the marker in cells that are non-naive-like cells and / or cells that are positive for one or more markers that indicate the cell is a non-naive T cell and / or a non-naive-like T cell and / or that the marker is associated with a non-naive state or a non-naive-like state in T cells. In certain embodiments, low expression of a marker associated with non-naive cells or non-naive-like cells is or includes at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% less expression of the marker in cells that are non-naive-like cells and / or cells that are positive for one or more markers that indicate the cell is a non-naive T cell and / or a non-naive-like T cell and / or that the marker is associated with a non-naive state or a non-naive-like state in T cells. EFF ) cells, memory T cells, central memory T cells (T CM ) and / or effect memory T(T EM ) cells by at least or at least about 10%, at least or at least about 20%, at least or at least about 30%, at least or at least about 40%, at least or at least about 50%, at least or at least about 60%, at least or at least about 70%, at least or at least about 80%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 99% less expression in the cell.

[0208] In some embodiments, the indicator cell is a non-naive T cell and / or a non-naive-like T cell and / or a marker associated with a non-naive state or a non-naive-like state in a T cell includes one or more cytokines. For example, in certain embodiments, non-naive T cells or non-naive-like T cells are negative for expression and / or production of one or more of IL-2, IFN-γ, IL-4, and IL-10. In some embodiments, the one or more cytokines are secreted. In specific embodiments, for example, during or after treatment with an agent that prevents, inhibits, or reduces secretion, non-naive-like T cells express the one or more cytokines internally.

[0209] In certain embodiments, naive-like T cells are positive for expression (e.g., surface expression) of CD45RA and CCR7. In specific embodiments, naive-like CD4+ T cells are positive for expression (e.g., surface expression) of CD45RA and CCR7. In some embodiments, naive-like CD8+ T cells are positive for expression (e.g., surface expression) of CD45RA and CCR7. In specific embodiments, naive-like T cells are positive for expression (e.g., surface expression) of CD45RA, CD27, and CCR7, and negative for expression (e.g., surface expression) of CD45RO. In specific embodiments, naive-like CD4+ T cells are positive for expression (e.g., surface expression) of CD45RA, CD27, and CCR7, and negative for expression (e.g., surface expression) of CD45RO. In some embodiments, the naive-like CD8+ T cells are positive for expression (eg, surface expression) of CD45RA, CD27, and CCR7, and negative for expression (eg, surface expression) of CD45RO.

[0210] In certain embodiments, CD4+ and / or CD8+ T cells are living cells. In certain embodiments, CD4+ and / or CD8+ T cells are living immature cells. The living cells are positive for the expression of a marker, which indicates that the cells undergo normal functional cell processes and / or have not yet undergone necrosis or programmed cell death, or are not in the process of undergoing necrosis or programmed cell death. In some embodiments, viability can be assessed by the redox potential of the cell, the integrity of the cell membrane, or the activity or function of mitochondria. In some embodiments, viability is the absence of specific molecules associated with cell death in the assay, or the absence of an indication of cell death.

[0211] In certain embodiments, cell viability is assessed by assays that may include, but are not limited to, dye uptake assays (e.g., calcein AM assays), XTT cell viability assays, and dye exclusion assays (e.g., trypan blue, eosin, or propidium dye exclusion assays). In specific embodiments, living cells have negative expression of one or more apoptosis markers (e.g., annexin V or active caspase 3). In some embodiments, living cells are negative for expression of one or more apoptosis markers that may include, but are not limited to, caspases (e.g., caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, and caspase 10), Bcl-2 family members (e.g., Bax, Bad, and Bid), annexin V, and / or TUNEL staining.

[0212] In some embodiments, expression is or includes the amount, level, concentration and / or existence of a label. In specific embodiments, the label is a polypeptide. In some embodiments, the label is an mRNA. In some embodiments, expression is or includes the amount, level, concentration and / or existence of a polypeptide (for example, a marker polypeptide). In certain embodiments, the amount, level, concentration and / or existence of the polynucleotide encoding the label (for example, mRNA or a cDNA derived from mRNA). In certain embodiments, expression is or includes the amount, level, concentration and / or existence of a label positioned on or exposed on a cell surface or positioned within a cell membrane. In certain embodiments, expression is or includes the amount, level, concentration and / or existence of a label positioned on or exposed on a cell surface or positioned within a cell membrane. In specific embodiments, expression is or includes internal expression, for example, in a cell interior, such as the amount, level, concentration and / or existence of a label in cytosol, nucleus, endoplasmic reticulum and / or Golgi body.

[0213] In some embodiments, by performing in vitro determination, the marker is measured, evaluated and / or quantified. In some examples, the in vitro determination is an immunoassay, an assay based on aptamers, a histological or cytological assay or an mRNA expression level assay. In some cases, the in vitro assay used can be an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry, surface plasmon resonance (SPR), chemiluminescence assay, lateral flow immunoassay, inhibition assay or affinity assay. In some embodiments, the expression of the marker is measured, evaluated and / or quantified by RNA-seq. In specific embodiments, the expression of the marker is measured, evaluated and / or quantified by immunostaining techniques. In specific embodiments, the expression of the marker is measured, evaluated and / or quantified by flow cytometry analysis. In some embodiments, the expression of the marker is measured, evaluated and / or quantified by internal cytokine staining.

[0214] In some embodiments, a marker is measured, assessed, and / or quantified in cells of a CD4+ T cell composition. In certain embodiments, at least or at least about 1%, 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%, at least or at least about 30%, at least or at least about 35%, at least or at least about 40%, at least or at least about 45%, at least or at least about 50%, at least or at least about 55%, at least or at least about 60%, at least or at least about 65%, at least or at least about 70%, at least or at least about 75%, at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 95%, at least or at least about 97%, or at least or at least about 99% of the CD4+ T cells are naive-like CD4+ T cells. In certain embodiments, between at or about 10% and at or about 50%, between at or about 20% and at or about 60%, between at or about 25% and at or about 75%, between at or about 30% and at or about 80%, between at or about 40% and at or about 90%, between at or about 50% and at or about 100%, between at or about 30% and at or about 50%, between at or about 40% and at or about 60%, between at or about 5 In some embodiments, 0% to 50% or about 70% of the CD4+ T cells are naive-like CD4+ T cells. In some embodiments, the naive-like CD4+ T cells are viable naive-like CD4+ T cells.

[0215] In certain embodiments, a marker is measured, assessed, and / or quantified in cells of a CD8+ T cell composition. In certain embodiments, at least or at least about 1%, 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%, at least or at least about 30%, at least or at least about 35%, at least or at least about 40%, at least or at least about 45%, at least or at least about 50%, at least or at least about 55%, at least or at least about 60%, at least or at least about 65%, at least or at least about 70%, at least or at least about 75%, at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 95%, at least or at least about 97%, or at least or at least about 99% of the CD8+ T cells are naive-like CD8+ T cells. In certain embodiments, between at or about 10% and at or about 50%, between at or about 20% and at or about 60%, between at or about 25% and at or about 75%, between at or about 30% and at or about 80%, between at or about 40% and at or about 90%, between at or about 50% and at or about 100%, between at or about 30% and at or about 50%, between at or about 40% and at or about 60%, between at or about 5 0% to 50% or about 70%, between 50% to 50% or about 70%, between 50% to 50% or about 75%, between 50% to 50% or about 75%, or between 50% to 50% or about 75% and 50% to 75% or about 99% of the CD8+ T cells are naive-like CD4+ T cells. In certain embodiments, the naive-like CD8+ T cells are viable naive-like CD8+ T cells.

[0216] In some embodiments, cells from a composition of CD4+ T cells are mixed or combined with cells from a composition of CD8+ T cells in amounts and / or ratios sufficient to produce an input composition having a ratio of CD4+ naive-like T cells to CD8+ naive-like T cells of between 10:1 and 0.05:1, between 8:1 and 0.1:1, between 5:1 and 0.2:1, between 2.5:1 and 0.25:1, between 2.2:1 and 0.8:1, between 2:1 and 0.5:1, or between 1.5:1 and 1:1, inclusive. In some embodiments, the cells are mixed in amounts and / or ratios sufficient to produce a ratio of CD4+ naive-like T cells to CD8+ naive-like T cells of between 2.2:1 and 0.8:1, inclusive. In certain embodiments, the cells are mixed or combined at a ratio of CD4+ naive-like T cells to CD8+ naive-like T cells of at or about 2.2: 1, 2.1: 1, 2.0: 1, 1.9: 1, 1.8: 1, 1.7: 1, 1.6: 1, 1.5: 1, 1.4: 1, 1.3: 1, 1.2: 1, 1.1: 1, 1.0: 1, 0.9: 1, or 0.8: 1. In certain embodiments, the cells are mixed or combined at a ratio of at or about 1.1: 1.

[0217] In some embodiments, the amount of the 6 , 5x 10 6 , 1x 10 7 , 5x 10 7 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x 10 8 , 1.5x 10 8 , 1.6x 10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x 10 8 , 2.0x10 8 , 2.1x 10 8 , 2.2x 10 8 , 2.3x 10 8 , 2.4x 10 8 , 2.5x 10 8 , 2.6x 10 8 , 2.7x 10 8 , 2.8x 10 8 , 2.9x10 8 , 3.0x 10 8, 3.5x 10 8 , 4.0x 10 8 , 4.5x 10 8 , 5x 10 8 , 5x 10 8 or 1x 10 9 The amount of total CD4+ T cells or total live CD4+ T cells is about 1x 10 6 , 5x 10 6 , 1x 10 7 , 5x 10 7 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x 10 8 , 1.5x 10 8 , 1.6x 10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x 10 8 , 2.0x 10 8 , 2.1x10 8 , 2.2x 10 8 , 2.3x 10 8 , 2.4x 10 8 , 2.5x 10 8 , 2.6x 10 8 , 2.7x 10 8 , 2.8x 10 8 , 2.9x 10 8 , 3.0x10 8 , 3.5x 10 8 , 4.0x 10 8 , 4.5x 10 8 , 5x 10 8 , 5.5x 10 8 or 1x 10 9 In certain embodiments, 1 x 10 6 1x 10 10 Between 1x 10 7 1x 10 9 Between 5x 10 7 and 5x 10 8 or between 1x 108 and 3x 10 8 Total CD4+ T cells or total live CD4+ T cells were between 1 x 10 6 1x 10 10 Between 1x 10 7 1x 10 9 Between 5x 10 7 and 5x 10 8 or between 1x 10 8 and 3x 10 8 An amount of total CD8+ T cells or total live CD8+ T cells between 1 and 200 μg / mL is mixed or combined to produce an input composition having a defined ratio of CD4+ naive-like T cells to CD8+ naive-like T cells.

[0218] In some embodiments, the amount of the 6 , 5x 10 6 , 1x 10 7 , 5x 10 7 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x 10 8 , 1.5x 10 8 , 1.6x 10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x 10 8 , 2.0x10 8 , 2.1x 10 8 , 2.2x 10 8 , 2.3x 10 8 , 2.4x 10 8 , 2.5x 10 8 , 2.6x 10 8 , 2.7x 10 8 , 2.8x 10 8 , 2.9x10 8 , 3.0x 10 8 , 3.5x 10 8 , 4.0x 10 8 , 4.5x 10 8 , 5x 10 8 , 5x 10 8 , or 1x 10 9 The amount of naive-like CD4+ T cells is about 1x 106 , 5x 10 6 , 1x 10 7 , 5x 10 7 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x10 8 , 1.5x 10 8 , 1.6x 10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x 10 8 , 2.0x 10 8 , 2.1x 10 8 , 2.2x 10 8 , 2.3x10 8 , 2.4x 10 8 , 2.5x 10 8 , 2.6x 10 8 , 2.7x 10 8 , 2.8x 10 8 , 2.9x 10 8 , 3.0x 10 8 , 3.5x 10 8 , 4.0x10 8 , 4.5x 10 8 , 5x 10 8 , 5.5x 10 8 , or 1x 10 9 In certain embodiments, 1 x 10 6 and 1x10 10 Between 1x 10 7 1x 10 9 Between 5x 10 7 and 5x 10 8 between or in 1x10 8 and 3x 10 8 The naive-like CD4+ T cells were between 10 and 10 6 1x 10 10 Between 1x10 7 1x 10 9 Between 5x 10 7 and 5x 108 or between 1x 10 8 and 3x 10 8 Naive-like CD8+ T cells are mixed or combined in an amount between 1 and 2 times that of the naive-like cells to produce an input composition having a defined ratio of CD4+ naive-like T cells to CD8+ naive-like T cells.

[0219] In certain embodiments, the ratio of naive CD4+ T cells to naive CD8+ T cells of the input composition has been adjusted, altered, and / or changed compared to the ratio of naive CD4+ T cells to naive CD8+ T cells of a sample (e.g., a biological sample). In certain embodiments, the adjustment, change, or alteration of the ratio of naive CD4+ T cells to naive CD8+ T cells compared to a biological sample is or is about or is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, 100 fold. In certain embodiments, a sample is a sample of cells from which the input composition is derived, separated, selected, and / or obtained.

[0220] In some embodiments, producing, generating and / or preparing the input composition includes one or more steps of mixing or combining cells of a CD4+ T cell composition with cells of a CD8+ T cell composition to produce an input composition of naive-like CD4+ T cells to naive-like CD8+ T cells at a ratio between 2.2:1 and 0.8:1. In certain embodiments, the number, number per volume, number per weight and / or amount, level or percentage of naive-like cells are measured, assessed and / or quantified in the CD4+ T cell composition and the CD8+ T cell composition prior to mixing or combining. In some embodiments, the amount, level, number, number per volume, number per weight and / or percentage of naive-like cells are measured, assessed and / or quantified by detecting CD45RA+; CCR7+ T cells. In certain embodiments, the input composition has a ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells between 2.2:1 and 0.8:1. In some embodiments, the infusion composition has a ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells of at or about 1.1:1.

[0221] In certain embodiments, the input cell composition contains a ratio (e.g., a defined ratio, a controlled ratio, and / or a fixed ratio) of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells. In specific embodiments, the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells is between 10:1 and 0.05:1, between 8:1 and 0.1:1, between 5:1 and 0.2:1, between 2.5:1 and 0.25:1, between 2.2:1 and 0.8:1, between 2:1 and 0.5:1, or between 1.5:1 and 1:1, inclusive. In certain embodiments, the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells is between 2:1 and 0.8:1, between 1.6:1 and 0.8:1, between 1.4:1 and 0.8:1, between 1.2:1 and 0.8:1, or between 1.2:1 and 0.8:1, inclusive. In some embodiments, the ratio is between 2.2:1 and 0.8:1, inclusive. In certain embodiments, the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells is or is about 2.2: 1, 2.1: 1, 2.0: 1, 1.9: 1, 1.8: 1, 1.7: 1, 1.6: 1, 1.5: 1, 1.4: 1, 1.3: 1, 1.2: 1, 1.1: 1, 1.0: 1, 0.9: 1, or 0.8: 1. In certain embodiments, the ratio is or is about 1.1: 1.

