T-cell activation marker and method for assessing T-cell activation

Abstract

The present disclosure relates to methods of assessing T cell activation status within a cellular composition, which may be used in conjunction with cell therapy or generating cells for use in cell therapy. The cells of the cell composition can express recombinant receptors, such as chimeric receptors, such as chimeric antigen receptors (CAR), or other transgenic receptors, such as T cell receptors (TCR). The methods provide markers and how the markers are used to assess the activation of T cells within cellular compositions.

Description

Cross Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 468,524, filed May 23, 2023, entitled “ACTIVATION MARKERS OF T CELLS AND METHODS FOR ASSESSING T CELL ACTIVATION,” the contents of which are incorporated by reference in their entirety. Reference to Electronic Sequence Listing

[0002] This application is filed with an electronic format sequence listing. The sequence listing is provided in a file named 735042027540SEQLIST.xml, created on May 21, 2024, which is 99,949 bytes in size. The information contained in the electronic format sequence listing is incorporated by reference in its entirety. TECHNICAL FIELD

[0003] The present invention relates to methods of assessing activation of T cells in a cell composition for use in conjunction with cell therapy or cell transduction. The T cells of the cell composition can express a recombinant receptor, such as a chimeric receptor, e.g., a chimeric antigen receptor (CAR), or other transgenic receptor, such as a T cell receptor (TCR). The methods provide assays for assessing activation of T cells using one or more markers and examining surface expression levels or percentage of cells positive for the marker to assess activation. BACKGROUND

[0004] There are a variety of methods for determining the activation status of T cells in culture or from a sample, which generally rely on markers present on the cell surface that are upregulated upon T cell activation. By examining these defined markers and their surface expression levels or percentage of T cells within a cell composition that are positive for the marker, the activation status is assessed. However, markers can only be present on certain subsets of T cells, and thus combinations of markers are used to assess T cell activation, resulting in the benefit of having multiple markers available to select from for assessing T cell activation. These methods of assessing T cell activation using markers rely on characterization results that demonstrate which surface markers expression levels change during T cell activation, and these methods benefit from a larger pool of available markers. Provided herein are methods that address such needs. SUMMARY

[0005] Provided herein is a method for assessing activation of T cells within a cellular composition, the method comprising: (a) detecting surface expression levels of one or more markers or percentage of cells positive for one or more markers in cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein: the one or more markers of group (i) are selected from: CD36L (SCARB1, SR-BI), CD120b, CD107b (LAMP-2), CD200 (OX2), CD357 (GITR), CD134 (OX40), CD83, CD155 (PVR), CD74, GPR56, Notch 1, CD119 (IFN-g R a chain), CD105 (Endoglin), CD262 (DR5, TRAIL-R2), CD170 (Siglec-5), CD73 (ecto-5'-nucleotidase), CD360 (IL-21R), CD20, CD107a (LAMP-1), CD109, CD132 (common g chain), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, bone marrow stromal cell antigen 2), CD323 (JAM3), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD97, integrin b7, mouse IgG3 k, Notch 2, CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, Ig light chain k, Ig light chain l, IgM, CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD266 (Fn14, TWEAKCD276 (B7-H3), CD326 (Ep-CAM), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-Rl), CD304 (Neuropilin-1), CD49d, DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R); and the one or more markers of group (ii) are selected from CD192 (CCR2), CCRL2, CD96 (TACTILE), CD195 (CCR5), CD124 (IL-4Ra), CD49f, CD314 (NKG2D), KLRG1 (MAFA), CD96 (TACTILE), HLA-E, CD195 (CCR5), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CD11b, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR; (b) comparing the surface expression level or percentage of positive cells of each of the one or more markers in the cell composition to the surface expression level or percentage of positive cells of each of the one or more markers in a reference, wherein a higher level or higher percentage of positive cells of a marker in (i) indicates that the T cell is activated compared to the reference, and a lower level or lower percentage of positive cells of a marker in (ii) indicates that the T cell is activated compared to the reference.

[0006] In some of any of the embodiments, the reference is comprised of an unstimulated control cell composition.

[0007] In some of any embodiment, the reference is an expression level or percentage of positive cells across a plurality of cell compositions, wherein each cell composition is from a different patient, subject, or donor. In some of any embodiment, the reference is an average expression level or average percentage of positive cells across a plurality of cell compositions, wherein each cell composition is from a different patient, subject, or donor. In some of any embodiment, the reference is a median expression level or median percentage of positive cells across a plurality of cell compositions, wherein each cell composition is from a different patient, subject, or donor.

[0008] Provided herein is a method for assessing surface expression of T cell activation markers of T cells, the method comprising detecting surface expression levels of one or more markers or percentage of cells positive for one or more markers in cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein: the one or more markers of group (i) are selected from: CD36L (SCARB1, SR-BI), CD120b, CD107b (LAMP-2), CD200 (OX2), CD357 (GITR), CD134 (OX40), CD83, CD155 (PVR), CD74, GPR56, Notch 1, CD119 (IFN-g R a chain), CD105 (Endoglin), CD262 (DR5, TRAIL-R2), CD170 (Siglec-5), CD73 (ecto-5'-nucleotidase), CD360 (IL-21R), CD20, CD107a (LAMP-1), CD109, CD132 (common g chain), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, bone marrow stromal cell antigen 2), CD323 (JAM3), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD97, integrin b7, mouse IgG3 k, Notch 2, CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, Ig light chain k, Ig light chain l, IgM, CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD266 (Fn14, TWEAKCD276 (B7-H3), CD326 (Ep-CAM), CD49e, CD87, CD8a, CD9, DcR1 (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin-1), CD49d, DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R); and the one or more markers of group (ii) are selected from CD192 (CCR2), CCRL2, CD96 (TACTILE), CD195 (CCR5), CD124 (IL-4Ra), CD49f, CD314 (NKG2D), KLRG1 (MAFA), CD96 (TACTILE), HLA-E, CD195 (CCR5), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CD11b, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR.

[0009] In some of any of the embodiments, the surface expression level or percentage of positive cells of the one or more markers of (i) is positively correlated with T cell activation. In some of any of the embodiments, the surface expression level or percentage of positive cells of the one or more markers of (ii) is negatively correlated with T cell activation.

[0010] Provided herein is a method of comparing activation of T cells in a donor, the method comprising: (a) detecting surface expression level of one or more markers or percentage of cells positive for one or more markers in cells of a composition comprising T cells, wherein the one or more markers is selected from group (i) and / or group (ii), wherein group (i) consists of: CD36L (SCARB1, SR-BI), CD120b, CD107b (LAMP-2), CD200 (OX2), CD357 (GITR), CD134 (OX40), CD83, CD155 (PVR), CD74, GPR56, Notch 1, CD119 (IFN-g R a chain), CD105 (Endoglin), CD262 (DR5, TRAIL-R2), CD170 (Siglec-5), CD73 (ecto-5'-nucleotidase), CD360 (IL-21R), CD20, CD107a (LAMP-1), CD109, CD132 (common g chain), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, bone marrow stromal cell antigen 2), CD323 (JAM3), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD97, integrin b7, mouse IgG3 k, Notch 2, CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, Ig light chain k, Ig light chain l, IgM, CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD266 (Fn14, TWEAKCD276 (B7-H3), CD326 (Ep-CAM), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin-1), CD49d, DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R); and group (ii) consists of CD192 (CCR2), CCRL2, CD96 (TACTILE), CD195 (CCR5), CD124 (IL-4Ra), CD49f, CD314 (NKG2D), KLRG1 (MAFA), CD96 (TACTILE), HLA-E, CD195 (CCR5), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CD11b, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR; (b) comparing the surface expression level or the percentage of positive cells to the level of each of the one or more markers or the percentage of positive cells in an unstimulated cell composition, wherein a higher level or a higher percentage of positive cells of a marker in (i) indicates that the T cell is activated, and a lower level or a lower percentage of positive cells of a marker in (ii) indicates that the T cell is activated compared to the unstimulated cell composition.

[0011] In some of any of the embodiments, the composition comprising T cells is incubated with a T cell stimulator under conditions to induce T cell activation prior to the detecting.

[0012] In some of any of the embodiments, the method comprises incubating the composition with a T cell stimulator prior to the detecting. In some of any of the embodiments, the method comprises incubating the composition with a T cell stimulator after the detecting.

