Method for producing a composition of manipulated T cells

Abstract

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 provided methods include pooling enriched CD4+ and CD8+ cells, such as in a 1:1 ratio, and then one or more steps for incubating the cells under stimulatory conditions, introducing a recombinant polypeptide into the cells through transduction or transfection, and / or culturing the cells under conditions that promote proliferation and / or expansion. In some aspects, the provided methods are efficient and reliable means for generating genetically engineered T cells with a high degree of success.

Description

[Technical Field]

[0001] Cross-reference of related applications This application is a U.S. Provisional Application No. 62 / 596,774 filed on December 8, 2017, titled "PROCESS FOR PRODUCING A COMPOSITION OF ENGINEERED T CELLS"; a U.S. Provisional Application No. 62 / 614,965 filed on January 8, 2018, titled "PROCESS FOR PRODUCING A COMPOSITION OF ENGINEERED T CELLS"; a U.S. Provisional Application No. 62 / 716,971 filed on August 9, 2018, titled "PROCESS FOR PRODUCING A COMPOSITION OF ENGINEERED T CELLS"; a U.S. Provisional Application No. 62 / 721,604 filed on August 22, 2018, titled "PROCESS FOR PRODUCING A COMPOSITION OF ENGINEERED T CELLS"; and a U.S. Provisional Application No. 62 / 721,604 filed on October 3, 2018, titled "PROCESS FOR PRODUCING A This invention claims priority to U.S. Provisional Application No. 62 / 740,903, titled “COMPOSITION OF ENGINEERED T CELLS”; U.S. Provisional Application No. 62 / 754,564, filed November 1, 2018, titled “PROCESS FOR PRODUCING A COMPOSITION OF ENGINEERED T CELLS”; U.S. Provisional Application No. 62 / 774,165, filed November 30, 2018, titled “PROCESS FOR PRODUCING A COMPOSITION OF ENGINEERED T CELLS”; and U.S. Provisional Application No. 62 / 774,855, filed December 3, 2018, titled “PROCESS FOR PRODUCING A COMPOSITION OF ENGINEERED T CELLS,” incorporating their entire contents by reference for any purpose.

[0002] Inclusion by referencing the sequence list This application is filed together with an electronic sequence listing. The sequence listing is provided as a file titled 735042014340SeqList.txt, created on December 7, 2018, and its size is 68 kilobytes. The electronic information of the sequence listing is incorporated in its entirety by reference.

[0003] field This disclosure provides a method for genetically engineering T cells, such as CD4+ T cells and / or CD8+ T cells, for use in cell therapy. In some aspects, the method provided comprises one or more steps for pooling enriched CD4+ and CD8+ cells in a 1:1 ratio, etc., then incubating the cells under stimulating conditions, introducing recombinant polypeptides into the cells through transduction or transfection, and / or culturing the cells under conditions that promote proliferation and / or expansion. In some aspects, the method provided is an efficient and reliable means for producing genetically engineered T cells with a high degree of success. [Background technology]

[0004] background Various cell therapy methods can be used to treat diseases and symptoms. Cell therapy involves immune cells, such as T cells, that have been genetically modified using recombinant receptors, such as chimeric antigen receptors. There is a need for improved methods for manufacturing and / or manipulating such cell therapies, including providing more efficient processes and / or improved cell composition products. [Overview of the Initiative]

[0005] overview In some embodiments, provided herein are methods for producing a composition of engineered cells, the method comprising: (a) combining a CD4+ T cell composition and a CD8+ T cell composition in a CD4+ T cell:CD8+ T cell ratio of 2:1 to 1:2 to generate an input composition; and (b) incubating the input composition under stimulating conditions to generate a stimulated composition, the stimulating conditions comprising the presence of a stimulating reagent 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 co-stimulatory molecules, the input composition being 5 × 10 6 At concentrations of less than 100 × 10⁻¹⁰ cells / ml 6 It contains a total of 100 CD4+ T cells and CD8+ T cells.

[0006] In some embodiments, provided herein are methods for producing a composition of manipulated cells, the method comprising the step of incubating an input composition under stimulating conditions to thereby produce a stimulated composition, wherein the input composition has a CD4+ T cell:CD8+ T cell ratio of 2:1 to 1:2, and the input composition has 5 × 10 6 At concentrations of less than 100 × 10⁻¹⁰ cells / ml 6 The system comprises a total of 100 CD4+ T cells and CD8+ T cells, and the stimulation condition includes the presence of a stimulating reagent 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.

[0007] In some embodiments, provided herein are methods for producing a composition of engineered cells, the method comprising the step of introducing recombinant receptors into the cells of the T cell composition, the T cell composition having at least 1 × 10 per 1 mL 6 individual or at least about 1 × 10 6having a concentration of viable cells, wherein at least 80%, at least 85%, at least 90% or at least 95% of the cells of the T cell composition are CD4+ T cells or CD8+ T cells.

[0008] 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 cells that are CD4+ T cells or CD8+ T cells. In certain embodiments, the input composition comprises 100×10 6 ~500×10 6 total CD4+ T cells and CD8+ T cells. In certain embodiments, the input composition comprises 300×10 6 or about 300×10 6 total CD4+ T cells and CD8+ T cells. In certain embodiments, the total CD4+ T cells and CD8+ T cells are viable cells. In certain embodiments, the input composition has a concentration of 1×10 6 cells / mL to 5×10 6 cells / mL. In certain embodiments, the input composition has a concentration of 3×10 6 cells / mL or about 3×10 6 cells / mL. In certain embodiments, the input composition has a CD4+ cell:CD8+ cell ratio of 1.5:1 to 1:1.5. In certain embodiments, the input composition has a CD4+ cell:CD8+ cell ratio of 1.2:1 to 0.8:1.

[0009] In certain embodiments, the input composition has a CD4+ cell:CD8+ cell ratio of 1:1 or about 1:1. In certain embodiments, the input composition comprises CD4+ and CD8+ that are surface positive for CD45RA and CCR7.

[0010] 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 1.1:1 or about 1.1:1.

[0011] In certain embodiments, the input composition comprises CD4+ and CD8+ cells that are surface-positive for CD27 and CCR7.

[0012] In certain embodiments, the ratio of CD4+ cells surface-positive for CD27 and CCR7 to CD8+ cells surface-positive for CD27 and CCR7 is 1.69:1 or approximately 1.69:1.

[0013] In certain embodiments, the input composition contains CD4+ cells and CD8+ cells that are surface-positive for CCR7 and surface-negative for CD62L, in an optional ratio of 2.0:1 to 1.5:1.

[0014] In some embodiments, the process further includes introducing recombinant receptors into cells from a stimulated composition to produce an engineered cell composition, wherein the introduction includes contacting cells from the stimulated composition with an active substance containing a polynucleotide encoding the recombinant receptor. In some embodiments, the process further includes introducing recombinant receptors into cells from a stimulated composition to produce an engineered cell composition, wherein the introduction includes transducing cells from the stimulated composition with a viral vector containing a polynucleotide encoding the recombinant receptor. In some embodiments, the introduction is carried out in serum-free medium.

[0015] In certain embodiments, the foregoing provides a method for producing a composition of engineered cells, the method comprising: (a) combining a CD4+ T cell composition and a CD8+ T cell composition in a CD4+ T cell:CD8+ T cell ratio of 2:1 to 1:2 to produce an input composition; and (b) incubating the input composition under stimulating conditions to produce a stimulated composition, the stimulating conditions comprising the presence of a stimulating reagent 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 co-stimulatory molecules, the input composition being 5 × 106 At concentrations of less than 100 × 10⁻¹⁰ cells / mL 6 It contains a total of 100 CD4+ T cells and CD8+ T cells.

[0016] In some aspects of the methods provided, the CD4+ cells and CD8+ cells in the input composition are enriched or selected from a primary sample obtained from the subject, and optionally, the CD4+ cells and CD8+ cells in the input composition are enriched or selected separately from a primary sample obtained from the subject. In certain aspects of the methods provided, the CD4+ T cell composition comprises at least 80%, at least 85%, at least 90%, or at least 95% CD4+ T cells. In certain aspects of the methods provided, the CD8+ T cell composition comprises at least 80%, at least 85%, at least 90%, or at least 95% CD8+ T cells.

[0017] In some embodiments, provided herein are methods for producing a composition of manipulated cells, the method comprising the step of incubating an input composition under stimulating conditions to thereby produce a stimulated composition, wherein the input composition has a CD4+ T cell:CD8+ T cell ratio of 2:1 to 1:2, and the input composition has 5 × 10 6 At concentrations of less than 100 × 10⁻¹⁰ cells / mL 6 The system comprises a total of 100 CD4+ T cells and CD8+ T cells, and the stimulation condition includes the presence of a stimulating reagent 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.

[0018] In any particular aspect of the method provided, incubation is carried out in serum-free medium. In any particular aspect of the method provided, the input composition comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells which are CD4+ T cells or CD8+ T cells. In any particular aspect of the method provided, the input composition comprises 100 × 106 ~500×10 6 The input composition contains a total of 300 × 10⁶ CD4+ T cells and CD8+ T cells. In any particular aspect of the method provided, the input composition contains 300 × 10⁶ CD4+ T cells. 6 1 or approximately 300 x 10 6 It contains a total of 100 CD4+ T cells and CD8+ T cells.

[0019] In some aspects of the method provided, the total CD4+ T cells and CD8+ T cells are viable cells. In some particular aspects of the method provided, the input composition is 1 × 10⁻⁶ 6 cells / mL~5×10 6 It has a concentration of cells / mL. In any particular aspect of the method provided, the input composition is 3 × 10 6 cells / mL or approximately 3 × 10⁶ 6 It has a concentration of cells / mL. In any particular aspect of the method provided, the input composition has a CD4+ cell:CD8+ cell ratio of 1.5:1 to 1:1.5. In any part of the method provided, the input composition has a CD4+ cell:CD8+ cell ratio of 1.2:1 to 0.8:1.

[0020] In any particular aspect of the method provided, the input composition has a CD4+ cell:CD8+ cell ratio of 1:1 or about 1:1. In any particular aspect of the method provided, the input composition comprises CD4+ and CD8+ cells that are surface-positive for CD45RA and CCR7. In some aspects of the method provided, 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 1.1:1 or about 1.1:1. In any particular aspect of the method provided, the input composition comprises CD4+ and CD8+ cells that are surface-positive for CD27 and CCR7. In any particular aspect of the method provided, 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 1.69:1 or about 1.69:1. In some aspect of the method provided, the input composition comprises CD4+ cells and CD8+ cells that are surface-positive for CCR7 and surface-negative for CD62L, in an optional ratio of 2.0:1 to 1.5:1.

[0021] In any particular aspect of the method provided, the method further comprises the step of introducing recombinant receptors into cells from a stimulated composition to produce an engineered cell composition, the introduction comprising the step of contacting cells from the stimulated composition with an active substance comprising a polynucleotide encoding the recombinant receptor.

[0022] In any particular aspect of the method provided, contact is contact by transfection using a vector, where the vector is a transposon, optionally a sleeping beauty (SB) transposon or a piggyback transposon, or contact is contact by transduction using a viral vector.

[0023] In some aspects of the methods provided, the method further comprises the step of introducing recombinant receptors into cells from a stimulated composition to produce an engineered cell composition, the introduction comprising the step of transducing a viral vector containing a polynucleotide encoding the recombinant receptor into cells from the stimulated composition. In certain aspects of the methods provided, the introduction is carried out in serum-free medium.

[0024] In any particular aspect of the method provided, the introduction of the stimulated composition is 300 × 10 6 Contains fewer than 10 cells. In any aspect of the method provided, for introduction, the stimulated composition is 50 × 10 6 Individual cells ~200 × 10 6 Contains individual cells. In any particular aspect of the method provided, for introduction, the stimulated composition is 100 × 10 6 1 or approximately 100 x 10 6 Contains individual cells. In any particular aspect of the method provided, for introduction, the stimulated composition is 3 × 10 6 It has a concentration of less than 0.5 × 10⁻¹⁶ cells / mL. In any of the embodiments of the method provided, for introduction, the stimulated composition is 0.5 × 10⁻¹⁶ 6 cells / mL~2×10 6 It has a concentration of cells / mL. In any particular aspect of the method provided, for introduction, the stimulated composition is 1 × 10 6 cells / mL or approximately 1 × 10⁶ 6 It has a concentration of cells / mL.

[0025] A particular aspect of any of the methods provided includes a step of preparing the composition of the stimulated composition after incubation under stimulating conditions, before introducing recombinant receptors into the cells of the stimulated composition. In some aspects of any of the methods provided, the cells of the stimulated composition are viable cells. In a particular aspect, provided herein is a method for producing a composition of engineered cells, the method comprising the step of introducing recombinant receptors into the cells of the T cell composition, the T cell composition having at least 1 × 10 per mL 6individual or at least about 1 × 10 6 The T cell composition has a concentration of viable cells, and 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.

[0026] In any particular aspect of the method provided, the concentration of the T cell composition is 5 × 10 per mL. 6 The number of viable cells is less than 100 × 10⁶. In some aspect of any of the methods provided, the T cell composition contains at least 100 × 10⁶ cells. 6 individual or at least about 100 x 10 6 1 or approximately 100 x 10 6 Contains 100 surviving cells. In any particular aspect of the method provided, the T cell composition contains 300 × 10 6 Includes fewer than 1 viable cell. In any particular aspect of the method provided, the introduction includes the step of contacting T cells by transducing a viral vector containing a polynucleotide encoding a recombinant receptor.

[0027] In some aspects of the method provided, the introduction is carried out in serum-free medium. In certain aspects of the method provided, one or more cells of the T cell composition are activated and / or have surface expression of the LDL receptor.

[0028] In any particular aspect of the method provided, 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 a surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L, and 4-1BB; (ii) include intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-γ, and TNF-α; (iii) be in a stage of the cell cycle G1 or later; and / or (iv) be able to proliferate.

[0029] In some aspects of the methods provided, the cells of the composition are generated by a process comprising the step of incubating an input composition containing CD4+ T cells and CD8+ T cells under stimulating conditions prior to introduction, wherein the stimulating conditions include the presence of a stimulating reagent that can activate 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 co-stimulatory molecules. In certain aspects of the methods provided, incubation was performed in serum-free medium.

[0030] In a particular embodiment, provided herein is a method for producing a composition of manipulated cells, the method comprising the steps of (a) incubating an input composition under stimulating conditions to thereby produce a stimulated composition, wherein the input composition has a CD4+ T cell:CD8+ T cell ratio of 2:1 to 1:2 and 5 x 10 6 At concentrations less than cells / mL, at least 100x10 6 (b) comprising 300 × 10¹ CD4+ T cells and CD8+ T cells, the stimulation condition comprising the presence of a stimulating reagent 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 (b) 300 × 10¹⁶ of the stimulated composition 6 The process includes introducing recombinant receptors into fewer than 100 cells, thereby generating an engineered cell composition, the introduction comprising contacting the cells of the stimulated composition with a viral vector containing a polynucleotide encoding the recombinant receptor.

[0031] In some aspects of the methods provided, incubation and / or introduction are performed in serum-free medium. In certain aspects of the methods provided, CD4+ T cells and CD8+ T cells are viable cells. In certain aspects of the methods provided, cells from the stimulated composition are viable cells.

[0032] In some aspects of the provided method, induction 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 aspects of the provided method, induction 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 certain aspects of the provided method, induction 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.

[0033] Some aspects of any of the methods provided further include the step of culturing the engineered composition under conditions that promote the proliferation and / or expansion of the engineered cells, thereby producing an output composition containing engineered T cells. In any particular aspect of any of the methods provided, the culturing is carried out in serum-free medium.

[0034] In a particular embodiment, provided herein is a method for producing a composition of manipulated cells, the method comprising the steps of (a) incubating an input composition under stimulating conditions to thereby produce a stimulated composition, wherein the input composition has a CD4+ T cell:CD8+ T cell ratio of 2:1 to 1:2, and the input composition is 5 × 10 6 At concentrations of less than 100 × 10⁻¹⁰ cells / mL 6 (b) comprising a total of 100 CD4+ T cells and CD8+ T cells, the stimulation condition comprising the presence of a stimulating reagent 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 (b) 300 × 10⁻¹⁶ from the stimulated composition 6(c) a step of introducing recombinant receptors into fewer than 10 cells, thereby producing an engineered cell composition, wherein the introduction includes transducing a viral vector containing a polynucleotide encoding recombinant receptors into cells of the stimulated composition, and (c) a step of culturing the engineered composition under conditions that promote the proliferation and / or expansion of the engineered cells, thereby producing an output composition containing engineered T cells.

[0035] In some aspects of the method provided, incubation, introduction and / or culture are carried out in serum-free medium.

[0036] In any particular aspect of the method provided, the input composition has a CD4+ cell:CD8+ cell ratio of 1.5:1 to 1:1.5, a CD4+ cell:CD8+ cell ratio of 1.2:1 to 0.8:1, and optionally a CD4+ T cell:CD8+ T cell ratio of 1:1 or about 1:1. In any particular aspect of the method provided, the input composition comprises CD4+ and CD8+ cells that are surface-positive for CD45RA and CCR7. In some aspects of the method provided, 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 1.1:1 or about 1.1:1. In any particular aspect of the method provided, the input composition comprises CD4+ and CD8+ cells that are surface-positive for CD27 and CCR7. In certain aspects of the method provided, the ratio of CD4+ cells surface-positive for CD27 and CCR7 to CD8+ cells surface-positive for CD27 and CCR7 is 1.69:1 or about 1.69:1. In some aspects of the method provided, the input composition comprises CD4+ cells and CD8+ cells surface-positive for CCR7 and surface-negative for CD62L.

[0037] In any particular aspect of the method provided, for introduction, the stimulated composition is 300 × 10 6Contains fewer than 10 cells. In any particular aspect of the method provided, for introduction, the stimulated composition is 50 × 10 6 Individual cells ~200 × 10 6 Individual cells, arbitrarily 100 x 10 6 1 or approximately 100 x 10 6 Contains individual cells. In any aspect of the method provided, for introduction, the stimulated composition is 3 × 10 6 It has a concentration of less than cells / mL. In any particular aspect of the method provided, for introduction, the stimulated composition is 0.5 × 10 6 cells / mL~2×10 6 cells / mL, optionally 1 × 10⁶ 6 cells / mL or approximately 1 × 10⁶ 6 It has a concentration of cells / mL. In any particular embodiment of the method provided, the method includes the step of adjusting the composition of the stimulated composition after incubation under stimulating conditions, before introducing recombinant receptors into the cells of the stimulated composition.

[0038] In any particular aspect of the method provided, incubation is carried out in the presence of one or more cytokines, for example, in serum-free medium. In any particular aspect of the method provided, one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and / or recombinant IL-15. In some aspects of the method provided, one or more cytokines include recombinant IL-2 at 10-200 IU / mL, recombinant IL-7 at 100 IU / mL-1,000 IU / mL, and / or recombinant IL-15 at 10-200 IU / mL. In any particular aspect of the method provided, one or more cytokines include recombinant IL-2 at 10-200 IU / mL, recombinant IL-7 at 100 IU / mL-1,000 IU / mL, and recombinant IL-15 at 10-200 IU / mL.

[0039] In any particular aspect of the method provided, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% of the cells of the stimulated composition (i) express a surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L, and 4-1BB; (ii) express intracellular cytokines selected from the group consisting of IL-2, IFN-γ, and TNF-α; (iii) be in a stage of the cell cycle G1 or later; and / or (iv) be able to proliferate.

[0040] In some aspects of the methods provided, the stimulating reagent comprises a main component that specifically binds to a member of the TCR complex, the main component optionally specifically binds to CD3. In certain aspects of the methods provided, the stimulating reagent further comprises an adjunct that specifically binds to a T cell costimulatory molecule, the costimulatory molecule optionally selected from CD28, CD137(4-1-BB), OX40, or ICOS. In certain aspects of the methods provided, the main component and / or adjunct comprises an antibody, the stimulating reagent optionally constitutes incubation with an anti-CD3 antibody and an anti-CD28 antibody or its antigen-binding fragment.

[0041] In some aspects of the methods provided, the main component and / or auxiliary component are present on the surface of a solid support. In certain aspects of the methods provided, the solid support is or contains beads. In certain aspects of the methods provided, the beads have a diameter greater than or about 3.5 μm but less than or equal to about 9 μm, less than or equal to about 8 μm, less than or equal to about 7 μm, less than or equal to about 6 μm, or less than or equal to about 5 μm. In some aspects of the methods provided, the beads have a diameter of 4.5 μm or about 4.5 μm. In certain aspects of the methods provided, the beads are inert. In certain aspects of the methods provided, the beads are a polystyrene surface or contain a polystyrene surface. In some aspects of the methods provided, the beads are magnetic or superparamagnetic.

[0042] In any particular aspect of the method provided, the bead:cell ratio is less than 3:1. In any particular aspect of the method provided, the bead:cell ratio is 2:1 to 0.5:1 or about 2:1 to about 0.5:1. In any part of the method provided, the bead:cell ratio is 1:1 or about 1:1.

[0043] In any particular aspect of the method provided, the input composition is incubated under stimulating conditions for less than 48 hours. In any particular aspect of the method provided, the input composition is incubated under stimulating conditions for 12 hours or more and 36 hours or less. In any several aspects of the method provided, the input composition is incubated under stimulating conditions for 18 hours or more and 30 hours or less. In any particular aspect of the method provided, the input composition is incubated under stimulating conditions for 24 hours or approximately 24 hours.

[0044] In any particular aspect of the method provided, contact, optionally transduction, takes place for less than 48 hours. In some aspects of the method provided, contact, optionally transduction, takes place for 12 hours or more but not exceeding 36 hours. In any particular aspect of the method provided, contact, optionally transduction, takes place for 18 hours or more but not exceeding 30 hours. In any particular aspect of the method provided, contact, optionally transduction, takes place for 24 hours or approximately 24 hours.

[0045] In some aspects of the methods provided, the viral vector is a retroviral vector. In certain aspects of the methods provided, the viral vector is a lentiviral vector or a gamma retroviral vector. In certain aspects of the methods provided, contact, optionally transduction, is carried out in the absence of a transduction adjuvant. In some aspects of the methods provided, transduction is carried out in the presence of one or more cytokines, for example, in serum-free medium. In certain aspects of the methods provided, one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and / or recombinant IL-15.

[0046] In any particular aspect of the method provided, one or more cytokines include recombinant IL-2 at 10-200 IU / mL, recombinant IL-7 at 100 IU / mL-1,000 IU / mL, and / or recombinant IL-15 at 10-200 IU / mL. In any part of the method provided, one or more cytokines include recombinant IL-2 at 10-200 IU / mL, recombinant IL-7 at 100 IU / mL-1,000 IU / mL, and recombinant IL-15 at 10-200 IU / mL.

[0047] In any particular aspect of the method provided, at least a portion of the culture is carried out using mixing and / or perfusion. In any particular aspect of the method provided, at least a portion of the culture is carried out using mixing and / or perfusion. The perfusion is carried out using perfusion rates of mL / day, approximately 1,600 mL / day, approximately 1,800 mL / day and / or approximately 2,000 mL / day, or at least 500 mL / day, at least 600 mL / day, at least 700 mL / day, at least 750 mL / day, at least 800 mL / day, at least 900 mL / day, at least 1,000 mL / day, at least 1,200 mL / day, at least 1,400 mL / day, at least 1,500 mL / day, at least 1,600 mL / day, at least 1,800 mL / day and / or at least 2,000 mL / day. In some aspect of the method provided, at least a first portion of the culture is carried out at a perfusion rate of 500 mL / day, 750 mL / day or 1,000 mL / day, about 500 mL / day, about 750 mL / day or about 1,000 mL / day, or at least 500 mL / day, at least 750 mL / day or at least 1,000 mL / day, and at least a second portion of the culture is carried out at a perfusion rate of 1,200 mL / day, 1,400 mL / day or 1,500 mL / day, about 1,200 mL / day, about 1,400 mL / day or about 1,500 mL / day, or at least 1,200 mL / day, at least 1,400 mL / day or at least 1,500 mL / day.

[0048] In any particular aspect of the method provided, perfusion is initiated and / or increased when the cells reach a certain density. In any particular aspect of the method provided, the certain density is 0.4 × 10⁻⁶ 6cells, 0.5×10 6 cells, 0.6×10 6 cells, 0.8×10 6 cells, 1.0×10 6 cells, 1.2×10 6 cells, 1.4x10 6 cells, 1.6x10 6 cells, 1.8x10 6 cells, 2.0x10 6 cells, 2.2x10 6 cells or 2.4x10 6 cells, approximately 0.4×10 6 cells, approximately 0.5×10 6 cells, approximately 0.6×10 6 cells, approximately 0.8×10 6 cells, approximately 1.0×10 6 cells, approximately 1.2×10 6 cells, approximately 1.4x10 6 cells, approximately 1.6x10 6 cells, approximately 1.8x10 6 cells, approximately 2.0x10 6 cells, approximately 2.2x10 6 cells or approximately 2.4x10 6 cells, or at least 0.4×10 6 cells, at least 0.5×10 6 cells, at least 0.6×10 6 cells, at least 0.8×10 6 cells, at least 1.0×10 6 cells, at least 1.2×10 6 cells, at least 1.4x10 6 cells, at least 1.6x10 6 cells, at least 1.8x10 6 cells, at least 2.0x10 6 cells, at least 2.2x10 6 cells or at least 2.4x10 6 cells. In some aspects of any of the provided methods, the cells are 0.6×10 6 cells / mL or approximately 0.6×106 When the density reaches cells / mL, perfusion is initiated and / or increased to a rate of 750 mL / day or about 750 mL / day. In certain embodiments of any of the provided methods, the cells are 2.0×10 6 cells / mL or about 2.0×10 6 When the density reaches cells / mL, perfusion is initiated and / or increased to a rate of 1500 mL / day or about 1500 mL / day.

[0049] In certain embodiments of any of the provided methods, the culture is performed in the presence of one or more cytokines, for example, in 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 certain embodiments of any of the provided methods, the one or more cytokines comprise recombinant IL-2 at 50 - 400 IU / mL, recombinant IL-7 at 100 IU / mL - 2,000 IU / mL, and / or recombinant IL-15 at 50 - 400 IU / mL. In certain embodiments of any of the provided methods, the culture is performed in serum-free medium, for example, in the presence of recombinant IL-2 at 50 - 400 IU / mL, recombinant IL-7 at 100 IU / mL - 2,000 IU / mL, and recombinant IL-15 at 50 - 400 IU / mL. In certain embodiments of any of the provided methods, the one or more cytokines comprise recombinant IL-2 at 50 - 400 IU / mL, recombinant IL-7 at 100 IU / mL - 1,000 IU / mL, and / or recombinant IL-15 at 10 - 200 IU / mL.

[0050] In some aspects of the provided method, culture 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 certain aspects of the provided method, culture 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 aspects of the provided method, culture 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.

[0051] In some aspects of the method provided, the culture is carried out until the composition contains at least a threshold number of T cells. In certain aspects of the method provided, the threshold number of T cells is 2400 × 10⁶ 6 pieces, approximately 2400×10 6 1 or at least 2400 × 10 6 These are individual cells. In any particular aspect of the method provided, the threshold number of T cells is 5500 × 10⁶ 6 pieces, approximately 5500×10 6 1 or at least 5500 × 10 6 It is an individual cell.

[0052] In any particular aspect of the method provided, the culture is continued for at least one day after the threshold number of T cells is reached. In any particular aspect of the method provided, the threshold number of T cells is 900 × 10⁶ 6 pieces, approximately 900×10 6 1 or at least 900 × 10 6 These are individual cells. In any particular aspect of the method provided, the threshold number of T cells is 1200 × 10⁶ 6 pieces, approximately 1200×10 6 individual or at least 1200 × 10 6 These are individual cells. In any particular aspect of the method provided, the culture is performed with a number of T cells of 2400 × 10⁶ 6 pieces, approximately 2400×106 1 or at least 2400 × 10 6 The process terminates when there are individual cells. In some aspect of the method provided, the threshold number of T cells is 3500 × 10⁶. 6 pieces, approximately 3500×10 6 1 or at least 3500 × 10 6 These are individual cells. In any particular aspect of the method provided, the culture is performed with a number of T cells of 5500 × 10⁶. 6 pieces, approximately 5500×10 6 1 or at least 5500 × 10 6 It ends when it is still a single cell.

[0053] Certain aspects of any of the methods provided include a step of collecting cells of the output composition after culture. Some aspects of any of the methods provided include a step of collecting cells of the output composition after culture, wherein the cells of the output composition are collected at least 9 days after the start of incubation under stimulating conditions. Certain aspects of any of the methods provided include a step of collecting cells of the output composition after culture, wherein the cells of the output composition are collected at least 10 days after the start of incubation under stimulating conditions.

[0054] Certain aspects of the methods provided include a 95% confidence interval for the period between the start of incubation and the collection of cells for the output composition, which is between 8 and 25 days. Certain aspects of the methods provided include a 95% confidence interval for the period between the start of incubation and the collection of cells for the output composition, which is between 9 and 21 days. Some aspects of the methods provided include a 95% confidence interval for the period between the start of incubation and the collection of cells for the output composition, which is between 9 and 16 days.

[0055] Certain aspects of any of the methods provided further include the step of formulating cells of the output composition for cryopreservation and / or administration to a subject, optionally in the presence of pharmaceutically acceptable excipients. In any of the certain aspects of any of the methods provided, the cells of the output composition are formulated in the presence of a cryoprotective substance. In some aspects of any of the methods provided, the cryoprotective substance includes DMSO. In any of the certain aspects of any of the methods provided, the cells of the output composition are formulated in a container, optionally in a vial or bag.

[0056] Certain aspects of any of the methods provided include a step of isolating CD4+ T cells and / or CD8+ T cells from a biological sample before incubation. In some aspects of any of the methods provided, isolation includes selecting cells, optionally by positive or negative selection, based on the surface expression of CD4 and / or CD8. In some aspects of any of the methods provided, isolation includes performing selection based on immunoaffinity. In some aspects of any of the methods provided, the biological sample includes primary T cells obtained from a subject. In some aspects of any of the methods provided, the subject is a human subject.

[0057] In any particular aspect of the method provided, the biological sample is or includes 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, or a leukocyte apheresis product. In any particular aspect of the method provided, the recombinant receptor can bind to a target antigen that is associated with, specific to, and / or expressed in cells or tissues of a disease, disorder, or symptom. In any particular aspect of the method provided, the disease, disorder, or symptom is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer.

[0058] In any particular aspect of the method provided, the target antigen is a tumor antigen. In any particular aspect of the method provided, the target antigens are 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 α, folate-binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, G250 / CAIX, GD2, GD3, gp100, Her2 / neu (receptor tyrosine kinase erbB2), HMW-MAA, IL-22R-α, IL-13 receptor α2 (IL-13Rα2), kinase insertion 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, mesoserine, Antigens are selected from mouse CMV, mucin 1 (MUC1), MUC16, NCAM, NKG2D, NKG2D ligand, NY-ESO-1, O-acetylated GD2 (OGD2), carcinoembryonic antigen, melanoma preferential expression antigen (PRAME), PSCA, progesterone receptor, Survivin, ROR1, TAG72, VEGF receptor, VEGF-R2, Wilms tumor 1 (WT-1), pathogen-specific antigens, and antigens associated with the universal tag.