[0222] In certain embodiments, the input cell composition has a density of at or about 1 x 10 6 , 5x 10 6 , 1x 10 7 , 5x 10 7 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x 10 8 , 1.5x 10 8 , 1.6x10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x 10 8 , 2.0x 10 8 , 2.1x 10 8 , 2.2x 10 8 , 2.3x 10 8, 2.4x 10 8 , 2.5x 10 8 , 2.6x 10 8 , 2.7x10 8 , 2.8x 10 8 , 2.9x 10 8 , 3.0x 10 8 , 3.5x 10 8 , 4.0x 10 8 , 4.5x 10 8 , 5x 10 8 , 5x 10 8 , or 1x 10 9 In certain embodiments, the input cell composition has a count of at or about 1 x 10 6 , 5x10 6 , 1x 10 7 , 5x 10 7 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x 10 8 , 1.5x 10 8 , 1.6x10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x 10 8 , 2.0x 10 8 , 2.1x 10 8 , 2.2x 10 8 , 2.3x 10 8 , 2.4x 10 8 , 2.5x10 8 , 2.6x 10 8 , 2.7x 10 8 , 2.8x 10 8 , 2.9x 10 8 , 3.0x 10 8 , 3.5x 10 8 , 4.0x 10 8 , 4.5x 10 8 , 5x10 8 , 5x 10 8 , or 1x 10 9 In some embodiments, the input cell composition has a count of at or about 1 x 10 6 , 5x106 , 1x 10 7 , 5x 10 7 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x10 8 , 1.5x 10 8 , 1.6x 10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x 10 8 , 2.0x 10 8 , 2.1x 10 8 , 2.2x 10 8 , 2.3x10 8 , 2.4x 10 8 , 2.5x 10 8 , 2.6x 10 8 , 2.7x 10 8 , 2.8x 10 8 , 2.9x 10 8 , 3.0x 10 8 , 3.5x 10 8 , 4.0x10 8 , 4.5x 10 8 , 5x 10 8 , 5x 10 8 , or 1x 10 9 The number of CD45RA+ / CCR7+ / CD4+ and CD45RA+ / CCR7+ / CD8+ T cells was determined.

[0223] In certain embodiments, the input cell composition has a 6 1x 10 10 Between 1x 10 7 1x 10 9 Between 5x 10 7 and 5x 10 8 or between 1x 10 8 and 3x10 8 In certain embodiments, the input cell composition has a count of at or about 1 x 10 6 1x 10 10 Between 1x10 7 1x 10 9 Between 5x 10 7and 5x 10 8 or between 1x 10 8 and 3x 10 8 In some embodiments, the input cell composition has an amount of between or about 1 x 10 6 1x 10 10 Between 1x 10 7 1x 10 9 Between 5x 10 7 and 5x 10 8 or between 1x 10 8 and 3x 10 8 The number of CD45RA+ / CCR7+ / CD4+ and CD45RA+ / CCR7+ / CD8+ T cells was between 1 and 2.

[0224] In some embodiments, the infused cell composition has or comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, 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 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD45RA+ / CCR7+ cells. In specific embodiments, the infused cell composition has or comprises no more than 100%, no more than 99%, no more than 98%, no more than 97%, no more than 96%, no more than 95%, no more than 90%, or no more than 85% CD45RA+ / CCR7+ cells.

[0225] In some embodiments, the amount of the 6 , 5x 10 6 , 1x 10 7 , 5x 10 7 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x 10 8 , 1.5x 10 8 , 1.6x 10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x 10 8 , 2.0x10 8 , 2.1x 10 8 , 2.2x 10 8, 2.3x 10 8 , 2.4x 10 8 , 2.5x 10 8 , 2.6x 10 8 , 2.7x 10 8 , 2.8x 10 8 , 2.9x10 8 , 3.0x 10 8 , 3.5x 10 8 , 4.0x 10 8 , 4.5x 10 8 , 5x 10 8 , 5x 10 8 or 1x10 9 The amount of total CD4+ T cells or total live CD4+ T cells is about 1x 10 6 , 5x 10 6 , 1x 10 7 , 5x 10 7 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x 10 8 , 1.5x 10 8 , 1.6x 10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x 10 8 , 2.0x 10 8 , 2.1x10 8 , 2.2x 10 8 , 2.3x 10 8 , 2.4x 10 8 , 2.5x 10 8 , 2.6x 10 8 , 2.7x 10 8 , 2.8x 10 8 , 2.9x 10 8 , 3.0x10 8 , 3.5x 10 8 , 4.0x 10 8 , 4.5x 10 8 , 5x 10 8 , 5.5x 10 8 or 1x 10 9In certain embodiments, 1 x 10 6 1x 10 10 Between 1x 10 7 1x 10 9 Between 5x 10 7 and 5x 10 8 Between or between 1x 10 8 and 3x 10 8 Total CD4+ T cells or total live CD4+ T cells were between 1 x 10 6 1x 10 10 Between 1x 10 7 1x 10 9 Between 5x 10 7 and 5x 10 8 or between 1x 10 8 and 3x 10 8 An amount of total CD8+ T cells or total live CD8+ T cells between 10 and 200 μg / mL is mixed or combined to produce an input cell composition having a defined ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells.

[0226] In certain embodiments, the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells of the input cell composition has been adjusted, altered, and / or changed compared to the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells of a sample (e.g., a biological sample). In certain embodiments, the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells is adjusted, altered, or changed by or about or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold compared to the biological sample. In certain embodiments, the sample is one from which cells of the input cell composition are derived, isolated, selected, and / or obtained.

[0227] In certain embodiments, the input cell composition contains a ratio (e.g., a defined ratio, a controlled ratio, and / or a fixed ratio) of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells. In specific embodiments, the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells is between 10:1 and 0.05:1, between 8:1 and 0.1:1, between 5:1 and 0.2:1, between 2.5:1 and 0.25:1, between 2.2:1 and 0.8:1, between 2:1 and 0.5:1, or between 2:1 and 1:1, inclusive. In certain embodiments, the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells is between 2:1 and 0.8:1, between 1.8:1 and 1:1, between 1.8:1 and 1.2:1, between 1.2:1 and 1.4:1, or between 1.8:1 and 1.6:1, inclusive. In some embodiments, the ratio is between 1.8:1 and 1.6:1, inclusive. In certain embodiments, the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells is or is about 2.2:1, 2.1:1, 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.69:1, 1.6:1, 1.5:1, 1.4:1, or 1.3:1. In certain embodiments, the ratio is or is about 1.69:1.

[0228] In certain embodiments, the input cell composition has a density of at or about 1 x 10 6 , 5x 10 6 , 1x 10 7 , 5x 10 7 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x 10 8 , 1.5x 10 8 , 1.6x10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x 10 8 , 2.0x 10 8 , 2.1x 10 8 , 2.2x 10 8 , 2.3x 10 8 , 2.4x 10 8 , 2.5x 10 8, 2.6x 10 8 , 2.7x10 8 , 2.8x 10 8 , 2.9x 10 8 , 3.0x 10 8 , 3.5x 10 8 , 4.0x 10 8 , 4.5x 10 8 , 5x 10 8 , 5x 10 8 , or 1x 10 9 In certain embodiments, the input cell composition has a count of at or about 1 x 10 6 , 5x10 6 , 1x 10 7 , 5x 10 7 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x 10 8 , 1.5x 10 8 , 1.6x10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x 10 8 , 2.0x 10 8 , 2.1x 10 8 , 2.2x 10 8 , 2.3x 10 8 , 2.4x 10 8 , 2.5x10 8 , 2.6x 10 8 , 2.7x 10 8 , 2.8x 10 8 , 2.9x 10 8 , 3.0x 10 8 , 3.5x 10 8 , 4.0x 10 8 , 4.5x 10 8 , 5x10 8 , 5x 10 8 , or 1x 10 9 In some embodiments, the input cell composition has a count of at or about 1 x 10 6 , 5x10 6 , 1x 10 7 , 5x 107 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x10 8 , 1.5x 10 8 , 1.6x 10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x 10 8 , 2.0x 10 8 , 2.1x 10 8 , 2.2x 10 8 , 2.3x10 8 , 2.4x 10 8 , 2.5x 10 8 , 2.6x 10 8 , 2.7x 10 8 , 2.8x 10 8 , 2.9x 10 8 , 3.0x 10 8 , 3.5x 10 8 , 4.0x10 8 , 4.5x 10 8 , 5x 10 8 , 5x 10 8 , or 1x 10 9 CD27+ / CCR7+ / CD4+ and CD27+ / CCR7+ / CD8+ T cells.

[0229] In certain embodiments, the input cell composition has a 6 1x 10 10 Between 1x 10 7 1x 10 9 Between 5x 10 7 and 5x 10 8 or between 1x 10 8 and 3x10 8 In certain embodiments, the input cell composition has a count of at or about 1 x 10 6 1x 10 10 Between 1x10 7 1x 10 9 Between 5x 10 7 and 5x 10 8 or between 1x 10 8and 3x 10 8 In some embodiments, the input cell composition has an amount of between or about 1 x 10 6 1x 10 10 Between 1x 10 7 1x 10 9 Between 5x 10 7 and 5x 10 8 or between 1x 10 8 and 3x 10 8 The number of CD27+ / CCR7+ / CD4+ and CD27+ / CCR7+ / CD8+ T cells was between 1 and 2.

[0230] In some embodiments, the input cell composition has or contains at least or about 1%, 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 50%, at least or about 60%, 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 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 99.5%, at least or about 99.9%, or 100% or about 100% CD27+ / CCR7+ cells. In certain embodiments, the input cell composition contains or comprises no more than or about no more than 100%, no more than or about no more than 99%, no more than or about no more than 98%, no more than or about no more than 97%, no more than or about no more than 96%, no more than or about no more than 95%, no more than or about no more than 90%, or no more than or about no more than 85% CD27+ / CCR7+ cells.

[0231] In some embodiments, the amount of the 6 , 5x 10 6 , 1x 10 7 , 5x 10 7 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x 10 8 , 1.5x 10 8 , 1.6x 10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x 10 8, 2.0x10 8 , 2.1x 10 8 , 2.2x 10 8 , 2.3x 10 8 , 2.4x 10 8 , 2.5x 10 8 , 2.6x 10 8 , 2.7x 10 8 , 2.8x 10 8 , 2.9x10 8 , 3.0x 10 8 , 3.5x 10 8 , 4.0x 10 8 , 4.5x 10 8 , 5x 10 8 , 5x 10 8 or 1x 10 9 The amount of total CD4+ T cells or total live CD4+ T cells is about 1x 10 6 , 5x 10 6 , 1x 10 7 , 5x 10 7 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x 10 8 , 1.5x 10 8 , 1.6x 10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x 10 8 , 2.0x 10 8 , 2.1x10 8 , 2.2x 10 8 , 2.3x 10 8 , 2.4x 10 8 , 2.5x 10 8 , 2.6x 10 8 , 2.7x 10 8 , 2.8x 10 8 , 2.9x 10 8 , 3.0x10 8 , 3.5x 10 8 , 4.0x 10 8 , 4.5x 10 8 , 5x 10 8 , 5.5x 10 8 or 1x 109 In some embodiments, 1 x 10 6 1x 10 10 Between 1x10 7 1x 10 9 Between 5x 10 7 and 5x 10 8 Between or between 1x 10 8 and 3x 10 8 Total CD4+ T cells or total live CD4+ T cells were between 1 x 10 6 1x 10 10 Between 1x 10 7 1x 10 9 Between 5x 10 7 and 5x 10 8 or between 1x 10 8 and 3x 10 8 An amount of total CD8+ T cells or total live CD8+ T cells between 10 and 200 μg / mL is mixed or combined to produce an input cell composition having a defined ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells.

[0232] In certain embodiments, the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells of the input cell composition has been adjusted, altered, and / or changed compared to the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells of a sample (e.g., a biological sample). In certain embodiments, the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells is adjusted, altered, or changed by or about or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold compared to a biological sample. In certain embodiments, the sample is one from which cells of the input cell composition are derived, isolated, selected, and / or obtained.

[0233] In certain embodiments, the input cell composition contains a ratio (e.g., a defined ratio, a controlled ratio, and / or a fixed ratio) of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells. In specific embodiments, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells is between 10:1 and 0.05:1, between 8:1 and 0.1:1, between 5:1 and 0.2:1, between 2.5:1 and 0.25:1, between 2.2:1 and 0.8:1, between 2:1 and 0.5:1, or between 1.5:1 and 1:1, inclusive. In certain embodiments, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells is between 2:1 and 0.8:1, between 1.6:1 and 0.8:1, between 1.4:1 and 0.8:1, between 1.2:1 and 0.8:1, or between 1.2:1 and 0.8:1, inclusive. In some embodiments, the ratio is between 2.2:1 and 0.8:1, inclusive. In certain embodiments, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells is or is about 2.2: 1, 2.1: 1, 2.0: 1, 1.9: 1, 1.8: 1, 1.7: 1, 1.6: 1, 1.5: 1, 1.4: 1, 1.3: 1, 1.2: 1, 1.1: 1, 1.0: 1, 0.9: 1, or 0.8: 1. In certain embodiments, the ratio is or is about 1.1: 1.

[0234] In certain embodiments, the input cell composition has a 6 1x 10 10 Between 1x 10 7 1x 10 9 Between 5x 10 7 and 5x 10 8 or between 1x 10 8 and 3x10 8 In certain embodiments, the input cell composition has a count of at or about 1 x 10 6 1x 10 10 Between 1x10 7 1x 10 9 Between 5x 10 7 and 5x 10 8 or between 1x 10 8 and 3x 10 8In some embodiments, the input cell composition has an amount of between or about 1 x 10 6 1x 10 10 Between 1x 10 7 1x 10 9 Between 5x 10 7 and 5x 10 8 Between 1x 10 8 and 3x 10 8 The amount of CD62L- / CCR7+ / CD4+ and CD62L- / CCR7+ / CD8+ T cells was between 4 and 8 months.