[0013] In some of any of the embodiments, the incubation with the T cell stimulator is performed in vivo in the subject. In some of any of the embodiments, the incubation with the T cell stimulator is performed in vitro or ex vivo.

[0014] In some of any of the embodiments, the incubation with the T cell stimulator is performed for 12-72 hours. In some of any of the embodiments, the incubation with the T cell stimulator is performed for about 24 hours.

[0015] In some of any of the embodiments, the one or more markers of group (i) is selected from CD20, CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (OX40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Bone marrow stromal cell antigen 2), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, integrin b7, mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain l, IgM, CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R); and the one or more markers of group (ii) is selected from CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD172g (SIRPg), CD127 (IL-7Ra), CD49f, a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR.

[0016] In some of any of the embodiments, the incubation with the T cell stimulator is performed for about 48 hours.

[0017] In some of any of the embodiments, the one or more markers of the group (i) are selected from CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (OX40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Bone marrow stromal antigen 2), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, integrin b7, mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain l, IgM, CD119 (IFN-g R a chain), CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Fn14, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcR1 (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin-1), CD49d, CD73 (ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), and GARP (LRRC32);and the one or more markers of group (ii) are selected from CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD11b, CX3CR1, NKp80, CD127 (IL-7Ra), and CD49f.

[0018] In some of any of the embodiments, the one or more markers of group (i) are selected from CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM), and GPR56, and the one or more markers of group (ii) are selected from CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CD11b, and CX3CR1.

[0019] In some of any of the embodiments, the one or more markers are of (i) and are selected from CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM), and GPR56.

[0020] Provided herein is a method of identifying an activated T cell, the method comprising detecting cell surface expression of one or more markers in a cell of a composition comprising T cells, wherein the one or more markers is selected from the group consisting of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM), and GPR56, and wherein a cell expressing a high level of the one or more markers is an activated T cell.

[0021] In some of any of the embodiments, the one or more markers is selected from group (i) and consists of CD200 (OX2), CD357 (GITR), CD120b, CD155 (PVR), CD107b (LAMP-2).

[0022] In some of any of the embodiments, the one or more markers is (ii) and is selected from CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CD11b, and CX3CR1.

[0023] Provided herein is a method of identifying an activated T cell, the method comprising detecting cell surface expression of one or more markers in a cell of a composition comprising T cells, wherein the one or more markers is selected from the group consisting of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CD11b, and CX3CR1, and wherein a cell expressing a low level of the one or more markers is an activated T cell.

[0024] In some of any of the embodiments, the detecting is detecting CD4+ or CD8+ T cells in the composition comprising T cells.

[0025] In some of any of the embodiments, the one or more markers from group (i) are selected from the group consisting of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), CD355 (CRTAM), and GPR56, and the one or more markers from group (ii) are selected from the group consisting of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD96 (TACTILE), and CD195 (CCR5).

[0026] In some of any of the embodiments, the detecting is detecting CD4+ T cells in the composition comprising T cells.

[0027] In some of any of the embodiments, the one or more markers from group (i) are selected from the group consisting of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, and CD165, and the one or more markers from group (ii) are selected from the group consisting of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), and CD127 (IL-7Ra).

[0028] Provided herein is a method for assessing surface expression of T cell activation markers of CD4+ T cells, the method comprising detecting surface expression levels of one or more markers or percentage of cells positive for one or more markers in CD4+ T cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein group (i) consists of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, and CD165, and group (ii) consists of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), and CD127 (IL-7Ra).

[0029] In some of any embodiments, the surface expression levels or percentage of positive cells of one or more markers of (i) are positively correlated with CD4+ T cell activation. In some of any embodiments, the surface expression levels or percentage of positive cells of one or more markers of (ii) are negatively correlated with CD4+ T cell activation.

[0030] Provided herein is a method for assessing activation of CD4+ T cells, the method comprising: (a) detecting surface expression levels of one or more markers or percentage of cells positive for one or more markers in CD4+ T cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein group (i) consists of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, and CD165, and group (ii) consists of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), and CD127 (IL-7Ra); and (b) comparing the surface expression levels or the percentage of positive cells to levels of each of the one or more markers or percentage of positive cells in cells of an unstimulated control cell composition, wherein a higher level or higher percentage of positive cells of a marker in (i) indicates that the CD4+ T cells are activated compared to the unstimulated control cell composition, and a lower level or lower percentage of positive cells of a marker in (ii) indicates that the CD4+ T cells are activated compared to the unstimulated control cell composition.

[0031] In some of any of the embodiments, the detecting is detecting CD8+ T cells in the composition comprising T cells.

[0032] In some of any of the embodiments, the one or more markers selected from group (i) are selected from CD120b, CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch 2, and the one or more markers from group (ii) are selected from CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CD11b, CX3CR1, and CD127 (IL-7Ra).

[0033] Provided herein is a method for assessing surface expression of T cell activation markers of CD8+ T cells, the method comprising detecting surface expression levels of one or more markers or percentage of cells positive for one or more markers in CD8+ T cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein group (i) consists of CD120b, CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch 2, and group (ii) consists of CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CD11b, CX3CR1, and CD127 (IL-7Ra).

[0034] In some of any embodiments, the surface expression levels or percentage of positive cells of one or more markers of (i) are positively correlated with CD8+ T cell activation. In some of any embodiments, the surface expression levels or percentage of positive cells of one or more markers of (ii) are negatively correlated with CD8+ T cell activation.

[0035] Provided herein is a method for assessing activation of CD8+ T cells, the method comprising: (a) detecting surface expression level of one or more markers or percentage of cells positive for one or more markers in CD8+ T cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein group (i) consists of CD120b, CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch2, and group (ii) consists of CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CD11b, CX3CR1, and CD127 (IL-7Ra); and (b) comparing the surface expression level or the percentage of positive cells to the level of each of the one or more markers or percentage of positive cells in cells of an unstimulated control cell composition, wherein a higher level or higher percentage of positive cells of a marker in (i) indicates that the CD8+ T cells are activated compared to the unstimulated control cell composition, and a lower level or lower percentage of positive cells of a marker in (ii) indicates that the CD8+ T cells are activated compared to the unstimulated control cell composition.

[0036] In some of any of the embodiments, the composition comprising T cells comprises T cells genetically engineered to express a recombinant receptor.

[0037] In some of any of the embodiments, the one or more markers selected from group (i) consists of CD120b, CD83, CD357 (GITR), CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD355 (CTRAM), CD155 (PVR), CD74, CD170 (Siglec-5), and GPR56, and the one or more markers selected from group (ii) consists of CD49f, CCRL2, CD124 (IL-4Ra), CD217, CD192 (CCR2), CD195 (CCR5), and CD96 (TACTILE).

[0038] Provided herein is a method for assessing activation of T cells, the method comprising: (a) detecting surface expression level of one or more markers or percentage of cells positive for one or more markers in CD8+ T cells of a composition comprising T cells, wherein the one or more markers is selected from group (i) and / or group (ii), wherein group (i) consists of CD120b, CD83, CD357 (GITR), CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD355 (CRTAM), CD155 (PVR), CD74, CD170 (Siglec-5), and GPR56, and group (ii) consists of CD120b, CD83, CD357 (GITR), CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD355 (CRTAM), CD155 (PVR), CD74, CD170 (Siglec-5), and GPR56.

[0039] In some of any of the embodiments, the one or more markers from group (i) is selected from CD134 (OX40), CD107b (LAMP-2), CD155 (PVR), CD74, and CD170 (Siglec-5), and the one or more markers selected from group (ii) is selected from CD49f, CCRL2, CD124 (IL-4Ra), CD217, CD355 (CRTAM), GPR56, and CD96 (TACTILE).

[0040] In some of any of the embodiments, the detecting is detecting recombinant receptor-expressing CD4+ T cells in the composition comprising T cells.

[0041] In some of any of the embodiments, the one or more markers from group (i) is selected from CD134 (OX40), CD107b (LAMP-2), CD155 (PVR), and CD74, and / or the one or more markers selected from group (ii) is selected from CD49f, CCRL2, and CD124 (IL-4Ra).

[0042] In some of any of the embodiments, the detecting is detecting recombinant receptor-expressing CD8+ T cells in the composition comprising T cells.