[0059] In some aspect of the method provided, the recombinant receptor is or comprises a functional non-TCR antigen receptor or a TCR or its antigen-binding fragment.

[0060] In any particular aspect of the method provided, the recombinant receptor is a chimeric antigen receptor (CAR). In any particular aspect of the method provided, the recombinant receptor is an anti-BCMA CAR. In some aspects of the method provided, the chimeric antigen receptor includes an extracellular domain containing an antigen-binding domain. In any particular aspect of the method provided, the antigen-binding domain is or includes an antibody, or optionally an antibody fragment which is a single-chain fragment. In any particular aspect of the method provided, the fragment includes an antibody variable region bound by a flexible linker. In some aspects of the method provided, the fragment includes an scFv. In any particular aspect of the method provided, the chimeric antigen receptor further includes a spacer and / or hinge region.

[0061] In any particular aspect of the method provided, the chimeric antigen receptor includes an intracellular signaling region. In some aspects of the method provided, the intracellular signaling region includes an intracellular signaling domain. In any particular aspect of the method provided, the intracellular signaling domain is or includes 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-activated tyrosine motif (ITAM). In any particular aspect of the method provided, the intracellular signaling domain is or includes an intracellular signaling domain or signaling portion of a CD3 chain, optionally a CD3-zeta (CD3ζ) chain.

[0062] In some aspects of the methods provided, the chimeric antigen receptor further includes a transmembrane domain located between an extracellular domain and an intracellular signaling domain. In certain aspects of the methods provided, the intracellular signaling domain further includes a co-stimulatory signaling domain. In certain aspects of the methods provided, the co-stimulatory signaling domain includes the intracellular signaling domain or signaling moiety of a T cell co-stimulatory molecule. In some aspects of the methods provided, the co-stimulatory signaling domain includes the intracellular signaling domain or signaling moiety of CD28, 4-1BB, or ICOS.

[0063] In any particular aspect of the method provided, the co-stimulatory signaling region is located between the transmembrane domain and the intracellular signaling region. In any particular aspect of the method provided, an output composition containing a number of cells greater than or equal to a threshold number is produced between more than 85% or about 85%, more than 90% or about 90%, or more than 95% or about 95% of the repeats of the method.

[0064] In some aspects of the method provided, the serum-free medium contains 0.5 mM to 5 mM of L-glutamine in dipeptide form, 0.5 mM to 5 mM of L-glutamine, and at least one protein in the base medium, and the medium is serum-free. In certain aspects of the method provided, the L-glutamine dipeptide form is L-alanyl-L-glutamine.

[0065] In certain aspects of the methods provided, the concentration of the dipeptide form of L-glutamine in the serum-free medium is 2 mM or about 2 mM. In some aspects of the methods provided, the concentration of L-glutamine in the serum-free medium is 2 mM or about 2 mM. In certain aspects of the methods provided, at least one protein comprises one or more of albumin, insulin, or transferrin, optionally one or more of human or recombinant albumin, human or recombinant insulin, or human or recombinant transferrin.

[0066] In some embodiments of the methods provided herein, cells are monitored for cell viability, concentration, density, number, or a combination thereof for at least a portion of the culture. In some such embodiments, monitoring is performed by optical methods, optionally by microscopy. In some such embodiments, monitoring is performed by bright-field microscopy, fluorescence microscopy, differential interference contrast microscopy, phase-contrast microscopy, digital holography microscopy (DHM), differential digital holography microscopy (DDHM), or a combination thereof. In some such embodiments, monitoring is performed by differential digital holography microscopy (DDHM). In some such embodiments, monitoring is performed intermittently or continuously for at least a portion of the culture, optionally at least every hour, at least every 6 hours, at least every 12 hours, at least every 18 hours, at least every 24 hours, or at least every 26 hours during the culture. In some of these embodiments, monitoring is performed until the cells reach a threshold number of T cells, a threshold number of viable T cells, a threshold concentration of T cells, or a threshold concentration of viable T cells. In some of these embodiments, monitoring and culture are performed in a closed system.

[0067] In some 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 more than 95% of the cells in the output composition are memory phenotypes, and 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 more than 95% of the cells in the output composition are central memory phenotypes, and at least 30%, at least 4 0%, 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 more than 95% are CD27+, CD28+, CCR7+, CD45RA-, CD45RO+, CD62L+, CD3+, CD95+, granzyme B- and / or CD127+, and / or 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 more than 95% of the cells in the output composition are CCR7+ / CD45RA- or CCR7+ / CD45RO+.

[0068] In some embodiments of the methods provided herein, by iteration of the method, optionally, multiple output compositions are produced from human biological samples in which the method is performed between multiple different individual subjects, and the average percentage of memory phenotypic cells in the multiple output compositions is about 40% to about 65%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, or about 60% to about 65%, and the average percentage of central memory phenotypic cells in the multiple output compositions is about 40% to about 65%, about 40% to about 45%, about 45% to about The average percentage of cells that are CD27+, CD28+, CCR7+, CD45RA-, CD45RO+, CD62L+, CD3+, CD95+, granzyme B- and / or CD127+ in multiple output compositions is approximately 40% to approximately 65%, approximately 40% to approximately 45%, approximately 45% to approximately 50%, approximately 50% to approximately 55%, approximately 55% to approximately 60%, or approximately 60% to approximately 65%, and that are CCR7+ / CD45RA- or CCR7+ / CD45RO+ in multiple output compositions. The average percentage of cells is approximately 40% to 65%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, or 60% to 65%, and the average percentage of central memory CD4+ T cells in multiple output composition engineered CD4+ T cells, optionally CAR+CD4+ T cells, is approximately 40% to 65%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, or 60% to 65%, and the average percentage of central memory CD4+ T cells in multiple output composition engineered CD8+ T cells, optionally CAR+CD8 The average percentage of central memory CD8+ T cells is approximately 40%–65%, 40%–45%, 45%–50%, 50%–55%, 55%–60%, or 60%–65%, and / or the average percentage of central memory T cells in multiple output compositions, optionally CAR+ T cells, optionally CD4+ central memory T cells, and CD8+ central memory T cells is approximately 40%–65%, 40%–45%, 45%–50%, 50%–55%, 55%–60%, or 60%–65%.

[0069] In some embodiments of the methods provided herein, the method produces an output composition exhibiting a predetermined characteristic, optionally a threshold number, of cells expressing CAR in the output composition in at least about 80%, about 90%, about 95%, about 97%, about 99%, about 100%, or 100% of human biological samples in which the method is performed among a plurality of different individual subjects. In some such embodiments, the plurality of different individual subjects include subjects having a disease or condition. In some such embodiments, the disease or condition is cancer. In some such embodiments, the cancer is a hematological cancer and optionally multiple myeloma. In some of 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 more than 95% of the cells in the composition are memory phenotypes, and 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 more than 95% of the cells in the composition are central memory phenotypes, and 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 more than 95% are CD27+, CD28+, CCR7+, CD45RA-, CD45RO+, CD62L+, CD3+, granzyme B- and / or CD127+, and / or 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 more than 95% of the cells in the output composition are CCR7+ / CD45RA- or CCR7+ / CD45RO+.

[0070] In certain embodiments, what is provided herein is a composition comprising manipulated cells prepared by any method provided herein. Some embodiments of any of the compositions provided include a pharmaceutically acceptable carrier. Certain embodiments of any of the compositions provided include a cryoprotective substance, optionally DMSO.

[0071] In certain embodiments, provided herein is a manufactured article comprising any composition provided herein and instructions for administering the output composition to a subject. In certain embodiments of the provided manufactured article, the subject has a disease or condition, and optionally, the recombinant receptor specifically recognizes or specifically binds to an antigen associated with or expressed on cells of the disease or condition. [Invention 1001] (a) A step of generating an input composition by combining a CD4+ T cell composition and a CD8+ T cell composition in a CD4+ T cell:CD8+ T cell ratio of 2:1 to 1:2. (b) A step of incubating an input composition under stimulating conditions to generate a stimulated composition, wherein the stimulating conditions include the presence of a stimulating reagent 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 co-stimulatory molecules. Includes, The input composition is 5 × 10 6 At concentrations of less than 100 × 10⁻¹⁰ cells / mL 6 Including a total of 100 CD4+ T cells and CD8+ T cells, A method for producing a composition of manipulated cells. [Invention 1002] The method of the present invention 1001, wherein CD4+ cells and CD8+ cells in the input composition are concentrated or selected from a primary sample obtained from a subject, and optionally, CD4+ cells and CD8+ cells in the input composition are concentrated or selected separately from a primary sample obtained from a subject. [Invention 1003] The method of the present invention 1001 or 1002, wherein the CD4+ T cell composition comprises at least 80%, at least 85%, at least 90%, or at least 95% CD4+ T cells. [Invention 1004] The method according to any one of the present invention 1001 to 1003, wherein the CD8+ T cell composition comprises at least 80%, at least 85%, at least 90%, or at least 95% CD8+ T cells. [Invention 1005] A process of incubating an input composition under stimulating conditions to generate a stimulated composition. Includes, The input composition has a CD4+ T cell:CD8+ T cell ratio of 2:1 to 1:2, and the input composition is 5 × 10 6 At concentrations of less than 100 × 10⁻¹⁰ cells / mL 6 It contains a total of 100 CD4+ T cells and CD8+ T cells, The stimulation condition includes the presence of a stimulating reagent that can activate 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. A method for producing a composition of manipulated cells. [Invention 1006] Any method 1001 to 1005 of the present invention, wherein incubation is performed in serum-free culture medium. [Invention 1007] A method according to any one of the present invention 1001 to 1006, wherein the input composition comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD4+ T cells or CD8+ T cells. [Invention 1008] The input composition is 100 × 10 6 ~500×10 6 A method of any of the present invention 1001 to 1007, comprising a total of 1000 CD4+ T cells and CD8+ T cells. [Invention 1009] The input composition is 300 × 10 6 1 or approximately 300 x 10 6 A method of any of the present invention 1001 to 1008, comprising a total of 1000 CD4+ T cells and CD8+ T cells. [Invention 1010] The method of the present invention 1009, wherein the total CD4+ T cells and CD8+ T cells are viable cells. [Invention 1011] The input composition is 1 × 10 6 cells / mL~5×10 6 A method according to any of the present invention 1001 to 1010, having a concentration of cells / mL. [Invention 1012] The input composition is 3 × 10 6 cells / mL or approximately 3 × 10⁶ 6 A method of the present invention, having a concentration of cells / mL, any one of the methods described in 1001 to 1011. [Invention 1013] A method according to any of the invention 1001 to 1012, wherein the input composition has a CD4+ cell:CD8+ cell ratio of 1.5:1 to 1:1.5. [Invention 1014] A method according to any one of the present invention 1001 to 1013, wherein the input composition has a CD4+ cell:CD8+ cell ratio of 1.2:1 to 0.8:1. [Invention 1015] A method according to any of the 1001 to 1014 of the present invention, wherein the input composition has a CD4+ cell:CD8+ cell ratio of 1:1 or approximately 1:1. [Invention 1016] A method according to any one of the invention 1001 to 1015, wherein the input composition comprises CD4+ and CD8+ which are surface positive for CD45RA and CCR7. [Invention 1017] The method of the present invention 1016, wherein the ratio of CD4+ cells surface-positive for CD45RA and CCR7 to CD8+ cells surface-positive for CD45RA and CCR7 is 1.1:1 or approximately 1.1:1. [Invention 1018] A method according to any one of the present invention 1001 to 1017, wherein the input composition comprises CD4+ cells and CD8+ cells that are surface-positive for CD27 and CCR7. [Invention 1019] The method of the present invention 1018, wherein the ratio of CD4+ cells surface-positive for CD27 and CCR7 to CD8+ cells surface-positive for CD27 and CCR7 is 1.69:1 or approximately 1.69:1. [Invention 1020] The method according to any one of the invention 1001 to 1019, wherein the input composition comprises CD4+ cells and CD8+ cells that are surface-positive to CCR7 and surface-negative to CD62L, in an optional ratio of 2.0:1 to 1.5:1. [Invention 1021] A step of introducing recombinant receptors into cells from a stimulated composition, thereby generating an engineered cell composition, wherein the introduction includes contacting cells from the stimulated composition with an active substance comprising a polynucleotide encoding a recombinant receptor. Any method of the present invention 1001 to 1020, further comprising the above. [Invention 1022] The contact is a contact by transfection using a vector, and the vector is a transposon, optionally a sleeping beauty (SB) transposon or a piggyback transposon. The contact is contact mediated by transduction using a viral vector. The method of the present invention 1021. [Invention 1023] A step of introducing recombinant receptors into cells from a stimulated composition, thereby producing an engineered cell composition, wherein the introduction includes transduction of cells from the stimulated composition with a viral vector containing a polynucleotide encoding a recombinant receptor. Any method of the present invention 1001 to 1022, further comprising the above. [Invention 1024] Any of the methods described in 1021 to 1023 of this invention, wherein the introduction is performed in serum-free medium. [Invention 1025] For introduction, the stimulated composition is 300 × 10 6 A method according to any of the present invention 1021 to 1024, comprising fewer than 1 cell. [Invention 1026] For introduction, the stimulated composition is 50 × 10 6 Individual cells ~200 × 10 6 Individual cells, arbitrarily approximately 100 x 10 6 Individual cells, for example, about 100 x 10⁶ 6 A method according to any one of the present invention 1021 to 1025, comprising individual CD4+ T cells and CD8+ T cells. [Invention 1027] For introduction, the stimulated composition should contain at least about 100 × 10 6 Individual cells, up to approximately 200 x 10⁻¹⁶ 6 Individual cells, for example, at least about 100 × 10⁶ 6 Each and a maximum of approximately 200 x 10 6 A method according to any one of the present invention 1021 to 1025, comprising individual CD4+ T cells and CD8+ T cells. [Invention 1028] For introduction, the stimulated composition is 3 × 10 6 Any method 1021 to 1027 of the present invention having a concentration of less than cells / mL. [Invention 1029] For introduction, the stimulated composition is 0.5 × 10 6 cells / mL~2×10 6 A method of the present invention, having a concentration of cells / mL, as described in any of items 1021 to 1028. [Invention 1030] For introduction, the stimulated composition is 1 × 10 6 cells / mL or approximately 1 × 10⁶ 6 A method of the present invention, having a concentration of cells / mL, as described in any of items 1021 to 1029. [Invention 1031] Any method of the present invention 1021 to 1030, comprising the step of incubating the stimulated composition under stimulating conditions and then adjusting the composition of the stimulated composition before introducing recombinant receptors into the cells of the stimulated composition. [Invention 1032] Any method of the present invention 1021 to 1031, wherein the cells of the stimulated composition are viable cells. [Invention 1033] A method for producing a composition of manipulated cells, comprising the step of introducing recombinant receptors into the cells of the T cell composition, wherein the T cell composition contains at least 1 × 10 per 1 mL. 6 individual or at least about 1 × 10 6 The method having a concentration of 100% of living cells, 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. [Invention 1034] The concentration of the T cell composition is 5 × 10 per 1 mL. 6 The method of the present invention 1033, which involves fewer than one viable cell. [Invention 1035] The T cell composition contains at least 100 × 10 6 individual or at least about 100 x 10 6 1 or approximately 100 x 10 6 The T cell composition contains at least 100 × 10⁶ viable cells. 6 Individual living cells, up to approximately 200 x 10⁶ 6 A method of the present invention, either 1033 or 1034, comprising a single viable cell. [Invention 1036] The T cell composition is 300 × 10 6 A method according to any of invention 1033 to 1035, comprising fewer than 1 viable cell. [Invention 1037] Any method of the present invention 1033 to 1036, wherein the introduction involves contacting T cells by transduction of a viral vector containing a polynucleotide encoding a recombinant receptor. [Invention 1038] Any method 1033 to 1037 of the present invention, wherein the introduction is performed in serum-free medium. [Invention 1039] Any method 1033 to 1038 of the present invention, wherein one or more cells of a T cell composition are activated and / or surface-expressed with an LDL receptor. [Invention 1040] 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 are (i) expressing a surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L, and 4-1BB, (ii) Intracellular expression of cytokines selected from the group consisting of IL-2, IFN-γ, and TNF-α, (iii) The cell cycle is in a stage G1 or later, and / or (iv) It can multiply, Any method described in invention 1033 to 1039. [Invention 1041] Prior to introduction, the cells of the composition are generated by a process comprising the step of incubating an input composition containing CD4+ T cells and CD8+ T cells under stimulating conditions, wherein the stimulating conditions include the presence of a stimulating reagent 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 co-stimulatory molecules, any method of the present invention 1033 to 1040. [Invention 1042] The method of the present invention 1041, wherein incubation is performed in serum-free medium, and optionally, introduction is performed in serum-free medium of the same or different composition as the serum-free medium used for incubation. [Invention 1043] A method for producing a composition of manipulated cells, comprising the following steps: (a) A step of incubating an input composition under stimulating conditions to produce a stimulated composition, The input composition has a CD4+ T cell:CD8+ T cell ratio of 2:1 to 1:2, and 5 × 10 6 At concentrations of less than 100 × 10⁻¹⁰ cells / mL 6 It contains individual CD4+ T cells and CD8+ T cells, The stimulation condition includes the presence of a stimulating reagent that can activate 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. Process, and (b) 300 × 10 of the stimulated composition 6 A step of introducing a recombinant receptor into fewer than 100 cells, thereby generating an engineered cell composition, wherein the introduction involves contacting the cells of the stimulated composition with a viral vector containing a polynucleotide encoding the recombinant receptor. [Invention 1044] Any method of the present invention 1043, wherein incubation and / or introduction is performed in serum-free medium. [Invention 1045] The method of either 1043 or 1044 of the present invention, wherein CD4+ T cells and CD8+ T cells are viable cells. [Invention 1046] Any method of the present invention 1044, wherein the cells from the stimulated composition are viable cells. [Invention 1047] Any method of the present invention 1033 to 1046, wherein 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. [Invention 1048] Any method of the present invention 1033 to 1047, wherein 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. [Invention 1049] Any method of the present invention 1033 to 1048, wherein 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. [Invention 1050] A process of culturing an engineered composition under conditions that promote the proliferation and / or expansion of engineered cells, thereby producing an output composition containing engineered T cells. Any method of the present invention 1033 to 1049, further comprising the above. [Invention 1051] The method of the present invention 1050, wherein the culture is performed in a serum-free medium. [Invention 1052] A method for producing a composition of manipulated cells, comprising the following steps: (a) A step of incubating an input composition under stimulating conditions, thereby generating a stimulated composition, wherein the input composition has a CD4+ T cell:CD8+ T cell ratio of 2:1 to 1:2, and the input composition has 5 × 10 6 At concentrations of less than 100 × 10⁻¹⁰ cells / mL 6 A step comprising a total of 100 CD4+ T cells and CD8+ T cells, wherein the stimulation condition includes the presence of a stimulating reagent 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, (b) 300 × 10 from the stimulated composition 6 A step of introducing recombinant receptors into fewer than 100 cells, thereby generating an engineered cell composition, the introduction comprising transduction of a viral vector containing a polynucleotide encoding a recombinant receptor into the cells of the stimulated composition, and (c) A step of culturing an engineered composition under conditions that promote the proliferation and / or expansion of engineered cells, thereby producing an output composition containing engineered T cells. [Invention 1053] The method of the present invention 1052, wherein one, two, or all of the incubation, introduction, and culture steps are performed in one or more serum-free media, and optionally the serum-free media have the same or different compositions. [Invention 1054] The method according to any of items 1041 to 1053 of the present invention, wherein the input composition has a CD4+ cell:CD8+ cell ratio of 1.5:1 to 1:1.5, a CD4+ cell:CD8+ cell ratio of 1.2:1 to 0.8:1, and optionally a CD4+ T cell:CD8+ T cell ratio of 1:1 or approximately 1:1. [Invention 1055] A method according to any one of the invention 1041 to 1054, wherein the input composition comprises CD4+ and CD8+ which are surface positive for CD45RA and CCR7. [Invention 1056] The method of the present invention 1055, wherein the ratio of surface-positive CD4+ cells to CD45RA and CCR7-positive CD8+ cells is 1.1:1 or approximately 1.1:1. [Invention 1057] A method according to any one of the present invention 1041 to 1056, wherein the input composition comprises CD4+ cells and CD8+ cells that are surface-positive for CD27 and CCR7. [Invention 1058] The method of the present invention 1057, wherein the ratio of CD4+ cells surface-positive for CD27 and CCR7 to CD8+ cells surface-positive for CD27 and CCR7 is 1.69:1 or approximately 1.69:1. [Invention 1059] A method according to any one of the invention 1041 to 1058, wherein the input composition comprises CD4+ cells and CD8+ cells that are surface-positive to CCR7 and surface-negative to CD62L. [Invention 1060] For introduction, the stimulated composition is 300 × 10 6 Less than one cell, optionally 50 x 10 6 Individual surviving cells ~200 × 10 6 individual living cells, and optionally 100 × 10⁶ 6 1 or approximately 100 x 10 6 A method according to any of the present invention 1043 to 1059, comprising a single living cell. [Invention 1061] For introduction, the stimulated composition should contain at least about 100 × 10 6 Individual living cells, up to approximately 200 x 10⁶ 6 A method according to any of the present invention 1043 to 1059, comprising a single living cell. [Invention 1062] For introduction, the stimulated composition is 3 × 10 6 A method of the present invention, having a concentration of less than cells / mL, as described in any of items 1043 to 1061. [Invention 1063] For introduction, the stimulated composition is 0.5 × 10 6 cells / mL~2×10 6 cells / mL, optionally 1 × 10⁶ 6 cells / mL or approximately 1 × 10⁶ 6 A method of the present invention, having a concentration of cells / mL, as described in any of items 1043 to 1062. [Invention 1064] Any method of the present invention 1043 to 1063, wherein the composition of the stimulated composition is adjusted after incubation under stimulating conditions, before introducing recombinant receptors into cells of the stimulated composition. [Invention 1065] Any method of the present invention 1001 to 1064, wherein incubation is optionally performed in serum-free medium in the presence of one or more cytokines. [Invention 1066] The method of the present invention 1065, wherein one or more cytokines are selected from recombinant IL-2, recombinant IL-7 and / or recombinant IL-15. [Invention 1067] The method of the present invention 1066, wherein one or more cytokines comprise recombinant IL-2 at 10-200 IU / mL, recombinant IL-7 at 100 IU / mL-1,000 IU / mL, and / or recombinant IL-15 at 10-200 IU / mL. [Invention 1068] The method of the present invention 1066 or 1067, wherein one or more cytokines comprise recombinant IL-2 at 10-200 IU / mL, recombinant IL-7 at 100 IU / mL-1,000 IU / mL, and recombinant IL-15 at 10-200 IU / mL. [Invention 1069] At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% of the cells of the stimulated composition, (i) expressing a surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L, and 4-1BB, (ii) Intracellular expression of cytokines selected from the group consisting of IL-2, IFN-γ, and TNF-α, (iii) The cell cycle is in a stage G1 or later, and / or (iv) It can multiply, Any method described in 1001 to 1068 of this invention. [Invention 1070] A method according to any of the present invention 1001 to 1069, wherein the stimulating reagent comprises a main component that specifically binds to a member of the TCR complex, and the main component optionally specifically binds to CD3. [Invention 1071] The method of the present invention 1070, wherein the stimulating agent further comprises an adjuvant that specifically binds to a T cell costimulatory molecule, and optionally the costimulatory molecule is selected from CD28, CD137(4-1-BB), OX40, or ICOS. [Invention 1072] The method of Invention 1070 or Invention 1071, wherein the main agent and / or auxiliary agent comprises an antibody, and optionally, a stimulating agent constitutes incubation with an anti-CD3 antibody and an anti-CD28 antibody or its antigen-binding fragment. [Invention 1073] Any method 1071 to 1072 of the present invention, wherein the main component and / or auxiliary component are present on the surface of a solid support. [Invention 1074] The method of the present invention 1073, wherein the solid support is beads or contains beads. [Invention 1075] The method of the present invention 1074, wherein the beads have a diameter greater than 3.5 μm or greater than approximately 3.5 μm but less than or equal to approximately 9 μm, or less than or equal to approximately 8 μm, or less than or equal to approximately 7 μm, or less than or equal to approximately 6 μm, or less than or equal to approximately 5 μm. [Invention 1076] The method of Invention 1074 or Invention 1075, wherein the beads have a diameter of 4.5 μm or about 4.5 μm. [Invention 1077] Any method 1074 to 1076 of the present invention, wherein the beads are inert. [Invention 1078] Any method of the present invention 1074 to 1077, wherein the beads have a polystyrene surface or include a polystyrene surface. [Invention 1079] Any method 1074 to 1078 of the present invention, wherein the beads are magnetic or superparamagnetic. [Invention 1080] A method according to any of invention 1074 to 1079, wherein the bead:cell ratio is less than 3:1. [Invention 1081] Any method of the present invention 1074 to 1080, wherein the bead:cell ratio is 2:1 to 0.5:1 or approximately 2:1 to approximately 0.5:1, and optionally, the bead:cell ratio is 1:1 or approximately 1:1. [Invention 1082] A method according to any one of the present invention 1071 to 1072, wherein the main agent and the auxiliary agent are reversibly bound to the surface of an oligomer particle reagent containing multiple streptavidin molecules or streptavidin mutein molecules. [Invention 1083] A method according to any one of the present invention 1001 to 1082, wherein the input composition is incubated under stimulating conditions for less than 48 hours. [Invention 1084] A method according to any one of the present invention 1001 to 1083, wherein the input composition is incubated under stimulating conditions for 12 hours or more and 36 hours or less. [Invention 1085] A method according to any one of the present invention 1001 to 1084, wherein the input composition is incubated under stimulating conditions for 18 to 30 hours. [Invention 1086] A method according to any of items 1001 to 1085 of the present invention, wherein the input composition is incubated under stimulating conditions for 24 hours or about 24 hours. [Invention 1087] Any method 1023 to 1086 of the present invention, wherein contact, and optionally transduction, are performed for less than 48 hours. [Invention 1088] A method according to any of the present invention 1023 to 1087, wherein contact and optionally transduction are performed for 12 to 36 hours. [Invention 1089] A method according to any of the present invention 1023 to 1088, wherein contact and optionally transduction are performed for 18 to 30 hours. [Invention 1090] Any method 1023 to 1089 of the present invention, wherein contact, optionally transduction, is performed for 24 hours or approximately 24 hours. [Invention 1091] A method according to any of the present invention 1023 to 1090, wherein the viral vector is a retroviral vector. [Invention 1092] A method according to any one of the present invention 1023 to 1091, wherein the viral vector is a lentiviral vector or a gamma retroviral vector. [Invention 1093] Any method of the present invention 1023 to 1092, wherein contact, and optionally transduction, are performed in the absence of a transduction adjuvant. [Invention 1094] Any method of the present invention 1023 to 1093, wherein the introduction is optionally carried out in serum-free medium in the presence of one or more cytokines. [Invention 1095] The method of the present invention 1094, wherein one or more cytokines are selected from recombinant IL-2, recombinant IL-7 and / or recombinant IL-15. [Invention 1096] The method of the present invention 1094 or 1095, wherein one or more cytokines comprise recombinant IL-2 at 10-200 IU / mL, recombinant IL-7 at 100 IU / mL-1,000 IU / mL, and / or recombinant IL-15 at 10-200 IU / mL. [Invention 1097] A method according to any one of the present invention 1094 to 1096, wherein one or more cytokines include recombinant IL-2 at 10 to 200 IU / mL, recombinant IL-7 at 100 IU / mL to 1,000 IU / mL, and recombinant IL-15 at 10 to 200 IU / mL. [Invention 1098] A method according to any one of the present invention 1050 to 1097, wherein at least a portion of the culture is carried out using mixing and / or perfusion. [Invention 1099] At least a portion of the culture is 500 mL / day, 600 mL / day, 700 mL / day, 750 mL / day, 800 mL / day, 900 mL / day, 1,000 mL / day, 1,200 mL / day, 1,400 mL / day, 1,500 mL / day, 1,600 mL / day, 1,800 mL / day and / or 2,000 mL / day, approximately 500 mL / day, approximately 600 mL / day, approximately 700 mL / day, approximately 750 mL / day, approximately 800 mL / day, approximately 900 mL / day, approximately 1,000 mL / day, approximately 1,200 mL / day, approximately 1,400 mL / day, approximately 1,500 mL / day, approximately 1,600 mL / day, The method of the present invention 1098, carried out using perfusion at a rate of approximately 1,800 mL / day and / or approximately 2,000 mL / day, or at least 500 mL / day, at least 600 mL / day, at least 700 mL / day, at least 750 mL / day, at least 800 mL / day, at least 900 mL / day, at least 1,000 mL / day, at least 1,200 mL / day, at least 1,400 mL / day, at least 1,500 mL / day, at least 1,600 mL / day, at least 1,800 mL / day and / or at least 2,000 mL / day. [Invention 1100] The method of the present invention 1098 or 1099, wherein at least a first portion of the culture is carried out at a perfusion rate of 500 mL / day, 750 mL / day or 1,000 mL / day, about 500 mL / day, about 750 mL / day or about 1,000 mL / day, or at least 500 mL / day, at least 750 mL / day or at least 1,000 mL / day, and at least a second portion of the culture is carried out at a perfusion rate of 1,200 mL / day, 1,400 mL / day or 1,500 mL / day, about 1,200 mL / day, about 1,400 mL / day or about 1,500 mL / day, or at least 1,200 mL / day, at least 1,400 mL / day or at least 1,500 mL / day. [Invention 1101] Any method of the present invention 1098-1100, wherein perfusion is initiated and / or increased when the cells reach a certain density. [Invention 1102] A specific density is 0.4 × 10 6 Individual cells, 0.5 × 10 6 Individual cells, 0.6 × 10 6 Individual cells, 0.8 × 10 6 Individual cells, 1.0 × 10 6 Individual cells, 1.2 × 10⁻⁶6 Individual cells, 1.4 × 10⁻⁶ 6 Individual cells, 1.6 × 10⁻⁶ 6 Individual cells, 1.8 × 10⁻⁶ 6 Individual cells, 2.0 × 10⁻⁶ 6 Individual cells, 2.2 × 10 6 Individual cells or 2.4 × 10⁻⁶ 6 A single cell, approximately 0.4 × 10⁻⁶ 6 Each cell is approximately 0.5 × 10⁻⁶ 6 Each cell is approximately 0.6 × 10⁻⁶ 6 Each cell is approximately 0.8 × 10⁻⁶. 6 Each cell is approximately 1.0 × 10⁻⁶ 6 Each cell is approximately 1.2 × 10⁻⁶ 6 Each cell is approximately 1.4 × 10⁻⁶ 6 Each cell is approximately 1.6 × 10⁻⁶ 6 Each cell is approximately 1.8 × 10⁻⁶ 6 Each cell is approximately 2.0 × 10⁻⁶ 6 Each cell is approximately 2.2 × 10⁻⁶ 6 Individual cells or approximately 2.4 × 10⁻⁶ 6 A single cell, or at least 0.4 × 10⁶ 6 A single cell, at least 0.5 × 10⁻⁶ 6 A single cell, at least 0.6 × 10⁻⁶ 6 Each cell, at least 0.8 × 10⁻⁶ 6 A single cell, at least 1.0 × 10⁻⁶ 6 Each cell, at least 1.2 × 10⁶ 6 Each cell, at least 1.4 × 10⁶ 6 Each cell, at least 1.6 × 10⁶ 6 Each cell, at least 1.8 × 10⁶ 6 Each cell, at least 2.0 × 10⁶ 6 Individual cells, at least 2.2 × 10⁻⁶ 6 A single cell or at least 2.4 × 10⁶ 6 The method of the present invention 1101, which involves individual cells. [Invention 1103] The cells are 0.6 × 10 6 Cells / mL or approximately 0.6 × 10⁶ 6 Any method according to Invention 1098-1102, wherein perfusion is initiated and / or increased to a rate of 750 mL / day or approximately 750 mL / day when a density of cells / mL is reached. [Invention 1104] The cells are 2.0 × 10 6 Cells / mL or approximately 2.0 × 10⁶ 6 The method of the present invention 1098, wherein perfusion is initiated and / or increased to a rate of 1500 mL / day or approximately 1500 mL / day once a density of cells / mL is reached. [Invention 1105] Any method of the present invention 1050 to 1104, wherein the culture is optionally performed in serum-free medium in the presence of one or more cytokines. [Invention 1106] The method of the present invention 1105, wherein one or more cytokines are selected from recombinant IL-2, recombinant IL-7 and / or recombinant IL-15. [Invention 1107] The method of the present invention 1105 or 1106, wherein one or more cytokines comprise recombinant IL-2 at 50-400 IU / mL, recombinant IL-7 at 100 IU / mL-2,000 IU / mL, and / or recombinant IL-15 at 50-400 IU / mL. [Invention 1108] A method according to any one of the present invention 1105 to 1107, wherein one or more cytokines include recombinant IL-2 at 50 to 400 IU / mL, recombinant IL-7 at 100 IU / mL to 2,000 IU / mL, and recombinant IL-15 at 50 to 400 IU / mL. [Invention 1109] Any method of the present invention 1050 to 1108, wherein culture 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. [Invention 1110] Any method of the present invention 1050 to 1109, wherein culture 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. [Invention 1111] Any method of the present invention 1050 to 1110, wherein culture 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. [Invention 1112] A method according to any one of the present invention 1050 to 1111, wherein the culture is carried out until the composition contains at least a threshold number of T cells, a threshold number of viable T cells, T cells at a threshold concentration, and viable T cells at a threshold concentration. [Invention 1113] The threshold number of T cells or the threshold number of viable T cells is 2400 × 10 6 individual or 5500 x 10 6 pieces, approximately 2400×10 6 individual or approximately 5500 x 10 6 10, or at least 2400 × 10 6 individual or at least 5500 x 10 6 The method of the present invention 1112, wherein the total number of nucleated cells is n, and optionally the survival rate of the total number of nucleated cells is about 75% or at least about 75%, or about 85% or at least about 85%. [Invention 1114] After reaching the threshold number of T cells, the threshold number of viable T cells, and the threshold concentration of T cells, the culture is continued for at least one day, and optionally, when the threshold number of T cells or the threshold number of viable T cells reaches 900 × 10⁶ 6 pieces, 1200×10 6 individual or 3500 x 10 6 pieces, approximately 900×10 6 pieces, approximately 1200×10 6 individual or approximately 3500 x 10 6 10, or at least 900 x 10 6 Each, at least 1200 x 10 6 individual or at least 3500 x 10 6 The method of the present invention 1112, which is the total number of nucleated cells. [Invention 1115] The threshold number of T cells is 2400 × 10⁻¹⁰, which has a survival rate of approximately 85% or at least approximately 85%. 6 Individual or approximately 2400 x 106 The total number of nucleated cells, according to any of the methods 1050 to 1114 of this invention. [Invention 1116] The threshold number of T cells is 5500 × 10 6 Individual or approximately 5500 x 10 6 The total number of nucleated cells, according to any of the methods 1050 to 1114 of this invention. [Invention 1117] A method of the present invention, any one of items 1050 to 1116, comprising the step of collecting cells of the output composition after culturing. [Invention 1118] A method according to any one of the present invention 1050 to 1117, comprising the step of collecting cells of the output composition after culturing, wherein the cells of the output composition are collected at least 9 days after the start of incubation under stimulating conditions. [Invention 1119] A method according to any one of the present invention 1050 to 1118, comprising the step of collecting cells of the output composition after culturing, wherein the cells of the output composition are collected at least 10 days after the start of incubation under stimulating conditions. [Invention 1120] The method of the present invention 1118 or 1119, comprising a 95% confidence interval for the period between the start of incubation and the collection of cells for the output composition, which is 8 to 25 days, optionally 14 to 18 days. [Invention 1121] The method of the present invention 1118 or 1119, comprising a 95% confidence interval of 9 to 21 days between the start of incubation and the collection of cells for the output composition. [Invention 1122] The method of the present invention 1118 or 1119, comprising a 95% confidence interval of 9 to 16 days between the start of incubation and the collection of cells for the output composition. [Invention 1123] Any method of the present invention 1052 to 1122 further comprising the step of formulating cells of the output composition for cryopreservation and / or administration to a subject in the presence of optionally pharmaceutically acceptable excipients. [Invention 1124] The method of the present invention 1123, wherein the cells of the output composition are formulated in the presence of a cryoprotective substance. [Invention 1125] The method of the present invention 1124, wherein the cryoprotective substance contains DMSO. [Invention 1126] Any method 1122 to 1125 of the present invention, wherein the cells of the output composition are formulated in a container, optionally a vial or bag. [Invention 1127] Any method of the present invention 1001 to 1126, further comprising the step of isolating CD4+ T cells and / or CD8+ T cells from a biological sample before incubation. [Invention 1128] The method of the present invention 1127, wherein isolation comprises selecting cells by optionally positive or negative selection based on the surface expression of CD4 and / or CD8. [Invention 1129] A method of the present invention 1127 or 1128, comprising isolation based on immunoaffinity. [Invention 1130] A method according to any one of the present invention 1127 to 1129, wherein the biological sample contains primary T cells obtained from the subject. [Invention 1131] The method of the present invention 1130, wherein the subject is a human subject. [Invention 1132] A method according to any one of the present invention 1127 to 1131, wherein 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, an apheresis product, or a leukocyte apheresis product. [Invention 1133] A method according to any of items 1001 to 1132 of the present invention, wherein a recombinant receptor can bind to a target antigen that is associated with, specific to, and / or expressed in cells or tissues of a disease, disorder, or symptom. [Invention 1134] The method of the present invention 1133, wherein the disease, disorder, or symptom is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer. [Invention 1135] The method of the present invention 1133 or 1134, wherein the target antigen is a tumor antigen. [Invention 1136] The target antigens are 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, CD133, CD 138, CD171, Chondroitin sulfate proteoglycan 4 (CSPG4), Carcinoembryonic antigen (CEA), CE7, Cyclin, Cyclin A2, c-Met, Biantigen, 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 α, folate-binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, G250 / CAIX, GD2, GD3, gp100, glypican 3 (GPC3), G protein-coupled receptor class C group 5 member D (GPRC5D), Her2 / neu (receptor tyrosine kinase erbB2), HMW-MAA, IL-22R-α, IL-13 receptor α2 (IL-13Rα2), kinase insertion 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, MAGE-A10, mesoserine (MSLN), ma Any method of the present invention 1133 to 1135, selected from Uss CMV, mucin 1 (MUC1), MUC16, NCAM, NKG2D, NKG2D ligand, NY-ESO-1, O-acetylated GD2 (OGD2), carcinoembryonic antigen, melanoma preferential expression antigen (PRAME), PSCA, progesterone receptor, Survivin, ROR1, TAG72, tyrosinase-related protein 1 (TRP1; also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2; also known as dopachrome tautomerase, dopachrome δ-isomerase or DCT), VEGF receptor, VEGF-R2, Wilms tumor 1 (WT-1), pathogen-specific antigen, and antigens associated with the universal tag. [Invention 1137] A method according to any one of the present invention 1001 to 1136, wherein the recombinant receptor is a functional non-TCR antigen receptor or a TCR or an antigen-binding fragment thereof, or comprises the same. [Invention 1138] A method according to any of the present invention 1001 to 1137, wherein the recombinant receptor is a chimeric antigen receptor (CAR). [Invention 1139] A method according to any of the present invention 1001 to 1138, wherein the recombinant receptor is anti-BCMA CAR. [Invention 1140] The method of the present invention 1138 or 1139, wherein the chimeric antigen receptor comprises an extracellular domain including an antigen-binding domain. [Invention 1141] The method of the present invention 1140, wherein the antigen-binding domain is an antibody, or optionally an antibody fragment which is a single-stranded fragment, or comprises the same. [Invention 1142] The method of the present invention 1141, wherein the fragment comprises an antibody variable region bound by a flexible linker. [Invention 1143] The method of the present invention 1141 or 1142, wherein the aforementioned fragment includes scFv. [Invention 1144] Any method of the present invention 1138 to 1143, wherein the chimeric antigen receptor further comprises a spacer and / or hinge region. [Invention 1145] A method according to any of the present invention 1138 to 1144, wherein the chimeric antigen receptor includes an intracellular signaling region. [Invention 1146] The method of the present invention 1145, wherein the intracellular signaling region includes an intracellular signaling domain. [Invention 1147] The method of the present invention 1146, wherein 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-activated tyrosine motif (ITAM). [Invention 1148] The method of the present invention 1146 or 1147, wherein the intracellular signaling domain is or comprises the intracellular signaling domain of a CD3 chain, optionally a CD3-zeta (CD3ζ) chain, or a signaling portion thereof. [Invention 1149] Any method according to invention 1145 to 1148, wherein the chimeric antigen receptor further comprises a transmembrane domain located between an extracellular domain and an intracellular signaling region. [Invention 1150] A method according to any one of the present invention 1145 to 1149, wherein the intracellular signaling region further comprises a co-stimulatory signaling region. [Invention 1151] The method of the present invention 1150, wherein the co-stimulatory signaling region includes the intracellular signaling domain or signaling portion of a T cell co-stimulatory molecule. [Invention 1152] The method of the present invention 1150 or 1151, wherein the co-stimulatory signaling region includes an intracellular signaling domain or signaling portion of CD28, 4-1BB, or ICOS. [Invention 1153] A method according to any one of the present invention 1150 to 1152, wherein the co-stimulatory signaling region is located between the transmembrane domain and the intracellular signaling region. [Invention 1154] Any method of the present invention 1113 to 1153, wherein an output composition containing a number of cells greater than or equal to a threshold number is produced during more than 85% or about 85%, more than 90% or about 90%, or more than 95% or about 95% of the iterations of the method. [Invention 1155] serum-free medium, The dipeptide form of L-glutamine at concentrations of 0.5 mM to 5 mM in the basic culture medium. 0.5 mM to 5 mM L-glutamine, and at least one protein A method of the present invention 1001 to 1154, comprising, wherein the culture medium does not contain serum. [Invention 1156] The method of the present invention 1155, wherein the dipeptide form of L-glutamine is L-alanyl-L-glutamine. [Invention 1157] The method of Invention 1155 or Invention 1156, wherein the concentration of the dipeptide form of L-glutamine in the serum-free medium is 2 mM or about 2 mM. [Invention 1158] The method according to any one of the present invention 1155 to 1157, wherein the concentration of L-glutamine in the serum-free medium is 2 mM or approximately 2 mM. [Invention 1159] Any method according to 1155 to 1158 of the present invention, wherein at least one protein comprises one or more of albumin, insulin, or transferrin, optionally one or more of human or recombinant albumin, human or recombinant insulin, or human or recombinant transferrin. [Invention 1160] A composition comprising manipulated cells prepared by any of the methods described in invention 1001-1159 or 1165-1177. [Invention 1161] The composition of the present invention 1160, further comprising a pharmaceutically acceptable carrier. [Invention 1162] A composition according to Invention 1160 or Invention 1161, comprising a cryoprotective substance, optionally DMSO. [Invention 1163] A manufactured article comprising any of the compositions of invention 1160 to 1162 and instructions for administering the output composition to a subject. [Invention 1164] A manufactured article of the present invention 1163, wherein the subject has a disease or symptom, and optionally, the recombinant receptor specifically recognizes or specifically binds to an antigen that is associated with or expressed on cells of the disease or symptom. [Invention 1165] A method according to any of the Invention 1055-1159, wherein the cells are monitored for cell viability, concentration, density, number, or a combination thereof for at least a portion of the culture. [Invention 1166] The method of the present invention 1165, wherein monitoring is performed by optical methods, optionally by microscopic observation. [Invention 1167] The method of Invention 1165 or Invention 1166, wherein monitoring is performed by bright-field microscopy, fluorescence microscopy, differential interference microscopy, phase-contrast microscopy, digital holography (DHM), differential digital holography (DDHM), or a combination thereof. [Invention 1168] The monitoring is performed by differential digital holography (DDHM) microscopy, according to any of the methods described in items 1165 to 1167 of this invention. [Invention 1169] The method according to any one of the Invention 1165 to 1168, wherein monitoring is performed intermittently or continuously for at least a portion of the culture, and optionally, at least every hour, at least every 6 hours, at least every 12 hours, at least every 18 hours, at least every 24 hours or at least every 26 hours during the culture. [Invention 1170] Any method 1165 to 1169 of the present invention, wherein monitoring is performed until the cells reach a threshold number of T cells, a threshold number of viable T cells, a threshold concentration of T cells, or a threshold concentration of viable T cells. [Invention 1171] Any method according to invention 1165 to 1170, wherein monitoring and culturing are performed in a closed system. [Invention 1172] 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 more than 95% of the cells in the output composition are of the memory phenotype. 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 more than 95% of the cells in the output composition are of the central memory phenotype. 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 more than 95% of the cells in the output composition are CD27+, CD28+, CCR7+, CD45RA-, CD45RO+, CD62L+, CD3+, CD95+, granzyme B- and / or CD127+, and / or 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 more than 95% of the cells in the output composition are CCR7+ / CD45RA- or CCR7+ / CD45RO+. Any method of the present invention 1050-1159 and 1165-1171. [Invention 1173] By repeating the above method, multiple output compositions can be optionally produced from a human biological sample, and the above method can be performed on multiple different individual subjects. The average percentage of memory phenotypic cells in multiple output compositions is approximately 40% to 65%, approximately 40% to 45%, approximately 45% to 50%, approximately 50% to 55%, approximately 55% to 60%, or approximately 60% to 65%. The average percentage of cells exhibiting the central memory phenotype in multiple output compositions is approximately 40% to 65%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, or 60% to 65%. The average percentage of cells that are CD27+, CD28+, CCR7+, CD45RA-, CD45RO+, CD62L+, CD3+, CD95+, granzyme B- and / or CD127+ in multiple output compositions is approximately 40% to approximately 65%, approximately 40% to approximately 45%, approximately 45% to approximately 50%, approximately 50% to approximately 55%, approximately 55% to approximately 60%, or approximately 60% to approximately 65%. The average percentage of cells that are CCR7+ / CD45RA- or CCR7+ / CD45RO+ in multiple output compositions is approximately 40% to 65%, approximately 40% to 45%, approximately 45% to 50%, approximately 50% to 55%, approximately 55% to 60%, or approximately 60% to 65%. In multiple output compositions of manipulated CD4+ T cells, the average percentage of central memory CD4+ T cells in the CAR+CD4+ T cells was approximately 40% to 65%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, or 60% to 65%. In multiple output compositions of manipulated CD8+ T cells, the average percentage of central memory CD8+ T cells in the CAR+CD8+ T cells is approximately 40% to 65%, approximately 40% to 45%, approximately 45% to 50%, approximately 50% to 55%, approximately 55% to 60%, or approximately 60% to 65%, and / or The average proportions of manipulated T cells from multiple output compositions, optionally CAR+ T cells containing central memory T cells, optionally CD4+ central memory T cells, and CD8+ central memory T cells, are approximately 40%–65%, 40%–45%, 45%–50%, 50%–55%, 55%–60%, or 60%–65%. Any method of the present invention 1050-1159 and 1165-1172. [Invention 1174] The method comprises producing an output composition that exhibits predetermined characteristics, optionally a threshold number, of cells expressing CAR in at least about 80%, about 90%, about 95%, about 97%, about 99%, about 100%, or 100% of a human biological sample, wherein the method is performed between a plurality of different individual subjects, according to any of the methods 1001-1159 and 1165-1173 of the present invention. [Invention 1175] The method of the present invention 1174, wherein multiple different individual subjects include subjects having a disease or symptoms. [Invention 1176] The method of the present invention 1175, wherein the disease or symptom is cancer. [Invention 1177] The method of the present invention 1176, wherein the cancer is a blood cancer, and optionally multiple myeloma. [Invention 1178] 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 more than 95% of the cells in the composition are of the memory phenotype. 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 more than 95% of the cells in the composition exhibit the central memory phenotype. 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 more than 95% of the cells in the composition are CD27+, CD28+, CCR7+, CD45RA-, CD45RO+, CD62L+, CD3+, granzyme B- and / or CD127+, and / or 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 more than 95% of the cells in the output composition are CCR7+ / CD45RA- or CCR7+ / CD45RO+. The composition of the present invention 1160. [Brief explanation of the drawing]