[0235] In some embodiments, the input cell composition has or contains at least or about 1%, 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 50%, at least or about 60%, 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 96%, at least or about 97%, at least or about 98%, at least or about 99%, at least or about 99.5%, at least or about 99.9%, or 100% or about 100% CD62L- / CCR7+ cells. In certain embodiments, the input cell composition contains or comprises no more than or about no more than 100%, no more than or about no more than 99%, no more than or about no more than 98%, no more than or about no more than 97%, no more than or about no more than 96%, no more than or about no more than 95%, no more than or about no more than 90%, or no more than or about no more than 85% CD62L- / CCR7+ cells.

[0236] In some embodiments, the amount of the 6 , 5x 10 6 , 1x 10 7 , 5x 10 7 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x 10 8 , 1.5x 10 8 , 1.6x 10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x 10 8 , 2.0x108 , 2.1x 10 8 , 2.2x 10 8 , 2.3x 10 8 , 2.4x 10 8 , 2.5x 10 8 , 2.6x 10 8 , 2.7x 10 8 , 2.8x 10 8 , 2.9x10 8 , 3.0x 10 8 , 3.5x 10 8 , 4.0x 10 8 , 4.5x 10 8 , 5x 10 8 , 5x 10 8 or 1x 10 9 The amount of total CD4+ T cells or total live CD4+ T cells is about 1x 10 6 , 5x 10 6 , 1x 10 7 , 5x 10 7 , 1.0x 10 8 , 1.1x 10 8 , 1.2x 10 8 , 1.3x 10 8 , 1.4x 10 8 , 1.5x 10 8 , 1.6x 10 8 , 1.7x 10 8 , 1.8x 10 8 , 1.9x 10 8 , 2.0x 10 8 , 2.1x10 8 , 2.2x 10 8 , 2.3x 10 8 , 2.4x 10 8 , 2.5x 10 8 , 2.6x 10 8 , 2.7x 10 8 , 2.8x 10 8 , 2.9x 10 8 , 3.0x10 8 , 3.5x 10 8 , 4.0x 10 8 , 4.5x 10 8 , 5x 10 8 , 5.5x 10 8 or 1x 10 9In some embodiments, 1 x 10 6 1x 10 10 Between 1x10 7 1x 10 9 Between 5x 10 7 and 5x 10 8 Between or between 1x 10 8 and 3x 10 8 Total CD4+ T cells or total live CD4+ T cells were between 1 x 10 6 1x 10 10 Between 1x 10 7 1x 10 9 Between 5x10 7 and 5x 10 8 or between 1x 10 8 and 3x 10 8 An amount of total CD8+ T cells or total live CD8+ T cells between 1 and 200 μg / mL is mixed or combined to produce an input cell composition having a defined ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells.

[0237] In certain embodiments, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells of the input cell composition has been adjusted, altered, and / or changed compared to the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells of a sample (e.g., a biological sample). In certain embodiments, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells is adjusted, altered, or changed by or about or by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold compared to the biological sample. In certain embodiments, the sample is one from which cells of the input cell composition are derived, isolated, selected, and / or obtained.

[0238] In some embodiments, generating, producing, and / or preparing an input cell composition comprises one or more steps of mixing or combining cells of a CD4+ T cell composition with cells of a CD8+ T cell composition to produce an input cell composition having a ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells between 2.2:1 and 0.8:1. In certain embodiments, the number, number per volume, number per weight, and / or amount, level, or percentage of CD45RA+ / CCR7+ cells in the CD4+ T cell composition and the CD8+ T cell composition are measured, assessed, and / or quantified prior to mixing or combining. In some embodiments, the amount, level, number, number per volume, number per weight, and / or percentage of CD45RA+ / CCR7+ cells is measured, assessed, and / or quantified by detecting CD45RA+;CCR7+ T cells. In certain embodiments, the input cell composition has a ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells of between 2.2: 1 and 0.8: 1. In some embodiments, the input cell composition has a ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells of at or about 1.1:1.

[0239] In some embodiments, generating, producing, and / or preparing an input cell composition comprises one or more steps of mixing or combining cells of a CD4+ T cell composition with cells of a CD8+ T cell composition to produce an input cell composition having a ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells between 2.4:1 and 1:1. In certain embodiments, the number, number per volume, number per weight, and / or amount, level, or percentage of CD27+ / CCR7+ cells are measured, assessed, and / or quantified in the CD4+ T cell composition and the CD8+ T cell composition prior to mixing or combining. In some embodiments, the amount, level, number, number per volume, number per weight, and / or percentage of CD27+ / CCR7+ cells are measured, assessed, and / or quantified by detecting CD45RA+;CCR7+ T cells. In certain embodiments, the input cell composition has a ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells of between 2.4:1 and 1:1. In some embodiments, the input cell composition has a ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells of at or about 1.69:1.

[0240] In some embodiments, generating, producing, and / or preparing an input cell composition comprises one or more steps of mixing or combining cells of a CD4+ T cell composition with cells of a CD8+ T cell composition to produce an input cell composition having a ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells between 2.2:1 and 0.8:1. In certain embodiments, the number, number per volume, number per weight, and / or amount, level, or percentage of CD62L- / CCR7+ cells in the CD4+ T cell composition and the CD8+ T cell composition are measured, assessed, and / or quantified prior to mixing or combining. In some embodiments, the amount, level, number, number per volume, number per weight, and / or percentage of CD62L- / CCR7+ cells are measured, assessed, and / or quantified by detecting CD62L- / CCR7+ T cells. In certain embodiments, the input cell composition has a ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells of between 2.2: 1 and 0.8: 1. In some embodiments, the input cell composition has a ratio of CD62L- / CCR7 / CD4+ T cells to CD62L- / CCR7 / CD8+ T cells of at or about 1.1:1.

[0241] B. Activation and stimulation

[0242] In some embodiments, the provided methods are used in conjunction with incubating cells under stimulating conditions. In some embodiments, stimulating conditions include conditions that activate or stimulate and / or can activate or stimulate signals in cells (e.g., CD4+T cells) (e.g., signals generated from TCR and / or co-receptors). In some embodiments, stimulating conditions include one or more steps of: cultivating, nurturing, incubating, activating, propagating cells together with and / or in the presence of stimulating reagents, such as reagents that activate or stimulate and / or can activate or stimulate signals in cells. In some embodiments, stimulating reagents stimulate and / or activate TCR and / or co-receptors. In specific embodiments, stimulating reagents are reagents provided herein, for example, as described in Chapter IB-1.

[0243] In certain embodiments, one or more compositions enriched for T cells are incubated under stimulating conditions prior to genetically engineering cells (e.g., transfecting and / or transducing cells), e.g., by the methods or techniques provided herein, e.g., the methods or techniques described in Section 1C. In certain embodiments, the composition enriched for T cells incubated under stimulating conditions is an input composition. In certain embodiments, the cells of the input composition have previously been isolated, selected, enriched, or obtained from a biological sample. In certain embodiments, the cells from the input composition have previously been cryogenically frozen and stored and thawed prior to incubation.

[0244] In some embodiments, the provided methods are used in conjunction with one or more treatment steps comprising a step of stimulating cells (e.g., cells from an input composition). In certain embodiments, incubation can be prior to or in conjunction with genetic engineering, e.g., genetic engineering resulting from an embodiment of transduction as described herein (e.g., the methods described in Section 1C). In some embodiments, stimulation results in activation and / or proliferation of cells, e.g., prior to engineering (e.g., transduction).

[0245] In some embodiments, the treating step comprises incubation of cells (e.g., input cells and / or cells of the input composition), wherein the incubation step can include culturing, growing, stimulating, activating, and / or propagating the cells. In some embodiments, the composition or cells are incubated in the presence of stimulating conditions or stimulating agents. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, simulate antigen exposure, and / or initiate cells for genetic engineering (e.g., for the introduction of recombinant antigen receptors).

[0246] In certain embodiments, for example, under stimulatory conditions, for example, in the presence of a stimulatory agent, the concentration of the stimulatory agent is less than or about 5 x 10 7 cells / mL, 4x 10 7 cells / mL, 3x 10 7 cells / mL, 2x 10 7 cells / mL, 1x 10 7 cells / mL, 9x 10 6 cells / mL, 8x 10 6 cells / mL, 7x 10 6 cells / mL, 6x 10 6 cells / mL, 5x 10 6 cells / mL, 4x 10 6 cells / mL, or 3x10 6Incubate cells (e.g., cells of the input composition) at a density of less than 5 x 10 cells / mL. 6 In some embodiments, cells are incubated at a density of 1x10 cells / mL. 3 cells / mL and 1x 10 9 cells / mL, 1x 10 4 cells / mL and 1x 10 8 cells / mL, 1x 10 5 cells / mL and 1x 10 7 cells / mL, 5x 10 5 cells / mL and 1x10 7 cells / mL, 1x 10 6 cells / mL and 5x 10 6 cells / mL, or 3 x 10 6 cells / mL and 5x 10 6 Incubate cells at a density between 1 x 10 cells / mL. In certain embodiments, cells are incubated at a density between 1 x 10 cells / mL and 1 x 10 cells / mL. 6 cells / mL, 1.5x 10 6 cells / mL, 2x 10 6 cells / mL, 2.5x10 6 cells / mL, 3x 10 6 cells / mL, 3.5 x 10 6 cells / mL, 4x 10 6 cells / mL, 4.5x10 6 cells / mL, or 5 x 10 6 In certain embodiments, the cells are incubated at a density of at least 3 x 10 cells / mL. 6 In some embodiments, the cells are incubated at a density of 10 cells / mL. In some embodiments, the cells are viable cells. In certain embodiments, the cells are negative for apoptosis markers (e.g., annexin V or active caspase 3). In specific embodiments, the cells are or include CD4+ T cells and CD8+ T cells.

[0247] In certain embodiments, the index of vigor includes but is not limited to the index of cell replication, mitochondrial function, energy balance, membrane integrity and cell mortality rate. In certain embodiments, the index of vigor also includes the index of oxidative stress, metabolic activation, metabolic stability, enzyme induction, enzyme inhibition and the interaction with cell membrane transporter. In some embodiments, living cells include cells that experience normal functional cell processes and / or do not experience necrosis or programmed cell death or are not in the cell under the process of experiencing necrosis or programmed cell death. In some embodiments, vigor can be assessed by the activity or function of the redox potential of the cell, the integrity of the cell membrane or the mitochondria. In some embodiments, vigor is the absence of specific molecules related to cell death in the assay, or the absence of an indication of cell death. In certain embodiments, the vigor of cells can be detected, measured and / or assessed by many conventional means. The non-limiting examples of such vigor assays include but are not limited to dye uptake assays (e.g., calcein AM assays), XTT cell viability assays and dye exclusion assays (e.g., trypan blue, eosin or propidium dye exclusion assays). Vigor assays can be used to determine the number or percentage (e.g., frequency) of viable cells in a cell dose, a cell composition and / or a cell sample.

[0248] In certain embodiments, apoptosis markers may include any known marker associated with apoptosis, and may include the expression of an active form of a gene, protein, or protein, or the appearance of features associated with apoptosis (such as blebbing and / or nuclear destruction). In certain embodiments, apoptosis markers are markers associated with apoptosis, including but not limited to pro-apoptotic factors known to initiate apoptosis, members of the death receptor pathway, activated members of the mitochondrial (intrinsic) pathway, Bcl-2 family members (such as Bax, Bad, and Bid, Fas, FADD), the presence of nuclear shrinkage (e.g., monitored by a microscope), the presence of chromosomal DNA fragmentation (e.g., the presence of chromosomal DNA ladders), or markers associated with apoptosis assays (e.g., TUNEL staining and annexin V staining). In some embodiments, apoptosis markers are expression of caspases, for example, expression of active forms of caspase-1, caspase-2, caspase-3, caspase-7, caspase-8, caspase-9, caspase-10, and / or caspase-13. In some embodiments, the apoptosis marker is Annexin V. In certain embodiments, the apoptosis marker is active caspase 3.

[0249] In some embodiments, for example, incubation at or about 1 x 10 5 or approximately 500,000 x 10 6 cells, or about 1 x 10 6or approximately 50,000 x 10 6 cells, or about 10 x 10 6 or about 5,000x10 6 cells, or about 1 x 10 6 or approximately 1,000 x 10 6 cells, or about 50 x 10 6 or approximately 5,000 x 10 6 cells, or about 10 x 10 6 or approximately 1,000 x 10 6 cells between 10 and 10 6 or about 2,500x10 6 In certain embodiments, for example, at least, about, or about 50 x 10 cells are incubated under stimulating conditions. 6 cells, 100 x 10 6 cells, 150 x 10 6 cells, 200 x 10 6 cells, 250 x 10 6 cells, 300 x 10 6 cells, 350 x 10 6 cells, 400 x 10 6 cells, 450 x 10 6 cells, or 500 x 10 6 In some embodiments, the cells are viable cells. In certain embodiments, the cells are negative for apoptosis markers (e.g., annexin V or active caspase 3). In specific embodiments, the cells are or include CD4+ T cells and CD8+ T cells.

[0250] In some embodiments, for example, incubation at or about 1 x 10 5 or approximately 25,000 x 10 6 Between or about 1x 10 6 or approximately 25,000 x 10 6 Between or about 10x 10 6 or approximately 2,500 x 10 6 Between or about 1x 10 6 or about 500x 10 6 Between or about 50x 10 6 or about 2,500x10 6Between or about 10x 10 6 or about 500x 10 6 Between or about 50x 10 6 or about 300 x 10 6 In certain embodiments, at least, about, or about 25 x 10 6 , 50x 10 6 , 75x10 6 , 100x 10 6 , 125x 10 6 , 150x 10 6 , 175x 10 6 , 200x 10 6 , 225x 10 6 , or 250x 10 6 In some embodiments, the CD4+ T cells are viable CD4+ T cells. In certain embodiments, the CD4+ T cells are negative for apoptosis markers (e.g., annexin V or active caspase 3).

[0251] In certain embodiments, for example, incubation at or about 1 x 10 5 or approximately 25,000 x 10 6 Between or about 1x 10 6 or approximately 25,000 x 10 6 Between or about 10x 10 6 or approximately 2,500 x 10 6 Between or about 1x 10 6 or about 500x 10 6 Between or about 50x 10 6 or about 2,500x10 6 Between or about 10x 10 6 or about 500x 10 6 Between or about 50x 10 6 or about 300 x 10 6 In some embodiments, for example, at least, about, or about 25 x 10 6 , 50x 10 6 , 75x10 6 , 100x 10 6, 125x 10 6 , 150x 10 6 , 175x 10 6 , 200x 10 6 , 225x 10 6 , or 250x 10 6 In some embodiments, the CD8+ T cells are viable CD8+ T cells. In certain embodiments, the CD8+ T cells are negative for apoptosis markers (e.g., annexin V or active caspase 3).