[0043] In some of any of the embodiments, the one or more markers from group (i) are selected from CD200 (OX2), CD107b (LAMP-2), CD155 (PVR), CD355 (CRTAM), and GPR56, and / or the one or more markers selected from group (ii) are selected from CCRL2, CD217, CD96 (TACTILE).

[0044] In some of any of the embodiments, the one or more markers of group (i) are selected from CD36L (SCARB1, SR-BI), CD262 (DR5, Trail-R2), CD105 (Endoglin), CD73 (ecto-5'-nucleotidase), CD83, CD119 (IFN-g R a chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R), and the one or more markers of group (i) are selected from CD96 (TACTILE).

[0045] Provided herein is a method for assessing activation of T cells, the method comprising: (a) detecting surface expression level of one or more markers or percentage of cells positive for one or more markers in a T cell composition comprising T cells expressing a recombinant receptor, wherein the one or more markers are selected from group (i) and / or group (ii), wherein group (i) consists of CD36L (SCARB1, SR-BI), CD262 (DR5, Trail-R2), CD105 (Endoglin), CD73 (ecto-5'-nucleotidase), CD83, CD119 (IFN-g R a chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R), and group (ii) consists of CD96 (TACTILE).

[0046] In some embodiments, the composition comprising T cells comprises cells expressing a recombinant receptor.

[0047] In some of any of the embodiments, the detecting is detecting recombinant expressing cells of the T cell composition.

[0048] In some of any of the embodiments, surface expression of the one or more markers of group (i) is increased on cells expressing the recombinant receptor compared to cells not expressing the recombinant receptor.

[0049] In some of any of the embodiments, surface expression of one or more markers of the group (ii) is reduced on cells expressing the recombinant receptor compared to cells not expressing the recombinant receptor.

[0050] In some of any of the embodiments, the composition comprising T cells comprises T cells genetically engineered to express a recombinant receptor, and wherein the T cell stimulator is a recombinant receptor stimulator that induces recombinant receptor-dependent T cell activation.

[0051] In some of any of the embodiments, the recombinant receptor is a chimeric antigen receptor (CAR).

[0052] In some of any of the embodiments, the recombinant receptor stimulator comprises a recombinant target antigen recognized by the recombinant receptor. In some of any of the embodiments, the recombinant receptor stimulator is an antibody specific for an extracellular antigen binding domain of the recombinant receptor. In some of any of the embodiments, the recombinant receptor stimulator is an anti-idiotypic antibody specific for an extracellular antigen binding domain of the recombinant receptor.

[0053] In some of any of the embodiments, the recombinant receptor stimulator is immobilized or attached to a solid support. In some of any of the embodiments, the solid support is a surface of a vessel, optionally a well of a microplate or a flask. In some of any of the embodiments, the solid support is a bead.

[0054] In some of any of the embodiments, the recombinant receptor stimulator is an antigen-expressing cell, optionally wherein the cell is a clone, from a cell line, or a primary cell taken from a subject. In some of any of the embodiments, the antigen-expressing cell is a cell line. In some of any of the embodiments, the cell line is a tumor cell line. In some of any of the embodiments, the antigen-expressing cell is a cell that has been engineered to express an antigen of the recombinant receptor.

[0055] In some of any of the embodiments, the detecting is detecting recombinant receptor-expressing T cells in the composition comprising T cells.

[0056] In some of any of the embodiments, the one or more markers from group (i) are selected from the group consisting of CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, CD165, CD83, CD357 (GITR), CD200 (OX2), and CD134 (OX40), and / or the one or more markers from group (ii) are selected from the group consisting of KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CD192 (CCR2), CD11b, and CX3CR1.

[0057] In some of any of the embodiments, the one or more markers from group (i) are selected from the group consisting of CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, CD165, and CD83, and / or the one or more markers from group (ii) are selected from the group consisting of KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CD11b, and CX3CR1.

[0058] In some of any of the embodiments, the detecting is detecting CD4+ T cells in the composition comprising T cells.

[0059] In some of any of the embodiments, the one or more markers from group (i) are selected from the group consisting of CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, and CD165, and / or the one or more markers from group (ii) are selected from the group consisting of KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), and CD127 (IL-7Ra).

[0060] In some of any of the embodiments, the detecting is detecting CD8+ T cells in the composition comprising T cells.

[0061] In some of any of the embodiments, the one or more markers from group (i) are selected from the group consisting of CD71, Notch 1, CD107a (LAMP-1), CD166, Notch 2, CD165, CD83, and / or the one or more markers from group (ii) are selected from the group consisting of CD11b, CX3CR1, and CD127 (IL-7Ra).

[0062] In some of any embodiment, the T cell stimulator is a pan-T cell activator. In some of any embodiment, the pan-T cell activation reagent comprises an anti-CD3 antibody and an anti-CD28 antibody, optionally wherein the pan-T cell activation reagent comprises an anti-CD3 Fab and an anti-CD28 Fab. In some of any embodiment, the pan-T cell activation reagent comprises anti-CD3 / anti-CD28 beads. In some of any embodiment, the pan-T cell activation reagent comprises a soluble anti-CD3 / anti-CD28 streptavidin oligomerization reagent.

[0063] In some of any embodiment, prior to the detecting of step (a), the method comprises contacting the cells of the composition comprising T cells with one or more binding agents that bind the one or more markers.

[0064] In some of any embodiment, prior to the detecting of step (a), the method comprises contacting the cells of the composition comprising T cells with one or more binding agents that comprise a substance for binding the one or more markers.

[0065] In some of any embodiment, the one or more binding agents is one or more antibodies or antigen-binding fragments. In some of any embodiment, the one or more binding agents is detectably labeled. In some of any embodiment, the one or more binding agents is fluorescently labeled.

[0066] In some of any embodiment, the detecting is by flow cytometry. In some of any embodiment, the detecting of step (a) is performed in conjunction with CITE-Seq or REAP-seq. In some of any embodiment, the detecting of step (a) is by immunohistochemistry, optionally by immunohistochemistry fluorescence.

[0067] In some of any embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 different markers are used in the detecting of step (a).

[0068] Provided herein is a kit for determining T cell activation, the kit comprising a substance for detecting the one or more markers in the method of some of any embodiment.

[0069] Provided herein is a kit for determining T cell activation, the kit comprising a binding agent comprising a substance for binding one or more markers in the methods of some of any embodiments.

[0070] In some embodiments, the substance for detecting each of the one or more markers is an antibody.

[0071] Provided herein is a kit for determining T cell activation, the kit comprising a means for detecting one or more markers selected from Group (i) and / or Group (ii),CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (OX40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Bone marrow stromal antigen 2), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, integrin b7, mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain l, IgM, CD119 (IFN-g R a chain), CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Fn14, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcR1 (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin-1), CD49d, CD73 (ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R),and group (ii) consists of: CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CD11b, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), CD49f, a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR.

[0072] Provided herein is a kit for determining T cell activation, the kit comprising a means for detecting one or more markers selected from Group (i) and / or Group (ii), wherein Group (i) consists of CD20, CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (OX40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Bone marrow stromal cell antigen 2), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, integrin b7, mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain l, IgM, CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R), and Group (ii) consists of CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD172g (SIRPg), CD127 (IL-7Ra), CD49f, a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR.

[0073] Provided herein is a kit for determining T cell activation comprising a means for detecting one or more markers selected from Group (i) and / or Group (ii), wherein Group (i) consists of: CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (OX40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Bone marrow stromal antigen 2), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, integrin b7, mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain l, IgM, CD119 (IFN-g R a chain), CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Fn14, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcR1 (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin-1), CD49d, CD73 (ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32),and group (ii) consists of: CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CD11b, and CX3CR1.

[0074] Provided herein is a kit for determining T cell activation, the kit comprising a means for detecting one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of: CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM), and GPR56, and group (ii) consists of: CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CD11b, and CX3CR1.

[0075] Provided herein is a kit for determining T cell activation, the kit comprising a means for detecting one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of: CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), CD355 (CRTAM), and GPR56, and group (ii) consists of: CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD96 (TACTILE), and CD195 (CCR5).

[0076] Provided herein is a kit for determining T cell activation comprising a means for detecting one or more markers selected from Group (i) and / or Group (ii), wherein Group (i) consists of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, and CD165, and Group (ii) consists of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), and CD127 (IL-7Ra).