[0072] [Figure 1]Figure 1A shows a plot of bivariate fit analysis of the ratio of viable CD4+ cells to viable CD8+ cells (viable 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 the T cell composition after T cell activation, transduction with a chimeric antigen receptor (CAR) construct, and expansion and proliferation. The curve represents the boundary of the bivariate normal ellipse at p=0.990. The data points represent the average ratio of four samples obtained from each subject, including healthy subjects (circles) and myeloma patients (plus signs). Figure 1B shows a plot of bivariate fit analysis of the CD45RA+ / CCR7+CD4 / CD8 ratio in a starting mixture of selected CD4 and CD8 cells compared to the CAR+CD4+ / CD8+ ratio in the T cell composition after T cell activation, transduction with a chimeric antigen receptor (CAR) construct, and expansion and proliferation. The curve represents the boundary of the bivariate normal ellipse at p=0.990. The data points represent the mean proportion of four samples obtained from each group, including healthy subjects (circles) and myeloma patients (plus signs). [Figure 2A]Figures 2A and 2C show plots of bivariate fitted analyses of the ratios of different phenotypic cells in a starting mixture of selected CD4 and CD8 cells compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+ T cell composition. Figure 2A shows a plot of bivariate fitted analyses of the ratio of CD45RA+ / CCR7+ / CD4+ cells versus CD45RA+ / CCR7+ / CD8+ cells in a starting mixture of selected CD4 and CD8 cells compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+ T cell composition. Figure 2B shows a plot of bivariate fitted analyses of the ratio of CD62L- / CCR7+ / CD4+ T cells versus CD62L- / CCR7+ / CD8+ T cells in a starting mixture of selected CD4 and CD8 cells compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+ T cell composition. Figure 2C shows a plot of bivariate fitted analysis of the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells in a starting mixture of selected CD4 and CD8 cells, compared to the CAR+CD4+ / CD8+ ratio in the manipulated CAR+ T cell composition. The curve represents the boundary of the bivariate normal ellipse at p=0.950. Data points represent the average ratio of multiple compositions derived from each subject, including healthy donors (circles) and multiple myeloma patients (plus signs). [Figure 2B] See the explanation in Figure 2A. [Figure 2C] See the explanation in Figure 2A. [Figure 3A]Figures 3A to 3C show plots of bivariate fitted analyses of the ratio of different phenotypic cells in a starting mixture of selected CD4 and CD8 cells obtained from seven multiple myeloma patients, compared to the CAR+CD4+ / CD8+ ratio in the generated engineered CAR+ T cell composition. Figure 3A shows a plot of bivariate fitted analyses of the ratio of CD27+ / CCR7+ / CD4+ cells versus CD27+ / CCR7+ / CD8+ cells in a starting mixture of selected CD4 and CD8 cells, compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+ T cell composition. Figure 3B shows a plot of bivariate fitted analyses of the ratio of CD27+ / CCR7+ / CD4+ T cells versus CD27+ / CCR7+ / CD8+ T cells in a starting mixture of selected CD4 and CD8 cells, compared to the CAR+CD4+ / CD8+ ratio in the engineered CAR+ T cell composition. Figure 3C shows a plot of bivariate fitted analysis of the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells in a starting mixture of selected CD4 and CD8 cells, compared to the CAR+CD4+ / CD8+ ratio in the manipulated CAR+ T cell composition. The curve represents the boundary of the bivariate normal ellipse at p=0.950. [Figure 3B] See the explanation in Figure 3A. [Figure 3C] See the explanation in Figure 3A. [Figure 4A] Figures 4A and 4B show the number of viable cells (VCC; x10⁶ cells / mL) and cell viability (%) assessed using continuous monitoring with differential DHM ("continuous", line) or manual sampling ("manual", dot) in Experiment Run 1 (Figure 4A) and Run 2 (Figure 4B). The upper panel shows the respective measurements, and the lower panel shows the linear regression analysis with R² and gradient(s) for comparing continuous monitoring and manual sampling. [Figure 4B] See the explanation in Figure 4A. [Figure 5] Figure 5 shows the number of viable cells (VCC; x10⁶ cells / mL) and cell viability (%) as assessed using continuous monitoring by differential DHM in the automated expansion process compared to the manual expansion process. [Figure 6A] Figures 6A–6D show exemplary phenotypic profiles of 40 engineered CAR+ T cell compositions obtained from multiple myeloma patients. CD45RA×CCR7 expression profiles between CAR+ T cell compositions are shown for the CD4+ population (Figure 6A) and CD8+ population (Figure 6B). CD27×CD28 expression profiles between CAR+ T cell compositions are shown for the CD4+ population (Figure 6C) and CD8+ population (Figure 6D). Each CAR+ T cell composition is indicated by a dot (●), cross (×), rhombus (◇), or triangle (△). [Figure 6B] See the explanation in Figure 6A. [Figure 6C] See the explanation in Figure 6A. [Figure 6D] See the explanation in Figure 6A. [Modes for carrying out the invention]

[0073] Detailed explanation Provided herein are methods for generating or producing compositions of engineered cells, such as engineered CD4+ T cells and CD8+ T cells, that express recombinant receptors. In certain embodiments, the methods are used in connection with processes that include incubating cells, such as a composition of input cells, under stimulating conditions, for example, by transfecting or transfecting them with polynucleotides encoding recombinant receptors, and / or culturing the engineered cells under conditions that promote cell proliferation and / or expansion.

[0074] In some embodiments, provided herein are methods for producing a composition of manipulated cells, comprising the steps of generating an input composition by combining CD4+ T cells and CD8+ T cells in a ratio of 2:1 to 1:2, and incubating the input composition under stimulating conditions. In certain embodiments, the method comprises the step of incubating an input composition having a CD4+ T cell:CD8+ T cell ratio of 2:1 to 1:2. In certain embodiments, the input composition is 5 × 10 6 At concentrations of less than 100 × 10⁻¹⁰ cells / mL 6 It contains a total of 100 CD4+ T cells and CD8+ T cells.

[0075] In certain embodiments, the Specified herein provides a set or fixed amount of cells, for example, at least 1 × 10 of a cell composition. 6 each, at least 10 x 10 6 pieces, at least 100 x 10 6 individual or at least 1,000 × 10 6 individual or approximately 1 x 10 6 pieces, about 10×10 6 pieces, approximately 100×10 6 individual or approximately 1,000 x 10 6 A method for producing a composition of engineered cells, comprising the step of introducing recombinant receptors into individual cells. In some embodiments, the cells are stimulated cells. In certain embodiments, the cells are viable cells. In certain embodiments, the introduction comprises the step of transducing a viral vector containing a polynucleotide encoding a recombinant receptor into T cells of the stimulated composition. In certain embodiments, the cell composition comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD4+ T cells or CD8+ T cells.

[0076] In some embodiments, the foregoing provides a step of (i) incubating an input composition under stimulating conditions to produce a stimulated composition, wherein the input composition has a CD4+ T cell:CD8+ T cell ratio of 2:1 to 1:2, and the input composition has a concentration of 5 × 10 6At concentrations of less than 100 × 10⁻¹⁰ cells / mL 6 (ii) containing a total of 300 × 10¹ CD4+ T cells and CD8+ T cells from the stimulated composition. 6 A method for producing an engineered cell composition, comprising the steps of (iii) introducing recombinant receptors into fewer than 100 cells, thereby generating an engineered cell composition, wherein the introduction comprises transduction of cells of the stimulated composition with a viral vector containing a polynucleotide encoding a recombinant receptor, and (iii) culturing the engineered composition under conditions that promote the proliferation and / or expansion of the engineered cells, thereby producing an output composition containing engineered T cells.

[0077] Various processes are available for generating genetically engineered T cell populations, including generating engineered T cells that express chimeric antigen receptors. However, in some embodiments, some of these processes may require long or relatively long periods of time to generate the engineered cells. In certain embodiments, some of these processes may differ in their ability to successfully generate therapeutically suitable engineered cells derived from a wide range of different subjects. In certain embodiments, some of these processes may produce genetically engineered T cell compositions with a high degree of variability in parameters such as cell health, viability, transduction efficiency, and / or cell activity.

[0078] The embodiments provided address one or more of these problems. In certain embodiments, the method provided generates engineered T cells suitable for therapeutic use, such as autologous cell therapy, in a short time or relatively short period compared to some existing processes. Furthermore, in some embodiments, the method provided results in a highly consistent and less volatile process with respect to the time required to produce engineered cells from samples collected from different subjects. In certain embodiments, the method provided can successfully generate 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 proportion of healthy cells, e.g., cells that are viable and / or do not express apoptosis markers, cells that express recombinant receptors, and / or cells that have high or relatively high activity in response to antigen stimulation, such as cytotoxicity, antitumor activity, and / or cytokine production. In some embodiments, the method provided provides a process for producing manipulated cell products, and in some aspects, it is possible to produce therapeutic cell compositions for a large or large proportion of samples, such as for all or a high proportion of samples, each originating from different individual subjects or patients, such as subjects or patients to be treated with the therapeutic composition (for example, in the context of autologous cell therapy), and having a specific success rate, such as a high success rate above a threshold rate, such as being able to produce such compositions having specific required or desired characteristics. In some aspects, the subjects or patients have a disease or condition, such as cancer, such as hematological cancer, such as multiple myeloma. In some aspects, the samples (for a high proportion of which it is possible to produce therapeutic cell compositions) are patient samples, such as those which are variable with respect to the cellular phenotype or other parameters of the sample or its cells.

[0079] In some embodiments, the method provided generates an engineered T cell composition having improved or superior cellular health compared to, for example, a cell composition produced by other processes. In some embodiments, the composition contains a high proportion of cells that are negative for apoptosis markers. In some embodiments, the method provided generates a T cell composition containing pluripotent cells that robustly produce cytokines. In some embodiments, the method provided generates a T cell composition that is rich in memory phenotypes, rich in central memory phenotypes, and / or rich in cells that are CD27+, CD28+, CCR7+, CD45RA-, CD45RO+, CD62L+, CD3+, CD95+, granzyme B-, and / or CD127+.In some embodiments, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the cells in the composition (or, for at least half or most of the samples produced using the method, or on average for the samples produced using the method, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%), T in the composition At least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the cells, or at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the manipulated T cells in the composition are central memory phenotype T cells, and are CD27+, CD28+, CCR7+, CD45RA-, and / or CCR7+, CD45RO+.In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, at least 85%, at least 90%, or at least 95% (or, for at least half or the majority of the samples produced using the method, or on average for the samples produced using the method, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, at least 85%, at least 90%, or less) At least 95% of the T cells in the composition, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, at least 85%, at least 90%, or at least 95%, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, at least 85%, at least 90%, or at least 95%, are memory phenotypic T cells, CD45RA-, and / or CD45RO+.

[0080] In certain embodiments, the methods provided are used in connection with a process for efficiently producing or generating manipulated cells suitable for use in cell therapy. In certain embodiments, the timing, conditions, and reagents used in each step of the process improve the efficiency of each subsequent step and / or the process as a whole. For example, in some embodiments, cells may be incubated, transduced, and / or cultured at a concentration high enough to achieve a desired effect, e.g., cell stimulation, or improved transduction efficiency, but low enough to avoid delayed growth or reduced viability in subsequent processing steps. Furthermore, in some embodiments, steps of the process are timed to start or end at specific points in time to improve the efficiency of subsequent process steps and / or the process as a whole. For example, in some embodiments, steps for incubation and manipulation (e.g., cell transduction or transfection) are completed earlier in the process than in other methods, which in certain embodiments improves the viability and / or health of cells during subsequent culture steps, and / or the rate of growth and expansion. Therefore, in one aspect, the specific timing, conditions, and reagents of each step affect the cells beyond the individual steps, and in certain aspects, affect the performance of the entire process.

[0081] In some embodiments, the method is used in connection with a process of generating or producing genetically engineered cells suitable for cell therapy in a manner that may be faster and more efficient than alternative processes. In certain embodiments, the method provided herein has a high success rate in generating or producing compositions of engineered cells from a broader target population than may be possible from alternative processes. In certain embodiments, engineered cells produced or generated by the provided method may have increased health, viability, and activation, and increased recombinant receptor expression, compared to cells produced by other methods. Thus, in some aspects, the speed and efficiency of the provided method for generating engineered cells for cell therapy makes it easier to plan and adapt cell therapies, such as autotherapy, for a broader target population than may be possible by some other methods.

[0082] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated by reference in whole for any purpose to the same extent as each individual publication is incorporated by reference individually. If any definitions set forth herein conflict with or otherwise contradict any definitions set forth herein in patents, applications, published applications, and other publications incorporated by reference herein, the definitions set forth herein shall prevail over the definitions incorporated by reference herein.

[0083] The section headings used in this specification are for organizational purposes only and should not be interpreted as limiting the subject matter described.

[0084] I. Process for generating manipulated cells Provided herein are methods for producing output compositions of engineered cells, such as engineered CD4+ T cells and CD8+ T cells, that express recombinant proteins, such as recombinant receptors, such as T cell receptors (TCRs) or chimeric antigen receptors (CARs). In certain embodiments, the methods provided herein may be used in connection with the manufacture, generation, or preparation of cell therapies and may be used in connection with additional processing steps such as cell isolation, separation, selection, activation or stimulation, transduction, washing, suspension, dilution, concentration, and / or formulation. In some embodiments, a method for generating or preparing engineered cells, such as engineered CD4+ T cells and CD8+ T cells, includes one or more of the following: isolation of cells obtained from a subject, preparation of cells under stimulating conditions, processing, incubation, and / or manipulation of cells (e.g., transduction). 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+ cells and primary CD8+ cells from a biological sample; incubating the input cells under stimulating conditions and manipulating them with vector particles, e.g., viral vector particles, to introduce recombinant polynucleotides into the cells, e.g., by transfection or induction; culturing the manipulated cells, e.g., transfected cells, e.g., to expand and grow the cells; and collecting, harvesting, and / or filling all or part of the cells into containers, e.g., bags or vials, to formulate the cells into an output composition. In some embodiments, the cells of the resulting output composition are reintroduced into the same subject before or after cryopreservation. In some embodiments, the output composition of the manipulated cells is suitable for use in therapies such as autologous cell therapy.