[0252] In some embodiments, the conditions for stimulation and / or activation may include one or more of the following: specific culture medium, temperature, oxygen content, carbon dioxide content, time, agents (e.g., 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 intended to activate cells)).

[0253] In some embodiments, stimulation conditions or stimulation reagents include one or more reagents (e.g., ligands) that can stimulate or activate the intracellular signaling domain of the TCR complex. In some aspects, the agent opens or starts the TCR / CD3 intracellular signaling cascade in T cells, for example, it is suitable for delivering primary signals to, for example, start ITAM-induced signals (e.g., those with specificity for TCR components (e.g., anti-CD3)), and / or promotes costimulatory signals (e.g., costimulatory signals with specificity for T cell costimulatory receptors), such as anti-CD28 or anti-4-1BB (e.g., it is bound to a solid support (e.g., beads) and / or one or more cytokines. Stimulating reagents include anti-CD3 / anti-CD28 beads (e.g., M-450 CD3 / CD28 T cell expander and / or Beads). Optionally, the expansion method can further comprise the step of adding anti-CD3 and / or anti-CD28 antibodies to the culture medium. In some embodiments, the stimulatory agent comprises a cytokine.

[0254] In certain embodiments, the stimulating conditions include incubating, culturing and / or cultivating cells together with a stimulating agent. In certain embodiments, the stimulating agent is a reagent as provided herein, for example, a reagent as described in chapters IB-1. In certain embodiments, the stimulating agent contains or comprises a bead. In certain embodiments, when a cell is contacted with a stimulating agent and / or incubated with a stimulating agent, the initiation and / or start of the cell incubated, cultivated and / or cultivated under stimulating conditions occurs. In certain embodiments, the cell is incubated before, during and / or after the cell is genetically engineered (e.g., by introducing a recombinant polynucleotide into the cell (e.g., by transduction or transfection)).

[0255] In some embodiments, the composition enriched for T cells is incubated at a ratio of stimulatory reagent and / or beads to cells of at or about 3: 1, 2.5: 1, 2: 1, 1.5: 1, 1.25: 1, 1.2: 1, 1.1: 1, 1: 1, 0.9: 1, 0.8: 1, 0.75: 1, 0.67: 1, 0.5: 1, 0.3: 1, or 0.2: 1. In specific embodiments, the ratio of stimulatory reagent and / or beads to cells is between 2.5: 1 and 0.2: 1, between 2: 1 and 0.5: 1, between 1.5: 1 and 0.75: 1, between 1.25: 1 and 0.8: 1, between 1.1: 1 and 0.9: 1. In specific embodiments, the ratio of stimulatory reagent to cells is about 1: 1 or is 1: 1.

[0256] In certain embodiments, the stimulation conditions include incubating, culturing and / or cultivating cells (e.g., cells from an input composition) using one or more cytokines and / or in the presence of one or more cytokines. In certain embodiments, one or more cytokines are recombinant cytokines. In some embodiments, the one or more cytokines are human recombinant cytokines. In certain embodiments, the one or more cytokines bind and / or are capable of binding to receptors expressed by T cells and / or endogenous to T cells. In certain embodiments, the one or more cytokines are or include members of the 4-α-helical bundle family of cytokines. In some embodiments, members of the 4-α-helical bundle family of cytokines include but are not limited to interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 7 (IL-7), interleukin 9 (IL-9), interleukin 12 (IL-12), interleukin 15 (IL-15), granulocyte colony stimulating factor (G-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). In some embodiments, the one or more cytokines are or include IL-15. In certain embodiments, the one or more cytokines are or include IL-7. In certain embodiments, the one or more cytokines are or include IL-2.

[0257] In certain embodiments, the amount or concentration of one or more cytokines are measured and / or quantitatively in international units (IU).International units can be used for quantitative vitamins, hormones, cytokines, vaccines, blood products and similar bioactive substances. In some embodiments, IU is or includes the unit of measure of the biological preparation effectiveness compared with the international reference standard (for example, the WHO first international standard (WHO 1st International Standard for Human IL-2) for IL-2, 86 / 504) with specific weight and intensity.International units are uniquely recognized and standardized methods for reporting biological activity units, and the biological activity units are open and derived from international collaborative research work. In specific embodiments, the compositions, sample or the IU in source of a cytokine can be obtained by comparing the test results with the product of similar WHO standard products. For example, in some embodiments, the IU / mg of a composition, sample, or source of human recombinant IL-2, IL-7, or IL-15 is compared to the WHO standard IL-2 product (NIBSC code: 86 / 500), the WHO standard IL-17 product (NIBSC code: 90 / 530), and the WHO standard IL-15 product (NIBSC code: 95 / 554).

[0258] In some embodiments, the biological activity in IU / mg is equivalent to (ED 50 , in ng / mL) -1 x10 6 In certain embodiments, the ED of recombinant human IL-2 or IL-15 50 The concentration equivalent to half-maximal stimulation of cell proliferation (XTT lysis) of CTLL-2 cells. In certain embodiments, the ED of recombinant human IL-7 is 50 Equivalent to the concentration required for half-maximal stimulation of PHA-activated human peripheral blood lymphocyte proliferation. Details related to the determination and calculation of IU of IL-2 are discussed in Wadhwa et al., Journal of Immunological Methods (2013), 379(1-2): 1-7; and Gearing and Thorpe, Journal of Immunological Methods (1988), 114(1-2): 3-9; and details related to the determination and calculation of IU of IL-15 are discussed in Soman et al., Journal of Immunological Methods (2009) 348(1-2): 83-94.

[0259] In some embodiments, cells (e.g., input cells) are incubated with a cytokine, e.g., a recombinant human cytokine, at a concentration between at or about 1 IU / mL and at or about 1,000 IU / mL, between at or about 10 IU / mL and at or about 50 IU / mL, between at or about 50 IU / mL and at or about 100 IU / mL, between at or about 100 IU / mL and at or about 200 IU / mL, between at or about 100 IU / mL and at or about 500 IU / mL, between at or about 250 IU / mL and at or about 500 IU / mL, or between at or about 500 IU / mL and at or about 1,000 IU / mL.

[0260] In some embodiments, cells (e.g., infused cells) are incubated with IL-2 (e.g., human recombinant IL-2) at a concentration of between at or about 1 IU / mL and at or about 500 IU / mL, between at or about 10 IU / mL and at or about 250 IU / mL, between at or about 50 IU / mL and at or about 200 IU / mL, between at or about 50 IU / mL and at or about 150 IU / mL, between at or about 75 IU / mL and at or about 125 IU / mL, between at or about 100 IU / mL and at or about 200 IU / mL, or between at or about 10 IU / mL and at or about 100 IU / mL, e.g., in serum-free medium. In certain embodiments, cells (e.g., cells of an infused composition) are incubated with recombinant IL-2 at a concentration of at or about 50 IU / mL, 60 IU / mL, 70 IU / mL, 80 IU / mL, 90 IU / mL, 100 IU / mL, 110 IU / mL, 120 IU / mL, 130 IU / mL, 140 IU / mL, 150 IU / mL, 160 IU / mL, 170 IU / mL, 180 IU / mL, 190 IU / mL, or 100 IU / mL. In some embodiments, cells (e.g., infused cells) are incubated in the presence of, for example, at or about 100 IU / mL of recombinant IL-2 (e.g., human recombinant IL-2).

[0261] In some embodiments, cells (e.g., input cells) are incubated with recombinant IL-7 (e.g., human recombinant IL-7) at a concentration of between at or about 100 IU / mL and at or about 2,000 IU / mL, between at or about 500 IU / mL and at or about 1,000 IU / mL, between at or about 100 IU / mL and at or about 500 IU / mL, between at or about 500 IU / mL and at or about 750 IU / mL, between at or about 750 IU / mL and at or about 1,000 IU / mL, or between at or about 550 IU / mL and at or about 650 IU / mL, e.g., in serum-free medium. In certain embodiments, cells (e.g., input cells) are incubated with IL-7 at a concentration of at or about 50 IU / mL, 100 IU / mL, 150 IU / mL, 200 IU / mL, 250 IU / mL, 300 IU / mL, 350 IU / mL, 400 IU / mL, 450 IU / mL, 500 IU / mL, 550 IU / mL, 600 IU / mL, 650 IU / mL, 700 IU / mL, 750 IU / mL, 800 IU / mL, 750 IU / mL, 750 IU / mL, 750 IU / mL, or 1,000 IU / mL. In certain embodiments, cells (e.g., input cells) are incubated in the presence of at or about 600 IU / mL of IL-7 (e.g., human recombinant IL-7).

[0262] In some embodiments, cells (e.g., infused cells) are incubated with recombinant IL-15 (e.g., human recombinant IL-15) at a concentration of between at or about 1 IU / mL and at or about 500 IU / mL, between at or about 10 IU / mL and at or about 250 IU / mL, between at or about 50 IU / mL and at or about 200 IU / mL, between at or about 50 IU / mL and at or about 150 IU / mL, between at or about 75 IU / mL and at or about 125 IU / mL, between at or about 100 IU / mL and at or about 200 IU / mL, or between at or about 10 IU / mL and at or about 100 IU / mL, e.g., in serum-free medium. In certain embodiments, cells (e.g., cells of an infusion composition) are incubated with recombinant IL-15 at a concentration of at or about 50 IU / mL, 60 IU / mL, 70 IU / mL, 80 IU / mL, 90 IU / mL, 100 IU / mL, 110 IU / mL, 120 IU / mL, 130 IU / mL, 140 IU / mL, 150 IU / mL, 160 IU / mL, 170 IU / mL, 180 IU / mL, 190 IU / mL, or 200 IU / mL. In some embodiments, cells (e.g., infusion cells) are incubated in the presence of, for example, at or about 100 IU / mL of recombinant IL-15 (e.g., human recombinant IL-15).

[0263] In a specific embodiment, cells (e.g., cells from an input composition) are incubated in the presence of IL-2, IL-7, and / or IL-15 under stimulating conditions, for example, in serum-free medium. In some embodiments, IL-2, IL-7, and / or IL-15 are recombinant. In certain embodiments, IL-2, IL-7, and / or IL-15 are human. In a specific embodiment, one or more cytokines are or include human recombinant IL-2, IL-7, and / or IL-15. In certain embodiments, cells are incubated in the presence of recombinant IL-2, IL-7, and IL-15 under stimulating conditions, for example, in serum-free medium.

[0264] The conditions may include one or more of the following: specific culture medium, temperature, oxygen content, carbon dioxide content, time, agents (e.g., 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 intended to activate cells)).

[0265] In some aspects, incubation is performed 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, incubation is performed in serum-free medium. In some embodiments, serum-free medium is a defined and / or well-defined cell culture medium. In certain embodiments, serum-free medium is a controlled medium that has been treated, for example, filtered, to remove inhibitors and / or growth factors. In some embodiments, serum-free medium contains protein. In certain embodiments, serum-free medium may contain serum albumin, hydrolysates, growth factors, hormones, carrier proteins, and / or attachment factors.

[0267] In some embodiments, at least a portion of the hatching in the presence of one or more stimulation conditions or stimulation reagents is for example carried out under centrifugal rotation in the internal cavity of a centrifugal chamber, as described in International Publication No. WO2016 / 073602. In some embodiments, at least a portion of the hatching carried out in a centrifugal chamber includes mixing with one or more reagents to induce stimulation and / or activation. In some embodiments, cells (such as selected cells) are mixed with stimulation conditions or stimulants in a centrifugal chamber. In some aspects of such processes, a certain volume of cells is mixed with a certain amount of one or more stimulation conditions or stimulants, and the stimulation conditions or stimulants are much smaller than the stimulation conditions or stimulants commonly used when carrying out similar stimulation in cell culture plates or other systems.

[0268] In some embodiments, stimulating agent is added to the cell in the chamber cavity, the amount added is selected in the tube or bag of periodic oscillation or rotation and not mixed in the centrifugal chamber, realizes that the roughly identical or similar selection efficiency of the cell of the same number or same volume of cell is usually used or the amount of the stimulating agent needed is compared significantly smaller (for example, not more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the amount). In some embodiments, incubation buffer is added to cells and stimulating agent, to realize the target volume of the hatching of for example 10mL to 200mL (for example, at least or at least about or about 10mL, 20mL, 30mL, 40mL, 50mL, 60mL, 70mL, 80mL, 90mL, 100mL, 150mL or 200mL) reagent. In some embodiments, incubation buffer and stimulating agent are pre-mixed before adding cell. In some embodiments, incubation buffer and stimulating agent are added to cells separately. In some embodiments, stimulation incubations are performed under periodic mild mixing conditions, which may help promote energetically favorable interactions and thereby allow for the use of less overall stimulatory agent while achieving both stimulation and activation of the cells.

[0269] In some embodiments, incubation is typically carried out under mixing conditions, such as in the presence of rotation, typically with relatively low force or speed, such as a speed lower than that used to pellet the cells, such as from or about 600 rpm to or about 1700 rpm (e.g., at or about or at least 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm), such as at an RCF of from or about 80 g to 100 g (e.g., at or about or at least 80 g, 85 g, 90 g, 95 g, or 100 g) at the sample or chamber wall or other container wall. In some embodiments, rotation is performed using repeated intervals of such low-speed rotation followed by a rest period, such as spinning and / or resting for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, such as spinning for about 1 or 2 seconds and then resting for about 5, 6, 7, or 8 seconds.

[0270] In some embodiments, the total duration of incubation under stimulating conditions (e.g., with a stimulating agent) is between or about 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, between 12 hours and 24 hours, between 18 hours and 30 hours, e.g., at least or about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours. In some embodiments, the total duration of incubation, e.g., with a stimulating agent, is between or about 18 hours and about 30 hours.

[0271] In some embodiments, cells are cultured, grown, and / or incubated under stimulating conditions before and / or during the step of introducing a polynucleotide (e.g., a polynucleotide encoding a recombinant receptor) into the cells (e.g., by transduction and / or transfection, e.g., as described in Section 1C). In certain embodiments, cells are cultured, grown, and / or incubated under stimulating conditions for an amount of time between 30 minutes and 2 hours, between 1 hour and 8 hours, between 6 hours and 12 hours, between 12 hours and 18 hours, between 16 hours and 24 hours, between 18 hours and 30 hours, between 24 hours and 48 hours, between 24 hours and 72 hours, between 42 hours and 54 hours, between 60 hours and 120 hours, between 96 hours and 120 hours, between 90 hours and between 1 day and 7 days, between 3 days and 8 days, between 1 day and 3 days, between 4 days and 6 days, or between 4 days and 5 days prior to genetic engineering. In some embodiments, the cells are incubated under stimulating conditions for at or about between 18 hours and 30 hours. In specific embodiments, the cells are incubated under stimulating conditions for at or about 24 hours.