[0077] Provided herein is a kit for determining T cell activation comprising a means for detecting one or more markers selected from Group (i) and / or Group (ii), wherein Group (i) consists of CD120b, CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch 2, and Group (ii) consists of CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CD11b, CX3CR1, and CD127 (IL-7Ra).

[0078] Provided herein is a kit for determining T cell activation comprising a means for detecting one or more markers selected from Group (i) and / or Group (ii), wherein Group (i) consists of CD120b, CD83, CD357 (GITR), CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD155 (PVR), CD74, and the one or more markers selected from Group (ii) consists of CD49f, CCRL2, CD124 (IL-4Ra), CD217, CD192 (CCR2), CD355 (CRTAM), GPR56, CD195 (CCR5), and CD96 (TACTILE).

[0079] Provided herein is a kit for determining T cell activation, the kit comprising a means for detecting one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, CD165, CD120b, CD83, CD357 (GITR), CD200 (OX2), and CD134 (OX40), and / or the one or more markers from group (ii) is selected from KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CD192 (CCR2), CD11b, and CX3CR1.

[0080] Provided herein is a kit for determining T cell activation, the kit comprising a means for detecting one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD262 (DR5, Trail-R2), CD105 (Endoglin), CD36L1 (SCARB1, SR-BI), CD73 (ecto-5'-nucleotidase), CD83, CD119 (IFN-g R a chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R), and / or the one or more markers from group (ii) is selected from CD96 (TACTILE).

[0081] Provided herein is a kit for determining T cell activation, the kit comprising a binding agent, the binding agent comprising a substance for binding one or more markers selected from group (i) and / or group (ii),CD36L (SCARB1, SR-BI), CD120b, CD107b (LAMP-2), CD200 (OX2), CD357 (GITR), CD134 (OX40), CD83, CD155 (PVR), CD74, GPR56, Notch 1, CD119 (IFN-g R a chain), CD105 (Endoglin), CD262 (DR5, TRAIL-R2), CD170 (Siglec-5), CD73 (ecto-5'-nucleotidase), CD360 (IL-21R), CD20, CD107a (LAMP-1), CD109, CD132 (common g chain), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, bone marrow stromal antigen 2), CD323 (JAM3), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD97, integrin b7, mouse IgG3 k, Notch 2, CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, Ig light chain k, Ig light chain l, IgM, CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD266 (Fn14, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD49e, CD87, CD8a, CD9, DcR1 (TRAIL-R3, CD263), ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin-1), CD49d, DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R),And group (ii) consists of: CD192 (CCR2), CCRL2, CD96 (TACTILE), CD195 (CCR5), CD124 (IL-4Ra), CD49f, CD314 (NKG2D), KLRG1 (MAFA), CD96 (TACTILE), HLA-E, CD195 (CCR5), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CD11b, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR.

[0082] Provided herein is a kit for determining T cell activation, the kit comprising a binding agent comprising a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD20, CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (OX40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, bone marrow stromal cell antigen 2), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, integrin b7, mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain l, IgM, CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R), and group (ii) consists of CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD172g (SIRPg), CD127 (IL-7Ra), CD49f, a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR.

[0083] Provided herein is a kit for determining T cell activation, the kit comprising a binding agent, the binding agent comprising a substance for binding one or more markers selected from group (i) and / or group (ii),CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (OX40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Bone marrow stromal antigen 2), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, integrin b7, mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain l, IgM, CD119 (IFN-g R a chain), CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Fn14, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcR1 (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin-1), CD49d, CD73 (ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32),and group (ii) consists of: CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CD11b, and CX3CR1.

[0084] Provided herein is a kit for determining T cell activation, the kit comprising a binding agent comprising a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of: CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM), and GPR56, and group (ii) consists of: CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CD11b, and CX3CR1.

[0085] Provided herein is a kit for determining T cell activation, the kit comprising a binding agent comprising a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of: CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), CD355 (CRTAM), and GPR56, and group (ii) consists of: CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD96 (TACTILE), and CD195 (CCR5).

[0086] Provided herein is a kit for determining T cell activation, the kit comprising a binding agent comprising a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, and CD165, and group (ii) consists of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), and CD127 (IL-7Ra).

[0087] Provided herein is a kit for determining T cell activation, the kit comprising a binding agent comprising a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD120b, CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch 2, and group (ii) consists of CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CD11b, CX3CR1, and CD127 (IL-7Ra).

[0088] Provided herein is a kit for determining T cell activation comprising a binding agent comprising a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD120b, CD83, CD357 (GITR), CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD155 (PVR), CD74, and the one or more markers selected from group (ii) consists of CD49f, CCRL2, CD124 (IL-4Ra), CD217, CD192 (CCR2), CD355 (CRTAM), GPR56, CD195 (CCR5), and CD96 (TACTILE).

[0089] Provided herein is a kit for determining T cell activation comprising a binding agent comprising a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, CD165, CD120b, CD83, CD357 (GITR), CD200 (OX2), and CD134 (OX40), and / or the one or more markers from group (ii) is selected from KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CD192 (CCR2), CD11b, and CX3CR1.

[0090] Provided herein is a kit for determining T cell activation comprising a binding agent comprising a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD36L (SCARB1, SR-BI), CD262 (DR5, Trail-R2), CD105 (Endoglin), CD73 (ecto-5'-nucleotidase), CD83, CD119 (IFN-g R a chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R), and / or the one or more markers from group (ii) is selected from CD96 (TACTILE).

[0091] In some of any of the embodiments, the agent for detecting the one or more markers is one or more binding agents that bind to the one or more markers. In some of any of the embodiments, the one or more binding agents is one or more antibodies or antigen-binding fragments. In some of any of the embodiments, the one or more binding agents is detectably labeled. In some of any of the embodiments, the one or more binding agents is fluorescently labeled.

[0092] Provided herein is a method of isolating activated T cells, the method comprising identifying a population of activated T cells and isolating the population according to the method in some of any of the embodiments.

[0093] Provided herein is a method of enriching activated T cells, the method comprising identifying a population of activated T cells and selecting the population according to the method in some of any of the embodiments, thereby obtaining a population of cells enriched for activated T cells.

[0094] Provided herein is a method of depleting a population of cells of activated T cells, the method comprising identifying a population of activated T cells and depleting the population of activated T cells according to the method in some of any of the embodiments.

[0095] Provided herein is a population of T cells produced according to the method in some of any of the embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0096] Figure 1A The difference in the percentage of positive cells for each marker for CD8+ CAR+ T cells calculated between the T0 and T48 hour stimulation time points is shown. The top 15 upregulated markers and the bottom 5 downregulated markers are shown here and are coded based on the literature as to whether they are canonical or non-canonical activation markers.

[0097] Figure 1B The difference in the percentage of positive cells for each marker for CD4+ CAR+ T cells calculated between the T0 and T48 hour stimulation time points is shown. In this case, a CD4 CAR-T product is shown. The top 15 upregulated markers and the bottom 5 downregulated markers are shown here and are coded based on the literature as to whether they are canonical or non-canonical activation markers.

[0098] Figure 2A The difference in the percentage of positive cells for each marker for CD8+ PBMC T cells calculated between the T0 and T48 hour stimulation time points is shown. The top 15 upregulated markers and the bottom 5 downregulated markers are shown here and are coded based on the literature as to whether they are canonical or non-canonical activation markers.

[0099] Figure 2BDifferences in the percentage of positive cells for each marker of CD4+ PBMC T cells calculated between the T0 and T48 hour stimulation time points are shown. The top 15 upregulated markers and the bottom 5 downregulated markers are shown here and are coded based on whether they are canonical or non-canonical activation markers according to the literature.

[0100] Figure 3A The same data from the previous four plots is shown, visualized differently, where the top 15 upregulated markers and the bottom 5 downregulated markers from each of the previous four plots are aggregated together and shown on the x-axis. The CD4 component of both PBMC and CAR+ T cells are shown together. If a marker is blank, this does not mean that the marker is not present in the CD4 component, but rather that it did not make it into the top 15 and bottom 5 marker lists.

[0101] Figure 3B The same data from the previous four plots is shown, visualized differently, where the top 15 upregulated markers and the bottom 5 downregulated markers from each of the previous four plots are aggregated together and shown on the x-axis. The CD8 component of both PBMC and CAR+ T cells are shown together. If a marker is blank, this does not mean that the marker is not present in the CD8 component, but rather that it did not make it into the top 15 and bottom 5 marker lists.