[0085] In certain embodiments, the method provided is used in connection with generating an output composition of cells expressing recombinant receptors from an initial composition of cells or an input composition. In certain embodiments, the input composition is produced, generated, and / or made up by combining, mixing, and / or pooling cells from a cell composition containing enriched T cells, enriched CD4+ T cells, and / or enriched CD8+ T cells (hereinafter also referred to herein as the enriched T cell composition, the enriched CD4+ T cell composition, and the enriched CD8+ T cell composition, respectively). In some embodiments, the cell input composition is a combined, mixed, and / or pooled composition of CD4+ T cells and CD8+ T cells. In certain embodiments, the method provided is used in connection with one or more of the following: activating and / or stimulating cells, e.g., cells of the input composition; genetically engineering the activated and / or stimulated cells to introduce polynucleotides encoding recombinant proteins, e.g., by transduction or transfection; and / or culturing the engineered cells under conditions that promote proliferation and / or rapid growth, e.g. In certain embodiments, the method may also be used in connection with isolating or selecting cells from a biological sample to generate an input composition of enriched T cells, for example, taken from a subject, collected, and / or obtained from the biological sample. In certain embodiments, the provided method may also be used in connection with taking, collecting, and / or formulating a composition of enriched T cells after the cells have been incubated, activated, stimulated, manipulated, transfected, transfected, and / or cultured.

[0086] In some embodiments, incubation of cells under stimulating conditions is or includes incubating cells with a stimulating reagent, e.g., a stimulating reagent described herein, such as in Section IB-1. In certain embodiments, under stimulating conditions, 5 × 10 6 At a set or fixed concentration, such as a concentration of less than 100 × 10⁶ cells / mL, 6A set or fixed quantity of cells, such as more than one cell, is incubated. In certain embodiments, incubation is carried out over a set or fixed period, such as less than two days, or over a period of 18 to 30 hours.

[0087] In certain embodiments, the methods provided herein are carried out in connection with cell manipulation, e.g., transduction or transfection. In some embodiments, a set or fixed amount of cells, e.g., viable CD4+ cells and viable CD8+ cells, are manipulated. In some embodiments, under stimulating conditions, 3 × 10⁻⁶ cells are manipulated. 6 At a set or fixed concentration, such as a concentration of less than 10 cells / mL, at least 10 × 10 6 A quantity of cells exceeding a single cell is incubated. In certain embodiments, the operation is performed over a set or fixed period, such as less than two days, or over a period of 18 to 30 hours.

[0088] In certain embodiments, at least part of the culture process is carried out using a closed bioreactor, for example, with constant mixing and / or perfusion. In certain embodiments, the mixing and / or perfusion incorporates the stable and / or stepwise replacement of used or old cell medium or solution with fresh medium or solution. In some embodiments, the culture is initiated, for example, within 2, 3, 4, or 5 days from the commencement or start of incubation under stimulating conditions; within 2, 3, 4, or 5 days from mixing, pooling, and / or combining cells, e.g., CD4+ cells and CD8+ cells, to produce the input composition; within 3, 4, 5, or 6 days from the collection of the biological sample; and / or within 3, 4, 5, or 6 days from the isolation, selection, and / or enrichment of a composition of enriched T cells, e.g., CD4+ T cells and / or CD8+ T cells, derived from the biological sample.

[0089] In some embodiments, one or more process steps are carried out at least partially in serum-free medium. In some embodiments, the serum-free medium is a defined and / or precisely defined cell culture medium. In certain embodiments, the serum-free medium is a treated, filtered, and controlled medium, for example, filtered to remove inhibitors and / or growth factors. In some embodiments, the serum-free medium contains proteins. In certain embodiments, the serum-free medium may contain serum albumin, hydrolysates, growth factors, hormones, carrier proteins and / or adhesion factors.

[0090] In some embodiments, the method provided is carried out such that one or more, or any, steps in the preparation of cells for clinical use, such as adoptive cell therapy, are performed without exposing the cells to non-sterile conditions. In some embodiments of such processes, the cells are isolated, separated or selected, transduced, washed, optionally activated or stimulated and formulated, all within a closed system. In some embodiments, one or more of the steps are performed separately from the closed system or sealed apparatus. In some such embodiments, the enriched cell composition is transferred away from the closed system or sealed apparatus under sterile conditions, such as by sterile transfer to a separate closed system.

[0091] In certain embodiments, enriched T cell compositions may be collected, formulated for cryoprotection, cryofrozen, and / or stored at or before, during, or after any step or process of the process for producing an enriched T cell output composition expressing recombinant receptors, at or below 0°C, below -20°C, or -70°C or -80°C, or below -70°C or -80°C. In some embodiments, cells may be stored for a period of less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 8 days, or less than 10 days, or for a period of less than 1 week, less than 2 weeks, less than 3 weeks, less than 4 weeks, less than 5 weeks, less than 6 weeks, less than 7 weeks, or less than 8 weeks, or for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks, or for more than 8 weeks. After storage, the enriched T cell composition may be thawed, and processing may be resumed from the same point in the process. In some embodiments, the enriched T cell input composition is cryopreserved and stored before subsequent processing, e.g., incubation under stimulating conditions. In certain embodiments, the enriched T cell culture and / or formulated composition is cryopreserved and stored before being administered to a subject, e.g., as autologous cell therapy.

[0092] In certain embodiments, the methods provided herein are used in connection with a process by which engineered cells are produced by a process comprising the steps of incubating cells under stimulating conditions, transducing cells to express recombinant receptors, such as CARs, and culturing cells under conditions that promote proliferation or growth. In certain embodiments, incubation is carried out for 18 to 30 hours, e.g., 24 hours or about 24 hours, followed by transduction over 18 to 30 hours, e.g., 24 hours or about 24 hours. In certain embodiments, cells are cultured under stimulating and / or growth-promoting conditions after the cells have been stimulated and transduced. In certain embodiments, incubation is initiated, for example, by contacting cells with a stimulating reagent, and transduction is initiated within 48 hours, 36 hours, or 30 hours after the incubation has started. In some embodiments, culture is carried out after incubation and transduction, and culture is initiated within 72 hours, 66 hours, or 60 hours after the incubation has started. In certain embodiments, the culture is carried out until a threshold quantity, density and / or growth of cells is achieved, until at least one day after the threshold quantity, density and / or growth of cells is achieved, and / or until at least eight, nine, ten, eleven or twelve days after the start of incubation.

[0093] In certain embodiments, a portion of the cells may be sampled or collected at any stage or step of the process, for example, while the composition remains in a closed system, cells may be taken from the enriched T cell composition, for example, during isolation, incubation, manipulation, culture and / or formulation. In certain embodiments, such cells may be analyzed for maker, characteristics or properties, including, but not limited to, viability, apoptosis, activation, stimulation, growth and / or depletion. In some embodiments, cells are sampled or collected by an automated process while the enriched T cell composition remains in a closed system. In some embodiments, the analysis of the sampled or collected cells is automated. In certain embodiments, the analysis is performed in a closed system under sterile conditions.

[0094] In some embodiments, provided herein is a process for generating engineered cells, comprising the steps of incubating an input cell composition under stimulating conditions (e.g., to activate T cells in the composition), manipulating the cell composition (e.g., transduction) to express recombinant receptors, such as CARs, culturing the cells under conditions that promote cell proliferation or expansion, and / or harvesting or collecting the cells to produce a cell composition containing engineered cells, such as engineered T cells for cell therapy. In some embodiments, the input cell composition is incubated under stimulating conditions including: a stimulating reagent (e.g., bead reagents as described in Section IB-1); a cell ratio of 1:1 or about 1:1; the input composition contains CD4+ T cells and CD8+ T cells in a ratio of 1:1 or about 1:1 (e.g., an input composition can be produced by pooling, mixing, and / or combining a composition enriched with CD4+ T cells and a composition enriched with CD8+ T cells in a ratio of 1:1 or about 1:1); the total duration of incubation with the stimulating reagent under stimulating conditions is about 12 hours to about 36 hours, e.g., about 18 hours to about 30 hours; and about 5 × 10⁻¹⁶ cells under stimulating conditions such as in the presence of the stimulating reagent. 5 cells / mL ~ approx. 5×10 7 Cell density / mL, e.g., 3 × 10⁻⁶ 6 cells / mL or approximately 3 × 10⁶ 6 Cells are incubated at a concentration of cells / mL, e.g., cells of the input composition; and / or cells are stimulated and / or activated in serum-free medium (e.g., serum-free medium containing one or more recombinant cytokines such as IL-2, IL-7, and IL-15). In some embodiments, cells are operated under conditions including: contacting cells with recombinant proteins, e.g., nucleic acid molecules encoding recombinant receptors, under centrifugation, e.g., at about 1600 × g for about 60 minutes, e.g., spinoculation (e.g., centrifugation inoculation); about 5 × 10 5 cells / mL ~ approx. 5×10 7Cell density / mL, e.g., 1 × 10⁻⁶ 6 cells / mL or approximately 1 × 10⁶ 6 Manipulate cells at a rate of cells / mL; approximately 100 × 10 from a composition cultured under stimulating conditions. 6 Manipulating individual cells; the total duration of the manipulation step, e.g., transduction, is about 12 to about 36 hours, e.g., about 18 to about 30 hours; and / or manipulating cells in serum-free medium (e.g., serum-free medium containing one or more recombinant cytokines such as IL-2, IL-7, and IL-15). In some embodiments, cells are cultured under conditions that promote cell proliferation or expansion, including: culturing cells under shaking and / or perfusion conditions; and / or culturing cells in serum-free medium (e.g., serum-free medium containing one or more recombinant cytokines such as IL-2, IL-7, and IL-15, optionally containing higher concentrations of recombinant cytokines than the serum-free medium used for stimulation / activation and / or manipulation). In some embodiments, cells are cultured to a threshold amount, concentration, and / or expansion, e.g., at least about 3500 × 10⁻⁶. 6 Individual cells or approximately 5500 × 10 6 When a threshold number of cells (e.g., total number of nucleated cells) is reached, the culture is terminated and the cells are harvested. In some embodiments, if the cells have not reached a target or threshold at a given point in time during the stimulation / activation, manipulation, culture, and / or harvesting process, the cells may be stimulated / activated, manipulated, and / or cultured until a later point in time when the target or threshold is reached.

[0095] In some embodiments, provided herein is a process for generating engineered cells, comprising the steps of incubating an input cell composition under stimulating conditions (e.g., to activate T cells in the composition), manipulating the cell composition (e.g., transduction) to express recombinant receptors, such as CARs, culturing the cells under conditions that promote cell proliferation or expansion, and / or harvesting or collecting the cells to produce a cell composition containing engineered cells, such as engineered T cells for cell therapy. In some embodiments, the input cell composition is incubated under stimulating conditions including: a stimulating reagent (e.g., bead reagents as described in Section IB-1); a cell ratio of 1:1 or about 1:1; the input composition contains CD4+ T cells and CD8+ T cells in a ratio of 1:1 or about 1:1 (e.g., an input composition can be produced by pooling, mixing, and / or combining a composition enriched with CD4+ T cells and a composition enriched with CD8+ T cells in a ratio of 1:1 or about 1:1); the total duration of incubation with the stimulating reagent under stimulating conditions is about 12 hours to about 36 hours, e.g., about 18 hours to about 30 hours; and about 5 × 10⁻¹⁶ cells under stimulating conditions such as in the presence of the stimulating reagent. 5 cells / mL ~ approx. 5×10 7 Cell density / mL, e.g., 3 × 10⁻⁶ 6 cells / mL or approximately 3 × 10⁶ 6 Cells are incubated at a concentration of cells / mL, e.g., cells of the input composition; and / or cells are stimulated and / or activated in serum-free medium (e.g., serum-free medium containing one or more recombinant cytokines such as IL-2, IL-7, and IL-15). In some embodiments, cells are operated under conditions including: contacting cells with recombinant proteins, e.g., nucleic acid molecules encoding recombinant receptors, under centrifugation, e.g., at about 1600 × g for about 60 minutes, e.g., spinoculation (e.g., centrifugation inoculation); about 5 × 10 5 cells / mL ~ approx. 5×10 7 Cell density / mL, e.g., 1 × 10⁻⁶ 6cells / mL or approximately 1 × 10⁶ 6 Manipulate cells at a rate of cells / mL; at least approximately 100 × 10⁶ cells derived from the composition cultured under stimulating conditions. 6 Individual cells, up to approximately 200 x 10⁻¹⁶ 6 Manipulating individual cells; the total duration of the manipulation step, e.g., transduction, is about 12 to about 36 hours, e.g., about 18 to about 30 hours; and / or manipulating cells in serum-free medium (e.g., serum-free medium containing one or more recombinant cytokines such as IL-2, IL-7, and IL-15). In some embodiments, cells are cultured under conditions that promote cell proliferation or expansion, including: culturing cells under shaking and / or perfusion conditions; and / or culturing cells in serum-free medium (e.g., serum-free medium containing one or more recombinant cytokines such as IL-2, IL-7, and IL-15, optionally containing higher concentrations of recombinant cytokines than the serum-free medium used for stimulation / activation and / or manipulation). In some embodiments, cells are cultured to a threshold amount, concentration, and / or expansion, e.g., at least about 2400 × 10⁻⁶. 6 The culture is terminated and the cells are harvested when a threshold cell number (e.g., total nucleated cell number) is achieved and the cells achieve a threshold viability, for example, when at least about 75% or at least about 85% of the cells are viable. In some embodiments, if the cells have not achieved the target or threshold at a given point in time during the stimulation / activation, manipulation, culture, and / or harvesting process, the cells may be stimulated / activated, manipulated, and / or cultured until a later point in time when the target or threshold is reached.

[0096] In some embodiments, cells or cell compositions prepared and / or processed by the provided method may be compared to cells or cell compositions processed or prepared by exemplary processes and / or alternative processes. In some embodiments, alternative processes and / or exemplary processes may differ in one or more specific aspects, but otherwise include similar or identical features, aspects, steps, stages, reagents and / or conditions to the aspects or aspects of the provided method being compared. For example, if the provided method is used in connection with incubating cells in the presence of a reagent, such cells may be compared to cells that are not incubated with the reagent in the exemplary process and / or alternative process. In some embodiments, unless otherwise specified, the provided method and exemplary processes and / or alternative processes will be similar and / or identical in other respects, such as using similar or identical steps for isolation, selection, concentration, activation, stimulation, manipulation, transfection, transduction, culture and / or formulation. In some embodiments, unless otherwise specified, the provided method and alternative processes isolate, select and / or concentrate cells from the same or similar types of biological samples and / or process cells of the same cell type and / or input cells.

[0097] Cells and compositions prepared by the method, including pharmaceutical compositions and formulations, as well as kits, systems, and apparatus for performing the method, are also provided. Methods for using cells, including therapeutic methods such as methods for adoptive cell therapy, as well as compositions prepared by the method, and pharmaceutical compositions for administration to a subject are also provided.

[0098] A. Sample and cell preparation In certain embodiments, the methods provided are used in connection with isolating, selecting and / or concentrating cells from a biological sample to produce one or more input compositions of concentrated cells, e.g., T cells. In some embodiments, the methods provided involve isolating cells or compositions thereof obtained from a biological sample, such as cells obtained from or derived from a subject, such as a subject having a particular disease or condition, requiring cell therapy, or a subject on which cell therapy is performed. In some embodiments, the subject is a human, e.g., a patient requiring a particular therapeutic intervention, e.g., adoptive cell therapy, for which cells are isolated, processed and / or manipulated. Thus, in some embodiments, the cells are primary cells, e.g., primary human cells. Samples include tissues, body fluids, and other samples taken directly from a subject. A biological sample may be a sample obtained directly from a biological source or a sample that has been processed. Biological samples include, but are not limited to, body fluids, e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, and processed samples derived therefrom.

[0099] In some contexts, a sample is blood or a blood-derived sample, an apheresis product or a leukocyte apheresis product, or derived therefrom. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemia, lymphoma, lymph nodes, intestinal-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lungs, stomach, intestines, colon, kidneys, pancreas, breasts, bones, prostate, cervix, testes, ovaries, tonsils or other organs, and / or cells derived therefrom. In the context of cell therapy, such as adoptive cell therapy, samples may include autologous and allogeneic source-derived samples.

[0100] In some cases, cells derived from the circulating blood of the subject are obtained, for example, by apheresis or leukocyte apheresis. The sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets, in some aspects, and cells other than erythrocytes and platelets, in others.

[0101] In some embodiments, blood cells collected from the subject are washed, for example, to remove the plasma fraction and to place the cells into a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the washing solution is devoid of calcium and / or magnesium and / or many or any divalent cations. In some embodiments, the washing step is achieved by a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some embodiments, the washing step is achieved by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, after washing, the cells are, for example, Ca 2+ / Mg 2+ The cells are resuspended in various biocompatible buffers, such as PBS, which do not contain [specific component]. In certain embodiments, components of the blood cell sample are removed, and the cells are resuspended directly in the culture medium.

[0102] In some embodiments, the preparation method includes a step of freezing, for example, cryopreserving, the cells either before or after isolation, selection and / or concentration and / or incubation for transduction and manipulation. In some embodiments, the freezing and subsequent thawing steps remove granulocytes and, to some extent, monocytes from the cell population. In some embodiments, the cells are suspended in a freezing solution after a washing step to remove, for example, plasma and platelets. In some aspects, any of a variety of known freezing solutions and parameters may be used. In some embodiments, the cells are, for example, 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%, or approximately 12.5%, approximately 12.0%, approximately 11.5%, approximately 11.0%, approximately 10.5%, approximately 10.0%, approximately 9.5%, approximately 9.0%, approximately 8.5%, or approximately 8.0%. The product is frozen, for example, cryopreserved or cryopreserved, in a medium and / or solution having a final concentration of DMSO of approximately 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or 5.0%, or 1% to 15%, 6% to 12%, 5% to 10%, 5% to 10%, or 6% to 8%. In certain embodiments, cells are frozen, for example, cryopreserved or cryopreserved in media and / or solutions having final concentrations of HSA of 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%, or approximately 5.0%, approximately 4.5%, approximately 4.0%, approximately 3.5%, approximately 3.0%, approximately 2.5%, approximately 2.0%, approximately 1.5%, approximately 1.25%, approximately 1.0%, approximately 0.75%, approximately 0.5%, or approximately 0.25%, or 0.1% to -5%, 0.25% to 4%, 0.5% to 2%, or 1% to 2%. One example involves using PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing medium. This is then diluted 1:1 with the medium to final concentrations of 10% and 4% DMSO and HSA, respectively.Next, the cells are generally frozen to -80°C or approximately -80°C at a rate of 1° or approximately 1° per minute and stored in the gas phase of a liquid nitrogen storage tank.

[0103] In some embodiments, the isolation of cells or populations includes one or more preparation and / or inaffinity-based cell separation steps. In some examples, cells are washed, centrifuged and / or incubated in the presence of one or more reagents to remove, for example, unwanted components, concentrate desired components, or lyse or remove cells sensitive to a particular reagent. In some examples, cells are separated based on one or more properties such as density, adhesion characteristics, size, sensitivity and / or resistance to a particular component. In some embodiments, the method includes the preparation of leukocytes from peripheral blood by lysing erythrocytes, and density-based cell separation methods such as centrifugation by Percoll or Ficoll gradient.

[0104] In some embodiments, at least part of the selection step includes incubation of cells with selection reagents. Incubation with one or more selection reagents as part of a selection method that may be performed using one or more selection reagents to select one or more different cell types based on the intracellular or cellular expression or presence of one or more specific molecules such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method using one or more selection reagents for separation based on such markers may be used. In some embodiments, one or more selection reagents result in separation which is separation based on affinity or immunoaffinity. For example, in some aspects, selection includes incubation with one or more reagents for separating cells and cell populations based on the cellular expression or expression level of one or more markers, typically cell surface markers, which is by incubation with an antibody, or with a binding partner that specifically binds to such markers, followed generally by a washing step, and separating cells bound to the antibody or binding partner from cells not bound to the antibody or binding partner.

[0105] In some aspects of such processes, a certain volume of cells is mixed with a certain amount of a selection reagent based on a desired affinity. Selection based on immunoaffinity can be carried out using any system or method that results in a favorable energetic interaction between the cells to be separated and a marker on the cell, e.g., an antibody on a solid surface or other binding partner, e.g., a molecule that specifically binds to a particle. In some embodiments, the method is carried out using particles such as beads coated with a selection agent (e.g., an antibody) specific to the cell marker, e.g., magnetic beads. The particles (e.g., beads) can be incubated or mixed with cells in a container such as a tube or bag, with shaking or mixing, at a constant cell density-to-particle (e.g., bead) ratio to promote an energetically favorable interaction. In other cases, the method involves cell selection in which all or part of the selection is carried out, for example, in the internal cavity of a centrifugal chamber under centrifugal rotation. In some embodiments, the incubation of cells with the selection reagent, e.g., an immunoaffinity-based selection reagent, is carried out in a centrifugal chamber. In certain embodiments, isolation or separation is performed using a system, device, or apparatus described in international patent application publication numbers WO2009 / 072003 or US20110003380A1. For example, the system is the one described in international publication number WO2016 / 073602.

[0106] In some embodiments, by performing such a selection process or part thereof (e.g., incubation with antibody-coated particles, e.g., magnetic beads) within the cavity of a centrifugal chamber, the user can control certain parameters such as the volume of various solutions, the addition of solution during processing, and the timing thereof, which can offer 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 the particles used for selection (e.g., bead reagents) and, consequently, the chemical potential of the solution, without affecting the total number of cells in the cavity. This can enhance the paired interaction between the cells being processed and the particles used for selection. In some embodiments, by performing the incubation process within the chamber, for example, in conjunction with the systems, circuits, and controls described herein, the user can agitate the solution at a desired time during incubation, thereby improving the interaction.

[0107] In some embodiments, at least part of the selection process, including incubation of cells with a selection reagent, is carried out in a centrifuge chamber. In some aspects of such a process, a certain volume of cells is mixed with a selection reagent based on a desired affinity in a much smaller amount than is typically used when performing a similar selection in a tube or container to select the same number and / or volume of cells according to the manufacturer's instructions. In some embodiments, one or more amounts of selection reagent are used, according to the manufacturer's instructions, which are 5% or less or about 5%, 10% or less or about 10%, 15% or less or about 15%, 20% or less or about 20%, 25% or less or about 25%, 50% or less or about 50%, 60% or less or about 60%, 70% or less or about 70%, or 80% or less or about 80%.

[0108] In some embodiments, cells are incubated in a composition that also contains a selection buffer containing a selection reagent, such as a molecule, polymer, or surface, optionally conjugated to a scaffold, such as magnetic beads, or magnetic beads, for selection, for example, selection based on the immunoaffinity of cells, where the cells are to be concentrated and / or depleted within the chamber cavity, but not to other surface markers on cells in the composition. In some embodiments, the selection reagent is added to the cells in the chamber cavity in a substantially smaller or nearly smaller amount (e.g., 5% or less, 10% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% or less or 80% or less of that amount) compared to the amount of selection reagent that would normally be used or required to achieve approximately the same or similar efficiency as selecting the same number of cells or the same volume of cells when selection is performed in the tube with shaking or rotation as described.In some embodiments, incubation is carried out by adding a selection buffer to the cells and selection reagent, for example, 10 mL to 200 mL or about 10 mL to about 200 mL, for example, at least 10 mL, at least 20 mL, at least 30 mL, at least 40 mL, at least 50 mL, at least 60 mL, at least 70 mL, at least 80 mL, at least 90 mL, at least 100 mL, at least 150 mL or at least 200 mL, or at least about 10 mL, at least about 20 mL, at least about 30 mL, at least about 40 mL, at least about The target volume is achieved by incubation of 50 mL, at least about 60 mL, at least about 70 mL, at least about 80 mL, at least about 90 mL, at least about 100 mL, at least about 150 mL or at least about 200 mL, or about 10 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 70 mL, about 80 mL, about 90 mL, about 100 mL, about 150 mL or about 200 mL, or 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL or 200 mL of reagents. In some embodiments, the selective buffer and selective reagents are pre-mixed before being added to the cells. In some embodiments, the selective buffer and selective reagents are added to the cells separately. In some embodiments, selective incubation is carried out under periodic, gentle mixing conditions that help promote energetically favorable interactions, thereby enabling the use of relatively small overall selective reagents while achieving high selective efficiency.

[0109] In some embodiments, the total incubation period with the selected reagent is 5 minutes to 6 hours or about 5 minutes to about 6 hours, for example, 30 minutes to 3 hours, for example, at least 30 minutes, at least 60 minutes, at least 120 minutes or at least 180 minutes, or at least about 30 minutes, at least about 60 minutes, at least about 120 minutes or at least about 180 minutes.

[0110] In some embodiments, incubation is generally carried out under mixed conditions such as the presence of spin, and generally at a relatively low force or speed, such as a speed slower than the speed used to pelletize the cells, for example, 600 rpm to 1700 rpm or about 600 rpm to about 1700 rpm (e.g., 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm, or about 600 rpm, about 1000 rpm, or about 1500 rpm or about 1700 rpm, or at least 6 The spinning is performed at RCF on the walls of a chamber or other container of a sample or chamber of 80g to 100g or about 80g to about 100g (e.g., 80g, 85g, 90g, 95g or 100g, or about 80g, about 85g, about 90g, about 95g or about 100g, or at least 80g, at least 85g, at least 90g, at least 95g or at least 100g) at 00 rpm, at least 1000 rpm, or at least 1500 rpm or at least 1700 rpm. In some embodiments, the spinning is performed using repeated intermittent periods of spinning at such low speeds followed by periods of stillness, for example, spinning and / or resting for 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds or 10 seconds, for example, spinning for about 1 second or 2 seconds followed by resting for about 5 seconds, about 6 seconds, about 7 seconds or about 8 seconds.

[0111] In some embodiments, such a process is carried out within a fully closed system in which the chambers are integrated. In some embodiments, this process (and in some aspects also one or more additional steps, such as a preceding washing step for washing a sample containing cells, such as an apheresis sample) is carried out automatically using an automated program to draw the cells, reagents, and other components into the chamber at appropriate times, extrude them, and centrifuge them to complete the washing and binding process in a single closed system.

[0112] In some embodiments, after incubation and / or mixing of cells and the selection reagent and / or reagent, the incubated cells are separated to select cells based on the presence or absence of a particular one or more reagents. In some embodiments, the separation is performed within the same closed system in which the incubation of cells and the selection reagent was performed. In some embodiments, after incubation with the selection reagent, the incubated cells, including cells bound to the selection reagent, are transferred to a system for separation of cells based on immunoaffinity. In some embodiments, the system for separation based on immunoaffinity is or includes a magnetic separation column.

[0113] Such separation steps can be based on positive selection, where cells bound to a reagent such as an antibody or binding partner are retained for subsequent use, and / or negative selection, where cells not bound to a reagent such as an antibody or binding partner are retained. In some cases, both fractions are retained for subsequent use. In some situations, negative selection that specifically identifies cell types within heterogeneous populations may be particularly useful when antibodies are unavailable, so that separation is best performed based on markers expressed by cells outside the desired population.

[0114] In some embodiments, the process steps further include negative and / or positive selection of incubated cells, for example, using a system or apparatus capable of performing affinity-based selection. In some embodiments, isolation is performed by enriching a particular cell population by positive selection or depleting a particular cell population by negative selection. In some embodiments, positive or negative selection involves selecting cells that are positively or negatively selected or that express a marker at a relatively high level. high ) expression (marker + This is achieved by incubating cells with one or more antibodies or other conjugates that specifically bind to one or more surface markers.

[0115] Isolation does not necessarily 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 cells expressing a marker, indicates an increase in the number or proportion of such cells, but does not necessarily 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 cells expressing a marker, indicates a decrease in the number or proportion of such cells, but none of such cells need to be completely removed.

[0116] In some cases, multiple separation steps are performed, in which fractions selected positively or negatively from one step are subjected to further separation steps, such as subsequent positive or negative selection. In some cases, a single separation step can deplete cells expressing multiple markers simultaneously, for example, by incubating cells with multiple antibodies or binding partners specific to each of the markers targeted for negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating cells with multiple antibodies or binding partners expressed on various cell types. In certain embodiments, the separation step is repeated and / or performed two or more times, in which fractions selected positively or negatively from one step are subjected to the same separation step, such as repeated positive or negative selection. In some cases, a single separation step is repeated and / or performed two or more times, for example, to increase the purity of selected cells and / or to further remove and / or deplete negatively selected cells from the negatively selected fraction. In certain embodiments, one or more separation steps are performed two, three, four, five, six, seven, eight, nine, ten, or more than ten times. In certain embodiments, one or more selection steps are performed one to ten times, one to five times, or three to five times, and / or repeated.

[0117] For example, in some aspects, a specific subpopulation of T cells, such as cells that are positive for or have high expression levels of one or more surface markers, e.g., CD28+ T cells, CD62L+ T cells, CCR7+ T cells, CD27+ T cells, CD95+ T cells, CD127+ T cells, CD4+ T cells, CD8+ T cells, CD45RA+ T cells, and / or CD45RO+ T cells, is isolated by positive selection techniques or negative selection techniques. In some embodiments, such cells are selected by incubation with one or more antibodies or binding partners that specifically bind to such markers. In some embodiments, the antibodies or binding partners can be conjugated, for example, directly or indirectly, to a solid support or matrix to result in selection, such as magnetic beads or paramagnetic beads. For example, CD3+ T cells and CD28+ T cells can be positively selected using magnetic beads conjugated with anti-CD3 / anti-CD28 (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander and / or Exp ACT® beads).

[0118] In some embodiments, T cells are isolated from PBMC samples by negative selection of markers expressed on B cells, monocytes, or other leukocytes, such as CD14, which are non-T cells. In some aspects, a CD4+ or CD8+ selection step is used to isolate CD4+ helper T cells and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further subdivided into subpopulations by positive or negative selection of markers expressed or relatively highly expressed on one or more naive, memory, and / or effector T cell subpopulations.

[0119] In some embodiments, for example, positive or negative selection based on surface antigens associated with each subpopulation further enriches or depletes naive stem cells, central memory stem cells, effector memory stem cells and / or central memory stem cells in CD8+ cells. In some embodiments, central memory T(T)CM Cell enrichment is performed to enhance the effect, such as improving long-term survival, expansion, and / or engraftment after administration, and this is particularly robust in some aspects, especially 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 aspects, T CM Combining CD8+ T cells and CD4+ T cells, which are rich in this compound, further enhances the effect.

[0120] In some embodiments, memory T cells are present in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. In PBMCs, the CD62L-CD8+ fraction and / or CD62L+CD8+ fraction can be enriched or depleted, for example, by using anti-CD8 antibodies and anti-CD62L antibodies.

[0121] In some embodiments, the central memory T(T CM Cell enrichment is based on positive expression or high surface expression of CD45RO, CD62L, CCR7, CD27, CD28, CD95, CD3 and / or CD127, and in some aspects, on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, T CM Isolation of a cell-enriched CD8+ population is performed by depleting cells expressing CD4, CD14, and CD45RA, and by positive selection or enrichment of cells expressing CD62L. In one aspect, central memory T(T) CMCell enrichment is carried out starting with a negative fraction of cells selected based on CD4 expression, which is then subjected to negative selection based on CD14 and CD45RA expression and positive selection based on CD62L. Such selections are carried out simultaneously in some aspects and sequentially in others. In some aspects, the same CD4 expression-based selection step used to prepare the CD8+ cell population or subpopulation is also used to generate a CD4+ cell population or subpopulation, so that both positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the method, optionally followed by one or more additional positive or negative selection steps. In some aspects, the selection of the CD4+ cell population and the selection of the CD8+ cell population are carried out simultaneously. In some aspects, the selection of the CD4+ cell population and the CD8+ cell population are carried out sequentially in either order. In some aspects, the method for selecting cells may include that described in published U.S. Patent Application No. 20170037369. In some embodiments, the selected CD4+ cell population and the selected CD8+ cell population may be combined after selection. In some aspects, the selected CD4+ cell population and the selected CD8+ cell population may be combined in a bioreactor bag as described herein.