[0272] In some embodiments, incubating cells under stimulating conditions comprises incubating the cells with a stimulating agent described in Section IB-1. In some embodiments, the stimulating agent contains or comprises beads, such as paramagnetic beads, and the cells are incubated with the stimulating agent at a ratio of less than 3:1 (beads:cells), such as a 1:1 ratio. In specific embodiments, the cells are incubated with the stimulating agent in the presence of one or more cytokines. In some embodiments, the cells are incubated with the stimulating agent in the presence of recombinant IL-2, IL-7, and IL-15 at a 1:1 (beads:cells) ratio.

[0273] In certain embodiments, the input composition comprising CD4+ and CD8+ T cells is incubated under stimulating conditions. In certain embodiments, the cells are incubated in serum-free medium. In certain embodiments, the input composition comprises CD4+ T cells to CD8+ T cells at a ratio of at or about 1:1. In certain embodiments, the input composition comprises CD4+ T cells to CD8+ T cells at a ratio of at or about 1:1 under stimulating conditions, for example, at a ratio of less than or about 5 x 10 6 Cells were incubated at a density of at least or about 100 x 10 cells / mL. 6 In certain embodiments, at least or about 50 x 10 cells are incubated under stimulating conditions. 6 CD4+ T cells and at least or about 50 x 10 6In some embodiments, the cells are incubated for 18 to 30 hours. In certain embodiments, the cells are incubated under stimulating conditions and include incubating the cells with a stimulating agent in the presence of IL-2, IL-7, and / or IL-15. In certain embodiments, the cells are incubated with a stimulating agent at a ratio of less than 3:1 to the cells. In some embodiments, the cells are incubated with an IL-2 at or about 50 IU / mL to or about 200 IU / mL, an IL-7 at or about 400 to or about 1,000 IU / mL, and / or an IL-15 at or about 50 IU / mL to or about 200 IU / mL.

[0274] In certain embodiments, the input composition is incubated under stimulating conditions at 100 x 10 6 500x 10 6 The input composition contains CD4+ and CD8+ T cells at a ratio of at or about 1:1. In certain embodiments, the cells are viable and / or negative for apoptosis markers. In some embodiments, the input composition is incubated at or about 300 x 10 6 In certain embodiments, the cells are incubated in serum-free medium. In certain embodiments, the cells are at or about 3 x 10 6 In some embodiments, the cells are incubated at a density of at or about 150 x 10 6 CD4+ T cells or about 150 x 10 6 In certain embodiments, the cells are incubated with the stimulating agent at a ratio of at or about 1:1 of stimulating agent to cells. In certain embodiments, the cells are incubated in the presence of at or about 100 IU / mL IL-2, at or about 600 IU / mL IL-7, and between 50 IU / mL and / or at or about 200 IU / mL IL-15.

[0275] 1. Stimulating Reagents

[0276] In some embodiments, the composition of incubating enriched cells under stimulating conditions is or includes incubating and / or contacting the composition of enriched cells with a stimulating agent capable of activating and / or amplifying T cells. In some embodiments, the stimulating agent is capable of stimulating and / or activating one or more signals in the cell. In some embodiments, one or more signals are mediated by receptors. In specific embodiments, one or more signals are or are associated with: changes in signal transduction and / or the level or amount of secondary messengers (e.g., cAMP and / or intracellular calcium); changes in the amount, cell localization, confirmation, phosphorylation, ubiquitination, and / or truncation of one or more cellular proteins; and / or changes in cell activity (e.g., transcription, translation, protein degradation, cell morphology, activation state, and / or cell division). In specific embodiments, the stimulating conditions include incubating, culturing, and / or cultivating cells with the stimulating agent. In certain embodiments, the stimulating agent contains or includes beads. In certain embodiments, the start of stimulation occurs when the cell is incubated or contacted with the stimulating agent. In specific embodiments, the stimulating agent contains or includes an oligomeric agent, such as a streptavidin mutant protein oligomer. In certain embodiments, the stimulatory agent activates and / or is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules.

[0277] In some embodiments, stimulation conditions or stimulation reagents include one or more agents (e.g., ligands) capable of activating the intracellular signaling domain of the TCR complex. In some embodiments, the agent considered herein may include but is not limited to RNA, DNA, protein (e.g., enzyme), antigen, polyclonal antibody, monoclonal antibody, antibody fragment, carbohydrate, lipid lectin or any other biomolecule with affinity for the desired target. In some embodiments, the desired target is a component of a T cell receptor and / or a T cell receptor. In certain embodiments, the desired target is CD3. In certain embodiments, the desired target is a T cell costimulatory molecule, such as CD28, CD137 (4-1-BB), OX40 or ICOS. The one or more agents may be directly or indirectly attached to the beads by various methods known and available in the art. Attachment may be covalent, non-covalent, electrostatic or hydrophobic, and may be achieved by various attachment means (including, for example, chemical means, mechanical means or enzymatic means). In some embodiments, the agent is an antibody or its antigen-binding fragment, such as Fab. In some embodiments, a biomolecule (eg, a biotinylated anti-CD3 antibody) can be indirectly attached to the beads through another biomolecule (eg, an anti-biotin antibody) that is directly attached to the beads.

[0278] In some embodiments, the stimulatory reagent contains one or more agents (e.g., antibodies or antigen-binding fragments thereof, such as Fab) that specifically bind to one or more of the following macromolecules on cells (e.g., T cells): CD2, CD3, CD4, CD5, CD8, CD25, CD27, CD28, CD29, CD31, CD44, CD45RA, CD45RO, CD54 (ICAM-1), CD127, MHC I, MHC II, CTLA-4, ICOS, PD- 1, OX40, CD27L (CD70), 4-1BB (CD137), 4-1BBL, CD30L, LIGHT, IL-2R, IL-12R, IL-1R, IL-15R; IFN-γR, TNF-αR, IL-4R, IL-10R, CD18 / CD11a (LFA-1), CD62L (L-selectin), CD29 / CD49d (VLA-4), Notch ligands (such as Delta-like ligand 1 / 4, Jagged 1 / 2, etc.), CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, and CXCR3 or fragments thereof, including the corresponding ligands of these macromolecules or fragments thereof. In some embodiments, the stimulating reagent contains one or more agents (e.g., antibodies or antigen-binding fragments thereof, such as Fab) that specifically bind to one or more of the following macromolecules on cells (e.g., T cells): CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA, and / or CD45RO. In some embodiments, the one or more agents are or are capable of being attached to beads (e.g., paramagnetic beads). In some embodiments, the one or more agents are or are capable of being attached (e.g., reversibly attached) to oligomeric agents (e.g., streptavidin mutein oligomers).

[0279] In some embodiments, one or more agents comprise an antibody or an antigen-binding fragment thereof, such as Fab. Antibodies can include polyclonal antibodies, monoclonal antibodies (including full-length antibodies with immunoglobulin Fc regions), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies, double antibodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab')2 and Fv). In some embodiments, the stimulating agent is or comprises an antibody fragment (including antigen-binding fragments), such as Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragments. It should be understood that any isotype of constant region can be used for the antibodies considered herein, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be obtained from any human or animal species (e.g., murine species). In some embodiments, the agent is or comprises an antibody that binds to and / or recognizes one or more components of a T cell receptor. In specific embodiments, the agent is or comprises an anti-CD3 antibody. In certain embodiments, the agent is or comprises an antibody that binds to and / or recognizes a co-receptor. In some embodiments, the stimulatory agent is or comprises an anti-CD28 antibody.

[0280] In some embodiments, cells (e.g., cells of an input population) are stimulated in the presence of a stimulatory agent to cell ratio of at or about 3: 1, 2.5: 1, 2: 1, 1.5: 1, 1.25: 1, 1.2: 1, 1.1: 1, 1: 1, 0.9: 1, 0.8: 1, 0.75: 1, 0.67: 1, 0.5: 1, 0.3: 1, or 0.2: 1. In specific embodiments, the ratio of stimulatory agent to cells is between 2.5: 1 and 0.2: 1, between 2: 1 and 0.5: 1, between 1.5: 1 and 0.75: 1, between 1.25: 1 and 0.8: 1, or between 1.1: 1 and 0.9: 1. In specific embodiments, the ratio of stimulatory agent to cells is about 1: 1 or is 1: 1.

[0281] In some embodiments, every 10 6 In some embodiments, cells are stimulated in the presence of at least 0.01 μg, about 0.02 μg, 0.03 μg, 0.04 μg, 0.05 μg, 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.75 μg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, or 10 μg of a stimulating agent per 10 cells. 6 In certain embodiments, cells are stimulated in the presence of 4 μg or about 4 μg per 10 cells. 6 In various embodiments, cells are stimulated in the presence of or about 0.8 μg per 10 cells. 6The cells were stimulated in the presence of about 0.8 μg of daptomycin per cell.

[0282] a. Bead reagent

[0283] In certain embodiments, the stimulating reagent contains particles (e.g., beads) conjugated or connected to one or more agents (e.g., biomolecules) that can activate and / or amplify cells (e.g., T cells). In some embodiments, the one or more agents are bound to beads. In some embodiments, beads are biocompatible, i.e., made of materials suitable for biological purposes. In some embodiments, beads are nontoxic to cultured cells (e.g., cultured T cells). In some embodiments, beads can be any particles that can attach agents in a manner that allows interactions between agents and cells.

[0284] In some embodiments, the stimulating reagent contains one or more agents capable of activating and / or amplifying cells (e.g., T cells), which are bound to or otherwise attached to beads, e.g., bound to or attached to the surface of beads. In certain embodiments, beads are non-cellular particles. In specific embodiments, beads can include colloidal particles, microspheres, nanoparticles, magnetic beads, etc. In some embodiments, beads are agarose beads. In certain embodiments, beads are agarose gel beads.

[0285] In certain embodiments, the stimulation reagent comprises monodisperse beads.In certain embodiments, the monodisperse beads comprise a size dispersion with a standard deviation of diameters from each other of less than 5%.

[0286] In some embodiments, pearl contains one or more medicaments, such as the medicament of being coupled, conjugated or connected (directly or indirectly) with the surface of pearl.In some embodiments, the medicament as considered herein can include but is not limited to RNA, DNA, protein (for example, enzyme), antigen, polyclonal antibody, monoclonal antibody, antibody fragment, carbohydrate, lipid lectin or any other biomolecule with affinity to desired target.In some embodiments, desired target is the component of T cell receptor and / or T cell receptor.In certain embodiments, desired target is CD3.In certain embodiments, desired target is T cell costimulatory molecule, such as CD28, CD137 (4-1-BB), OX40 or ICOS.Described one or more medicaments can be directly or indirectly attached to pearl by various methods known in the art and available.Attachment can be covalent, non-covalent, electrostatic or hydrophobic, and can be realized by various attachment means (including such as chemical means, mechanical means or enzymatic means). In some embodiments, a biomolecule (eg, a biotinylated anti-CD3 antibody) can be indirectly attached to the beads through another biomolecule (eg, an anti-biotin antibody) that is directly attached to the beads.

[0287] In some embodiments, the stimulating reagent contains beads and one or more agents that directly interact with macromolecules on the cell surface. In certain embodiments, beads (e.g., paramagnetic beads) interact with cells via one or more agents (e.g., antibodies) that are specific to one or more macromolecules (e.g., one or more cell surface proteins) on the cell. In certain embodiments, beads (e.g., paramagnetic beads) are labeled with a first agent (e.g., an anti-biotin antibody) or other biomolecule as described herein, and then a second agent (e.g., a second antibody (e.g., a biotinylated anti-CD3 antibody) or other second biomolecule (e.g., streptavidin) is added, whereby the second antibody or other second biomolecule is specifically bound to such a first antibody or other biomolecule on the particle.

[0288] In some embodiments, the stimulatory reagent contains one or more agents (e.g., antibodies) attached to beads (e.g., paramagnetic beads) that specifically bind to one or more of the following macromolecules on cells (e.g., T cells): CD2, CD3, CD4, CD5, CD8, CD25, CD27, CD28, CD29, CD31, CD44, CD45RA, CD45RO, CD54 (ICAM-1), CD127, MHC I, MHC II, CTLA-4, ICOS, PD- 1, OX40, CD27L (CD70), 4-1BB (CD137), 4-1BBL, CD30L, LIGHT, IL-2R, IL-12R, IL-1R, IL-15R; IFN-γR, TNF-αR, IL-4R, IL-10R, CD18 / CD11a (LFA-1), CD62L (L-selectin), CD29 / CD49d (VLA-4), Notch ligands (such as Delta-like ligand 1 / 4, Jagged 1 / 2, etc.), CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, and CXCR3 or fragments thereof, including the corresponding ligands of these macromolecules or fragments thereof. In some embodiments, the agent (e.g., an antibody) attached to the beads specifically binds to one or more of the following macromolecules on cells (e.g., T cells): CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA, and / or CD45RO.

[0289] In some embodiments, one or more agents attached to the beads are antibodies. Antibodies can include polyclonal antibodies, monoclonal antibodies (including full-length antibodies with immunoglobulin Fc regions), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies, double antibodies and single-chain molecules) and antibody fragments (e.g., Fab, F(ab')2 and Fv). In some embodiments, the stimulating agent is an antibody fragment (including antigen-binding fragments), such as Fab, Fab'-SH, Fv, scFv or (Fab')2 fragments. It should be understood that the constant region of any isotype can be used for the antibodies considered herein, including IgG, IgM, IgA, IgD and IgE constant regions, and such constant regions can be obtained from any human or animal species (e.g., murine species). In some embodiments, the agent is an antibody that binds and / or recognizes one or more components of a T cell receptor. In specific embodiments, the agent is an anti-CD3 antibody. In certain embodiments, the agent is an antibody that binds and / or recognizes a co-receptor. In some embodiments, the stimulating agent comprises an anti-CD28 antibody. In some embodiments, the beads have a diameter of greater than or about 0.001 μm, greater than or about 0.01 μm, greater than or about 0.1 μm, greater than or about 1.0 μm, greater than or about 10 μm, greater than or about 50 μm, greater than or about 100 μm, or greater than or about 1000 μm, and no more than or about 1500 μm. In some embodiments, the beads have a diameter of between at or about 1.0 μm and at or about 500 μm, between at or about 1.0 μm and at or about 150 μm, between at or about 1.0 μm and at or about 30 μm, between at or about 1.0 μm and at or about 10 μm, between at or about 1.0 μm and at or about 5.0 μm, between at or about 2.0 μm and at or about 5.0 μm, or between at or about 3.0 μm and at or about 5.0 μm. In some embodiments, the beads have a diameter of at or about 3 μm to at or about 5 μm. In some embodiments, the beads have a diameter of at least or about 0.001 μm, 0.01 μm, 0.1 μm, 0.5 μm, 1.0 μm, 1.5 μm, 2.0 μm, 2.5 μm, 3.0 μm, 3.5 μm, 4.0 μm, 4.5 μm, 5.0 μm, 5.5 μm, 6.0 μm, 6.5 μm, 7.0 μm, 7.5 μm, 8.0 μm, 8.5 μm, 9.0 μm, 9.5 μm, 10 μm, 12 μm, 14 μm, 16 μm, 18 μm, or 20 μm. In certain embodiments, the beads have a diameter of at or about 4.5 μm. In certain embodiments, the beads have a diameter of at or about 2.8 μm.