[0102] Figure 4 The abundance of 5 representative non-canonical activation markers within the CAR+ T product is shown, depending on whether the cell also expresses at least one canonical activation marker. Cells are divided into two bins: cells that express at least one canonical activation marker and cells that do not express any canonical activation markers. The percentage of cells that express each of the five non-canonical activation markers is shown separately in the two bins. Cells that express at least one canonical marker are more likely to also express each of the non-canonical markers.

[0103] Figures 5A-5S Pseudo-color plots of upregulated non-canonical markers generated in Figures 1-3 are shown, filtered only on CAR+ T cells, and plotted against the three canonical activation markers.

[0104] Figures 6A-6J Pseudo-color plots of other canonical markers generated in Figures 1-3 are shown, filtered only on CAR+ T cells, and plotted against Figure 4 the three canonical activation markers used in Figure 5.

[0105] Figures 7A-7K Pseudo-color plots of downregulated non-canonical markers generated in Figures 1-3 are shown, filtered only on CAR+ T cells, and plotted against Figure 4Three canonical activation markers used in the mapping.

[0106] Figure 8 A Venn diagram showing the number of markers with >10% change filtered across all T cells (both CAR-T and PBMC) in each time point pair is shown. Some representative markers from each group are shown.

[0107] Figures 9A-9N Histograms showing the expression of 14 different cell surface markers in CAR+ or CAR- cells stimulated for 48 hours (T = 48h), 24 hours (T-24h), or unstimulated (T = 0h), grouped by CD4+ T cells and CD8+ T cells.

[0108] Figures 10A-10B Area under the curve (AUC) of different markers for CAR+ and CAR- cells, grouped by each manufacturing process, is shown, where the AUC for each sample is calculated by summing the percent positive for each marker across all time points.

[0109] Figures 11A-11B Data and percent positive for each marker in CAR+ chambers plotted on the y-axis, and time (in hours post stimulation) plotted on the x-axis is shown. Each sample is represented as a separate line for each marker within each process. DETAILED DESCRIPTION

[0110] Provided herein are methods for assessing T cell activation in a composition of cells, the methods comprising detecting surface expression levels of one or more markers or the percentage of cells positive for one or more markers. In some aspects, the provided methods allow for determining an incubation duration or time setting for use with T cells based on activation of the T cells. The provided embodiments are based on the identification of T cell markers that are upregulated or downregulated in activated T cells. The T cell markers include, in many aspects, non-canonical markers that have not previously been reported to be associated with T cell activation or that are not typically used to assess T cell activation. The provided embodiments include markers that can be used to assess or determine activation of particular subsets of T cells, such as CD4+ or CD8+ T cells. The provided embodiments also include markers that can be used to assess or determine activation of T cells engineered with T cell signaling recombinant receptors, such as chimeric antigen receptors (CARs) or recombinant T cell receptors (TCRs). Also provided herein are kits for assessing T cell activation in a composition of cells, the kits containing materials for detecting the one or more markers.

[0111] Existing methods for assessing T cell activation, for example during in vitro or ex vivo T cell culture, generally rely on defined markers or surface proteins expressed on the cell surface upon T cell activation. Existing methods rely on defined canonical markers of T cell activation. Often, one marker does not fully coincide with whether all existing T cells are activated, leading to benefits in assessing T cell activation using multiple markers.

[0112] The provided methods allow for the assessment of T cell activation using the markers provided herein. In some aspects, the markers are non-canonical markers. The results provided herein demonstrate that markers, e.g., non-canonical markers, are upregulated in stimulated cells when compared to unstimulated cells. The results also demonstrate that certain markers are expressed in subsets of cells, such as T cells from peripheral blood mononuclear cells (PBMCs) and / or T cells with chimeric antigen receptors (CARs), indicating that such markers can be used to assess activation of T cells in such subsets. In some embodiments, certain markers disclosed herein are downregulated upon T cell activation. The results provided herein demonstrate that certain markers increase or decrease expression of these markers based on whether the cell is activated after the T cell has been stimulated, e.g., 72 hours, 48 hours, or 24 hours before detecting its activation status by examining surface expression of surface markers on the T cell, as verified using canonical activation markers. The results provided herein indicate that if cells are distinguished by whether they express canonical markers, then cells expressing canonical markers are more likely to be positive for other markers, such as CD200 (OX2), CD357 (GITR), CD120b, CD155 (PVR), and CD107b (LAMP-2). Furthermore, if markers identified as differentially expressed on stimulated cell populations are plotted against canonical markers of cell activation, then a double positive cell population emerges for non-canonical markers that increase upon activation, but not for non-canonical markers that decrease upon stimulation. In some embodiments, the change in marker expression is time sensitive and shows expression differences based on the amount of time after cell stimulation. In some embodiments, the change in marker expression is cell type specific (e.g., depending on whether the cell is a CD4 or CD8 T cell). In some embodiments, the change in marker expression is specific to cell treatment, where the change in cell marker expression occurs more significantly in PBMCs or CAR+ T cells.

[0113] In some aspects, the provided methods involve assessing T cell activation based on expression or surface levels of the following markers: CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (OX40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, bone marrow stromal antigen 2), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, integrin b7, mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain l, IgM, CD119 (IFN-g R a chain), CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Fn14, TWEAKR), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcR1 (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin-1), CD49d, CD73 (ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, TSLPR (TSLP-R), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CD11b, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), CD49f, a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR. In some embodiments, the provided methods involve assessing T cell activation in engineered T cells expressing a recombinant receptor. In some embodiments, the receptor is a chimeric antigen receptor (CAR). In some embodiments, the provided methods involve assessing T cell activation in CD4+ T cells. In some embodiments, the provided methods involve assessing T cell activation in CD8+ T cells. In some embodiments, the provided methods involve assessing T cell activation in CD4+ CAR+ T cells. In some embodiments, the provided methods involve assessing T cell activation in CD8+ CAR+ T cells.

[0114] In some aspects, the provided methods can be used to determine whether a cell is ready to be transduced. In some aspects, the provided methods further comprise selecting or isolating a T cell for engineering of the T cell after determining activation. In some aspects, the provided methods further comprise engineering a T cell determined and / or monitored for activation to produce a cell therapy product. In some embodiments, the provided methods can be used to monitor the kinetics of T cell activation, stimulation, or during incubation, such as under expansion conditions. In some aspects, the provided methods can be used to identify relationships between activation status and outcome, such as how the cells evolve over time. In some aspects, the provided methods allow for determining when a cell can be administered to a patient.

[0115] In some embodiments, the provided methods can be used to predict the quality of a T cell undergoing a manufacturing process, such as the quality of a T cell during or after a manufacturing process. In some aspects, the activation status determined by the provided methods can be used as a readout during manufacturing, such as a readout of manufacturing success or success of a manufacturing step, such as cell stimulation. In some embodiments, the provided methods can be used to monitor whether a T cell is sufficiently activated, such as to expand a T cell to a desired threshold number, such as a number required for a clinical dose of T cells for a T cell therapy.

[0116] In some aspects, activation of a T cell can result in differentiation of the T cell. A higher proportion of early memory T cells, such as naive-like T cells, in a T cell therapy can improve patient outcomes (see, e.g., Jiang et al., Journal of Pharmaceutical Sciences (2021) 110: 1871-1876). In some embodiments, the provided methods can be used to monitor the memory status of a T cell, either directly or by monitoring the activation status of the T cell. In some embodiments, the activation status of a T cell is monitored to predict the memory status of the T cell.

[0117] In some aspects, the cell phenotype information obtained by the provided methods can be used during process development to optimize the manufacturing process or the duration or other conditions of steps thereof, in order to improve the quality of the treated T cells. In some cases, the information can be used to develop a process control strategy, wherein, for example, conditions of one or more (e.g., current or subsequent) manufacturing steps can be changed, such as the duration of a current or subsequent manufacturing step, when a predicted cell phenotype, such as activation status, falls outside of a determined range, in order to improve the final quality of the manufactured T cells. For example, when the activation status falls outside of a determined range during incubation, in some cases, a subsequent incubation can be performed under perfusion conditions and / or in the presence of a small molecule, such as to modulate the T cell phenotype to a desired profile.