[0122] In some embodiments, central memory CD8+ cells are CD27+, CD28+, CD62L+, CCR7+, CD45RA-, and / or CD45RO+. In some embodiments, central memory CD8+ cells are CD62L+ and CD45RO+. In some embodiments, central memory CD8+ cells are CCR7+ and CD45RO+. In some embodiments, central memory CD8+ cells are CCR7+ and CD45RA-. In some embodiments, central memory CD8+ cells are CD62L+ and CCR7+. In some embodiments, central memory CD8+ cells are CD62L+ / CD45RA-, CCR7+ / CD45RA-, CD62L+ / CCR7+, or CD62L+ / CCR7+ / CD45RA-, and moderately to highly express 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-.

[0123] In certain embodiments, a biological sample, such as a PBMC or other leukocyte sample, is used to select CD4+ T cells that retain both negative and positive fractions. In certain embodiments, CD8+ T cells are selected from the negative fraction. In some embodiments, a biological sample is used to select CD8+ T cells that retain both negative and positive fractions. In certain embodiments, CD4+ T cells are selected from the negative fraction.

[0124] In certain cases, PBMC samples or other leukocyte samples are subjected to selection of CD4+ cells that retain both negative and positive fractions. The negative fraction is then subjected to negative selection based on the expression of CD14 and CD45RA or CD19, and positive selection based on markers characteristic of central memory T cells such as CD62L or CCR7, in which case positive and negative selection are performed in either order.

[0125] In some embodiments, CD4+ T helper cells are classified into naive cells, central memory cells, and effector cells by identifying cell populations possessing cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO- T cells, CD45RA+ T cells, CD62L+ T cells, 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-, CCR7+ / CD45RA-, CD62L+ / CCR7+, or CD62L+ / CCR7+ / CD45RA-, expressing CD44 to a moderate to high degree. In some embodiments, central memory CD4+ cells are CD27+ / CD28+ / CD62L+ / CD45RA-, CD27+ / CD28+ / CCR7+ / CD45RA-, CD27+ / CD28+ / CD62L+ / CCR7+, or CD27+ / CD28+ / CD62L+ / CCR7+ / CD45RA-. In some embodiments, effector CD4+ cells are CD62L- and CD45RO-.

[0126] In one example, to enrich CD4+ cells by negative selection, a monoclonal antibody cocktail typically contains antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner binds to a solid support or matrix, such as magnetic or paramagnetic beads, to enable cell separation for positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (as outlined in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, pp. 17-25 Edited by: SABrooks and U.Schumacher (copyright) Humana Press Inc., Totowa, NJ).

[0127] In some cases, the sample to be incubated, or the cell composition to be separated, is incubated with a selective reagent containing small magnetizable or magnetically responsive materials, such as magnetically responsive particles or microparticles, e.g., paramagnetic beads (e.g., Dynalbeads or MACS® beads). The magnetically responsive material, e.g., particles, is generally bound directly or indirectly to a binding partner, such as an antibody, that specifically binds to molecules, such as surface markers, present in a single cell, a group of cells, or a cell population that is to be separated, e.g., selected to be negative or positive.

[0128] In some embodiments, magnetic particles or magnetic beads comprise a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. Many well-known magnetically responsive materials exist for use in magnetic separation methods. Suitable magnetic particles include those described in Molday's U.S. Patent No. 4,452,773 and European Patent Specification EP 452342 B, which are incorporated herein by reference. Other examples include colloidal-sized particles, such as those described in Owen's U.S. Patent No. 4,795,698 and Liberti et al.'s U.S. Patent No. 5,200,084.

[0129] Incubation is generally carried out under conditions in which an antibody or binding partner, or a secondary antibody or other reagent that specifically binds to such an antibody or binding partner attached to a molecule, such as magnetic particles or magnetic beads, specifically binds to cell surface molecules if present in the cells of the sample.

[0130] In certain embodiments, magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, magnetic particles are attached to cells via a coating of primary antibodies specific to one or more markers. In certain embodiments, cells are labeled with a primary antibody or binding partner, rather than beads, and then magnetic particles coated with a cell-type-specific secondary antibody or other binding partner (e.g., streptavidin) are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with a biotinylated primary antibody or a biotinylated secondary antibody.

[0131] In some cases, separation is achieved by placing the sample in a magnetic field and attracting cells to which magnetically responsive or magnetizable particles have been attached to a magnet, thereby separating them from unlabeled cells. In the case of positive selection, cells attracted to the magnet are retained. In the case of negative selection, cells that are not attracted (unlabeled cells) are retained. In some cases, a combination of positive and negative selection is performed during the same selection process, where the positive and negative fractions are retained and further processed or subjected to additional separation steps.

[0132] In some embodiments, affinity-based selection is achieved by magnetically activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetically activated cell sorting (MACS), such as the CliniMACS system, allows for high-purity selection of cells with attached magnetized particles. In certain embodiments, MACS operates in a mode in which non-target and target species are successively eluted after the application of an external magnetic field. That is, cells attached to magnetized particles are retained in place while non-attached species are being eluted. Then, after this initial elution step is complete, species that were trapped in the magnetic field and whose elution was prevented are released in some way so that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from heterogeneous cell populations.

[0133] In some embodiments, the magnetically responsive particles remain attached to cells that are subsequently incubated, cultured, and / or manipulated. In some embodiments, the particles remain attached to 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 a cleavable linker. In some embodiments, the magnetizable particles are biodegradable.

[0134] In some scenarios, separation and / or other processes are performed using the CliniMACS system (Miltenyi Biotec), for example, to automatically separate cells at a clinical scale in a closed, sterile system. Components may include a built-in microcomputer, a magnetic separation unit, a peristaltic pump, and various pinch valves. The built-in computer, in some scenarios, controls all components of the instrument and commands the system to perform repetitive procedures in a unified sequence. The magnetic separation unit, in some scenarios, includes a movable permanent magnet and a holder for the selective column. The peristaltic pump controls the flow rate across the entire tubing set and, together with the pinch valves, ensures that the buffer flows through the system in a controlled manner and that the cells remain continuously suspended.

[0135] The CliniMACS system, in some aspects, uses magnetizable particles bound to antibodies, supplied dissolved in a sterile, non-pyrogenic solution. In some embodiments, cells are labeled with magnetic particles, and then washed to remove excess particles. The cell preparation bag is then connected to a tube set, and the tube set is connected to a buffer-containing bag and a cell collection bag. The tube set consists of pre-assembled sterile tubes, including a pre-column and a separation column, and is for single use only. After the separation program is started, the system automatically applies the cell sample to the separation column. Labeled cells are retained in the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell population for use with the methods described herein is unlabeled and not retained in the column. In some embodiments, the cell population for use with the methods described herein is labeled and retained in the column. In some embodiments, the cell population for use with the methods described herein is eluted from the column after the magnetic field is removed and collected in the cell collection bag.

[0136] In certain embodiments, separation and / or other steps are performed using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system includes a cell processing unit that, in some aspects, automatically washes cells and fractionates them by centrifugation. The CliniMACS Prodigy system may also include a built-in camera and image recognition software that determines the optimal cell fractionation endpoint by identifying macroscopic layers of the cell product of the source. For example, peripheral blood is automatically separated into layers of red blood cells, white blood cells, and plasma. The CliniMACS Prodigy system may also include an internal cell culture chamber for performing cell culture protocols, such as cell differentiation and proliferation, antigen addition, and long-term cell culture. An input port allows for aseptic removal and replenishment of culture medium, and cells can be monitored using an internal microscope. For example, see 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.

[0137] In some embodiments, the cell populations described herein are collected and concentrated (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are transported into a fluid flow. In some embodiments, the cell populations described herein are collected and concentrated (or depleted) via preparative scale (FACS) sorting. In certain embodiments, the cell populations described herein are collected and concentrated (or depleted) by using a microelectromechanical system (MEMS) chip in combination with a FACS-based detection system (see, e.g., WO2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376). In any case, cells can be labeled with multiple markers, thereby enabling the isolation of precisely defined T cell subsets with high purity.

[0138] In some embodiments, antibodies or binding partners are labeled with one or more detectable markers to facilitate separation for positive and / or negative selection. For example, separation may be based on binding to a fluorescently labeled antibody. In some examples, cell separation based on the binding of antibodies or other binding partners specific to one or more cell surface markers is performed in a fluid flow, for example, by a fluorescently labeled cell sorter (FACS) including a preparative scale (FACS) and / or a microelectromechanical system (MEMS) chip, in combination with, for example, a flow cytometry detection system. Such methods make it possible to perform positive and negative selection simultaneously based on multiple markers.

[0139] In some embodiments, isolation and / or selection yield one or more compositions of enriched T cells, e.g., CD3+ T cells, CD4+ T cells, and / or CD8+ T cells. In some embodiments, two or more distinct compositions of enriched T cells are isolated, selected, enriched, or obtained from a single biological sample. In some embodiments, distinct compositions are collected, extracted, and / or obtained from the same subject and isolated, selected, enriched, and / or obtained from distinct biological samples.

[0140] In certain embodiments, isolation and / or selection yield one or more compositions of enriched T cells comprising at least 60% or about 60%, at least 65% or about 65%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%, at least 98% or about 98%, at least 99% or about 99%, at least 99.5% or about 99.5%, at least 99.9% or about 99.9%, or 100% or about 100%. In certain embodiments, the enriched T cell composition consists essentially of CD3+ T cells.

[0141] In certain embodiments, isolation and / or concentration yield a composition of enriched CD4+ T cells comprising at least 60% or about 60%, at least 65% or about 65%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%, at least 98% or about 98%, at least 99% or about 99%, at least 99.5% or about 99.5%, at least 99.9% or about 99.9%, or 100% or about 100%. In certain embodiments, the CD4+ T cell input composition contains less than 40% or about 40%, less than 35% or about 35%, less than 30% or about 30%, less than 25% or about 25%, less than 20% or about 20%, less than 15% or about 15%, less than 10% or about 10%, less than 5% or about 5%, less than 1% or about 1%, less than 0.1% or about 0.1%, or less than 0.01% or about 0.01% of CD8+ T cells, and / or does not contain CD8+ T cells, and / or does not contain or substantially contain CD8+ T cells. In some embodiments, the enriched T cell composition consists essentially of CD4+ T cells.

[0142] In certain embodiments, isolation and / or concentration yield a composition of enriched CD8+ T cells comprising at least 60% or about 60%, at least 65% or about 65%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%, at least 98% or about 98%, at least 99% or about 99%, at least 99.5% or about 99.5%, at least 99.9% or about 99.9%, or 100% or about 100%. In certain embodiments, the CD8+ T cell composition contains less than 40% or about 40%, less than 35% or about 35%, less than 30% or about 30%, less than 25% or about 25%, less than 20% or about 20%, less than 15% or about 15%, less than 10% or about 10%, less than 5% or about 5%, less than 1% or about 1%, less than 0.1% or about 0.1%, or less than 0.01% or about 0.01% of CD4+ T cells, and / or does not contain CD4+ T cells, and / or does not contain or substantially contain CD4+ T cells. In some embodiments, the enriched T cell composition consists essentially of CD8+ T cells.

[0143] In some embodiments, one or more compositions of enriched T cells are frozen, e.g., cryopreserved and / or cryo-freezed after isolation, selection and / or enrichment. In certain embodiments, compositions of enriched CD4+ T cells are frozen, e.g., cryopreserved and / or cryo-freezed after isolation, selection and / or enrichment. In certain embodiments, compositions of enriched CD8+ T cells are frozen, e.g., cryopreserved and / or cryo-freezed after isolation, selection and / or enrichment. In some embodiments, one or more compositions of enriched T cells are frozen, e.g., cryopreserved and / or cryo-freezed before any step of incubation, activation, stimulation, manipulation, transduction, transfection, culture, expansion, harvesting and / or formulation of the cell composition. In certain embodiments, compositions of enriched CD4+ T cells are frozen, e.g., cryopreserved and / or cryo-freezed before any step of incubation, activation, stimulation, manipulation, transduction, transfection, culture, expansion, harvesting and / or formulation of the cell composition. In some embodiments, enriched CD8+ T cell compositions are frozen, e.g., cryopreserved and / or cryo-frozen, before any step of incubation, activation, stimulation, manipulation, transduction, transfection, culture, expansion, harvesting and / or formulation of the cell composition. In certain embodiments, one or more cryo-frozen input compositions are stored, e.g., at -80°C or about -80°C for 12 to 7 days, 24 to 120 hours or 2 to 5 days. In certain embodiments, one or more cryo-frozen input compositions are stored at -80°C or about -80°C for a period of less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, or less than 5 days, less than 4 days, less than 3 days, less than 2 days or less than 1 day. In some embodiments, one or more cryo-freezed input compositions are stored at -80°C or about -80°C for 1 day or about 1 day, 2 days or about 2 days, 3 days or about 3 days, 4 days or about 4 days, 5 days or about 5 days, or 6 days or about 6 days.

[0144] In some embodiments, a sample containing cells (e.g., apheresis product or leukocyte apheresis product) is washed to remove one or more anticoagulants, such as heparin, that were added during apheresis or leukocyte apheresis.

[0145] In some embodiments, cell-containing samples (e.g., whole blood samples, buffy coat samples, peripheral blood mononuclear cell (PBMC) samples, unfractionated T cell samples, lymphocyte samples, leukocyte samples, apheresis products, or leukocyte apheresis products) are cryopreserved and / or cryoprotected (e.g., frozen) and then thawed before any step of isolation, selection, activation, stimulation, manipulation, transduction, transfection, incubation, culture, harvesting, formulation, and / or administration of the formulated cell population to a subject.

[0146] In certain embodiments, the apheresis product or leukocyte apheresis product is cryopreserved and / or cryoprotected (e.g., frozen) as described below, and then thawed before being subjected to a cell selection or isolation step (e.g., a T cell selection or isolation step). In some embodiments, after the cryopreserved and / or cryoprotected apheresis product or leukocyte apheresis product has been subjected to a T cell selection or isolation step, no additional cryopreservation and / or cryoprotection steps are performed during or between subsequent steps such as activation, stimulation, manipulation, transduction, transfection, incubation, culture, harvesting, formulation, and / or administration of the formulated cell population to a subject. For example, T cells selected from a thawed cryopreserved and / or cryoprotected apheresis product or leukocyte apheresis product are not cryopreserved and / or cryoprotected again before being thawed for downstream processes such as T cell activation / stimulation or transduction.

[0147] In certain embodiments, cryopreserved and / or cryoprotected apheresis products or leukocyte apheresis products are banked (e.g., without selecting T cells before freezing the sample), which in some aspects can provide greater flexibility to subsequent manufacturing processes. In one aspect, banking cells before selection may increase cell yield in downstream processes, and banking cells early may mean improved health of the cells, making it easier to meet manufacturing success criteria. In another aspect, cryopreserved and / or cryoprotected apheresis products or leukocyte apheresis products, once thawed, can be subjected to one or more different selection methods. The advantages of this approach include, among other things, enhancing the availability, efficacy, and / or other aspects of cells in cell therapies for the treatment of target diseases or conditions, such as donors and / or other recipients of the sample.

[0148] In some embodiments, the sample (e.g., apheresis sample or leukocyte apheresis sample) is collected after the donor has been diagnosed with a disease or condition, before or without prior cell selection (e.g., without prior T cell selection such as chromatographic selection), and cryopreserved and / or cryoprotected. In some embodiments, the timing of cryopreservation is also before the donor receives one or more of the following: any initial treatment for the disease or condition, any targeted therapy or any therapy labeled for the treatment of the disease or condition, or any therapy other than radiation and / or chemotherapy. In some embodiments, the sample is collected after the first relapse of the disease following initial treatment for the disease, and before the donor or subject receives any subsequent treatment for the disease. The initial treatment and / or subsequent treatment may be therapies other than cell therapy. In some embodiments, the collected cells may be used for cell therapy after the initial treatment and / or subsequent treatment. In one aspect, cryopreserved and / or cryoprotected samples without prior cell selection may help reduce initial costs, such as those associated with untreated patients in randomized clinical trials that may cross over and subsequently require treatment.

[0149] In some embodiments, samples (e.g., apheresis samples or leukocyte apheresis samples) are collected after second-line treatment for the disease and after a second relapse of the disease before the donor or subject receives further treatment for the disease, prior to or without prior cell selection (e.g., without prior T-cell selection such as chromatographic selection), and cryopreserved and / or cryoprotected. In some embodiments, patients are identified as being at high risk of relapse after second-line treatment, for example, by assessing specific risk factors. In some embodiments, risk factors are based on disease type and / or genetic characteristics, such as double-hit lymphoma, primary refractory cancer, or activated B-cell lymphoma. In some embodiments, risk factors are based on clinical features such as early relapse after first-line treatment or other poor prognostic indicators after treatment (e.g., IPI (International Prognostic Index) > 2).

[0150] In some embodiments, samples (e.g., apheresis samples or leukocyte apheresis samples) are collected before the donor or subject is diagnosed with any disease, prior to or without prior cell selection (e.g., without prior T cell selection such as chromatographic selection), and cryopreserved and / or cryoprotected. In some embodiments, the donor or subject may be determined to be at risk of developing any disease. In some embodiments, the donor or subject may be a healthy subject. In some cases, the donor or subject may choose to bank or store cells if cell therapy is needed at a later stage of life, even if they are not considered to be at risk of developing or being diagnosed with any disease. In some embodiments, the donor or subject may be considered to be at risk of developing any disease based on factors such as gene mutations, genetic abnormalities, gene disruption, family history, protein abnormalities (such as defects in protein production and / or processing), and lifestyle choices that may increase the risk of developing any disease. In some embodiments, cells are collected as a prophylactic agent.

[0151] In some embodiments, cryopreserved and / or cryoprotected cell samples (e.g., apheresis samples or leukocyte apheresis samples), such as cell samples that have not undergone prior cell selection (e.g., without prior T cell selection such as chromatographic selection), are stored or banked for periods of 12 hours or more, 24 hours or more, 36 hours or more, or 48 hours or more, or approximately 12 hours, approximately 24 hours, approximately 36 hours or approximately 48 hours. In some embodiments, samples are stored or banked for periods of 1 week or more, 2 weeks or more, 3 weeks or more, or 4 weeks or more. In some embodiments, samples are stored or banked for extended periods. In some cases, the sample is kept for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 7 months or more, 8 months or more, 9 months or more, 10 months or more, 11 months or more, 1 year or more, 2 years or more, 3 years or more, 4 years or more, 5 years or more, 6 years or more, 7 years or more, 8 years or more, 9 years or more, 10 years or more, 11 years or more, 12 years or more, 13 years or more, 14 years or more, 15 years or more, 16 years or more, 17 years or more, 18 years or more, 19 years or more, 20 years or more, 25 years or more, 30 years or more, and 35 years. The above information will be preserved for 40 years or more, or for a period of approximately 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.

[0152] In some embodiments, apheresis samples or leukocyte apheresis samples collected from a donor are transported to a storage or processing facility in a cooled environment and / or cryogenically stored in the storage facility or processed in the processing facility. In some embodiments, the samples are processed before transport by selecting T cells, such as CD4+ T cells and / or CD8+ T cells. In some embodiments, such processing is performed after transport and before cryogenic storage of the samples. In some embodiments, processing is performed after thawing the samples following cryogenic storage.

[0153] By allowing donors, and by extension their cells, to preserve their cells before extensive treatment for the disease and / or before the onset or diagnosis of the disease or symptoms, such cells may have certain advantages for use in cell therapy compared to cells collected after one or more treatments. For example, cells collected before one or more treatments may be healthier, exhibit higher levels of specific cellular activity, proliferate more rapidly, and / or be more receptive to genetic manipulation than cells that have undergone several treatments. Another example of the advantages of 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 become readily available when recipients later require them. This increases the capacity of the apheresis laboratory and provides greater flexibility for technicians in scheduling the apheresis collection process.

[0154] Exemplary methods and systems for cryogenic preservation and processing of cells obtained from samples such as apheresis samples may include those described in International Publication Application No. WO2018170188. In some embodiments, the methods and systems include collecting apheresis before a patient requires cell therapy and then cryopreserving the apheresis sample for later use in a process of manipulating cells, such as T cells, using recombinant receptors (e.g., CARs). In some cases, such a process may include those described herein. In some embodiments, an apheresis sample is collected from a subject and cryopreserved before subsequent T cell selection, cell population activation, stimulation, manipulation, transduction, transfection, incubation, culture, harvesting, formulation, and / or administration of the formulated cell population to the subject. In such examples, the cryopreserved apheresis sample is thawed before the sample is subjected to one or more selection steps, such as those described herein.

[0155] In some embodiments, cryopreserved and / or cryoprotected cell samples (e.g., apheresis samples or leukocyte apheresis samples), for example, cell samples that have not been subjected to prior cell selection (e.g., without prior T cell selection such as chromatographic selection), are thawed before use in downstream processes for producing cell populations for cell therapy, for example, T cell populations containing CAR+ T cells. In some embodiments, such cryopreserved and / or cryoprotected cell samples (e.g., apheresis samples or leukocyte apheresis samples) are used in connection with processes provided herein for engineered T cell therapies such as CAR+ T cell therapy. In certain examples, no additional cryopreservation steps are performed before or during the collection / formulation steps.

[0156] 1. Input composition In certain embodiments, the method provided is used in connection with the manufacture and / or preparation of cell input compositions. In certain embodiments, the input cell composition is a composition of cells for use in genetic engineering, e.g., cells to be genetically engineered and / or cells to be produced. In certain embodiments, the cells are treated with, contacted with, 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 certain embodiments, the input cell composition contains CD4+ T cells and CD8+ T cells that are naive T cells and / or naive-like T cells.

[0157] In some embodiments, the desired, fixed, and / or controlled ratio is the ratio or number of two types of cells or isolated cell populations contained in an input cell composition designed to yield an output cell composition having a desired, specified, and / or controlled ratio of manipulated CD4+ T cells to CD8+ T cells, or a ratio within an acceptable error rate or its difference, at the completion of incubation and / or manipulation or other processing steps, and / or at thawing and / or immediately before administration to the subject.

[0158] In certain embodiments, the input composition is a composition of enriched CD3+ T cells. In some embodiments, the input composition is or comprises at least 60% or about 60%, at least 65% or about 65%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%, at least 98% or about 98%, at least 99% or about 99%, at least 99.5% or about 99.5%, at least 99.9% or about 99.9%, or 100% or about 100% CD3+ T cells. In some embodiments, the input composition is essentially made up of CD3+ T cells. In certain embodiments, the input composition is a composition of cells enriched with respect to enriched CD4+ T cells and enriched CD8+ T cells. In certain embodiments, the input composition is or comprises at least 60% or about 60%, at least 65% or about 65%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%, at least 98% or about 98%, at least 99% or about 99%, at least 99.5% or about 99.5%, at least 99.9% or about 99.9%, or 100% or about 100% of cells, which are CD4+ T cells or CD8+ T cells. In some embodiments, the input composition essentially consists of CD4+ T cells and CD8+ T cells.

[0159] In certain embodiments, the input composition contains 30% to 70% or about 30% to about 70%, 35% to 65% or about 35% to about 65%, 40% to 60% or about 40% to about 60%, 45% to 55% or about 45% to about 55%, or about 50% or 50% CD4+ T cells and 30% to 70% or about 30% to about 70%, 35% to 65% or about 35% to about 65%, 40% to 60% or about 40% to about 60%, 45% to 55% or about 45% to about 55%, or about 50% or 50% CD8+ T cells. In certain embodiments, the input composition contains 45% to 55% or about 45% to about 55%, about 50%, or 50% CD4+ T cells and 45% to 55% or about 45% to about 55%, about 50%, or 50% CD8+ T cells.

[0160] In some embodiments, at least one distinct composition of enriched CD4+ T cells and at least one distinct composition of enriched CD8+ T cells are isolated, selected, enriched, or obtained from a single biological sample, e.g., a sample of PBMCs or other leukocyte cells from the same donor, such as a patient or a healthy individual. In some embodiments, for example, the distinct composition of source enriched CD4+ T cells and the distinct composition of enriched CD8+ T cells are initially isolated, selected, and / or enriched from the same biological sample, e.g., a single biological sample obtained, collected, and / or extracted from a single subject. In some embodiments, the biological sample is first subjected to the selection of CD4+ T cells, where both 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, where both negative and positive fractions are retained, and the negative fraction is further subjected to the selection of CD4+ T cells. In some embodiments, the selection method is carried out as described in international PCT publication number WO2015 / 164675. In some embodiments, a biological sample is first positively selected for CD8+ T cells to produce at least one composition of enriched CD8+ T cells, and then a fraction negative for CD4+ T cells is positively selected to produce at least one composition of enriched CD4+ T cells, such that at least one composition of enriched CD8+ T cells and at least one composition of enriched CD4+ T cells are distinct compositions from the same biological sample, e.g., from the same donor patient or a healthy individual; and then fractions negative for CD4+ T cells are positively selected to produce at least one composition of enriched CD4+ T cells. In some embodiments, two or more distinct compositions of enriched T cells, such that at least one is a composition of enriched CD4+ T cells and at least one is a distinct composition of enriched CD8+ T cells from the same donor, are separately frozen, e.g., cryopreserved or cryopreserved in cryopreservation medium. In some embodiments, the separately cryopreserved cell compositions are stored and / or transported in separate containers in one or more transports. In some cases, separately cryopreserved cell compositions are thawed and optionally washed.

[0161] In some aspects, two or more separate compositions of enriched T cells, such that at least one is a composition of enriched CD4+ T cells and at least one separate composition of enriched CD8+ T cells derived from the same biological sample, are thawed and mixed, combined, and / or pooled, and the compositions may optionally be washed before or after mixing, combining, and / or pooling. In some aspects, the mixed, combined, and / or pooled, and optionally washed compositions of enriched T cells form an input composition. In some aspects, the input composition (e.g., containing CD4+ T cells and CD8+ T cells in a 1:1 or approximately 1:1 ratio) is activated and / or stimulated by contact with a stimulating reagent (e.g., by incubation with CD3 / CD28 conjugate magnetic beads for T cell activation), and the volume of the cell composition from activation / stimulation is optionally adjusted, e.g., reduced, to achieve a target volume. In some aspects, the activated / stimulated cell composition is manipulated, transduced, and / or transfected, for example, using a retroviral vector encoding a recombinant protein (e.g., CAR), so that the same recombinant protein is expressed in the CD4+ T cells and CD8+ T cells of the cell composition. In some aspects, the volume of the cell composition from the manipulation is optionally adjusted, for example, reduced, to achieve a target volume. In some aspects, the method includes removing the stimulating reagent, for example, magnetic beads, from the cell composition. In some aspects, the cell composition containing the manipulated CD4+ T cells and manipulated CD8+ T cells is cultured, for example, for expansion and proliferation of the CD4+ T cell and / or CD8+ T cell population therein. In certain embodiments, the cell composition from the culture is harvested and / or collected and / or formulated, for example, by washing the cell composition in a formulation buffer. In certain embodiments, the formulated cell composition containing CD4+ T cells and CD8+ T cells is frozen, for example, cryopreserved or cryopreserved in cryopreservation medium. In some cases, the cryopreserved formulations may be stored and / or transported in one or more containers.In some scenarios, the engineered CD4+ T cells and CD8+ T cells in the formulation originate from the same donor or biological sample, express the same recombinant protein (e.g., CAR), and the formulation is administered to the target recipient, such as the same donor, who needs it.

[0162] In certain embodiments, the input composition has a CD4+ T cell:CD8+ T cell ratio of 3:1 to 1:3, 2:1 to 1:2, 1.5 to 0.75, 1.25 to 0.75, or 1.2 to 0.8. In certain embodiments, the input composition has a CD4+ T cell:CD8+ T cell ratio of 1:1 or approximately 1:1.

[0163] In some embodiments, cells from a composition of enriched CD4+ T cells and cells from a composition of enriched CD8+ T cells are mixed, combined, and / or pooled to produce an input composition containing CD4+ T cells and CD8+ T cells. In certain embodiments, the compositions of enriched CD4+ T cells and enriched CD8+ T cells are pooled, mixed, and / or combined before incubating the cells under stimulating conditions. In certain embodiments, the compositions of enriched CD4+ T cells and enriched CD8+ T cells are pooled, mixed, and / or combined after isolating, enriching, and / or selecting CD4+ T cells and CD8+ T cells from a biological sample. In certain embodiments, the compositions of enriched CD4+ T cells and enriched CD8+ T cells are pooled, mixed, and / or combined following freezing, e.g., cryo-freezing and thawing of the compositions of enriched CD4+ T cells and enriched CD8+ T cells.

[0164] In certain embodiments, the input composition is manufactured, generated, or produced by mixing, pooling, and / or combining cells from a composition of enriched CD4+ cells with cells from a composition of enriched CD8+ cells. In certain embodiments, the composition of enriched CD4+ T cells contains at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.9%, or about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 99.9% CD4+ T cells. In certain embodiments, the composition of enriched CD4+ T cells contains 100% CD4+ T cells or about 100% CD4+ T cells. In certain embodiments, the enriched T cell composition contains, and / or does not contain, and / or does not contain CD8+ T cells in amounts of less than 20% or about 20%, less than 10% or about 10%, less than 5% or about 5%, less than 1% or about 1%, less than 0.1% or about 0.1%, or less than 0.01% or about 0.01%. In some embodiments, the cell population consists essentially of CD4+ T cells. In certain embodiments, a composition of enriched CD8+ T cells contains at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.9%, or about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 99.9%, or contains 100% CD8+ T cells, or contains about 100% CD8+ T cells. In certain embodiments, a composition of enriched CD8+ T cells contains less than or about 20%, 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 does not contain CD4+ T cells, and / or does not contain or substantially contains CD4+ T cells. In some embodiments, the cell population is essentially composed of CD8+ T cells.

[0165] In certain embodiments, CD4+ T cells and CD8+ T cells are pooled, mixed, and / or combined in CD4+ T cell:CD8+ T cell ratios between 1:10–10:1, 1:5–5:1, 4:1–1:4, 3:1, 1:3–3:1, 2:1–1:2, 1.5:1–1:1.5, 1.25:1–1:1.25, 1.2:1–1:1.2, 1.1:1–1:1.1, or approximately 1:1 or 1:1. In certain embodiments, CD4+ T cells and CD8+ T cells are pooled, mixed, and / or combined in CD4+ T cell:CD8+ T cell ratios of 2:1 to 1:2, 1.5:1 to 1:1.5, 1.25:1 to 1:1.25, 1.2:1 to 1:1.2, 1.1:1 to 1:1.1, or approximately 1:1 or 1:1. In some embodiments, CD4+ T cells and CD8+ T cells are pooled, mixed, and / or combined in CD4+ T cell:CD8+ T cell ratios of 1:1 or approximately 1:1.