[0290] In some embodiments, the beads have a g / cm 3 , greater than or greater than about 0.01 g / cm3 , greater than or about 0.05 g / cm 3 , greater than or greater than about 0.1 g / cm 3 , greater than or greater than about 0.5 g / cm 3 , greater than or about 0.6 g / cm 3 , greater than or greater than about 0.7 g / cm 3 , greater than or greater than about 0.8 g / cm 3 , greater than or greater than about 0.9 g / cm 3 , greater than or greater than about 1 g / cm 3 , greater than or greater than about 1.1 g / cm 3 , greater than or greater than about 1.2 g / cm 3 , greater than or greater than about 1.3 g / cm 3 , greater than or greater than about 1.4 g / cm 3 , greater than or greater than about 1.5 g / cm 3 , greater than or greater than about 2 g / cm 3 , greater than or greater than about 3 g / cm 3 , greater than or greater than about 4 g / cm 3 , or greater than or greater than about 5 g / cm 3 In some embodiments, the beads have a density of at or about 0.001 g / cm 3 With or about 100g / cm 3 Between, or about 0.01 g / cm 3 With or about 50g / cm 3 Between, or about 0.1 g / cm 3 With or about 10g / cm 3 Between, or about 0.1 g / cm 3 With or about 5g / cm 3 Between, or about 0.5 g / cm 3 and about 1g / cm 3 Between, or about 0.5 g / cm 3 and about 1.5 g / cm 3 Between, about or about 1g / cm 3 and about 1.5 g / cm 3 Between, about or about 1g / cm 3 and about 2g / cm 3 Between, or about 1g / cm 3 With or about 5g / cm 3 In some embodiments, the beads have a density of at or about 0.5 g / cm 3 , or about 0.5 g / cm 3 , or about 0.6 g / cm3 , or about 0.7 g / cm 3 , or about 0.8 g / cm 3 , or about 0.9 g / cm 3 , or about 1.0 g / cm 3 , or about 1.1 g / cm 3 , or about 1.2 g / cm 3 , or about 1.3 g / cm 3 , or about 1.4 g / cm 3 , or about 1.5 g / cm 3 , or about 1.6 g / cm 3 , or about 1.7 g / cm 3 , or about 1.8 g / cm 3 , or about 1.9 g / cm 3 , or about 2.0 g / cm 3 In certain embodiments, the beads have a density of at or about 1.6 g / cm 3 In certain embodiments, the beads or particles have a density of at or about 1.5 g / cm 3 In certain embodiments, the particles have a density of at or about 1.3 g / cm 3 density.

[0291] In certain embodiments, the plurality of beads has a uniform density.In certain embodiments, uniform density comprises a density standard deviation of less than or about 10%, less than or about 5%, or less than or about 1% of the average bead density.

[0292] In some embodiments, the beads have a diameter at or about 0.001 μm. 2 / g particles (m 2 / g) to about 1,000m 2 / g, or about 0.10m 2 / g to about 100m 2 / g, or about 0.1m 2 / g to about 10m 2 / g, or about 0.1m 2 / g to about 1m 2 / g, or about 1m 2 / g to about 10m 2 / g, or about 10m 2 / g to about 100m 2 / g, or about 0.5m 2 / g to about 20m 2 / g, or about 0.5m 2 / g to about 5m 2 / g, or about 1m 2 / g to about 4m 2 In some embodiments, the particles or beads have a surface area of ​​or about 1 m 2 / g to about 4m 2 / g of surface area.

[0293] In some embodiments, the beads contain at least one material that can be coupled, connected or conjugated to a pharmaceutical agent at or near the surface of the beads. In some embodiments, the beads are surface functionalized, i.e., contain functional groups that can form covalent bonds with binding molecules (e.g., polynucleotides or polypeptides). In specific embodiments, the beads include surface-exposed carboxyl groups, amino groups, hydroxyl groups, tosyl groups, epoxy groups and / or chloromethyl groups. In specific embodiments, the beads include surface-exposed agarose and / or sepharose. In certain embodiments, the beads surface includes attached stimulating reagents that can bind or attach binding molecules. In specific embodiments, the biomolecule is a polypeptide. In some embodiments, the beads include surface-exposed protein A, protein G or biotin.

[0294] In some embodiments, the beads react in a magnetic field. In some embodiments, the beads are magnetic beads. In some embodiments, the magnetic beads are paramagnetic. In specific embodiments, the magnetic beads are superparamagnetic. In certain embodiments, the beads do not exhibit any magnetic properties unless they are exposed to a magnetic field.

[0295] In certain embodiments, the beads comprise a magnetic core, a paramagnetic core, or a superparamagnetic core. In some embodiments, the magnetic core contains a metal. In some embodiments, the metal can be, but is not limited to, iron, nickel, copper, cobalt, gadolinium, manganese, tantalum, zinc, zirconium, or any combination thereof. In certain embodiments, the magnetic core comprises a metal oxide (e.g., iron oxide), a ferrite (e.g., manganese ferrite, cobalt ferrite, nickel ferrite, etc.), hematite, and a metal alloy (e.g., CoTaZn). In some embodiments, the magnetic core comprises one or more of a ferrite, a metal, a metal alloy, iron oxide, or chromium dioxide. In some embodiments, the magnetic core comprises elemental iron or a compound thereof. In some embodiments, the magnetic core comprises one or more of magnetite (Fe3O4), magnetic hematite (γFe2O3), or pyrite (Fe3S4). In some embodiments, the kernel comprises iron oxide (e.g., Fe3O4).

[0296] In certain embodiments, bead contains magnetic core, paramagnetic core and / or superparamagnetic core covered by surface functionalized coating (coat or coating).In some embodiments, coating can contain such material, it can include but not limited to polymer, polysaccharide, silicon dioxide, fatty acid, protein, carbon, agarose, sepharose or its combination.In some embodiments, polymer can be polyethylene glycol, poly (lactic acid-to-glycolic acid), polyglutaraldehyde, polyurethane, polystyrene or polyvinyl alcohol.In certain embodiments, outer coating (coat or coating) comprises polystyrene.In specific embodiments, outer coating is surface functionalized.

[0297] In some embodiments, the stimulating agent comprises a bead containing a metal oxide core (e.g., an iron oxide core) and a coating, wherein the metal oxide core comprises at least one polysaccharide (e.g., dextran), and wherein the coating comprises at least one polysaccharide (e.g., aminodextran), at least one polymer (e.g., polyurethane) and silica. In some embodiments, the metal oxide core is a colloidal iron oxide core. In certain embodiments, the one or more agents comprise an antibody or an antigen-binding fragment thereof. In specific embodiments, the one or more agents comprise anti-CD3 antibodies and anti-CD28 antibodies. In some embodiments, the stimulating agent comprises anti-CD3 antibodies, anti-CD28 antibodies and anti-biotin antibodies. In some embodiments, the stimulating agent comprises anti-biotin antibodies. In some embodiments, the beads have a diameter of about 3 μm to about 10 μm. In some embodiments, the beads have a diameter of about 3 μm to about 5 μm. In certain embodiments, the beads have a diameter of about 3.5 μm.

[0298] In some embodiments, the stimulating reagent comprises one or more agents attached to beads, the beads comprising a metal oxide core (e.g., an iron oxide core) and a coating (e.g., a protective coating), wherein the coating comprises polystyrene. In certain embodiments, the beads are monodisperse paramagnetic (e.g., superparamagnetic) beads comprising a paramagnetic (e.g., superparamagnetic) iron core (e.g., a core comprising magnetite (Fe3O4) and / or maghemite (γFe2O3)) and a polystyrene coating (coat or coating). In some embodiments, the beads are non-porous. In some embodiments, the beads contain a functionalized surface to which the one or more agents are attached. In certain embodiments, the one or more agents are covalently bound to the beads on the surface. In some embodiments, the one or more agents comprise an antibody or an antigen-binding fragment thereof. In some embodiments, the one or more agents comprise an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the one or more agents include anti-CD3 antibodies and / or anti-CD28 antibodies, and antibodies or antigenic fragments thereof that can bind to labeled antibodies (e.g., biotinylated antibodies) such as labeled anti-CD3 or anti-CD28 antibodies. In certain embodiments, the beads have a density of about 1.5 g / cm 3 The density and about 1m 2 / g to about 4m 2 In a specific embodiment, the beads are monodisperse superparamagnetic beads having a diameter of about 4.5 μm and a surface area of ​​about 1.5 g / cm 3 In some embodiments, the beads have an average diameter of about 2.8 μm and a density of about 1.3 g / cm 3 The density of monodisperse superparamagnetic beads.

[0299] In some embodiments, the T cell enriched composition is incubated with a stimulating agent at a beads to cell ratio of at or about 3: 1, 2.5: 1, 2: 1, 1.5: 1, 1.25: 1, 1.2: 1, 1.1: 1, 1: 1, 0.9: 1, 0.8: 1, 0.75: 1, 0.67: 1, 0.5: 1, 0.3: 1, or 0.2: 1. In specific embodiments, the beads to cell ratio is between 2.5: 1 and 0.2: 1, between 2: 1 and 0.5: 1, between 1.5: 1 and 0.75: 1, between 1.25: 1 and 0.8: 1, between 1.1: 1 and 0.9: 1. In specific embodiments, the beads to cell ratio is about 1: 1 or is 1: 1.

[0300] b. Oligomerization Reagents

[0301] In certain embodiments, the stimulating reagent contains an oligomeric reagent (e.g., a streptavidin mutant reagent) that is conjugated, connected, or attached to one or more agents (e.g., ligands) capable of activating the intracellular signaling domain of the TCR complex. In some embodiments, the one or more agents have an attached binding domain or binding partner (e.g., binding partner C) that can bind to an oligomeric reagent at a specific binding site (e.g., binding site Z). In some embodiments, a variety of agents reversibly bind to oligomeric reagents. In various embodiments, the oligomeric reagent has multiple specific binding sites that, in certain embodiments, reversibly bind to multiple agents at a binding domain (e.g., binding partner C). In some embodiments, the amount of the binding agent is reduced or decreased in the presence of a competing reagent (e.g., a reagent that is also capable of binding to a specific binding site (e.g., binding site Z)). Among the oligomeric stimulating reagents, anti-CD3 / anti-CD28 oligomeric streptavidin mutant reagents are included, described in International PCT Publication No. WO2018 / 197949.

[0302] In some embodiments, the stimulating reagent is or includes a reversible system in which at least one agent (e.g., an agent capable of generating a signal in a cell (e.g., a T cell)) is associated with an oligomeric agent (e.g., reversibly associated). In some embodiments, the reagent contains multiple binding sites capable of binding to the reagent (e.g., reversibly binding). In some cases, the reagent is an oligomeric particle reagent with at least one attached agent capable of generating a signal in a cell (e.g., a T cell). In some embodiments, the agent contains at least one binding site (e.g., binding site B) that can specifically bind to an epitope or region of a molecule and also contains a binding partner (also referred to herein as binding partner C) that is bound to at least one binding site (e.g., binding site Z) of the agent. In some embodiments, the binding interaction between binding partner C and the at least one binding site Z is a non-covalent interaction. In some cases, the binding interaction between binding partner C and the at least one binding site Z is a covalent interaction. In some embodiments, the binding interaction (e.g., non-covalent interaction) between binding partner C and the at least one binding site Z is reversible.

[0303] Substances that can be used as oligomerization agents in such reversible systems are known, see, for example, U.S. Patent Nos. 5,168,049; 5,506,121; 6,103,493; 7,776,562; 7,981,632; 8,298,782; 8,735,540; 9,023,604; and International Published PCT Application Nos. WO2013 / 124474 and WO2014 / 076277. Non-limiting examples of agents and binding partners capable of forming reversible interactions, as well as substances capable of reversing such binding (e.g., competitive agents), are described below.

[0304] In some embodiments, the oligomeric agent is an oligomer of streptavidin, a streptavidin mutein or analog, avidin, an avidin mutein or analog (such as neutravidin), or a mixture thereof, wherein such an oligomeric agent contains one or more binding sites for reversibly associating with the binding domain of the agent (e.g., binding partner C). In some embodiments, the binding domain of the agent can be biotin, a biotin derivative or analog, or a streptavidin binding peptide or other molecule capable of specifically binding to streptavidin, a streptavidin mutein or analog, avidin, or an avidin mutein or analog.

[0305] In certain embodiments, one or more agents (e.g., agents capable of generating a signal in a cell, such as a T cell) associate (e.g., reversibly bind) with an oligomeric agent, for example, via multiple specific binding sites (e.g., binding site Z) present on the oligomeric agent. In some cases, this results in the agents being closely arranged with one another such that if a target cell having a cell surface molecule (at least two copies) that is bound or recognized by the agent is brought into contact with the agent, an avidity effect can occur.

[0306] In some embodiments, the oligomeric agent is a streptavidin oligomer, a streptavidin mutein oligomer, a streptavidin analog oligomer, an avidin oligomer, an oligomer composed of an avidin mutein or an avidin analog (such as neutravidin), or a mixture thereof. In certain embodiments, the oligomeric agent contains a specific binding site capable of binding to the binding domain of the agent (e.g., binding partner C). In some embodiments, the binding domain can be biotin, a biotin derivative or analog, or a streptavidin binding peptide or other molecules capable of specifically binding to streptavidin, a streptavidin mutein or analog, avidin or avidin mutein or analog.

[0307] In some embodiments, streptavidin can be wild-type streptavidin, streptavidin mutant protein or analog (such as streptavidin-like polypeptide). Likewise, in some aspects, avidin includes wild-type avidin or a mutant protein or analog (such as neutravidin, which is a deglycosylated avidin with modified arginine, which generally exhibits a more neutral pI and can be used as a substitute for natural avidin). Typically, deglycosylated neutral forms of avidin include, for example, those commercially available forms such as "Extravidin" available from Sigma Aldrich, or "NeutrAvidin" available from ThermoScientific or Invitrogen.