[0118] In some aspects, the activation status information obtained by the provided methods can be used to assess or reduce lot-to-lot variability of T cells subjected to a manufacturing process. In some aspects, the activation status information can be used to assess or reduce lot-to-lot variability of a drug product produced using a manufacturing process. For example, by ensuring that T cells across different cell therapy manufacturing runs are in comparable activation states, the differentiation and memory status of the T cells can be kept consistent. This can reduce variability (e.g., patient-to-patient variability) in the resulting T cell therapy (see, e.g., Jiang et al., Journal of Pharmaceutical Sciences (2021) 110: 1871-1876).

[0119] In some embodiments, the provided methods can be used to monitor the activation status of T cells before or after the T cells are engineered. In some aspects, transgene expression can be higher in activated T cells than in non-activated T cells, such as after viral transduction of the T cells (see, e.g., Ghassemi et al., Nature Biomedical Engineering (2022) 6: 118-128). In some aspects, the efficiency of electroporation for engineering can be higher in activated T cells than in non-activated T cells (see, e.g., Zhang et al., BMC Biotechnology (2018) 18: 4). In some embodiments, the T cells are monitored according to the provided methods before engineering, e.g., such that engineering can be initiated once the provided methods predict that the T cells are sufficiently activated for improved transgene expression. In some embodiments, the T cells are monitored according to the provided methods after engineering, e.g., to determine whether the T cells are sufficiently activated or remain sufficiently activated after engineering for improved transgene expression.

[0120] All publications mentioned in this application (including patents, scientific articles, and databases) are incorporated by reference in their entirety for all purposes, to the same extent as if each individual publication were incorporated by reference individually. If there is a conflict between the definitions described herein and those described in patents, applications, published applications, and other publications incorporated herein by reference, the definitions described herein are intended to prevail.

[0121] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. I. Methods of Assessing T Cell Activation

[0122] In some embodiments, the provided methods involve assessing surface expression of a T cell activation marker on T cells in the cells of the composition. In some embodiments, the provided methods are used to assess T cell activation within the cells of the composition. Exemplary compositions are described in Section II. In some embodiments, the provided methods involve performing any of the cell processing steps described in Section II. In some embodiments, the provided methods involve determining T cell activation with the T cells described in Section II or Section III.

[0123] In some embodiments, the T cells are assessed by detecting surface expression of a marker. In some embodiments, the T cells are assessed by detecting expression of one or more markers. In some embodiments, the one or more markers are expressed on the surface of the T cells. In some embodiments, the methods involve determining the presence or absence of the one or more markers on the T cells on the cells of the composition. In some embodiments, T cell activation is determined by expression of one or more markers. In some embodiments, an increase in the one or more markers indicates T cell activation. In some embodiments, a decrease in the one or more markers indicates T cell activation. In some embodiments, the one or more markers are used to determine the extent of T cell activation within the cells of the composition. In some embodiments, the one or more markers are used to determine the number or percentage of activated T cells within the cells of the composition.

[0124] In some embodiments, activation of T cells is determined based on the expression of a combination of one or more markers in the cells of the composition. In some embodiments, activation of T cells is determined based on an increase in expression of some of the one or more markers and a decrease in expression of other of the one or more markers on the cells of the composition. In some embodiments, activation of T cells is determined based on the expression of a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 markers. In some embodiments, activation of T cells is determined based on the expression of a combination of 2 markers. In some embodiments, activation of T cells is determined based on the expression of a combination of 3 markers. In some embodiments, activation of T cells is determined based on the expression of a combination of 4 markers. In some embodiments, activation of T cells is determined based on the expression of a combination of 5 markers. In some embodiments, activation of T cells is determined based on the expression of a combination of 6 markers. In some embodiments, activation of T cells is determined based on the expression of a combination of 7 markers. In some embodiments, activation of T cells is determined based on the expression of a combination of 8 markers. In some embodiments, activation of T cells is determined based on the expression of a combination of 9 markers. In some embodiments, activation of T cells is determined based on the expression of a combination of 10 markers. In some embodiments, activation of T cells is determined based on the expression of a combination of 15 markers. In some embodiments, activation of T cells is determined based on the expression of a combination of 20 markers. In some embodiments, activation of T cells is determined based on the expression of a combination of between 2 and 20 markers. In some embodiments, activation of T cells is determined based on the expression of a combination of between 2 and 10 markers. In some embodiments, activation of T cells is determined based on the expression of a combination of between 2 and 5 markers.

[0125] In some embodiments, the provided methods involve determining CD4+ T cell activation in the cells of the composition. In some embodiments, the provided methods involve determining CD8+ T cell activation in the cells of the composition. In some embodiments, the provided methods involve determining activation of T cells containing a recombinant receptor in the cells of the composition. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR). In some embodiments, the provided methods involve determining activation of CD4+ T cells containing a recombinant receptor in the cells of the composition. In some embodiments, the provided methods involve determining activation of CD8+ T cells containing a recombinant receptor in the cells of the composition.

[0126] In some embodiments, the cells are present in a subject. In some embodiments, the cells are isolated from a subject and their activation status is assessed ex vivo. In some embodiments, the T cells are stimulated or activated in vitro and surface expression of the one or more markers is assessed according to the provided methods. Exemplary stimulation reagents for activating cells include any reagent as described in Section III.

[0127] In some embodiments, the stimulation conditions can include one or more of: a particular medium, temperature, oxygen content, carbon dioxide content, time, agents (e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agent intended to activate the cells). In some embodiments, the cells are stimulated and the phenotype is determined by whether or not a soluble factor (e.g., a cytokine or chemokine) is produced or secreted. In some embodiments, the stimulation is non-specific, i.e., not antigen-specific stimulation. In some embodiments, the cells are incubated in the presence of the stimulation conditions or stimulation agents for a duration of about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours, about 24 hours, about 48 hours, about 72 hours, or for a duration between 1 hour and 4 hours, between 1 hour and 12 hours, between 12 hours and 24 hours, between 12 and 48 hours, or between 12 and 72 hours, each inclusive, or for more than 24 hours.

[0128] In some embodiments, the cells are stimulated with an agent that is an antigen or an epitope thereof specific for the recombinant receptor or is an antibody or fragment thereof that binds and / or recognizes the recombinant receptor, or a combination thereof. In some embodiments, the recombinant receptor is a CAR and the agent is an antigen or an epitope thereof specific for the CAR or is an antibody or fragment thereof that binds and / or recognizes the CAR, or a combination thereof. In particular embodiments, the cells are stimulated by incubating the cells in the presence of target cells having surface expression of an antigen recognized by the CAR. In certain embodiments, the recombinant receptor is a CAR and the agent is an antibody or active fragment, variant, or portion thereof that binds the CAR. In certain embodiments, the antibody or active fragment, variant, or portion thereof that binds the CAR is an anti-idiotypic (anti-ID) antibody.

[0129] In some embodiments, the stimulatory conditions or stimulatory agents include one or more agents (e.g., ligands) capable of activating the intracellular signaling domains of the TCR complex. In some aspects, the agents turn on or initiate the TCR / CD3 intracellular signaling cascade in T cells. Such agents can include, for example, antibodies bound to a solid support (such as beads), such as those specific for TCR components and / or costimulatory receptors (e.g., anti-CD3, anti-CD28); and / or one or more cytokines. In some embodiments, the one or more agents are PMA and ionomycin. A. Markers

[0130] In some embodiments, the provided methods involve assessing T cell activation based on the presence of one or more markers on the T cells. In some embodiments, the one or more markers are present on the surface of the T cells and can be used to measure activation of the T cells. In some embodiments, the markers are identified by comparing surface expression of the markers on stimulated T cells to unstimulated T cells. In some embodiments, the markers are identified by comparing expression of potential markers to expression of canonical markers on stimulated and unstimulated T cells to identify markers that correspond to activation. In some embodiments, flow cytometry is used to compare potential markers to canonical markers to identify markers. In some embodiments, screening is performed using infinity flow cytometry to identify markers by utilizing canonical markers of T cell activation as a backbone for cross-sample staining, then screening potential markers across wells, each well having a unique potential marker. In some embodiments, infinity flow cytometry screening can be used to associate potential activation markers with canonical activation to assess which potential activation markers can be used as markers for assessing T cell activation.