[0166] In some embodiments, cells from enriched CD4+ T cell compositions and enriched CD8+ T cell compositions are pooled, mixed, and / or combined in CD4+ T cell:CD8+ T cell ratios between 1:10-10:1, 1:5-5:1, 4:1-1:4, 3:1, 1:3-3:1, 2:1-1:2, 1.5:1-1:1.5, 1.25:1-1:1.25, 1.2:1-1:1.2, 1.1:1-1:1.1, or approximately 1:1 or 1:1. In certain embodiments, cells from enriched CD4+ T cells and enriched CD8+ T cells are pooled, mixed, and / or combined in CD4+ T cell:CD8+ T cell ratios of 2:1 to 1:2, 1.5:1 to 1:1.5, 1.25:1 to 1:1.25, 1.2:1 to 1:1.2, 1.1:1 to 1:1.1, or approximately 1:1 or 1:1. In some embodiments, cells from enriched CD4+ T cells and enriched CD8+ T cells are pooled, mixed, and / or combined in CD4+ T cell:CD8+ T cell ratios of 1:1 or approximately 1:1.

[0167] In certain embodiments, the input composition has a ratio of CD4+ naive-like T cells to CD8+ naive-like T cells, e.g., a specified, controlled, and / or fixed ratio. In certain embodiments, the ratio of CD4+ naive-like T cells to CD8+ naive-like T cells is 10:1 to 0.05:1, 8:1 to 0.1:1, 5:1 to 0.2:1, 2.5:1 to 0.25:1, 2.2:1 to 0.8:1, 2:1 to 0.5:1, or 1.5:1 to 1:1. 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. In some embodiments, the ratio is between 2.2:1 and 0.8:1. In certain embodiments, the ratio of CD4+ naive-like T cells to CD8+ naive-like T cells is 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, or approximately 2.2:1, approximately 2.1:1, approximately 2.0:1, approximately 1.9:1, approximately 1.8:1, approximately 1.7:1, approximately 1.6:1, approximately 1.5:1, approximately 1.4:1, approximately 1.3:1, approximately 1.2:1, approximately 1.1:1, approximately 1.0:1, approximately 0.9:1 or approximately 0.8:1. In certain embodiments, the ratio is 1.1:1 or approximately 1.1:1.

[0168] In certain embodiments, the input composition is 1 x 10 6 , 5x10 6 , 1x10 7 , 5x10 7 , 1.0x10 8 , 1.1x10 8 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8 , 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x10 8, 2.2x10 8 , 2.3x10 8 , 2.4x10 8 , 2.5x10 8 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 , 5x10 8 Or 1x10 9 , or approximately 1x10 6 , about 5x10 6 Approximately 1x10 7 , about 5x10 7 , about 1.0x10 8 Approximately 1.1 x 10 8 , about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8 , about 2.6x10 8 Approximately 2.7 x 10 8 , about 2.8x10 8 Approximately 2.9 x 10 8 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5x10 8 Or approximately 1x10 9 It has a total amount of cells or total viable cells. In a particular embodiment, the input composition is 1 x 10 6 , 5x106 , 1x10 7 , 5x10 7 , 1.0x10 8 , 1.1x10 8 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8 , 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x10 8 , 2.2x10 8 , 2.3x10 8 , 2.4x10 8 , 2.5x10 8 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 , 5x10 8 Or 1x10 9 , or approximately 1x10 6 , about 5x10 6 Approximately 1x10 7 , about 5x10 7 , about 1.0x10 8 Approximately 1.1 x 10 8 , about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8, about 2.6x10 8 Approximately 2.7 x 10 8 , about 2.8x10 8 Approximately 2.9 x 10 8 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5x10 8 Or approximately 1x10 9 The input composition has cells expressing CD4 or CD8 in an amount of 1x10 6 , 5x10 6 , 1x10 7 , 5x10 7 , 1.0x10 8 , 1.1x10 8 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8 , 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x10 8 , 2.2x10 8 , 2.3x10 8 , 2.4x10 8 , 2.5x10 8 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 , 5x10 8 Or 1x10 9 , or approximately 1x10 6 , about 5x10 6 Approximately 1x10 7 , about 5x10 7 , about 1.0x10 8 Approximately 1.1 x 10 8, about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8 , about 2.6x10 8 Approximately 2.7 x 10 8 , about 2.8x10 8 Approximately 2.9 x 10 8 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5x10 8 Or approximately 1x10 9 It has a certain amount of naive-like CD4+ T cells and naive-like CD8+ T cells.

[0169] In certain embodiments, the input composition is 1 × 10 6 pieces~1×10 10 individual or approximately 1 x 10 6 pieces~approx. 1×10 10 pieces, 1×10 7 pieces~1×10 9 individual or approximately 1 x 10 7 pieces~approx. 1×10 9 pieces, 5×10 7 pieces~5x10 8 pieces or approximately 5 x 10 7 pieces ~ approx. 5x10 8 pieces, or 1x10 8 pieces~3x10 8 pieces or approximately 1 x 10 8 pieces~approx.3x10 8 It has a total of 1 × 10⁶ cells or total viable cells. In a particular embodiment, the input composition is 1 × 10⁶ 6 ~1 × 1010 Or approximately 1 x 10 6 ~Approx. 1×10 10 , 1 x 10 7 ~1 × 10 9 Or approximately 1 x 10 7 ~Approx. 1×10 9 , 5×10 7 ~5x10 8 Or approximately 5 x 10 7 ~about 5x10 8 , or 1x10 8 ~3x10 8 Or approximately 1x10 8 ~about 3x10 8 The input composition has cells expressing CD4 or CD8 in quantities of 1 × 10⁶. 6 ~1 × 10 10 Or approximately 1 x 10 6 ~Approx. 1×10 10 , 1 x 10 7 ~1 × 10 9 Or approximately 1 x 10 7 ~Approx. 1×10 9 , 5×10 7 ~5x10 8 Or approximately 5 x 10 7 ~about 5x10 8 , or 1x10 8 ~3x10 8 Or approximately 1x10 8 ~about 3x10 8 It has a certain amount of naive-like CD4+ T cells and naive-like CD8+ T cells.

[0170] In some embodiments, the input composition has or contains at least 1% or about 1%, at least 5% or about 5%, at least 10% or about 10%, at least 20% or about 20%, at least 30% or about 30%, at least 40% or about 40%, at least 50% or about 50%, at least 60% or about 60%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, at least 99% or about 99%, at least 99.5% or about 99.5%, at least 99.9% or about 99.9%, or 100% or about 100% of naive-like cells. In certain embodiments, the input composition contains or includes naive-like cells in the following proportions: 100% or less or about 100%, 99% or less or about 99%, 98% or less or about 98%, 97% or less or about 97%, 96% or less or about 96%, 95% or less or about 95%, 90% or less or about 90%, or 85% or less or about 85%.

[0171] In certain embodiments, the methods provided herein include one or more steps of manufacturing, generating and / or preparing an input composition. In certain embodiments, the manufacturing, 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.

[0172] In some embodiments, the cells of the input composition, e.g., CD4+ T cells and CD8+ T cells, are isolated and / or selected from a sample, e.g., a biological sample. In certain embodiments, the source of cells for the input composition is a composition of cells, e.g., a composition of CD4+ T cells and CD8+ T cells isolated and / or selected from a sample. In certain embodiments, the composition of CD4+ T cells and the composition of CD8+ T cells are isolated 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 isolated and / or selected from the same sample. In certain embodiments, the composition of CD4+ T cells and the composition of CD8+ T cells are isolated and / or selected from a sample taken from or obtained from the same subject.

[0173] In certain embodiments, the CD4+ T cell composition contains or comprises at least 60% or about 60%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, at least 99% or about 99%, at least 99.5% or about 99.5%, at least 99.9% or about 99.9%, or 100% or about 100% CD4+ T cells. In some embodiments, the CD4+ T cell composition contains or includes 100% or less or about 100%, 99% or less or about 99%, 98% or less or about 98%, 97% or less or about 97%, 96% or less or about 96%, 95% or less or about 95%, 90% or less or about 90%, or 85% or less or about 85% of CD4+ T cells.

[0174] In certain embodiments, the CD8+ T cell composition contains or comprises at least 60% or about 60%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, at least 99% or about 99%, at least 99.5% or about 99.5%, at least 99.9% or about 99.9%, or 100% or about 100% CD8+ T cells. In certain embodiments, the CD8+ T cell composition contains or includes 100% or less or about 100%, 99% or less or about 99%, 98% or less or about 98%, 97% or less or about 97%, 96% or less or about 96%, 95% or less or about 95%, 90% or less or about 90%, or 85% or less or about 85% of CD8+ T cells.

[0175] In certain embodiments, the production, generation, and / or preparation of an input composition includes, for example, one or more steps of measuring, determining, and / or quantifying the amount, portion, number, number per volume, number per weight, and / or percentage of viable CD4+ T cells and / or viable CD8+ T cells present in a CD4+ T cell composition and / or CD8+ T cell composition before combining or mixing the cells of the cell composition. In certain embodiments, the production, generation, and / or preparation of an input composition includes, for example, 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 CD4+ T cell composition and / or CD8+ T cell composition. In some embodiments, naive-like CD4+ T cells and / or naive-like CD8+ T cells are viable naive-like cells.

[0176] In certain embodiments, the production, generation, and / or preparation of an 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 viable CD4+ T cells and / or viable CD8+ T cells present in a sample, e.g., a biological sample. In certain embodiments, the production, generation, and / or preparation of an 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 a sample. In some embodiments, naive-like CD4+ T cells and / or naive-like CD8+ T cells are viable naive-like cells.

[0177] In some embodiments, the cells of the input composition are isolated and / or selected from a sample, e.g., a biological sample. In certain embodiments, a portion of naive-like cells in the sample, e.g., a portion of naive-like CD4+ T cells and naive-like CD8+ T cells, is known, determined, measured, or evaluated. In some embodiments, cells are isolated and / or selected from the sample to directly produce a cell composition, e.g., an input composition, having a defined, fixed, or controlled ratio of naive-like CD4+ T cells to naive-like CD8+ T cells. In certain embodiments, cells are isolated and / or selected by immunoaffinity bead selection. In some embodiments, cells are isolated and / or selected using an affinity column. In certain embodiments, cells are isolated or selected from the sample according to one of the methods described in WO2015 / 164675 to produce a cell composition having a defined, controlled, and / or fixed ratio of naive-like CD4+ cells to naive-like CD8+ cells.

[0178] In certain embodiments, the input composition contains cells directly isolated and / or selected from a sample by first and second isolation or selection. In certain embodiments, the input composition is prepared by performing first and second selection to isolate CD4+ T cells and CD8+ T cells in sufficient quantities, numbers, or concentrations to yield a defined, fixed, and / or controlled ratio of naive-like CD4+ T cells to naive-like CD8+ T cells.

[0179] In some embodiments, cells are directly isolated, selected, and / or concentrated from a sample to produce an input composition enriched with CD4+ and CD8+ cells. In some embodiments, the quantity, number, percentage, number per volume, and / or number per weight of naive-like CD4+ and naive-like CD8+ cells is measured, evaluated, and / or determined in the sample, and the CD4+ and CD8+ cells are isolated, selected, and / or concentrated in sufficient quantities to obtain an input composition having a specified, fixed, and / or controlled ratio of naive-like CD4+ T cells to naive-like CD8+ T cells. In some embodiments, the cells directly isolated, selected, and / or concentrated from the sample are the input composition and are used in subsequent processing steps, e.g., subsequent processing steps including incubation, stimulation, activation, manipulation, and / or formulation of the concentrated cells.

[0180] In some embodiments, cells isolated, selected and / or enriched from a sample such as an input composition have a CD4+ cell to CD8+ cell ratio in a defined, fixed, or controlled ratio of naive-like CD4+ cells to naive-like CD8+ cells. In embodiments of the methods provided herein, the first and / or second selection of samples, or their subpopulations, can be carried out in such a manner that it results in an input composition having a desired ratio of naive-like CD4+ T cells to naive-like CD8+ cells.

[0181] In some embodiments, the ratio of CD4+ T cells to CD8+ T cells in a sample, e.g., a biological sample, is determined before performing a first and / or second selection from the sample. In certain embodiments, the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells in the sample is determined before performing a first and / or second selection. Based on the specific ratio of CD4+ T cells to CD8+ T cells and / or naive-like CD4+ T cells to naive-like CD8+ T cells in the sample, which may vary between samples, a specific selection method can be adapted to the sample by, for example, determining the size of the chromatography column or selecting the amount or concentration of the immunoaffinity reagent to achieve the desired, fixed, or controlled ratio. The relative levels or frequencies of various cell populations in a subject can be determined based on evaluating the surface expression of one or more markers present in such populations or subpopulations. For example, several well-known methods for evaluating the expression levels of surface markers or proteins in terms of cell surface proteins, such as flow cytometry, may be used, e.g., affinity-based methods, e.g., immunoaffinity-based detection.

[0182] In some situations, the appropriate ratio of naive-like CD4+ T cells to naive-like CD8+ T cells can vary depending on the situation, e.g., the specific disease, symptoms, or previous treatment of the subject from which the cells originate, and / or the specific antigenic specificity of the cells, the relative representation among cells of a particular type (e.g., CD4+ cells) of various subpopulations, e.g., effector cells vs. memory cells vs. naive cells, and / or one or more conditions under which the cells are incubated, e.g., culture medium, stimulant, culture time, buffer, oxygen content, carbon dioxide content, antigen, cytokine, antibody, and other components. Therefore, cell types that are typically or generally known to proliferate or expand more rapidly than other cell types do not necessarily possess such properties in all situations. Thus, in some cases, the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells is determined based on the known capabilities of the cell type in normal or typical situations, coupled with an assessment of the phenotype or state of the cells, or the subject from which the cells originate, and / or empirical evidence.

[0183] In some embodiments, the separation and / or process is carried out using immunomagnetic beads. In some embodiments, a cell sample containing CD4+ cells and CD8+ cells is brought into contact with magnetic beads containing a first immunoaffinity reagent that binds to CD4 or CD8, and magnetic beads containing a second immunoaffinity reagent that binds to the other of CD4 or CD8. The separation and / or process may be carried out simultaneously and / or sequentially.

[0184] In some embodiments, the first and / or second immunoaffinity reagents are present in the incubation composition at suboptimal yield concentrations, thereby the enriched composition contains less than the total CD4+ cells in the incubation composition, e.g., 70%, and / or less than the total CD8+ cells in the incubation composition, e.g., 70%, thereby producing a composition enriched with CD4+ T cells and CD8+ T cells.

[0185] In some embodiments, a suboptimal yield concentration of an affinity reagent is a concentration below the concentration used or required to achieve an optimal or maximum yield of bound cells in a given selection or enrichment, which includes incubating cells with the reagent and recovering or separating the cells bound to the reagent ("yield" is, for example, the number of cells thus recovered or selected, compared to the total number of cells in incubation that are targeted by the reagent, to which the reagent is specific, or have a marker to which the reagent is specific and to which it can bind). A suboptimal yield concentration is generally a concentration or amount of the reagent that, in such a process or step, results in a yield of less than the total number of bound cells, e.g., 70% or less, when recovering the cells bound to the reagent. In some embodiments, a suboptimal concentration of an affinity reagent results in a yield of 50% or less, 45% or less, 40% or less, 30% or less, or 25% or less, or about 50%, about 45%, about 40%, about 30% or about 25%. The concentration may be expressed in terms of the number or mass of particles or surfaces per cell, and / or the mass or number of molecules of the active substance (e.g., antibody fragments) per cell. In certain embodiments, suboptimal yield concentrations are sufficient to derive or achieve the fixation, control, and / or defined ratio of naive-like CD4+ T cells to naive-like CD8+ T cells.

[0186] In some embodiments, for example, when working with one or more of two or more selective reagents having affinity for CD4+ T cells and / or CD8+ T cells at suboptimal yield concentrations, one or more of such reagents are used at a higher concentration than one or more of the other such reagents to bias the ratio of cell types recognized by that reagent compared to the cell types recognized by the other. For example, a reagent that specifically binds to a marker whose ratio is to be biased may be included at a concentration (e.g., active substance or mass per cell) that is increased by half, 1x, 2x, 3x, 4x, 5x, 10x or more compared to the others, depending on the degree to which the ratio increase is desired.

[0187] In some embodiments, when operating with cells in a range below optimal and / or sufficient to achieve reagent saturation, the amount of immunoaffinity reagent is proportional to the approximate yield of enriched cells. In certain embodiments, the appropriate amount or concentration of immunoaffinity reagent, depending on the desired ratio of enriched or selected CD4+ T cells and CD8+ T cells in the resulting composition, can be determined as a matter of practice.

[0188] In some embodiments, the separation and / or isolation step is carried out using magnetic beads to which an immunoaffinity reagent is reversibly bound, via peptide ligand interaction with streptavidin mutein, for example, as described in WO2015 / 164675. An example of such magnetic beads is Streptamers®. In some embodiments, the separation and / or step is carried out using magnetic beads, such as those commercially available from Miltenyi Biotec.

[0189] In some embodiments, the first selection or enrichment of CD4+ and CD8+ cells from a sample is performed using an immunoaffinity-based reagent comprising, respectively, at least first and second affinity chromatography matrices immobilized with antibodies. In some embodiments, one or both of the first and / or second selections may use multiple affinity chromatography matrices and / or antibodies, thereby connecting multiple matrices and / or antibodies used for the same selection, i.e., the first or second selection, in series. In some embodiments, the one or more affinity chromatography matrices used for the first and / or second selections are at least 50 × 10⁻⁶ 6 Cells / mL, at least 100 × 10⁶ 6 Cells / mL, at least 200 × 10⁶ 6 cells / mL or at least 400x10 6 Cells / mL, or approximately 50 × 10 6 cells / mL, approximately 100×10 6cells / mL, approximately 200×10 6 cells / mL or approximately 400 x 10 6 Cells / mL can be adsorbed, selected, or concentrated. 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 concentrated composition is achieved by selecting a sufficient amount of matrix and / or a sufficient relative amount, assuming, for example, the adsorption capacity of one or more columns for selecting cells.

[0190] In one exemplary embodiment, CD4+ T cells and CD8+ T cells have equal or similar portions of naive-like cells, and the adsorption capacity of one or more matrix cells is the same between the first and second selections, for example, 1 x 10⁶ each. 8 Cells / mL or approximately 1x10 8 The cells / mL concentration is such that, by enriching or selecting cells in the first and second selections, a composition having a 1:1 or approximately 1:1 ratio of CD4+ cells to CD8+ cells, including naive-like CD4+ T cells versus naive-like CD8+ T cells, is obtained. In certain embodiments, depending on the portion of naive-like cells and the desired ratio of the resulting input composition, the appropriate volume, diameter, or number of affinity matrix chromatography columns for the first and / or second selections can be selected or determined as a common issue.

[0191] In some embodiments, the adsorption capacities of one or more column matrices are adjusted to reveal differences in the frequency of naive-like cells, e.g., naive-like CD4+ cells or naive-like CD8+ cells, compared to the frequency of cells in the respective CD4+ or CD8+ parent populations in the starting sample from the subject. The relative levels or frequencies of various cell populations in the subject can be determined based on evaluating the surface expression of one or more markers present in such populations or subpopulations. For example, several well-known methods for evaluating the expression levels of surface markers or proteins, such as flow cytometry, may be used, such as affinity-based methods or immunoaffinity-based detection methods.

[0192] In some embodiments, naive-like cells, e.g., naive-like CD4+ T cells and / or naive-like CD8+ T cells, are evaluated, measured, and / or detected in cell compositions, e.g., CD4+ T cell compositions and / or CD8+ T cell compositions, or in samples, e.g., biological samples. In some embodiments, naive-like T cells are T cells that are positive for the expression of one or more markers indicating that a cell is a naive cell and / or a naive-like cell. In certain embodiments, naive-like T cells are cells that are positive for the expression of markers associated with the naive or naive-like state of a T cell. In certain embodiments, naive-like T cells are T cells that are negative for the expression of one or more markers indicating that a cell is not a naive cell and / or not a naive-like cell. In certain embodiments, naive-like T cells are cells that are negative for the expression of markers associated with the non-naive or non-naive-like state of a T cell. In certain embodiments, the non-naive or non-naive-like state within a T cell may include, for example, an effector T(T) EFF ) cells, memory T cells, central memory T cells (T CM ), Effector Memory T (T EM This includes cells and combinations thereof.

[0193] In some embodiments, naive-like T cells are positive for the expression of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more than ten markers indicating that the cell is a naive cell and / or a naive-like cell and / or associated with the naive or naive state of the T cell. In some embodiments, the markers are expressed on the cell surface. In certain embodiments, naive-like T cells are negative for the expression of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more than ten markers indicating that the cell is a non-naive cell and / or a non-naive-like cell and / or associated with the non-naive or non-naive state of the T cell.

[0194] Markers indicating that T cells are naive T cells and / or naive-like T cells, and / or associated with the naive or naive-like state of 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+ T cells and / or naive-like CD8+ T cells, are positive for the expression of CD27, CD28, CD45RA, CD62L, and / or CCR7. In certain embodiments, naive-like T cells are positive for the surface expression of one or more of CD27, CD28, CD45RA, CD62L, and / or CCR7.

[0195] Markers indicating that cells are non-naive T cells and / or non-naive-like T cells, and / or associated with the non-naive or non-naive-like state of T cells, include, but are not limited to, CD25, CD45RO, CD56, KLRG1, and / or CD95. In some embodiments, naive-like T cells, e.g., naive-like CD4+ T cells and / or naive-like CD8+ T cells, are negative for the expression of CD25, CD45RO, CD56, and / or KLRG1. In certain embodiments, naive-like T cells, e.g., naive-like CD4+ T cells and / or naive-like CD8+ T cells, have low expression of markers associated with non-naive or non-naive-like cells. In certain embodiments, naive-like T cells have low expression of CD95. In certain embodiments, naive-like T cells are negative for the surface expression of one or more of the following: CD25, CD45RO, CD56, and / or KLRG1.

[0196] In some embodiments, low expression of markers associated with non-naive cells or non-naive-like cells is 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 intracellular markers that are positive for one or more markers associated with the non-naive or non-naive state of T cells, or includes at least 99% less expression of such markers. In certain embodiments, low expression of markers associated with non-naive cells or non-naive-like cells is associated with effector T(T) EFF ) cells, memory T cells, central memory T cells (T CM ) and / or effector memory T(T EM) Expression that is at least 10% or about 10%, at least 20% or about 20%, at least 30% or about 30%, at least 40% or about 40%, at least 50% or about 50%, at least 60% or about 60%, at least 70% or about 70%, at least 80% or about 80%, at least 90% or about 90%, at least 95% or about 95%, or at least 99% or about 99% less than the expression of the intracellular marker, or including such expression.

[0197] In some embodiments, markers indicating that cells are non-naive T cells and / or non-naive-like T cells, and / or associated with the non-naive or non-naive-like state of T cells, include one or more cytokines. For example, in certain embodiments, non-naive T cells or non-naive-like T cells are negative for the expression and / or production of one or more of IL-2, IFN-γ, IL-4, and IL-10. In some embodiments, one or more cytokines are secreted. In certain embodiments, one or more cytokines are internally expressed by non-naive-like T cells, for example, during or after treatment with agents that prevent, inhibit, or reduce secretion.

[0198] In certain embodiments, naive-like T cells are positive for the expression of CD45RA and CCR7, e.g., surface expression. In certain embodiments, naive-like CD4+ T cells are positive for the expression of CD45RA and CCR7, e.g., surface expression. In some embodiments, naive-like CD8+ T cells are positive for the expression of CD45RA and CCR7, e.g., surface expression. In certain embodiments, naive-like T cells are positive for the expression of CD45RA, CD27, and CCR7, e.g., surface expression, and negative for the expression of CD45RO, e.g., surface expression. In certain embodiments, naive-like CD4+ T cells are positive for the expression of CD45RA, CD27, and CCR7, e.g., surface expression, and negative for the expression of CD45RO, e.g., surface expression. In some embodiments, naive-like CD8+ T cells are positive for the expression of CD45RA, CD27, and CCR7, e.g., surface expression, and negative for the expression of CD45RO, e.g., surface expression.

[0199] In certain embodiments, CD4+ T cells and / or CD8+ T cells are viable cells. In certain embodiments, CD4+ T cells and / or CD8+ T cells are viable naive-like cells. Viable cells are positive for the expression of markers indicating that the cell has undergone normal functional cellular processes and / or has not undergone or is not undergoing necrosis or programmed cell death. In some embodiments, viability can be assessed by the cell's redox potential, cell membrane integrity, or mitochondrial activity or function. In some embodiments, viability is the absence of certain molecules associated with cell death or the absence of signs of cell death in the assay.

[0200] In certain embodiments, cell viability is associated with assays that may include, but are not limited to, dye uptake assays (e.g., calcein AM assay), XTT cell viability assays, and dye exclusion assays (e.g., trypan blue dye exclusion assay, eosin dye exclusion assay, or propidium dye exclusion assay). In certain embodiments, viable cells have negative expression of one or more apoptotic markers, e.g., annexin V or activated caspase 3. In some embodiments, viable cells are negative for the expression of one or more apoptotic markers, which 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.

[0201] In some embodiments, expression is the amount, level, concentration and / or presence of a marker. In certain embodiments, the marker is a polypeptide. In some embodiments, the marker is mRNA. In some embodiments, expression is the amount, level, concentration and / or presence of a polypeptide, e.g., a marker polypeptide. In certain embodiments, expression is the amount, level, concentration and / or presence of a polynucleotide encoding a marker, e.g., mRNA, or cDNA derived from mRNA. In certain embodiments, expression is the amount, level, concentration and / or presence of a marker on the cell surface, or a marker exposed on the cell surface, or a marker within the cell membrane. In certain embodiments, expression is the amount, level, concentration and / or presence of a marker on the cell surface, or a marker exposed on the cell surface, or a marker within the cell membrane. In certain embodiments, expression is internal expression, e.g., the amount, level, concentration and / or presence of an intracellular marker, such as in the cytosol, nucleus, endoplasmic reticulum and / or Golgi apparatus.

[0202] In some embodiments, markers are measured, evaluated, and / or quantified by performing in vitro assays. In some examples, the in vitro assay is an immunoassay, an aptamer-based assay, a histological or cytological assay, or an mRNA expression level assay. In some cases, the in vitro assay used may be enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry assay, surface plasmon resonance (SPR), chemiluminescence assay, lateral flow immunoassay, inhibitory assay, or avidity assay. In some embodiments, marker expression is measured, evaluated, and / or quantified by RNA-seq. In certain embodiments, marker expression is measured, evaluated, and / or quantified by immunostaining techniques. In certain embodiments, marker expression is measured, evaluated, and / or quantified by flow cytometry analysis. In some embodiments, marker expression is measured, evaluated, and / or quantified by internal cytokine staining.

[0203] In some embodiments, the marker is measured, evaluated and / or quantified intracellularly in the CD4+ T cell composition. In certain embodiments, at least 1% or about 1%, at least 5% or about 5%, at least 10% or about 10%, at least 15% or about 15%, at least 20% or about 20%, at least 25% or about 25%, at least 30% or about 30%, at least 35% or about 35%, at least 40% or about 40%, at least 45% or about 45%, at least 50% or about 50%, at least 55% or about 55%, and less Naive-like CD4+ T cells are those in which 60% or approximately 60%, at least 65% or approximately 65%, at least 70% or approximately 70%, at least 75% or approximately 75%, at least 80% or approximately 80%, at least 85% or approximately 85%, at least 90% or approximately 90%, at least 95% or approximately 95%, at least 95% or approximately 95%, at least 97% or approximately 97%, or at least 99% or approximately 99% of the CD4+ T cells are defined as naive-like CD4+ T cells. In certain aspects, 10% to 50% or approximately 10% to 50%, 20% to 60% or approximately 20% to 60%, 25% to 75% or approximately 25% to 75%, 30% to 80% or approximately 30% to 80%, 40% to 90% or approximately 40% to 90%, 50% to 100% or approximately 50% to 100%, 30% to 50% or approximately 30% to 50%, 40% to 60% or approximately 40% to 60%, and 50% to 70% are also included. Alternatively, approximately 50% to 70%, 60% to 80%, 70% to 90%, 80% to 100%, 5% to 25%, 25% to 50%, 50% to 75%, 75% to 75%, or 75% to 99% are naive-like CD4+ T cells. In certain aspects, naive-like CD4+ T cells are viable naive-like CD4+ T cells.

[0204] In certain embodiments, the marker is measured, evaluated and / or quantified intracellularly in the CD8+ T cell composition. In certain embodiments, at least 1% or about 1%, at least 5% or about 5%, at least 10% or about 10%, at least 15% or about 15%, at least 20% or about 20%, at least 25% or about 25%, at least 30% or about 30%, at least 35% or about 35%, at least 40% or about 40%, at least 45% or about 45%, at least 50% or about 50%, at least 55% or about 55%, and less Naive-like CD8+ T cells are those in which 60% or approximately 60%, at least 65% or approximately 65%, at least 70% or approximately 70%, at least 75% or approximately 75%, at least 80% or approximately 80%, at least 85% or approximately 85%, at least 90% or approximately 90%, at least 95% or approximately 95%, at least 95% or approximately 95%, at least 97% or approximately 97%, or at least 99% or approximately 99% of the CD8+ T cells are defined as naive-like CD8+ T cells. In certain aspects, 10% to 50% or approximately 10% to 50%, 20% to 60% or approximately 20% to 60%, 25% to 75% or approximately 25% to 75%, 30% to 80% or approximately 30% to 80%, 40% to 90% or approximately 40% to 90%, 50% to 100% or approximately 50% to 100%, 30% to 50% or approximately 30% to 50%, 40% to 60% or approximately 40% to 60%, and 50% to 70% are also included. Alternatively, approximately 50% to 70%, 60% to 80%, 70% to 90%, 80% to 100%, 5% to 25%, 25% to 50%, 50% to 75%, 75% to 75%, or 75% to 99% are naive-like CD4+ T cells. In certain aspects, naive-like CD8+ T cells are viable naive-like CD8+ T cells.

[0205] In some embodiments, cells from a CD4+ T cell composition are mixed or combined with cells from a CD8+ T cell composition in an amount and / or proportion sufficient to produce an input composition having a CD4+ naive-like T cell:CD8+ naive-like T cell ratio of 10:1 to 0.05:1, 8:1 to 0.1:1, 5:1 to 0.2:1, 2.5:1 to 0.25:1, 2.2:1 to 0.8:1, 2:1 to 0.5:1, or 1.5:1 to 1:1. In some embodiments, the cells are mixed in an amount and / or proportion sufficient to achieve a CD4+ naive-like T cell:CD8+ naive-like T cell ratio of 2.2:1 to 0.8:1. In certain embodiments, cells are mixed or combined to achieve CD4+ naive-like T cell:CD8+ naive-like T cell ratios of 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, or approximately 2.2:1, approximately 2.1:1, approximately 2.0:1, approximately 1.9:1, approximately 1.8:1, approximately 1.7:1, approximately 1.6:1, approximately 1.5:1, approximately 1.4:1, approximately 1.3:1, approximately 1.2:1, approximately 1.1:1, approximately 1.0:1, approximately 0.9:1 or approximately 0.8:1. In certain embodiments, cells are mixed or combined in a ratio of 1.1:1 or approximately 1.1:1.