[0308] In some embodiments, reagent is streptavidin or streptavidin mutant protein or analog.In some embodiments, wild-type streptavidin (wt-streptavidin) has by Argarana et al., Nucleic Acids Res.14 (1986) 1871-1882 disclosed amino acid sequence (SEQ ID NO:72).Usually, streptavidin exists naturally as a tetramer of four identical subunits, that is, it is a homotetramer, wherein each subunit contains a single binding site for biotin, biotin derivatives or analogs or biotin mimetics.The exemplary sequence of streptavidin subunit is the amino acid sequence shown in SEQ ID NO:72, but such sequence can also be included in the sequence present in the homologue from other Streptomyces (Streptomyces) species.In particular, each subunit of streptavidin can exhibit a strong binding affinity to biotin, and the equilibrium dissociation constant (K D ) is approximately or about 10 -14 M. In some cases, streptavidin can exist as a monovalent tetramer in which only one of the four binding sites is functional (Howarth et al. (2006) Nat. Methods, 3:267-73; Zhang et al. (2015) Biochem. Biophys. Res. Commun., 463:1059-63), as a divalent tetramer in which two of the four binding sites are functional (Fairhead et al. (2013) J. Mol. Biol., 426:199-214), or can exist as a monomer or dimer (Wu et al. (2005) J. Biol. Chem., 280:23225-31; Lim et al. (2010) Biochemistry, 50:8682-91).

[0309] In some embodiments, streptavidin can be in any form, such as wild-type or unmodified streptavidin, such as streptavidin from a species of Streptomyces, or a functionally active fragment thereof, wherein the functionally active fragment comprises at least one functional subunit comprising a binding site for biotin, a biotin derivative or analog, or a biotin mimetic, such as at least one functional subunit of wild-type streptavidin from Streptomyces avidinii, generally comprising SEQ ID NO: 72, or a functionally active fragment thereof. For example, in some embodiments, streptavidin can include a fragment of wild-type streptavidin that is shortened at the N-terminus and / or C-terminus. Such minimal streptavidin includes any streptavidin that begins at the N-terminus in the region of amino acid positions 10 to 16 of SEQ ID NO: 72 and terminates at the C-terminus in the region of amino acid positions 133 to 142 of SEQ ID NO: 72. In some embodiments, the functionally active fragment of streptavidin comprises the amino acid sequence set forth in SEQ ID NO: 73. In some embodiments, the streptavidin as shown in SEQ ID NO: 73 may further contain an N-terminal methionine at a position corresponding to Ala13 (numbered as shown in SEQ ID NO: 72). The positions of residues in streptavidin or streptavidin muteins are referenced to the numbering of residues in SEQ ID NO: 72.

[0310] Examples of streptavidin or streptavidin mutant proteins are mentioned, for example, in WO 86 / 02077, DE 19641876A1, US 6,022,951, WO 98 / 40396 or WO 96 / 24606. Examples of streptavidin mutant proteins are known in the art, see, for example, U.S. Patent Nos. 5,168,049; 5,506,121; 6,022,951; 6,156,493; 6,165,750; 6,103,493; or 6,368,813; or International Published PCT Application No. WO 2014 / 076277.

[0311] In some embodiments, the streptavidin mutein may contain amino acids that are not part of unmodified or wild-type streptavidin, or may include only a portion of wild-type or unmodified streptavidin. In some embodiments, the streptavidin mutein contains at least one subunit that has one or more amino acid substitutions (replacements) compared to a subunit of unmodified or wild-type streptavidin (e.g., a wild-type streptavidin subunit as set forth in SEQ ID NO: 72 or a functionally active fragment thereof as set forth in SEQ ID NO: 73 or SEQ ID NO: 94).

[0312] In some embodiments, the binding affinity (e.g., dissociation constant (K)) of streptavidin or a streptavidin mutein to the binding domain is d )) is less than or less than about 1 x 10 -4 M, 5x 10 -4 M, 1x10 -5 M, 5x 10 -5 M, 1x 10 -6 M, 5x10 -6 M or 1x 10 -7 M, but usually larger than 1x 10 -13 M, 1x 10 -12 M or 1x 10 -11 M. For example, a peptide sequence (Strep-tag) (as disclosed in U.S. Pat. No. 5,506,121) can be used as a biotin mimetic and demonstrate binding affinity to streptavidin, such as K D Around 10 -4 with 10 -5 Between M. In some cases, binding affinity can be further improved by mutations within the streptavidin molecule, see, for example, U.S. Patent No. 6,103,493 or International Published PCT Application No. WO 2014 / 076277. In some embodiments, binding affinity can be determined by methods known in the art (e.g., any method described herein).

[0313] In some embodiments, the agent (e.g., streptavidin or a streptavidin mutein) exhibits binding affinity for a peptide ligand binding partner, which can be a binding partner C present in an agent (e.g., a receptor binding agent or a selective agent). In some embodiments, the peptide sequence comprises a sequence having the general formula His-Pro-Xaa, wherein Xaa is glutamine, asparagine, or methionine, such as, for example, the sequence shown in SEQ ID NO: 89. In some embodiments, the peptide sequence has the general formula shown in SEQ ID NO: 90, such as, for example, that shown in SEQ ID NO: 80. In one example, the peptide sequence is Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (also known as a Strep-tag). In one example, the peptide sequence is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also known as II, as set forth in SEQ ID NO:75). In some embodiments, the peptide ligand comprises a sequential arrangement of at least two streptavidin binding modules, wherein the distance between the two modules is at least 0 and no more than 50 amino acids, wherein one binding module has 3 to 8 amino acids and comprises at least the sequence His-Pro-Xaa, wherein Xaa is glutamine, asparagine, or methionine, and wherein the other binding module comprises the same or a different streptavidin peptide ligand as set forth in SEQ ID NO:90 (see, e.g., International Published PCT Application No. WO 02 / 077018; U.S. Patent No. 7,981,632). In some embodiments, the peptide ligand comprises a sequence having the formula set forth in any one of SEQ ID NOs:82 or 83. In some embodiments, the peptide ligand has an amino acid sequence set forth in any one of SEQ ID NOs:76-78 and 84-85. In most cases, all of these streptavidin binding peptides bind to the same binding site, i.e., the biotin binding site of streptavidin. If one or more such streptavidin binding peptides are used as binding partners C (e.g., C1 and C2), the multimeric and / or oligomeric particle reagents that bind to the one or more agents via binding partners C are typically composed of one or more streptavidin muteins.

[0314] In some embodiments, the streptavidin mutein is a mutant as described in U.S. Patent No. 6,103,493. In some embodiments, based on the amino acid sequence of wild-type streptavidin (as shown in SEQ ID NO: 72), the streptavidin mutein contains at least one mutation in the region of amino acid positions 44 to 53. In some embodiments, the streptavidin mutein contains mutations at one or more residues 44, 45, 46, and / or 47. In some embodiments, the streptavidin mutein contains a substitution of Glu at position 44 of wild-type streptavidin with a hydrophobic aliphatic amino acid (e.g., Val, Ala, Ile, or Leu), any amino acid at position 45, an aliphatic amino acid at position 46 (e.g., a hydrophobic aliphatic amino acid), and / or a basic amino acid (e.g., Arg or Lys, such as typically Arg) for Val at position 47. In some embodiments, Ala is at position 46 and / or Arg is at position 47 and / or Val or Ile is at position 44. In some embodiments, the streptavidin mutant contains residues Val44-Thr45-Ala46-Arg47, such as shown in the exemplary streptavidin mutein containing the amino acid sequence set forth in SEQ ID NO:86 or SEQ ID NO:87 or 88 (also referred to as streptavidin mutant 1, SAM1). In some embodiments, the streptavidin mutein contains residues Ile44-Gly45-Ala46-Arg47, such as shown in the exemplary streptavidin mutein containing the amino acid sequence set forth in SEQ ID NO:91, 74 or 79 (also referred to as SAM2). In some cases, such streptavidin muteins are described, for example, in U.S. Patent No. 6,103,493 and are available under the trademark In some embodiments, the mutein streptavidin comprises the amino acid sequence set forth in SEQ ID NO: 92 or SEQ ID NO: 93. In certain embodiments, the molecule is a tetramer of streptavidin or a mutein streptavidin comprising the sequence set forth in any one of SEQ ID NOs: 73, 87, 74, 92, 94, 88, or 79, which, as a tetramer, is a molecule containing 20 primary amines (each monomer comprising 1 N-terminal amine and 4 lysines).

[0315] In some embodiments, the streptavidin mutein exhibits an equilibrium dissociation constant (K D ) binding affinity, the K D For the peptide ligand (Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; also known as as shown in SEQ ID NO: 81), is equal to or less than or less than about 3.7 x 10 -5 M, and / or for peptide ligands (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys; also known as II, as shown in SEQ ID NO: 75) is equal to or less than or about 7.1 x 10 -5 M, and / or equal to or less than or about 7.0 x 10 -5 M, 5.0x 10 -5 M, 1.0x 10 -5 M, 5.0x10 -6 M, 1.0x 10 -6 M, 5.0x 10 -7 M or 1.0x 10 -7 M, but usually larger or greater than about 1x 10 -13 M, 1x 10 -12 M or 1x10 -11 M.

[0316] In some embodiments, the resulting streptavidin mutein exhibits an equilibrium association constant (K A ) binding affinity, the K A For the peptide ligand (Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; also known as as shown in SEQ ID NO: 81), is equal to or greater than or greater than about 2.7 x 10 4 M -1 , and / or for peptide ligands (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys; also known as II, as shown in SEQ ID NO: 75) is equal to or greater than or greater than about 1.4 x 10 4 M -1 , and / or equal to or greater than or about 1.43 x 10 4 M -1 , 1.67x 10 4 M -1 , 2x 10 4 M -1 , 3.33x 10 4 M -1 , 5x 104 M -1 , 1x 10 5 M -1 , 1.11x 10 5 M -1 , 1.25x 10 5 M -1 , 1.43x 10 5 M -1 , 1.67x10 5 M -1 , 2x 10 5 M -1 , 3.33x 10 5 M -1 , 5x 10 5 M -1 , 1x 10 6 M -1 , 1.11x 10 6 M -1 , 1.25x10 6 M -1 , 1.43x10 6 M -1 , 1.67x 10 6 M -1 , 2x 10 6 M -1 , 3.33x 10 6 M -1 , 5x 10 6 M -1 , 1x 10 7 M -1 , but usually less than 1x 10 13 M -1 , 1x 10 12 M -1 or 1x 10 11 M -1 .

[0317] In certain embodiments, provided herein are oligomeric particle reagents consisting of and / or containing a variety of streptavidin or streptavidin mutant protein tetramers. In certain embodiments, provided herein are oligomeric particle reagents containing a plurality of binding sites that reversibly bind or can reversibly bind to one or more agents (e.g., stimulants and / or selectors). In some embodiments, the oligomeric particles have a radius (e.g., average or mean radius) of between 70 nm and 125 nm (including end values); a radius of between 10 nm and 25 nm; ... 7 g / mol is about 1x 10 9In some embodiments, the oligomeric particle reagent is bound (e.g., reversibly bound) to one or more agents, such as agents that bind to molecules (e.g., receptors) on the cell surface. In certain embodiments, the one or more agents are or comprise an antibody or antigen-binding fragment thereof, such as a Fab. In some embodiments, the one or more agents specifically bind to one or more of the following macromolecules on a cell (e.g., a T cell): CD2, CD3, CD4, CD5, CD8, CD25, CD27, CD28, CD29, CD31, CD44, CD45RA, CD45RO, CD54 (ICAM-1), CD127, MHC I, MHC II, CTLA-4, ICOS, PD-1, OX40, CD27L (CD70), 4-1BB (CD137), 4-1BBL, CD30L, LIGHT, IL-2R, IL-12R, IL-1R, IL-15R; IFN-γR, TNF-αR, IL-4R, IL-10R, CD18 / CD11a (LFA-1), CD62L (L-selectin), CD29 / CD49d (VLA-4), Notch ligands (e.g., Delta-like ligand 1 / 4, Jagged 1 / 2, etc.), CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, and CXCR3 or fragments thereof, including the corresponding ligands or fragments thereof of these macromolecules. In some embodiments, the one or more agents specifically bind to one or more of the following macromolecules on cells (e.g., T cells): CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA, and / or CD45RO. In some embodiments, the one or more agents comprise antibodies or antigen-binding fragments thereof, such as Fab, and the antibodies may include polyclonal antibodies, monoclonal antibodies (including full-length antibodies with immunoglobulin Fc regions), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies, double antibodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab')2, and Fv). In some embodiments, the one or more agents are or comprise antibody fragments (including antigen-binding fragments), such as Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragments. It will be understood that constant regions of any isotype may be used in the antibodies contemplated herein, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions may be obtained from any human or animal species (e.g., murine species).In some embodiments, the one or more agents are or comprise antibodies that bind to and / or recognize one or more components of a T cell receptor. In specific embodiments, the one or more agents are or comprise anti-CD3 antibodies. In certain embodiments, the one or more agents are or comprise antibodies that bind to and / or recognize a co-receptor. In some embodiments, the one or more agents are or comprise anti-CD28 antibodies. In some embodiments, the one or more agents are or comprise anti-CD3 and / or anti-CD28 antibodies or antigen-binding fragments thereof, e.g., containing a binding partner (e.g., a streptavidin binding peptide, e.g., II) or an antigenic fragment thereof. In certain embodiments, the one or more agents are or comprise a peptide containing a binding partner (e.g., a streptavidin binding peptide, e.g., II) anti-CD3 and / or anti-CD28 Fab.

[0318] In some embodiments, provided herein are oligomeric particle reagents consisting of and / or containing a plurality of streptavidin or streptavidin mutant protein tetramers. In certain embodiments, provided herein are oligomeric particle reagents containing a plurality of binding sites that reversibly bind or can reversibly bind to one or more agents (e.g., stimulants and / or selection agents). In some embodiments, the oligomeric particles have a radius (e.g., average radius) of between 80 nm or about 120 nm (including end values); a radius of between 7.5 x 10 6 g / mol is about 2x 10 8 g / mol (inclusive); and / or a tetramer of streptavidin or a streptavidin mutein in an amount (e.g., an average amount) between at or about 500 and at or about 10,000, inclusive. In some embodiments, the oligomeric particle reagent is bound (e.g., reversibly bound) to one or more agents, e.g., agents that bind to molecules (e.g., receptors) on the surface of a cell. In some embodiments, the agent is an anti-CD3 and / or anti-CD28 Fab, e.g., containing a binding partner (e.g., a streptavidin binding peptide (e.g., II)). In certain embodiments, the one or more agents are Fabs containing a binding partner (e.g., a streptavidin binding peptide, e.g., II) anti-CD3 and / or anti-CD28 Fab.