[0131] In some embodiments, the one or more markers for assessing T cell activation are selected from CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (OX40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, bone marrow stromal cell antigen 2), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, integrin b7, mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain l, IgM, CD119 (IFN-g R a chain), CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Fn14, TWEAKCD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcR1 (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin-1), CD49d, CD73 (ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, TSLPR (TSLP-R), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CD11b, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), CD49f, a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR.

[0132] In some embodiments, the marker is a marker that is (i) upregulated upon T cell stimulation or activation or (ii) downregulated upon T cell stimulation or activation. In some embodiments, the one or more markers are selected from group (i) and / or group (ii). In some embodiments, the T cell is a CD3+ T cell. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the T cell is a T cell obtained from the peripheral blood of a subject, such as a T cell obtained from a PBMC sample. In some embodiments, the T cell is an isolated T cell that is not engineered. In some embodiments, the T cell is engineered with a recombinant receptor, such as a CAR.

[0133] In some embodiments, the sample is CD4+ CAR+ T cells, CD8+ CAR+ T cells, CD4+ PBMC T cells, and CD8+ PBMC T cells.In some embodiments, the marker of group (i) is selected from CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (OX40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Bone marrow stromal antigen 2), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, integrin b7, mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain l, IgM, CD119 (IFN-g R a chain), CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Fn14, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcR1 (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin-1), CD49d, CD73 (ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R).In some embodiments, the marker of group (ii) is selected from CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CD11b, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), CD49f, a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR.

[0134] In some embodiments, the one or more markers are selected from Group (i) and / or Group (ii), wherein the one or more markers of Group (i) are selected from CD20, CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (OX40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Bone marrow stromal cell antigen 2), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, integrin b7, mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain l, IgM, CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R); and / or the one or more markers of Group (ii) are selected from CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD172g (SIRPg), CD127 (IL-7Ra), CD49f, a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR. In some embodiments, the one or more markers are evaluated 12-36 hours after stimulation. In some embodiments, the one or more markers are evaluated about 24 hours after stimulation. In some embodiments, the T cells are CD3+ T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are T cells obtained from a subject’s peripheral blood, such as T cells obtained from a PBMC sample. In some embodiments, the T cells are unengineered isolated T cells.In some embodiments, the T cells are engineered with a recombinant receptor, such as a CAR.

[0135] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (OX40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Bone marrow stromal cell antigen 2), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, integrin b7, mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain l, IgM, CD119 (IFN-g R a chain), CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Fn14, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcR1 (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin-1), CD49d, CD73 (ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), and GARP (LRRC32);and / or one or more markers of group (ii) is selected from CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD11b, CX3CR1, NKp80, CD127 (IL-7Ra), and CD49f. In some embodiments, the one or more markers is assessed 24-72 hours after stimulation. In some embodiments, the one or more markers is assessed about 48 hours after stimulation. In some embodiments, the T cells are CD3+ T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are T cells obtained from the peripheral blood of a subject, such as T cells obtained from a PBMC sample. In some embodiments, the T cells are isolated T cells that are not engineered. In some embodiments, the T cells are engineered with a recombinant receptor, such as a CAR.

[0136] In some embodiments, the one or more markers are selected from Group (i) and / or Group (ii), wherein the one or more markers of Group (i) are selected from CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), CD355 (CRTAM), GPR56, CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD96 (TACTILE), and CD195 (CCR5); and the one or more markers of Group (ii) are selected from CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CD11b, and CX3CR1. In some embodiments, the one or more markers are assessed 24-72 hours after stimulation. In some embodiments, the one or more markers are assessed 48 hours after stimulation. In some embodiments, the T cells are CD3+ T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are T cells obtained from the peripheral blood of a subject, such as T cells obtained from a PBMC sample. In some embodiments, the T cells are unengineered isolated T cells. In some embodiments, the T cells are engineered with a recombinant receptor, such as a CAR.

[0137] In some embodiments, the one or more markers are selected from CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), CD355 (CRTAM), GPR56, CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD96 (TACTILE), and CD195 (CCR5).

[0138] In some embodiments, the one or more markers are selected from CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CD11b, and CX3CR1.

[0139] In some embodiments, the one or more markers are selected from Panel (i) and / or Panel (ii), wherein the one or more markers of Panel (i) are selected from CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), CD355 (CRTAM), and GPR56; and / or the one or more markers of Panel (ii) are selected from CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD96 (TACTILE), and CD195 (CCR5). In some embodiments, the one or more markers are assessed 24-72 hours after stimulation. In some embodiments, the one or more markers are assessed 48 hours after stimulation. In some embodiments, the T cells are engineered with a recombinant receptor, such as a CAR. In some embodiments, the T cells are CD3+CAR+ T cells. In some embodiments, the T cells are CD4+CAR+ T cells. In some embodiments, the T cells are CD8+CAR+ T cells. In some embodiments, the sample contains CD4+CAR+ T cells and CD8+CAR+ T cells. In some embodiments, the one or more markers are used to assess T cell activation on CD4+CAR+ and / or CD8+CAR+ T cells.

[0140] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from CD120b, CD83, CD357 (GITR), CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD355 (CTRAM), CD155 (PVR), CD74, CD170 (Siglec-5), and GPR56; and / or the one or more markers of group (ii) are selected from CD49f, CCRL2, CD124 (IL-4Ra), CD217, CD192 (CCR2), CD195 (CCR5), and CD96 (TACTILE). In some embodiments, the one or more markers are assessed 24-72 hours after stimulation. In some embodiments, the one or more markers are assessed 48 hours after stimulation. In some embodiments, the T cells are engineered with a recombinant receptor, such as a CAR. In some embodiments, the T cells are CD3+CAR+ T cells. In some embodiments, the T cells are CD4+CAR+ T cells. In some embodiments, the T cells are CD8+CAR+ T cells. In some embodiments, the sample contains CD4+CAR+ T cells and CD8+CAR T cells. In some embodiments, the one or more markers are used to assess T cell activation on CD4+CAR+ and / or CD8+CAR T cells.

[0141] In some embodiments, the one or more markers are selected from Group (i) and / or Group (ii), wherein the one or more markers of Group (i) are selected from CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, and CD165; and / or the one or more markers of Group (ii) are selected from CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), and CD127 (IL-7Ra). In some embodiments, the one or more markers are assessed 24-72 hours after stimulation. In some embodiments, the one or more markers are assessed 48 hours after stimulation. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are T cells obtained from the peripheral blood of a subject, such as T cells obtained from a PBMC sample. In some embodiments, the T cells are unengineered isolated T cells. In some embodiments, the T cells are engineered with a recombinant receptor, such as a CAR. In some embodiments, the T cells are CD4+ CAR+ T cells.

[0142] In some embodiments, the one or more markers are selected from Group (i) and / or Group (ii), wherein the one or more markers of Group (i) are selected from CD120b, CD83, CD357 (GITR), CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD155 (PVR), CD74, and CD170 (Siglec-5); and / or the one or more markers of Group (ii) are selected from CD49f, CCRL2, CD124 (IL-4Ra), CD217, and CD192 (CCR2). In some embodiments, the one or more markers are assessed 24-72 hours after stimulation. In some embodiments, the one or more markers are assessed 48 hours after stimulation. In some embodiments, the T cells are CD4+ T cells engineered with a recombinant receptor, such as a CAR. In some embodiments, the T cells are CD4+ CAR+ T cells.

[0143] In some embodiments, the one or more markers are selected from Group (i) and / or Group (ii), wherein the one or more markers of Group (i) are selected from CD120b, CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch 2; and / or the one or more markers of Group (ii) are selected from CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CD11b, CX3CR1, and CD127 (IL-7Ra). In some embodiments, the one or more markers are assessed 24-72 hours after stimulation. In some embodiments, the one or more markers are assessed 48 hours after stimulation. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are T cells obtained from the peripheral blood of a subject, such as T cells obtained from a PBMC sample. In some embodiments, the T cells are unengineered isolated T cells. In some embodiments, the T cells are engineered with a recombinant receptor, such as a CAR. In some embodiments, the T cells are CD8+ CAR+ T cells.