[0206] In some embodiments, 1x10 6 , 5x10 6 , 1x10 7 , 5x10 7 , 1.0x10 8 , 1.1x10 8 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8 , 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x10 8 , 2.2x10 8 , 2.3x108 , 2.4x10 8 , 2.5x10 8 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 , 5x10 8 Or 1x10 9 , or approximately 1x10 6 , about 5x10 6 Approximately 1x10 7 , about 5x10 7 , about 1.0x10 8 Approximately 1.1 x 10 8 , about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8 , about 2.6x10 8 Approximately 2.7 x 10 8 , about 2.8x10 8 Approximately 2.9 x 10 8 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5x10 8 Or approximately 1x10 9 The amount of total CD4+ T cells or total viable CD4+ T cells is 1 x 10⁻¹⁰ 6 , 5x10 6 , 1x10 7 , 5x107 , 1.0x10 8 , 1.1x10 8 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8 , 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x10 8 , 2.2x10 8 , 2.3x10 8 , 2.4x10 8 , 2.5x10 8 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 , 5.5x10 8 Or 1x10 9 , or approximately 1x10 6 , about 5x10 6 Approximately 1x10 7 , about 5x10 7 , about 1.0x10 8 Approximately 1.1 x 10 8 , about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8 , about 2.6x10 8 Approximately 2.7 x 108 , about 2.8x10 8 Approximately 2.9 x 10 8 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5.5x10 8 Or approximately 1x10 9 An input composition having a specified ratio of CD4+ naive-like T cells to CD8+ naive-like T cells is prepared by mixing or combining a certain amount of total CD8+ T cells or total viable CD8+ T cells. In a particular embodiment, 1 x 10 6 ~1x10 10 , 1x10 7 ~1x10 9 , 5x10 7 ~5x10 8 or 1x10 8 ~3x10 8 Total CD4+ T cells or total viable CD4+ T cells, 1 x 10 6 ~1x10 10 , 1x10 7 ~1x10 9 , 5x10 7 ~5x10 8 Or 1x10 8 ~3x10 8 Or approximately 1x10 6 ~approximately 1x10 10 Approximately 1x10 7 ~approximately 1x10 9 , about 5x10 7 ~about 5x10 8 Or approximately 1x10 8 ~about 3x10 8 An input composition having a specified ratio of CD4+ naive-like T cells to CD8+ naive-like T cells is prepared by mixing or combining a certain amount of total CD8+ T cells or total viable CD8+ T cells.

[0207] In some embodiments, 1x10 6 , 5x10 6 , 1x10 7 , 5x10 7 , 1.0x10 8, 1.1x10 8 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8 , 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x10 8 , 2.2x10 8 , 2.3x10 8 , 2.4x10 8 , 2.5x10 8 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 , 5x10 8 Or 1x10 9 , or approximately 1x10 6 , about 5x10 6 Approximately 1x10 7 , about 5x10 7 , about 1.0x10 8 Approximately 1.1 x 10 8 , about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8 , about 2.6x10 8 Approximately 2.7 x 10 8 , about 2.8x10 8Approximately 2.9 x 10 8 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5x10 8 Or approximately 1x10 9 This amount of naive-like CD4+ T cells, 1 x 10 6 , 5x10 6 , 1x10 7 , 5x10 7 , 1.0x10 8 , 1.1x10 8 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8 , 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x10 8 , 2.2x10 8 , 2.3x10 8 , 2.4x10 8 , 2.5x10 8 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 , 5.5x10 8 Or 1x10 9 , or approximately 1x10 6 , about 5x10 6 Approximately 1x10 7 , about 5x10 7 , about 1.0x10 8 Approximately 1.1 x 10 8 , about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8, about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8 , about 2.6x10 8 Approximately 2.7 x 10 8 , about 2.8x10 8 Approximately 2.9 x 10 8 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5.5x10 8 Or approximately 1x10 9 An input composition having a specified ratio of CD4+ naive-like T cells to CD8+ naive-like T cells is prepared by mixing or combining it with an amount of naive-like CD8+ T cells. In a particular embodiment, 1 x 10 6 ~1x10 10 , 1x10 7 ~1x10 9 , 5x10 7 ~5x10 8 or 1x10 8 ~3x10 8 Naive-like CD4+ T cells, 1 x 10 6 ~1x10 10 , 1x10 7 ~1x10 9 , 5x10 7 ~5x10 8 Or 1x10 8 ~3x10 8 , or approximately 1x10 6 ~approximately 1x10 10 Approximately 1x10 7 ~approximately 1x10 9 , about 5x10 7 ~about 5x10 8 Or approximately 1x10 8 ~about 3x10 8An input composition having a specified ratio of CD4+ naive-like T cells to CD8+ naive-like T cells is prepared by mixing or combining with an amount of naive-like CD8+ T cells.

[0208] In certain embodiments, the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells in the input composition is adjusted, altered, and / or modified compared to the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells in a sample, e.g., a biological sample. In certain embodiments, the ratio of naive-like CD4+ T cells to naive-like CD8+ T cells from a biological sample is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1x, 1.5x, 2x, 2.5x, 3x, 4x, 5x, 10x, 20x, 50x, 100x, or approximately 5%, approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 95%, approximately 100%, approximately 1x, approximately 1.5x, approximately 2x, approximately 2.5x, approximately 3x, approximately 4x, approximately 5x, approximately 10x, approximately 2x The sample is adjusted, altered, or modified by 0x, approximately 50x, approximately 100x, or by 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 80%, at least 90%, at least 95%, at least 100%, at least 1x, at least 1.5x, at least 2x, at least 2.5x, at least 3x, at least 4x, at least 5x, at least 10x, at least 20x, at least 50x, or at least 100x. In certain embodiments, the sample is a sample from which cells of the input composition are derived, isolated, selected, and / or obtained.

[0209] In some embodiments, the production, generation, and / or preparation of an input composition includes one or more steps of mixing or combining cells from a CD4+ T cell composition and cells from a CD8+ T cell composition to produce an input composition having a naive-like CD4+ T cell:naive-like CD8+ T cell ratio of 2.2:1 to 0.8:1. In certain embodiments, the number, number per volume, number per weight, and / or quantity, level, or percentage of naive-like cells are measured, evaluated, and / or quantified in the CD4+ T cell composition and the CD8+ T cell composition before mixing or combining. In some embodiments, the quantity, level, number, number per volume, number per weight, and / or percentage of naive-like cells are measured, evaluated, and / or quantified by detecting CD45RA+;CCR7+ T cells. In certain embodiments, the input composition has a CD45RA+ / CCR7+ / CD4+ T cell:CD45RA+ / CCR7+ / CD8+ T cell ratio of 2.2:1 to 0.8:1. In some embodiments, the input composition has a CD45RA+ / CCR7+ / CD4+ T cell:CD45RA+ / CCR7+ / CD8+ T cell ratio of 1.1:1 or about 1.1:1.

[0210] In certain embodiments, the input cell composition has a ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells, e.g., a specified, controlled, and / or fixed ratio. In certain embodiments, the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells is 10:1 to 0.05:1, 8:1 to 0.1:1, 5:1 to 0.2:1, 2.5:1 to 0.25:1, 2.2:1 to 0.8:1, 2:1 to 0.5:1, or 1.5:1 to 1:1. 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. In some embodiments, the ratio is between 2.2:1 and 0.8:1. In certain embodiments, the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells is 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, or approximately 2.2:1, approximately 2.1:1, approximately 2.0:1, approximately 1.9:1, approximately 1.8:1, approximately 1.7:1, approximately 1.6:1, approximately 1.5:1, approximately 1.4:1, approximately 1.3:1, approximately 1.2:1, approximately 1.1:1, approximately 1.0:1, approximately 0.9:1, or approximately 0.8:1. In certain embodiments, the ratio is 1.1:1 or approximately 1.1:1.

[0211] In certain embodiments, the input cell composition is 1 x 10 6 , 5x10 6 , 1x10 7 , 5x10 7 , 1.0x10 8 , 1.1x10 8 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8, 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x10 8 , 2.2x10 8 , 2.3x10 8 , 2.4x10 8 , 2.5x10 8 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 , 5x10 8 Or 1x10 9 , or approximately 1x10 6 , about 5x10 6 Approximately 1x10 7 , about 5x10 7 , about 1.0x10 8 Approximately 1.1 x 10 8 , about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8 , about 2.6x10 8 Approximately 2.7 x 10 8 , about 2.8x10 8 Approximately 2.9 x 10 8 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5x10 8or about 1x10 9 and has a total number of cells or total viable cells in an amount of. In certain embodiments, the input cell composition is 1x10 6 , 5x10 6 , 1x10 7 , 5x10 7 , 1.0x10[[ID=eleven]] 8 , 1.1x10 8 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8 , 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x10 8 , 2.2x10 8 , 2.3x10 8 , 2.4x10 8 , 2.5x10 8 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10[[ID=eleven]] 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 ]>, 5x*10 8 or 1x10 9 or about 1x10 6 , about 5x10 6 , about 1x10<* 7 , about 5x10 7 , about 1.0x10 8 , about 1.1x10 8 , about 1.2x10[[ID=eleven]] 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 , about 1.7x10 8 , about 1.8x10 8 , about 1.9x10<* 8 , about 2.0x10 8 , about 2.1x10 8 It should be noted that there are some tags in the original text that seem to be in an incorrect format (e.g., <* 7 and <* 8 ), which may cause issues during translation or further processing. If possible, it would be beneficial to correct these tags to ensure accurate interpretation., about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8 , about 2.6x10 8 , about 2.7x10 8 , about 2.8x10 8 , about 2.9x10 8 , about 3.0x10 8 , about 3.5x{10} 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5x10 8 or about 1x10 9 of cells expressing CD4 or CD8. In some embodiments, the input cell composition is 1x10[[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​6 Approximately 1x10 7 , about 5x10 7 , about 1.0x10 8 Approximately 1.1 x 10 8 , about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8 , about 2.6x10 8 Approximately 2.7 x 10 8 , about 2.8x10 8 Approximately 2.9 x 10 8 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5x10 8 Or approximately 1x10 9 It has a quantity of CD45RA+ / CCR7+ / CD4+ T cells and CD45RA+ / CCR7+ / CD8+ T cells.

[0212] In a particular embodiment, the input cell composition is 1 × 10 6 ~1 × 10 10 pieces, 1×10 7 ~1 × 10 9 pieces, 5×10 7 ~5×10 8 individual or 1 x 10 8 ~3×10 8 It has a total of 10⁵ cells or total viable cells. In a particular embodiment, the input cell composition is 1 x 10⁵ 6 ~1x10 10 , 1x10 7 ~1x10 9 , 5x10 7~5x10 8 Or 1x10 8 ~3x10 8 , or approximately 1x10 6 ~approximately 1x10 10 Approximately 1x10 7 ~approximately 1x10 9 , about 5x10 7 ~about 5x10 8 Or approximately 1x10 8 ~about 3x10 8 The input cell composition has a quantity of cells expressing CD4 or CD8. In some embodiments, the input cell composition is 1 x 10 6 ~1x10 10 , 1x10 7 ~1x10 9 , 5x10 7 ~5x10 8 Or 1x10 8 ~3x10 8 , or approximately 1x10 6 ~approximately 1x10 10 Approximately 1x10 7 ~approximately 1x10 9 , about 5x10 7 ~about 5x10 8 Or approximately 1x10 8 ~about 3x10 8 It has a quantity of CD45RA+ / CCR7+ / CD4+ T cells and CD45RA+ / CCR7+ / CD8+ T cells.

[0213] In some embodiments, the input cell composition has or contains 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% of CD45RA+ / CCR7+ cells. In certain embodiments, the input cell composition contains or contains 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 90% or less, or 85% or less of CD45RA+ / CCR7+ cells.

[0214] In some embodiments, 1x10 6 , 5x10 6 , 1x10 7 , 5x10 7 , 1.0x10 8 , 1.1x10 8 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8 , 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x10 8 , 2.2x10 8 , 2.3x10 8 , 2.4x10 8 , 2.5x10 8 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 , 5x10 8 Or 1x10 9 , or approximately 1x10 6 , about 5x10 6 Approximately 1x10 7 , about 5x10 7 , about 1.0x10 8 Approximately 1.1 x 10 8 , about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x108 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8 , about 2.6x10 8 Approximately 2.7 x 10 8 , about 2.8x10 8 Approximately 2.9 x 10 8 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5x10 8 Or approximately 1x10 9 The amount of total CD4+ T cells or total viable CD4+ T cells is 1 x 10⁻¹⁰ 6 , 5x10 6 , 1x10 7 , 5x10 7 , 1.0x10 8 , 1.1x10 8 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8 , 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x10 8 , 2.2x10 8 , 2.3x10 8 , 2.4x10 8 , 2.5x10 8 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 , 5.5x10 8 Or 1x10 9 , or approximately 1x10 6 , about 5x10 6 Approximately 1x10 7, about 5x10 7 , about 1.0x10 8 Approximately 1.1 x 10 8 , about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8 , about 2.6x10 8 Approximately 2.7 x 10 8 , about 2.8x10 8 Approximately 2.9 x 10 8 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5.5x10 8 Or approximately 1x10 9 An input cell composition having a specified ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells is prepared by mixing or combining a certain amount of total CD8+ T cells or total viable CD8+ T cells. In a particular embodiment, 1 x 10 6 ~1x10 10 , 1x10 7 ~1x10 9 , 5x10 7 ~5x10 8 or 1x10 8 ~3x10 8 Total CD4+ T cells or total viable CD4+ T cells, 1 x 10 6 ~1x10 10 , 1x10 7 ~1x10 9 , 5x10 7 ~5x10 8 Or 1x10 8~3x10 8 , or approximately 1x10 6 ~approximately 1x10 10 Approximately 1x10 7 ~approximately 1x10 9 , about 5x10 7 ~about 5x10 8 Or approximately 1x10 8 ~about 3x10 8 A certain amount of total CD8+ T cells or total viable CD8+ T cells is mixed or combined with an input cell composition having a specified ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells.

[0215] In certain embodiments, the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells in the input cell composition is adjusted, altered, and / or modified compared to the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells in a sample, e.g., a biological sample. In certain embodiments, the ratio of CD45RA+ / CCR7+ / CD4+ T cells to CD45RA+ / CCR7+ / CD8+ T cells from a biological sample is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1x, 1.5x, 2x, 2.5x, 3x, 4x, 5x, 10x, 20x, 50x, 100x, or approximately 5%, approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 95%, approximately 100%, approximately 1x, approximately 1.5x, approximately 2x, approximately 2.5x, approximately 3x, approximately 4x, approximately 5 The sample is adjusted, altered, or modified by a factor of 1, 10, 20, 50, 100, or by 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 80%, at least 90%, at least 95%, at least 100%, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, or at least 100. In certain embodiments, the sample is a sample from which cells of an input cell composition are derived, isolated, selected, and / or obtained.

[0216] In certain embodiments, the input cell composition has a ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells, e.g., a specified, controlled, and / or fixed ratio. In certain embodiments, the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells is 10:1 to 0.05:1, 8:1 to 0.1:1, 5:1 to 0.2:1, 2.5:1 to 0.25:1, 2.2:1 to 0.8:1, 2:1 to 0.5:1, or 2:1 to 1:1. 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. In some embodiments, the ratio is between 1.8:1 and 1.6:1. In certain embodiments, the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells is 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, or approximately 2.2:1, approximately 2.1:1, approximately 2.0:1, approximately 1.9:1, approximately 1.8:1, approximately 1.7:1, approximately 1.69:1, approximately 1.6:1, approximately 1.5:1, approximately 1.4:1, or approximately 1.3:1. In certain embodiments, the ratio is 1.69:1 or approximately 1.69:1.

[0217] In certain embodiments, the input cell composition is 1 x 10 6 , 5x10 6 , 1x10 7 , 5x10 7 , 1.0x10 8 , 1.1x10 8 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8 , 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x108 , 2.2x10 8 , 2.3x10 8 , 2.4x10 8 , 2.5x10 8 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 , 5x10 8 Or 1x10 9 , or approximately 1x10 6 , about 5x10 6 Approximately 1x10 7 , about 5x10 7 , about 1.0x10 8 Approximately 1.1 x 10 8 , about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8 , about 2.6x10 8 Approximately 2.7 x 10 8 , about 2.8x10 8 Approximately 2.9 x 10 8 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5x10 8 Or approximately 1x10 9 It has a total amount of cells or total viable cells. In a particular embodiment, the input cell composition is 1 x 106 , 5x10 6 , 1x10 7 , 5x10 7 , 1.0x10 8 , 1.1x10 8 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8 , 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x10 8 , 2.2x10 8 , 2.3x10 8 , 2.4x10 8 , 2.5x10 8 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 , 5x10 8 Or 1x10 9 Or approximately 1x10 6 , about 5x10 6 Approximately 1x10 7 , about 5x10 7 , about 1.0x10 8 Approximately 1.1 x 10 8 , about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8, about 2.5x10 8 , about 2.6x10 8 Approximately 2.7 x 10 8 , about 2.8x10 8 Approximately 2.9 x 10 8 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5x10 8 Or approximately 1x10 9 The input cell composition has a quantity of cells expressing CD4 or CD8. In some embodiments, the input cell composition is 1 x 10 6 , 5x10 6 , 1x10 7 , 5x10 7 , 1.0x10 8 , 1.1x10 8 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8 , 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x10 8 , 2.2x10 8 , 2.3x10 8 , 2.4x10 8 , 2.5x10 8 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 , 5x10 8 Or 1x10 9 , or approximately 1x10 6 , about 5x10 6 Approximately 1x10 7 , about 5x10 7 , about 1.0x108 Approximately 1.1 x 10 8 , about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8 , about 2.6x10 8 Approximately 2.7 x 10 8 , about 2.8x10 8 Approximately 2.9 x 10 8 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5x10 8 Or approximately 1x10 9 It has a quantity of CD27+ / CCR7+ / CD4+ T cells and CD27+ / CCR7+ / CD8+ T cells.

[0218] In a particular embodiment, the input cell composition is 1 × 10 6 ~1 × 10 10 pieces, 1×10 7 ~1 × 10 9 pieces, 5×10 7 ~5×10 8 individual or 1 x 10 8 ~3×10 8 It has a total of 10⁵ cells or total viable cells. In a particular embodiment, the input cell composition is 1 x 10⁵ 6 ~1x10 10 , 1x10 7 ~1x10 9 , 5x10 7 ~5x10 8 Or 1x10 8 ~3x10 8, or approximately 1x10 6 ~approximately 1x10 10 Approximately 1x10 7 ~approximately 1x10 9 , about 5x10 7 ~about 5x10 8 Or approximately 1x10 8 ~about 3x10 8 The input cell composition has a quantity of cells expressing CD4 or CD8. In some embodiments, the input cell composition is 1 x 10 6 ~1x10 10 , 1x10 7 ~1x10 9 , 5x10 7 ~5x10 8 Or 1x10 8 ~3x10 8 , or approximately 1x10 6 ~approximately 1x10 10 Approximately 1x10 7 ~approximately 1x10 9 , about 5x10 7 ~about 5x10 8 Or approximately 1x10 8 ~about 3x10 8 It has a quantity of CD27+ / CCR7+ / CD4+ T cells and CD27+ / CCR7+ / CD8+ T cells.

[0219] In some embodiments, the input cell composition has or contains at least 1% or about 1%, at least 5% or about 5%, at least 10% or about 10%, at least 20% or about 20%, at least 30% or about 30%, at least 40% or about 40%, at least 50% or about 50%, at least 60% or about 60%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, at least 99% or about 99%, at least 99.5% or about 99.5%, at least 99.9% or about 99.9%, or 100% or about 100% of CD27+ / CCR7+ cells. In certain embodiments, the input cell composition contains or includes CD27+ / CCR7+ cells in the following proportions: 100% or less or about 100%, 99% or less or about 99%, 98% or less or about 98%, 97% or less or about 97%, 96% or less or about 96%, 95% or less or about 95%, 90% or less or about 90%, or 85% or less or about 85%.

[0220] In some embodiments, 1x10 6 , 5x10 6 , 1x10 7 , 5x10 7 , 1.0x10 8 , 1.1x10 8 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8 , 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x10 8 , 2.2x10 8 , 2.3x10 8 , 2.4x10 8 , 2.5x108 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 , 5x10 8 Or 1x10 9 , or approximately 1x10 6 , about 5x10 6 Approximately 1x10 7 , about 5x10 7 , about 1.0x10 8 Approximately 1.1 x 10 8 , about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8 , about 2.6x10 8 Approximately 2.7 x 10 8 , about 2.8x10 8 Approximately 2.9 x 10 8 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5x10 8 Or approximately 1x10 9 The amount of total CD4+ T cells or total viable CD4+ T cells is 1 x 10⁻¹⁰ 6 , 5x10 6 , 1x10 7 , 5x10 7 , 1.0x10 8 , 1.1x108 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8 , 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x10 8 , 2.2x10 8 , 2.3x10 8 , 2.4x10 8 , 2.5x10 8 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 , 5.5x10 8 Or 1x10 9 , or approximately 1x10 6 , about 5x10 6 Approximately 1x10 7 , about 5x10 7 , about 1.0x10 8 Approximately 1.1 x 10 8 , about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8 , about 2.6x10 8 Approximately 2.7 x 10 8 , about 2.8x10 8 Approximately 2.9 x 108 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5.5x10 8 Or approximately 1x10 9 An input cell composition having a specified ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells is produced by mixing or combining a certain amount of total CD8+ T cells or total viable CD8+ T cells. In a particular embodiment, 1 x 10 6 ~1x10 10 , 1x10 7 ~1x10 9 , 5x10 7 ~5x10 8 or 1x10 8 ~3x10 8 Total CD4+ T cells or total viable CD4+ T cells, 1 x 10 6 ~1x10 10 , 1x10 7 ~1x10 9 , 5x10 7 ~5x10 8 Or 1x10 8 ~3x10 8 , or approximately 1x10 6 ~approximately 1x10 10 Approximately 1x10 7 ~approximately 1x10 9 , about 5x10 7 ~about 5x10 8 Or approximately 1x10 8 ~about 3x10 8 A certain amount of total CD8+ T cells or total viable CD8+ T cells is mixed or combined with an input cell composition having a specified ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells.

[0221] In certain embodiments, the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells in the input cell composition is adjusted, altered, and / or modified compared to the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells in a sample, e.g., a biological sample. In certain embodiments, the ratio of CD27+ / CCR7+ / CD4+ T cells to CD27+ / CCR7+ / CD8+ T cells from a biological sample is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1x, 1.5x, 2x, 2.5x, 3x, 4x, 5x, 10x, 20x, 50x, 100x, or approximately 5%, approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 95%, approximately 100%, approximately 1x, approximately 1.5x, approximately 2x, approximately 2.5x, approximately 3x, approximately 4x, approximately 5x, The sample is adjusted, altered, or modified by approximately 10 times, approximately 20 times, approximately 50 times, approximately 100 times, or by 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 80%, at least 90%, at least 95%, at least 100%, at least 1 time, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, or at least 100 times. In certain embodiments, the sample is a sample from which cells of the input cell composition are derived, isolated, selected, and / or obtained.

[0222] In certain embodiments, the input cell composition has a ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells, e.g., a specified, controlled, and / or fixed ratio. In certain embodiments, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells is 10:1 to 0.05:1, 8:1 to 0.1:1, 5:1 to 0.2:1, 2.5:1 to 0.25:1, 2.2:1 to 0.8:1, 2:1 to 0.5:1, or 1.5:1 to 1:1. 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. In some embodiments, the ratio is between 2.2:1 and 0.8:1. In certain embodiments, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells is 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, or approximately 2.2:1, approximately 2.1:1, approximately 2.0:1, approximately 1.9:1, approximately 1.8:1, approximately 1.7:1, approximately 1.6:1, approximately 1.5:1, approximately 1.4:1, approximately 1.3:1, approximately 1.2:1, approximately 1.1:1, approximately 1.0:1, approximately 0.9:1, or approximately 0.8:1. In certain embodiments, the ratio is 1.1:1 or approximately 1.1:1.

[0223] In a particular embodiment, the input cell composition is 1 × 10 6 ~1 × 10 10 pieces, 1×10 7 ~1 × 10 9 pieces, 5×10 7 ~5×10 8 individual or 1 x 10 8 ~3×10 8 It has a total of 10⁵ cells or total viable cells. In a particular embodiment, the input cell composition is 1 x 10⁵ 6 ~1x10 10 , 1x10 7 ~1x109 , 5x10 7 ~5x10 8 Or 1x10 8 ~3x10 8 , or approximately 1x10 6 ~approximately 1x10 10 Approximately 1x10 7 ~approximately 1x10 9 , about 5x10 7 ~about 5x10 8 Or approximately 1x10 8 ~about 3x10 8 The input cell composition has a quantity of cells expressing CD4 or CD8. In some embodiments, the input cell composition is 1 x 10 6 ~1x10 10 , 1x10 7 ~1x10 9 , 5x10 7 ~5x10 8 Or 1x10 8 ~3x10 8 , or approximately 1x10 6 ~approximately 1x10 10 Approximately 1x10 7 ~approximately 1x10 9 , about 5x10 7 ~about 5x10 8 Or approximately 1x10 8 ~about 3x10 8 It has a quantity of CD62L- / CCR7+ / CD4+ T cells and CD62L- / CCR7+ / CD8+ T cells.

[0224] In some embodiments, the input cell composition has or contains CD62L- / CCR7+ cells in the following proportions: at least 1% or about 1%, at least 5% or about 5%, at least 10% or about 10%, at least 20% or about 20%, at least 30% or about 30%, at least 40% or about 40%, at least 50% or about 50%, at least 60% or about 60%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, at least 99% or about 99%, at least 99.5% or about 99.5%, at least 99.9% or about 99.9%, or 100% or about 100%. In certain embodiments, the input cell composition contains or includes CD62L- / CCR7+ cells in amounts of 100% or less or about 100%, 99% or less or about 99%, 98% or less or about 98%, 97% or less or about 97%, 96% or less or about 96%, 95% or less or about 95%, 90% or less or about 90%, or 85% or less or about 85%.

[0225] In some embodiments, 1x10 6 , 5x10 6 , 1x10 7 , 5x10 7 , 1.0x10 8 , 1.1x10 8 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8 , 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x10 8 , 2.2x10 8 , 2.3x10 8 , 2.4x10 8 , 2.5x108 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 , 5x10 8 Or 1x10 9 , or approximately 1x10 6 , about 5x10 6 Approximately 1x10 7 , about 5x10 7 , about 1.0x10 8 Approximately 1.1 x 10 8 , about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8 , about 2.6x10 8 Approximately 2.7 x 10 8 , about 2.8x10 8 Approximately 2.9 x 10 8 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5x10 8 Or approximately 1x10 9 The amount of total CD4+ T cells or total viable CD4+ T cells is 1 x 10⁻¹⁰ 6 , 5x10 6 , 1x10 7 , 5x10 7 , 1.0x10 8 , 1.1x108 , 1.2x10 8 , 1.3x10 8 , 1.4x10 8 , 1.5x10 8 , 1.6x10 8 , 1.7x10 8 , 1.8x10 8 , 1.9x10 8 , 2.0x10 8 , 2.1x10 8 , 2.2x10 8 , 2.3x10 8 , 2.4x10 8 , 2.5x10 8 , 2.6x10 8 , 2.7x10 8 , 2.8x10 8 , 2.9x10 8 , 3.0x10 8 , 3.5x10 8 , 4.0x10 8 , 4.5x10 8 , 5x10 8 , 5.5x10 8 Or 1x10 9 , or approximately 1x10 6 , about 5x10 6 Approximately 1x10 7 , about 5x10 7 , about 1.0x10 8 Approximately 1.1 x 10 8 , about 1.2x10 8 , about 1.3x10 8 , about 1.4x10 8 , about 1.5x10 8 , about 1.6x10 8 Approximately 1.7 x 10 8 , about 1.8x10 8 Approximately 1.9 x 10 8 , about 2.0x10 8 Approximately 2.1 x 10 8 , about 2.2x10 8 , about 2.3x10 8 , about 2.4x10 8 , about 2.5x10 8 , about 2.6x10 8 Approximately 2.7 x 10 8 , about 2.8x10 8 Approximately 2.9 x 108 , about 3.0x10 8 , about 3.5x10 8 , about 4.0x10 8 , about 4.5x10 8 , about 5x10 8 , about 5.5x10 8 Or approximately 1x10 9 An input cell composition having a specified ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells is prepared by mixing or combining a certain amount of total CD8+ T cells or total viable CD8+ T cells. In a particular embodiment, 1 x 10 6 ~1x10 10 , 1x10 7 ~1x10 9 , 5x10 7 ~5x10 8 or 1x10 8 ~3x10 8 Total CD4+ T cells or total viable CD4+ T cells, 1 x 10 6 ~1x10 10 , 1x10 7 ~1x10 9 , 5x10 7 ~5x10 8 Or 1x10 8 ~3x10 8 , or approximately 1x10 6 ~approximately 1x10 10 Approximately 1x10 7 ~approximately 1x10 9 , about 5x10 7 ~about 5x10 8 Or approximately 1x10 8 ~about 3x10 8 A certain amount of total CD8+ T cells or total viable CD8+ T cells is mixed or combined with an input cell composition having a specified ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells.

[0226] In certain embodiments, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells in the input cell composition is adjusted, altered, and / or modified compared to the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells in a sample, e.g., a biological sample. In certain embodiments, the ratio of CD62L- / CCR7+ / CD4+ T cells to CD62L- / CCR7+ / CD8+ T cells from a biological sample is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1x, 1.5x, 2x, 2.5x, 3x, 4x, 5x, 10x, 20x, 50x, 100x, or approximately 5%, approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 95%, approximately 100%, approximately 1x, approximately 1.5x, approximately 2x, approximately 2.5x, approximately 3x, approximately 4x, approximately 5x. , approximately 10 times, approximately 20 times, approximately 50 times, approximately 100 times, or 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 80%, at least 90%, at least 95%, at least 100%, at least 1 time, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times. In certain embodiments, the sample is a sample from which cells of the input cell composition are derived, isolated, selected, and / or obtained.

[0227] In some embodiments, the production, generation, and / or preparation of an input cell composition includes one or more steps of mixing or combining cells from a CD4+ T cell composition and cells from a CD8+ T cell composition to produce an input cell composition having a CD45RA+ / CCR7+ / CD4+ T cell:CD45RA+ / CCR7+ / CD8+ T cell ratio of 2.2:1 to 0.8:1. In certain embodiments, the number, number per volume, number per weight, and / or amount, level, or percentage of CD45RA+ / CCR7+ cells is measured, evaluated, and / or quantified in the CD4+ T cell composition and the CD8+ T cell composition before 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, evaluated, and / or quantified by detecting CD45RA+;CCR7+ T cells. In certain embodiments, the input cell composition has a CD45RA+ / CCR7+ / CD4+ T cell:CD45RA+ / CCR7+ / CD8+ T cell ratio of 2.2:1 to 0.8:1. In some embodiments, the input cell composition has a CD45RA+ / CCR7+ / CD4+ T cell:CD45RA+ / CCR7+ / CD8+ T cell ratio of 1.1:1 or approximately 1.1:1.