[0319] In some embodiments, every 10 6In some embodiments, cells are stimulated in the presence of at least about 0.01 μg, 0.02 μg, 0.03 μg, 0.04 μg, 0.05 μg, 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.75 μg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, or 10 μg of an oligomerization stimulating agent per 10 cells. 6 In certain embodiments, cells are stimulated in the presence of 4 μg or about 4 μg per 10 cells. 6 In some aspects, 4 μg of oligomeric stimulatory agent is or includes at or about 3 μg of oligomeric particles and at or about 1 μg of an attached agent, such as at or about 0.5 μg of anti-CD3 Fab and at or about 0.5 μg of anti-CD28 Fab.

[0320] 2. Removal of stimulating agents from cells

[0321] In some embodiments, before collecting, gathering in the crops or preparing cells, stimulating reagent is removed or separated from cell or cell mass. In some embodiments, after or during hatching (for example, the hatching described in this article (as in chapter ID)), stimulating reagent is removed or separated from cell or cell mass. In certain embodiments, cell or cell mass are after hatching but before the step of collecting, gathering in the crops or preparing cells, experience the process, program, step or technology of removing stimulating reagent. In specific embodiments, cell or cell mass are after hatching, experience the process, program, step or technology of removing stimulating reagent. In some aspects, when during incubation, from cell separation or removal of stimulating reagent, for the remaining duration of hatching, cell is returned to the incubation conditions identical with separation or removal before.

[0322] In certain embodiments, the stimulating agent is removed and / or separated from the cell. In specific embodiments, in some cases, during incubation, the binding and / or association between the stimulating agent and the cell may decrease over time. In certain embodiments, one or more agents may be added to reduce the binding and / or association between the stimulating agent and the cell. In specific embodiments, changes in cell culture conditions (such as addition of agents and / or changes in culture medium temperature and / or pH) may reduce the binding and / or association between the stimulating agent and the cell. Therefore, in some embodiments, the stimulating agent can be removed from incubation, cell culture system and / or solution separately from the cell, for example, without removing the cell from incubation, cell culture system and / or solution at the same time.

[0323] In certain embodiments, after a certain amount of time, the stimulating agent is separated and / or removed from the cell. In specific embodiments, the amount of time is the amount of time from the start of stimulation. In specific embodiments, the start of hatching is considered to be at or about when the cell contacts the stimulating agent and / or the culture medium or solution containing the stimulating agent. In specific embodiments, the stimulation starts for or about 120 hours, 108 hours, 96 hours, 84 hours, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours or 12 hours (comprising end values), remove or separate the stimulating agent from the cell. In specific embodiments, after stimulation starts or after stimulation for approximately 48 hours, the stimulating agent is removed or separated from the cell. In certain embodiments, after stimulation starts or after stimulation for approximately 72 hours, the stimulating agent is removed or separated from the cell. In some embodiments, after stimulation starts or after stimulation for approximately 96 hours, the stimulating agent is removed or separated from the cell.

[0324] Methods for removing stimulating agents (e.g., stimulating agents that are or contain particles such as bead particles or magnetizable particles) from cells are known. In certain embodiments, bead stimulating agents, such as paramagnetic beads conjugated to anti-CD3 / anti-CD28 antibodies, are separated or removed from cells or cell populations. In some embodiments, a competing antibody (e.g., an unlabeled antibody) can be used, which, for example, binds to the primary antibody of the stimulating agent and changes its affinity for its antigen on the cell, thereby allowing gentle detachment. In some cases, after detachment, the competing antibody can remain associated with the particle (e.g., bead particle), and the unreacted antibody is washed away or can be washed away, and the cell does not contain antibodies for separation, selection, enrichment, and / or activation. An example of such a reagent is DETACaBEAD (Friedl et al. 1995; Entschladen et al. 1997). In some embodiments, particles (e.g., bead particles) can be removed in the presence of a cleavable linker (e.g., a DNA linker), thereby conjugating the particle-bound antibody to a linker (e.g., CELLection, Dynal). In some cases, the linker region provides a cleavable site to allow removal of the particle (e.g., bead particle) from the cell after isolation, for example, by addition of DNase or other release buffer. In some embodiments, other enzymatic methods can also be used to release the particle (e.g., bead particle) from the cell. In some embodiments, the particle (e.g., bead particle or magnetizable particle) is biodegradable.

[0325] In some embodiments, the stimulating agent is magnetic, paramagnetic and / or superparamagnetic, and / or contains beads that are magnetic, paramagnetic and / or superparamagnetic, and the stimulating agent can be removed from the cells by exposing the cells to a magnetic field. Examples of suitable devices containing magnets for generating a magnetic field include the DynaMag CTS (Thermo Fisher), the Magnetic Separator (Takara), and the EasySep magnet (Stem Cell Technologies).

[0326] In a specific embodiment, before completing the method provided, such as before harvesting, collecting and / or preparing the engineered cells produced by the method provided herein, stimulating agents are removed or separated from the cell. In some embodiments, after engineered (such as transduction or transfection) cells, stimulating agents are removed and / or separated from the cell. In certain embodiments, after cultivating cells, such as before cultivating engineered (such as transfection or transduction) cells under conditions that promote proliferation and / or amplification, stimulating agents are removed. In a specific embodiment, after cultivating cells, cells realize threshold value number, density and / or amplification, stimulating agents are removed. In some embodiments, before preparing cells, such as before forming the cells cultivated, such as before the cultivation cells having realized threshold value number, concentration or amplification, stimulating agents are removed.

[0327] In some embodiments, before collecting, gathering in the crops or preparing cells, stimulating bead reagents (such as stimulating magnetic bead reagents) are removed or separated from cells or cell groups. In some embodiments, by being exposed to a magnetic field during or after incubation (such as, the incubation described in chapter ID herein), stimulating bead reagents such as stimulating magnetic bead reagents are removed or separated from cells or cell groups. In certain embodiments, after incubation but before the step of collecting, gathering in the crops or preparing cells, cells or cell groups are exposed to a magnetic field to remove stimulating bead reagents, such as stimulating magnetic bead reagents. In specific embodiments, after incubation, cells or cell groups are exposed to a magnetic field to remove stimulating bead reagents, such as stimulating magnetic bead reagents. In some aspects, when stimulating bead reagents are separated or removed from cells or cell groups during incubation, for the remaining duration of incubation, cells or cell groups are returned to the same incubation conditions as before being exposed to the magnetic field.

[0328] In certain embodiments, the stimulating bead reagent (e.g., a stimulating magnetic bead reagent) is removed or separated from the cells within at or about 120 hours, 108 hours, 96 hours, 84 hours, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, or 12 hours (inclusive) of the start of stimulation, for example, by exposure to a magnetic field. In certain embodiments, the stimulating bead reagent (e.g., a stimulating magnetic bead reagent) is removed or separated from the cells at or about 72 hours after the start of stimulation, for example, by exposure to a magnetic field. In some embodiments, the stimulating bead reagent (e.g., a stimulating magnetic bead reagent) is removed or separated from the cells at or about 96 hours after the start of stimulation, for example, by exposure to a magnetic field.

[0329] In certain embodiments, after a certain amount of time, the stimulating agent is separated and / or removed from the cell. In a specific embodiment, the amount of time is the amount of time from the start and / or start of incubation under stimulating conditions. In a specific embodiment, the start of incubation is considered to be at or about when the cell contacts the stimulating agent and / or the culture medium or solution containing the stimulating agent. In a specific embodiment, the stimulating agent is removed or separated from the cell within or about 28 days, 21 days, 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days and 9 days after the incubation is started or started. In some embodiments, the stimulating agent is removed or separated from the cell within or about 28 days, 21 days, 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days after CD4+T cells and CD8+T cells are merged, combined and / or mixed into the input composition. In certain embodiments, the stimulatory agent is removed or separated from the cells within or about 28 days, 21 days, 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, or 9 days after obtaining, isolating, enriching, and / or selecting CD4+ T cells and CD8+ T cells from the biological sample.

[0330] In some embodiments, removing a stimulating agent (such as an oligomeric stimulating agent as described) comprises adding a substance, such as a competitor, to the incubated T cell population to disrupt (such as reduce and / or terminate) signaling of one or more stimulating agents. In some embodiments, the incubated T cell population contains the presence of a substance, such as a competitor, such as biotin or a biotin analog (such as D-biotin). In some embodiments, the substance (such as a competitor, such as biotin or a biotin analog (such as D-biotin) is present in an amount that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 100 times, at least 1000 times, or more than the amount of the substance in a reference population or in a cultured T cell preparation to which the substance is not exogenously added during incubation. In some embodiments, the substance (such as a competitor) in the cultured T cell population is present in an amount that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 100 times, at least 1000 times, or more than the amount of the substance in the reference population or in a cultured T cell preparation to which the substance is not exogenously added during incubation. In some embodiments, the amount of the oligomerization stimulating agent, such as biotin or a biotin analog (e.g., D-biotin), is from at or about 10 μM to at or about 100 μM, at or about 100 μM to at or about 1 mM, at or about 100 μM to at or about 500 μM, or at or about 10 μM to at or about 100 μM. In some embodiments, 10 μM or about 10 μM of biotin or a biotin analog (e.g., D-biotin) is added to the cell or cell population to separate or remove the oligomerization stimulating agent from the cell or cell population.

[0331] In certain embodiments, the one or more agents (such as agents that stimulate or activate TCR and / or co-receptors) are, for example, associated (such as reversibly bound) with oligomeric agents via multiple specific binding sites (such as binding site Z) present on oligomeric agents. In some cases, this causes the agents to be arranged closely to each other, so that if a target cell with a cell surface molecule (at least two copies) bound or identified by the agent is contacted with the agent, an avidity effect can occur. In some aspects, receptor binding agents have low affinity for the receptor molecules of cells at binding site B, so that receptor binding agents dissociate with cells in the presence of a competitive agent. Therefore, in some embodiments, the agent is removed from the cell in the presence of a competitive agent.

[0332] In some embodiments, oligomeric stimulating reagents are streptavidin mutein oligomers with reversibly attached anti-CD3 and anti-CD28 Fab. In some embodiments, the attached Fab contains a streptavidin binding domain, for example, which allows reversible attachment to a streptavidin mutein oligomer. In some cases, anti-CD3 and anti-CD28 Fab are closely arranged to each other so that if T cells expressing CD3 and / or CD28 are contacted with oligomeric stimulating reagents with reversibly attached Fab, avidity effects may occur. In some aspects, Fab has low affinity for CD3 and CD28, so that Fab is dissociated from cells in the presence of a competitive reagent (such as biotin or biotin variants or analogs). Therefore, in some embodiments, Fab is removed or dissociated from cells in the presence of a competitive reagent (such as D-biotin).

[0333] In some embodiments, the stimulatory oligomeric agent (e.g., a stimulatory oligomeric streptavidin mutein agent) is removed or separated from the cells or cell population prior to collection, harvesting, or formulation of the cells. In some embodiments, the stimulatory oligomeric agent (e.g., a stimulatory oligomeric streptavidin mutein agent) is removed or separated from the cells or cell population after or during incubation, e.g., an incubation described herein (e.g., in Section ID) by contacting or exposing to a competing agent (e.g., biotin or a biotin analog (e.g., D-biotin)). In certain embodiments, the cells or cell population are contacted or exposed to a competing agent (e.g., biotin or a biotin analog (e.g., D-biotin)) after incubation but prior to the step for collection, harvesting, or formulation of the cells to remove the stimulatory oligomeric agent, e.g., a stimulatory oligomeric streptavidin mutein agent. In certain embodiments, following incubation, the cells or cell populations are contacted with or exposed to a competing agent (e.g., biotin or a biotin analogue such as D-biotin) to remove the stimulatory oligomeric agent (e.g., a stimulatory oligomeric streptavidin mutein agent). In some aspects, when the stimulatory oligomeric agent (e.g., a stimulatory oligomeric streptavidin mutein agent) is separated or removed from the cells during incubation, e.g., by contacting with or exposing to a competing agent (e.g., biotin or a biotin analogue such as D-biotin), the cells are returned to the same incubation conditions as before separation or removal for the remainder of the incubation.

[0334] In some embodiments, the cells are contacted with a competing agent at, about, or at least about 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 100 μM, 500 μM, 0.01 μM, 1 mM, or 10 mM to remove or isolate the oligomerization stimulating agent from the cells. In various embodiments, the cells are contacted with at least abou...

Claims

1. A method for producing an engineered cell composition, the method comprising: (a) combining a CD4+ T cell composition and a CD8+ T cell composition at a ratio of CD4+ T cells to CD8+ T cells between 2:1 and 1:2, thereby producing an input composition; (b) incubating the input composition under stimulatory conditions, thereby producing a stimulatory composition, wherein the stimulatory conditions include the presence of a stimulatory agent that is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules; and wherein the input composition comprises a concentration of less than 5 x 10 6 At least 100x10 cells / mL 6 Total CD4+ and CD8+ T cells.

2. The method of claim 1, wherein the CD4+ and CD8+ cells in the input composition are enriched or selected from a primary sample from a subject, optionally wherein the CD4+ and CD8+ cells in the input composition are separately enriched or selected from a primary sample from a subject.

3. The method of claim 1 or claim 2, wherein the CD4+ T cell composition comprises at least 80%, at least 85%, at least 90% or at least 95% CD4+ T cells.

4. The method of any one of claims 1-3, wherein the CD8+ T cell composition comprises at least 80%, at least 85%, at least 90% or at least 95% CD8+ T cells.

5. A method for producing an engineered cell composition, the method comprising incubating an input composition under stimulating conditions, thereby producing a stimulating composition, wherein: The input composition comprises CD4+ and CD8+ T cells at a ratio between 2:1 and 1:2, and wherein the input composition comprises a concentration of less than 5 x 10 6 At least 100x10 cells / mL 6 Total CD4+ and CD8+ T cells; and The stimulatory conditions include the presence of a stimulatory agent that is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules.

6. The method according to any one of claims 1 to 5, wherein the incubation is performed in a serum-free medium.

7. The method of any one of claims 1-6, wherein the input composition comprises at least 80%, at least 85%, at least 90% or at least 95% of the cells that are CD4+ T cells or CD8+ T cells.

8. The method according to any one of claims 1 to 7, wherein the input composition comprises 100 x 10 6 500x 10 6 Total CD4+ and CD8+ T cells between .

9. The method of any one of claims 1 to 8, wherein the input composition comprises at or about 300 x 10 6 Total CD4+ and CD8+ T cells.

10. The method of claim 9, wherein the total CD4+ and CD8+ T cells are viable cells.

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