[0228] In some embodiments, the production, generation, and / or preparation of an input cell composition includes one or more steps of mixing or combining cells from a CD4+ T cell composition and cells from a CD8+ T cell composition to produce an input cell composition having a CD27+ / CCR7+ / CD4+ T cell:CD27+ / CCR7+ / CD8+ T cell ratio of 2.4:1 to 1:1. In certain embodiments, the number, number per volume, number per weight, and / or amount, level, or percentage of CD27+ / CCR7+ cells is measured, evaluated, and / or quantified in the CD4+ T cell composition and the CD8+ T cell composition before mixing or combining. In some embodiments, the amount, level, number, number per volume, number per weight, and / or percentage of CD27+ / CCR7+ cells is measured, evaluated, and / or quantified by detecting CD45RA+;CCR7+ T cells. In certain embodiments, the input cell composition has a CD27+ / CCR7+ / CD4+ T cell:CD27+ / CCR7+ / CD8+ T cell ratio of 2.4:1 to 1:1. In some embodiments, the input cell composition has a CD27+ / CCR7+ / CD4+ T cell:CD27+ / CCR7+ / CD8+ T cell ratio of 1.69:1 or approximately 1.69:1.

[0229] In some embodiments, the production, generation, and / or preparation of an input cell composition includes one or more steps of mixing or combining cells from a CD4+ T cell composition and cells from a CD8+ T cell composition to produce an input cell composition having a CD62L- / CCR7+ / CD4+ T cell:CD62L- / CCR7+ / CD8+ T cell ratio of 2.2:1 to 0.8:1. In certain embodiments, the number, number per volume, number per weight, and / or amount, level, or percentage of CD62L- / CCR7+ cells is measured, evaluated, and / or quantified in the CD4+ T cell composition and CD8+ T cells before mixing or combining. In some embodiments, the amount, level, number, number per volume, number per weight, and / or percentage of CD62L- / CCR7+ cells is measured, evaluated, and / or quantified by detecting CD62L- / CCR7+ T cells. In certain embodiments, the input cell composition has a CD62L- / CCR7+ / CD4+ T cell:CD62L- / CCR7+ / CD8+ T cell ratio of 2.2:1 to 0.8:1. In some embodiments, the input cell composition has a CD62L- / CCR7 / CD4+ T cell:CD62L- / CCR7 / CD8+ T cell ratio of 1.1:1 or approximately 1.1:1.

[0230] B. Activation and Stimulation In some embodiments, the methods provided are used in connection with incubating cells under stimulating conditions. In some embodiments, the stimulating conditions include conditions that activate or stimulate, and / or can activate or stimulate, signals within cells, e.g., CD4+ T cells, such as signals generated from TCRs and / or co-receptors. In some embodiments, the stimulating conditions include one or more steps of culturing, cultivating, incubating, activating, and growing cells with and / or in the presence of a stimulating reagent, e.g., a reagent that activates or stimulates, and / or can activate or stimulate, intracellular signals. In some embodiments, the stimulating reagent stimulates and / or activates TCRs and / or co-receptors. In certain embodiments, the stimulating reagent is a reagent provided herein, for example, as described in Section IB-1.

[0231] In certain embodiments, one or more compositions of enriched T cells are incubated under stimulating conditions before genetically engineering the cells, e.g., transfecting and / or transducing them, by methods or techniques provided herein, e.g., methods or techniques described in Section IC. In certain embodiments, the compositions of enriched T cells incubated under stimulating conditions are input compositions. In certain embodiments, the cells of the input composition are previously isolated, selected, enriched, or obtained from a biological sample. In certain embodiments, the cells from the input composition are previously cryopreserved and stored and thawed before incubation.

[0232] In some embodiments, the provided method is used in connection with one or more processing steps that include a step of stimulating cells, such as cells from an input composition. In certain embodiments, incubation may precede or be related to embodiments of transduction described herein, such as genetic manipulation resulting from the methods described in Section IC. In some embodiments, stimulation results in cell activation and / or proliferation, for example, before the manipulation, such as transduction.

[0233] In some embodiments, the processing step includes incubation of cells such as input cells and / or cells of the input composition, and the incubation step may include cell culture, cultivation, stimulation, activation and / or proliferation. In some embodiments, the composition or cells are incubated in the presence of stimulating conditions or stimulants. Such conditions include those designed to induce proliferation, expansion, activation and / or survival of cells in a population, mimic antigen exposure and / or prime cells for genetic manipulation such as the introduction of recombinant antigen receptors.

[0234] In certain embodiments, cells, for example, cells of an input composition, are subjected to stimulating conditions, such as in the presence of a stimulating reagent, for example, 5x10 7 Cells / mL < 4x10 7 Cells / mL < , 3 x 10 7 Cells / mL < 2x10 7 Cells / mL < 1x10 7 Cells / mL < 9x10 6 Cells / mL < , 8x10 6 Cells / mL < 7x10 6 Cells / mL <6x10 6 Cells / mL <5x10 6 Cells / mL < 4x10 6 cells / mL or less or 3x10 6 Less than cells / mL, or approximately 5x10 7 cells / mL, approximately 4x10 7 cells / mL, approximately 3x107 cells / mL, approximately 2x10 7 cells / mL, approximately 1x10 7 cells / mL, approximately 9x10 6 cells / mL, approximately 8x10 6 cells / mL, approximately 7x10 6 cells / mL, approximately 6x10 6 cells / mL, approximately 5x10 6 cells / mL, approximately 4x10 6 cells / mL or approximately 3 x 10 6 The cells are incubated at a density of cells / mL. In certain embodiments, the cells are 5 × 10⁶ 6 The cells are incubated at a density of less than 1 × 10⁶ cells / mL. In some embodiments, the cells are incubated at a density of less than 1 × 10⁶ cells / mL. 3 cells / mL~1×10 9 cells / mL, 1×10 4 cells / mL~1×10 8 cells / mL, 1×10 5 cells / mL~1×10 7 cells / mL, 5x10 5 cells / mL~1x10 7 cells / mL, 1x10 6 cells / mL~5x10 6 Cells / mL or 3x10 6 cells / mL~5x10 6 The cells are incubated at a density of cells / mL. In certain embodiments, the cells are 1 × 10⁶ 6 cells / mL, 1.5×10 6 cells / mL, 2×10 6 cells / mL, 2.5×10 6 cells / mL, 3×10 6 cells / mL, 3.5x10 6 cells / mL, 4x10 6 cells / mL, 4.5x10 6 cells / mL or 5x10 6 cells / mL, or approximately 1 × 10⁶ 6 cells / mL, approximately 1.5×10 6 cells / mL, approximately 2×10 6 cells / mL, approximately 2.5×10 6 cells / mL, approximately 3×10 6 cells / mL, approximately 3.5x10 6 cells / mL, approximately 4x106 cells / mL, approximately 4.5x10 6 Cells / mL or approximately 5 x 10 6 The cells are incubated at a density of cells / mL. In certain embodiments, the cells are 3 × 10⁶ 6 cells / mL or approximately 3 × 10⁶ 6 The cells are incubated at a density of 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 activated caspase 3. In certain embodiments, the cells are CD4+ T cells and CD8+ T cells, or contain them.

[0235] In certain embodiments, indicators of viability include, but are not limited to, indicators of cell replication, mitochondrial function, energy balance, membrane integrity, and cell death. In certain embodiments, indicators of viability further include indicators of oxidative stress, metabolic activation, metabolic stability, enzyme induction, enzyme inhibition, and interactions with cell membrane transporters. In some embodiments, viable cells include cells undergoing normal functional cellular processes and / or cells that have not undergone or are not undergoing necrosis or programmed cell death. In some embodiments, viability can be assessed by the redox potential of cells, cell membrane integrity, or mitochondrial activity or function. In some embodiments, viability is the absence of certain molecules associated with cell death or the absence of signs of cell death in an assay. In certain embodiments, the viability of cells can be detected, measured, and / or assessed by several common means. Non-limiting examples of such viability assays include, but are not limited to, dye uptake assays (e.g., calcein AM assay), XTT cell viability assays, and dye exclusion assays (e.g., trypan blue dye exclusion assay, eosin dye exclusion assay, or propidium dye exclusion assay). Viability assays are useful for determining the number or proportion (e.g., frequency) of viable cells in cell dose, cell composition, and / or cell samples.

[0236] In certain embodiments, apoptosis markers may include any known marker associated with apoptosis, including the expression of genes, proteins or active proteins, or the appearance of apoptosis-related features such as vesicle formation and / or nuclear disruption. In certain embodiments, apoptosis markers may include, but are not limited to, pro-apoptotic factors known to initiate apoptosis, members of the death receptor pathway, activating members of the mitochondrial (intrinsic) pathway, Bcl-2 family members such as Bax, Bad, and Bid, Fas, FADD, the presence of nuclear contraction (e.g., monitored by microscopy), the presence of chromosomal DNA fragmentation (e.g., the presence of a chromosomal DNA ladder), or markers associated with apoptosis assays such as TUNEL staining and annexin V staining. In some embodiments, apoptosis markers are caspase expression, e.g., the expression of active caspases-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 activated caspase-3.

[0237] In some embodiments, 1 × 10 5 ~500,000 × 10 6 individual or approximately 1 x 10 5 ~About 500,000×10 6 pieces, 1×10 6 ~50,000 x 10 6 individual or approximately 1 x 10 6 ~About 50,000×10 6 pieces, 10×10 6 ~5,000 x 10 6 1 or approximately 10 x 10 6 ~Approx. 5,000×10 6 pieces, 1x10 6 ~1,000x10 6 pc or approximately 1x10 6 ~Approx. 1,000x10 6 pieces, 50x10 6 ~5,000x10 61 or approximately 50x10 6 ~Approx. 5,000x10 6 pieces, 10x10 6 ~1,000x10 6 1 or approximately 10x10 6 ~Approx. 1,000x10 6 pieces, 100×10 6 ~2500×10 6 1 or approximately 100 x 10 6 ~About 2500×10 6 Individual cells, for example, cells of an input composition, are incubated under stimulating conditions, such as in the presence of a stimulating reagent. In certain embodiments, At least 50x10 6 Individual cells, at least 100 x 10 6 Individual cells, at least 150 x 10 6 Individual cells, at least 200 x 10 6 Individual cells, at least 250 x 10 6 Individual cells, at least 300 x 10 6 Individual cells, at least 350 x 10 6 Individual cells, at least 400 x 10 6 Individual cells, at least 450 × 10 6 A single cell, or at least 500 x 10⁶ 6 Individual cells, 50 x 10 6 Individual cells, 100 x 10 6 Individual cells, 150 x 10 6 Individual cells, 200 x 10 6 Individual cells, 250 x 10 6 Individual cells, 300 x 10 6 Individual cells, 350 x 10 6 Individual cells, 400 x 10 6 Individual cells, 450 x 10 6 A single cell, or 500 x 10 6 Individual cells, or approximately 50 x 10 6 Each cell is approximately 100 x 10⁻¹⁶ 6 Each cell is approximately 150 x 10⁻¹⁰ 6 Each cell is approximately 200 x 10⁻¹⁶ 6 Each cell is approximately 250 x 10⁻¹⁶. 6 Each cell is approximately 300 x 10⁻¹⁰. 6 Each cell is approximately 350 x 10⁻¹⁰. 6 Each cell is approximately 400 x 10⁻¹⁰.6 Each cell is approximately 450 x 10⁻¹⁶. 6 A single cell, or approximately 500 x 10⁻¹⁶ cells. 6 individual cells However, they are incubated under stimulating conditions, for example. In some embodiments, the cells are viable cells. In certain embodiments, the cells are negative for apoptosis markers, such as annexin V or activated caspase 3. In certain embodiments, the cells are or contain CD4+ T cells and CD8+ T cells.

[0238] In some embodiments, 1 × 10 5 ~25,000 x 10 6 individual or approximately 1 x 10 5 ~About 25,000×10 6 pieces, 1×10 6 ~25,000 x 10 6 individual or approximately 1 x 10 6 ~About 25,000×10 6 pieces, 10×10 6 ~2,500 x 10 6 1 or approximately 10 x 10 6 ~Approx. 2,500×10 6 pieces, 1x10 6 ~500x10 6 pc or approximately 1x10 6 ~about 500x10 6 pieces, 50x10 6 ~2,500x10 6 1 or approximately 50x10 6 ~Approx. 2,500x10 6 pieces, 10x10 6 ~500x10 6 1 or approximately 10x10 6 ~about 500x10 6 pieces, 50x10 6 ~300x10 6 1 or approximately 50x10 6 ~about 300x10 6 Individual CD4+ T cells, for example, CD4+ T cells of an input composition, are incubated under stimulating conditions, such as in the presence of a stimulating reagent. In certain embodiments, At least 25x10 6 1 piece, at least 50x10 6 Each, at least 75x106 pieces, at least 100x10 6 10 pieces, at least 125x10 6 150x10 6 Each, at least 175x10 6 1, at least 200x10 6 1, at least 225x10 6 1, or at least 250x10 6 pieces, 25x10 6 pieces, 50x10 6 pieces, 75x10 6 pieces, 100x10 6 pieces, 125x10 6 pieces, 150x10 6 pieces, 175x10 6 pieces, 200x10 6 pieces, 225x10 6 1 piece, or 250x10 6 1 piece, or approximately 25 x 10 6 pieces, about 50x10 6 pieces, about 75x10 6 pieces, about 100x10 6 pieces, about 125x10 6 pieces, about 150x10 6 pieces, about 175x10 6 pieces, about 200x10 6 pieces, approx. 225x10 6 1, or approximately 250 x 10 6 pieces CD4+ T cells are incubated, for example, under stimulating conditions. In some embodiments, the CD4+ T cells are viable CD4+ T cells. In certain embodiments, the CD4+ T cells are negative for apoptosis markers, such as annexin V or activated caspase 3.

[0239] In certain embodiments, 1 × 10 5 ~25,000 x 10 6 individual or approximately 1 x 10 5 ~About 25,000×10 6 pieces, 1×10 6~25,000 x 10 6 individual or approximately 1 x 10 6 ~About 25,000×10 6 pieces, 10×10 6 ~2,500 x 10 6 1 or approximately 10 x 10 6 ~Approx. 2,500×10 6 pieces, 1x10 6 ~500x10 6 pc or approximately 1x10 6 ~about 500x10 6 pieces, 50x10 6 ~2,500x10 6 1 or approximately 50x10 6 ~Approx. 2,500x10 6 pieces, 10x10 6 ~500x10 6 1 or approximately 10x10 6 ~about 500x10 6 pieces, 50x10 6 ~300x10 6 1 or approximately 50x10 6 ~about 300x10 6 Individual CD8+ T cells, for example, CD8+ T cells of an input composition, are incubated under stimulating conditions, such as in the presence of a stimulating reagent. In some embodiments, At least 25x10 6 1 piece, at least 50x10 6 Each, at least 75x10 6 pieces, at least 100x10 6 10 pieces, at least 125x10 6 150x10 6 Each, at least 175x10 6 1, at least 200x10 6 1, at least 225x10 6 1, or at least 250x10 6 pieces, 25x10 6 pieces, 50x10 6 pieces, 75x10 6 pieces, 100x10 6 pieces, 125x10 6 pieces, 150x10 6 pieces, 175x106 pieces, 200x10 6 pieces, 225x10 6 1 piece, or 250x10 6 1 piece, or approximately 25 x 10 6 pieces, about 50x10 6 pieces, about 75x10 6 pieces, about 100x10 6 pieces, about 125x10 6 pieces, about 150x10 6 pieces, about 175x10 6 pieces, about 200x10 6 pieces, approx. 225x10 6 1, or approximately 250 x 10 6 pieces CD8+ T cells are incubated, for example, under stimulating conditions. In some embodiments, the CD8+ T cells are viable CD8+ T cells. In certain embodiments, the CD8+ T cells are negative for apoptosis markers, such as annexin V or activated caspase 3.

[0240] In some embodiments, the conditions for stimulation and / or activation may include one or more of the following: a specific culture medium, temperature, oxygen content, carbon dioxide content, time, active agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulating factors, e.g., cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other active agents designed to activate cells.

[0241] In some embodiments, the stimulating conditions or stimulating reagents include one or more reagents, e.g., ligands, that can stimulate or activate the intracellular signaling domain of the TCR complex. In some embodiments, activators suitable for delivering primary signals, e.g., suitable for initiating activation of ITAM-induced signals, e.g., activators specific to TCR components, e.g., anti-CD3, and / or activators that promote costimulatory signals, e.g., activators specific to T cell costimulatory receptors, e.g., anti-CD28 or anti-4-1BB bound to a solid support such as beads, and / or activators such as one or more cytokines, turn on or initiate the TCR / CD3 intracellular signaling cascade within the T cell. Among the stimulating reagents are anti-CD3 beads / anti-CD28 beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander, and / or ExpACT® beads). Optionally, the expansion method may further include the step of adding anti-CD3 antibodies and / or anti-CD28 antibodies to the culture medium. In some embodiments, the stimulators include cytokines.

[0242] In certain embodiments, the stimulating conditions include incubating, culturing, and / or cultivating cells with a stimulating reagent. In certain embodiments, the stimulating reagent is a reagent provided herein, e.g., the reagent described in Section IB-1. In certain embodiments, the stimulating reagent contains or includes beads. In certain embodiments, the commencement and / or initiation of incubation, culturing, and / or cultivating of cells under stimulating conditions occurs when the cells come into contact with the stimulating reagent and / or are incubated with the stimulating reagent. In certain embodiments, the cells are incubated before, during, and / or after genetically engineering the cells, e.g., introducing recombinant polynucleotides into the cells by transduction or transfection.

[0243] In some embodiments, the enriched T cell composition is incubated with a stimulating reagent and / or beads:cell ratio of 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, or approximately 3:1, approximately 2.5:1, approximately 2:1, approximately 1.5:1, approximately 1.25:1, approximately 1.2:1, approximately 1.1:1, approximately 1:1, approximately 0.9:1, approximately 0.8:1, approximately 0.75:1, approximately 0.67:1, approximately 0.5:1, approximately 0.3:1 or approximately 0.2:1. In certain embodiments, the stimulating reagent a...

Claims

1. A method for producing a composition of manipulated cells, wherein the method is (a) A step of incubating an input composition under stimulating conditions to produce a stimulated composition, The input composition contains a CD4+ T cell:CD8+ T cell ratio of 3:1 to 1:3, and 1 × 10⁻⁶ 6 cells / mL ~ 5×10 6 100 × 10 at a concentration of cells / mL 6 ~500 x 10 6 The input composition comprises a total of 1000 CD4+ T cells and CD8+ T cells, wherein the T cells in the input composition are primary T cells obtained from a human subject with disease or symptoms, and A step comprising a stimulating condition that includes a main agent that specifically binds to CD3 and an adjunct agent that specifically binds to CD28; (b) A step of introducing a recombinant receptor, which is a chimeric antigen receptor (CAR), into T cells from a stimulated composition, thereby generating an engineered cell composition, The introduction is 100 × 10 from the stimulated composition. 6 The process involves contacting 6 T cells (approximately 500 x 10⁶) with an active substance containing a polynucleotide encoding a recombinant receptor. Incubation and introduction were performed in a first serum-free medium containing 0.5 mM–5 mM L-glutamine, 0.5 mM–5 mM L-alanyl-L-glutamine, 50 IU / mL–500 IU / mL recombinant IL-2, 100 IU / mL–2,000 IU / mL recombinant IL-7, and 50 IU / mL–500 IU / mL recombinant IL-15, respectively. The process involves initiating the introduction under stimulating conditions within two days of the start of incubation; and (c) A step of culturing the manipulated composition to produce an output composition containing manipulated T cells, The culture was performed in a second serum-free medium containing 0.5 mM to 5 mM L-glutamine, 0.5 mM to 5 mM L-alanyl-L-glutamine, 50 IU / mL to 500 IU / mL recombinant IL-2, 100 IU / mL to 2,000 IU / mL recombinant IL-7, and 50 IU / mL to 500 IU / mL recombinant IL-15. The culture is initiated within 3 days of the start of incubation under stimulating conditions. The culture is carried out under steady-state shaking conditions. At least a portion of the culture is performed by perfusion using the second serum-free medium, The culture is performed such that the manipulated composition contains at least 2,000 × 10 6 The incubation is carried out until a threshold number of viable T cells is reached, and this threshold number of viable T cells is achieved within 9 days of the start of incubation, and the culture is performed for less than 1 week, and The output composition is suitable for cell therapy to treat the disease or symptoms, process Methods that include...

2. The method according to claim 1, wherein the input composition comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells, which are CD4+ T cells and CD8+ T cells.

3. The input composition is 100 × 10 6 ~300 x 10 6 The method according to claim 1 or 2, comprising a total of 100 CD4+ T cells and CD8+ T cells.

4. The input composition has a concentration of 3×10 6 cells / mL to 5×10 6 cells / mL, and the method according to any one of claims 1 to 3.

5. The method according to any one of claims 1 to 4, wherein the input composition comprises a CD4+ T cell:CD8+ T cell ratio of 2:1 to 1:

2.

6. From the stimulated composition, 300 × 10 6 Less than one cell, or 100 × 10 6 Individual T cells ~ 200 × 10 6 The method according to any one of claims 1 to 5, comprising contacting individual T cells with an active substance containing the polynucleotide.

7. From the stimulated composition, 150 × 10 6 Individual cells: 500 x 10⁶; 6 cells: 200 x 10⁶ 6 A single cell can be up to 500 x 10⁶ cells, or 250 x 10⁶ cells. 6 The method according to any one of claims 1 to 5, comprising contacting 6 cells (up to 500 × 10⁶) with the active substance containing the polynucleotide.

8. During contact, cells from the stimulated composition decreased by 3 × 10 6 Concentrations less than cells / mL, or 0.5 × 10⁻⁶ 6 cells / mL ~ 2×10 6 The method according to any one of claims 1 to 7, wherein the cells are cultured at a concentration of cells / mL.

9. The method according to any one of claims 1 to 8, wherein the contact is initiated within 2 days, within 36 hours, or within 30 hours after the start of incubation.

10. The method according to any one of claims 1 to 9, wherein the culture is carried out under conditions that promote the expansion and proliferation of the manipulated cells.

11. The method according to any one of claims 1 to 10, wherein the incubation comprises recombinant IL-2 in a concentration of 50 to 200 IU / mL, recombinant IL-7 in a concentration of 100 IU / mL to 1,000 IU / mL, and recombinant IL-15 in a concentration of 50 to 200 IU / mL.

12. The method according to any one of claims 1 to 11, wherein the main component and / or auxiliary component are present on the surface of the beads.

13. The method according to claim 12, wherein the ratio of beads to cells is less than 3:1, or between 2:1 and 0.5:

1.

14. The method according to any one of claims 1 to 11, wherein the main agent and the auxiliary agent are reversibly bound to the surface of an oligomer particle reagent containing a plurality of streptavidin molecules or streptavidin mutein molecules.

15. The method according to any one of claims 1 to 14, wherein the incubation is performed for less than 48 hours, 12 hours or more and 36 hours or less, or 18 hours or more and 30 hours or less.

16. The method according to any one of claims 1 to 15, wherein the active substance containing the polynucleotide is a viral vector.

17. The method according to claim 16, wherein the viral vector is a retroviral vector.

18. The method according to claim 16 or 17, wherein the viral vector is a lentiviral vector or a gamma-retroviral vector.

19. At least a portion of the culture is 500 mL / day, 600 mL / day, 700 mL / day, 750 mL / day, 800 mL / day, 900 mL / day, 1,000 mL / day, 1,200 mL / day, 1,400 mL / day, 1,500 mL / day, 1,600 mL / day, 1,800 mL / day and / or 2,000 mL / day, or at least 500 mL / day, at least 600 mL / day, at least 700 mL / day, less The method according to any one of claims 1 to 18, wherein the perfusion is carried out using a rate of at least 750 mL / day, at least 800 mL / day, at least 900 mL / day, at least 1,000 mL / day, at least 1,200 mL / day, at least 1,400 mL / day, at least 1,500 mL / day, at least 1,600 mL / day, at least 1,800 mL / day and / or at least 2,000 mL / day.

20. At least the first portion of the culture is carried out at a perfusion rate of 500 mL / day, 750 mL / day, or 1,000 mL / day, or at least 500 mL / day, at least 750 mL / day, or at least 1,000 mL / day. The method according to any one of claims 1 to 19, wherein at least a second portion of the culture is carried out at a perfusion rate of 1,200 mL / day, 1,400 mL / day, or 1,500 mL / day, or at least 1,200 mL / day, at least 1,400 mL / day, or at least 1,500 mL / day.

21. When the cells reach a first specific density, perfusion is initiated, and the first specific density is 0.4 × 10⁻⁶ 6 Individual cells, 0.5 × 10 6 Individual cells, 0.6 × 10 6 Individual cells, 0.8 × 10 6 Individual cells, 1.0 × 10 6 Individual cells, 1.2 × 10⁻⁶ 6 Individual cells, 1.4 × 10⁻⁶ 6 Individual cells, 1.6 × 10⁻⁶ 6 Individual cells, 1.8 × 10⁻⁶ 6 Individual cells, 2.0 × 10⁻⁶ 6 Individual cells, 2.2 × 10 6 Individual cells or 2.4 × 10⁻⁶ 6 A single cell, or at least 0.4 × 10⁶ 6 A single cell, at least 0.5 × 10⁻⁶ 6 A single cell, at least 0.6 × 10⁻⁶ 6 Each cell, at least 0.8 × 10⁻⁶ 6 A single cell, at least 1.0 × 10⁻⁶ 6 Each cell, at least 1.2 × 10⁶ 6 Each cell, at least 1.4 × 10⁶ 6 Each cell, at least 1.6 × 10⁶ 6 Each cell, at least 1.8 × 10⁶ 6 Each cell, at least 2.0 × 10⁶ 6 Individual cells, at least 2.2 × 10⁻⁶ 6 A single cell or at least 2.4 × 10⁶ 6 It is an individual cell, When the cells reach a second specific density, perfusion is increased, and the second specific density is 0.4 × 10⁻⁶. 6 Individual cells, 0.5 × 10 6 Individual cells, 0.6 × 10 6 Individual cells, 0.8 × 10 6 Individual cells, 1.0 × 10 6 Individual cells, 1.2 × 10⁻⁶ 6 Individual cells, 1.4 × 10⁻⁶ 6 Individual cells, 1.6 × 10⁻⁶ 6 Individual cells, 1.8 × 10⁻⁶ 6 Individual cells, 2.0 × 10⁻⁶ 6 Individual cells, 2.2 × 10 6 Individual cells or 2.4 × 10⁻⁶ 6 A single cell, or at least 0.4 × 10⁶ 6 A single cell, at least 0.5 × 10⁻⁶ 6 A single cell, at least 0.6 × 10⁻⁶ 6 Each cell, at least 0.8 × 10⁻⁶ 6 A single cell, at least 1.0 × 10⁻⁶ 6 Each cell, at least 1.2 × 10⁶ 6 Each cell, at least 1.4 × 10⁶ 6 Each cell, at least 1.6 × 10⁶ 6 Each cell, at least 1.8 × 10⁶ 6 Each cell, at least 2.0 × 10⁶ 6 Individual cells, at least 2.2 × 10⁻⁶ 6 A single cell or at least 2.4 × 10⁶ 6 Individual cells, The method according to any one of claims 1 to 20.

22. The method according to any one of claims 1 to 21, wherein the culture comprises recombinant IL-2 at 100 to 400 IU / mL, recombinant IL-7 at 100 IU / mL to 2,000 IU / mL, and recombinant IL-15 at 100 to 400 IU / mL.

23. The method according to any one of claims 1 to 22, wherein the concentrations of recombinant IL-2, recombinant IL-7, and recombinant IL-15 for the culture are twice the concentrations of recombinant IL-2, recombinant IL-7, and recombinant IL-15 for the incubation, respectively.

24. The method according to any one of claims 1 to 23, wherein the culture is started within 60 hours or within 48 hours after the start of incubation.

25. The method according to any one of claims 1 to 24, wherein the culture is performed for 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 4 days, 5 days, or 6 days, or for 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 4 days, 5 days, or less than 6 days.

26. The threshold number of viable T cells is 2400 × 10⁶ 6 pieces, 3500×10 6 10 units, or 5500 x 10 6 The method according to any one of claims 1 to 25, wherein the T cell is a single living T cell.

27. The method according to any one of claims 1 to 26, wherein the viability of a threshold number of cells is 75% or at least 75%, or 85% or at least 85%.

28. The method according to any one of claims 1 to 27, wherein the threshold number of viable T cells is achieved 5 to 9 days after the start of incubation, or 5 to 8 days after the start of incubation.

29. The method according to any one of claims 1 to 28, further comprising the step of formulating cells of the output composition for cryopreservation and / or administration to a subject.

30. The method according to any one of claims 1 to 29, wherein CD4+ T cells and CD8+ T cells of the input composition are isolated from a biological sample derived from the subject before incubation.

31. The method according to claim 30, wherein the biological sample is 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, or a leukocyte apheresis product.

32. The method according to any one of claims 1 to 31, wherein a recombinant receptor can bind to a target antigen that is associated with, specific to, and / or expressed in cells or tissues of the disease or symptom.

33. The method according to any one of claims 1 to 32, wherein the disease or symptom is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer.

34. The method according to any one of claims 1 to 33, wherein the disease or symptom is multiple myeloma.

35. The method according to any one of claims 1 to 34, wherein the recombinant receptor is anti-BCMA CAR.

36. (A) 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%, or at least 95% of the cells in the output composition are of the memory phenotype. 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%, or at least 95% of the cells in the output composition are of the central memory phenotype. 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%, or at least 95% of the cells in the output composition are CD27+, CD28+, CCR7+, CD45RA-, CD45RO+, CD62L+, CD3+, CD95+, granzyme B- and / or CD127+, and / or 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%, or at least 95% of the cells in the output composition are CCR7+ / CD45RA- or CCR7+ / CD45RO+; and / or (B) Among the multiple iterations of the method for producing multiple output compositions, (i) The average percentage of memory phenotypic cells in multiple output compositions is 40%–65%, 40%–45%, 45%–50%, 50%–55%, 55%–60%, or 60%–65%. (ii) The average percentage of cells with a central memory phenotype in multiple output compositions is 40%–65%, 40%–45%, 45%–50%, 50%–55%, 55%–60%, or 60%–65%. (iii) The average percentage of cells in multiple output compositions that are CD27+, CD28+, CCR7+, CD45RA-, CD45RO+, CD62L+, CD3+, CD95+, granzyme B-, and / or CD127+ is 40%–65%, 40%–45%, 45%–50%, 50%–55%, 55%–60%, or 60%–65%. (iv) The average percentage of cells that are CCR7+ / CD45RA- or CCR7+ / CD45RO+ in multiple output compositions is 40%–65%, 40%–45%, 45%–50%, 50%–55%, 55%–60%, or 60%–65%. (v) The average percentage of central memory CD4+ T cells in manipulated CD4+ T cells of multiple output compositions is 40%–65%, 40%–45%, 45%–50%, 50%–55%, 55%–60%, or 60%–65%. (vi) The average percentage of central memory CD8+ T cells in manipulated CD8+ T cells of multiple output compositions is 40%–65%, 40%–45%, 45%–50%, 50%–55%, 55%–60%, or 60%–65%, and / or (vii) The average percentage of central memory T cells in the manipulated T cells of multiple output compositions is 40%–65%, 40%–45%, 45%–50%, 50%–55%, 55%–60%, or 60%–65%. The method according to any one of claims 1 to 35.

Images