[0144] In some embodiments, the one or more markers are selected from Group (i) and / or Group (ii), wherein the one or more markers of Group (i) are selected from CD120b, CD83, CD357 (GITR), CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD355 (CTRAM), CD155 (PVR), and GPR56; and / or the one or more markers of Group (ii) are selected from CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), and CD96 (TACTILE). In some embodiments, the one or more markers are assessed 24-72 hours after stimulation. In some embodiments, the one or more markers are assessed 48 hours after stimulation. In some embodiments, the T cells are CD8+ T cells engineered with a recombinant receptor, such as a CAR. In some embodiments, the T cells are CD8+ CAR+ T cells.

[0145] In some embodiments, the one or more markers are selected from Group (i) and / or Group (ii), wherein the one or more markers of Group (i) are selected from CD134 (OX40), CD107b (LAMP-2), CD155 (PVR), CD74, and CD170 (Siglec-5); and the one or more markers of Group (ii) are selected from CD49f, CCRL2, CD124 (IL-4Ra), CD217, CD355 (CRTAM), GPR56, and CD96 (TACTILE). In some embodiments, the one or more markers are selective for activation of CAR+ T cells. In some embodiments, the T cells are selective for activation of CAR+ T cells upon stimulation with a CAR-dependent agent, such as an anti-idiotypic antibody, or by antigen-expressing cells. In some embodiments, the one or more markers are assessed 24-72 hours after stimulation. In some embodiments, the one or more markers are assessed 48 hours after stimulation. In some embodiments, the T cells are CD3+ CAR+ T cells. In some embodiments, the T cells are CD4+ CAR+ T cells. In some embodiments, the T cells are CD8+ CAR+ T cells. In some embodiments, the sample contains CD4+ CAR+ T cells and CD8+ CAR T cells. In some embodiments, the one or more markers are used to assess T cell activation on CD4+ CAR+ and / or CD8+ CAR T cells.

[0146] In some embodiments, the one or more markers are selected from Group (i) and / or Group (ii), wherein the one or more markers of Group (i) are selected from CD134 (OX40), CD107b (LAMP-2), CD155 (PVR), and CD74; and / or the one or more markers of Group (ii) are selected from CD49f, CCRL2, and CD124 (IL-4Ra). In some embodiments, the one or more markers are selective for activation of CD4+ CAR+ T cells. In some embodiments, the T cells are selective for activation of CD4+ CAR+ T cells upon stimulation with a CAR-dependent agent, such as an anti-idiotypic antibody, or by antigen-expressing cells. In some embodiments, the one or more markers are assessed 24-72 hours after stimulation. In some embodiments, the one or more markers are assessed 48 hours after stimulation. In some embodiments, the T cells are CD4+ CAR+ T cells. In some embodiments, the one or more markers are used to assess T cell activation on CD4+ CAR+ T cells.

[0147] In some embodiments, the one or more markers are selected from Group (i) and / or Group (ii), wherein the one or more markers of Group (i) are selected from CD107b (LAMP-2), CD155 (PVR), CD355 (CRTAM), and GPR56; and the one or more markers of Group (ii) are selected from CCRL2, CD217, CD96 (TACTILE). In some embodiments, the one or more markers are selective for activation of CD8+CAR+T cells. In some embodiments, the T cells are selective for activation of CD8+CAR+T cells upon stimulation with a CAR-dependent reagent, such as an anti-idiotype antibody. In some embodiments, the one or more markers are assessed 24-72 hours after stimulation. In some embodiments, the one or more markers are assessed 48 hours after stimulation. In some embodiments, the T cells are CD8+CAR+T cells. In some embodiments, the one or more markers are used to assess T cell activation on CD4+CAR+T cells.

[0148] In some embodiments, the one or more markers are selected from Group (i) and / or Group (ii), wherein the one or more markers of Group (i) are selected from CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, CD165, CD120b, CD83, CD357 (GITR), CD200 (OX2), and CD134 (OX40); and / or the one or more markers of Group (ii) are selected from KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CD192 (CCR2), CD11b, and CX3CR1. In some embodiments, the one or more markers are assessed 24-72 hours after stimulation. In some embodiments, the one or more markers are assessed 48 hours after stimulation. In some embodiments, the T cells are CD3+T cells. In some embodiments, the T cells are CD4+T cells. In some embodiments, the T cells are CD8+T cells. In some embodiments, the T cells are T cells obtained from the peripheral blood of a subject, such as T cells obtained from a PBMC sample.

[0149] In some embodiments, the one or more markers are selected from Group (i) and / or Group (ii), wherein the one or more markers of Group (i) are selected from CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, CD165, and CD83); and / or the one or more markers of Group (ii) are selected from KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CD11b, and CX3CR1. In some embodiments, the T cells are selective for assessing stimulation of non-engineered T cells. In some embodiments, the T cells are selective for stimulation using pan-T cell activation such as anti-CD3 / anti-CD28. In some embodiments, the one or more markers are assessed 24-72 hours after stimulation. In some embodiments, the one or more markers are assessed 48 hours after stimulation. In some embodiments, the T cells are CD3+ T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are T cells obtained from the peripheral blood of a subject, such as T cells obtained from a PBMC sample.

[0150] In some embodiments, the one or more markers are selected from Group (i) and / or Group (ii), wherein the one or more markers of Group (i) are selected from CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, and CD165; and / or the one or more markers of Group (ii) are selected from KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), and CD127 (IL-7Ra). In some embodiments, the T cells are selective for assessing stimulation of non-engineered CD4+ T cells. In some embodiments, the T cells are selective for stimulation of CD4+ T cells using pan-T cell activation such as anti-CD3 / anti-CD28. In some embodiments, the one or more markers are assessed 24-72 hours after stimulation. In some embodiments, the one or more markers are assessed 48 hours after stimulation. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are T cells obtained from the peripheral blood of a subject, such as CD4+ T cells obtained from a PBMC sample.

[0151] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from CD71, Notch 1, CD107a (LAMP-1), CD166, Notch 2, CD165, CD83; and / or the one or more markers of group (ii) are selected from CDl lb, CX3CR1, and CD127 (IL-7Ra). In some embodiments, the T cells are selective for stimulation of non-engineered CD8+ T cells. In some embodiments, the T cells are selective for stimulation of CD8+ T cells using pan-T cell activation such as anti-CD3 / anti-CD28 pairs. In some embodiments, the one or more markers are evaluated 24-72 hours after stimulation. In some embodiments, the one or more markers are evaluated 48 hours after stimulation. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are T cells obtained from the peripheral blood of a subject, such as CD8+ T cells obtained from a PBMC sample.

[0152] In some embodiments, the one or more markers are differentially expressed on cells expressing a recombinant receptor. In some embodiments, the one or more markers are differentially expressed on cells expressing a chimeric antigen receptor (CAR). In some embodiments, the recombinant receptor is a CAR. In some embodiments, the surface expression of the one or more markers is increased on cells expressing a recombinant receptor compared to surface expression on cells not expressing a recombinant receptor. In some embodiments, the surface expression of the one or more markers is decreased on cells expressing a recombinant receptor compared to surface expression on cells not expressing a recombinant receptor.

[0153] In some embodiments, the one or more markers are selected from Group (i) or Group (ii), wherein the one or more markers of Group (i) are selected from CD262 (DR5, Trail-R2), CD105 (Endoglin), CD36L1 (SCARB1, SR-BI), CD73 (ecto-5'-nucleotidase), CD83, CD119 (IFN-g R a chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R), and / or the one or more markers of Group (ii) are selected from CD96 (TACTILE). In some embodiments, the one or more markers are expressed in a T cell composition comprising T cells expressing a recombinant receptor. In some embodiments, the one or more markers of Group (i) are increased in surface expression on cells expressing a recombinant receptor compared to surface expression on cells that do not express a recombinant receptor. In some embodiments, the one or more markers of Group (ii) are decreased in surface expression on cells expressing a recombinant receptor compared to surface expression on cells that do not express a recombinant receptor.

[0154] In some embodiments, the one or more markers are surface proteins. In some embodiments, the one or more markers can be involved in a variety of cellular functions. In some embodiments, the one or more markers can be involved in, among others, metabolism, cell proliferation, cell signaling, immune response, cell apoptosis, etc. In some embodiments, the one or more markers are selected from Group (i) or Group (ii), wherein the one or more markers of Group (i) are selected from CD262 (DR5, Trail-R2), CD105 (Endoglin), CD36L1 (SCARB1, SR-BI), CD73 (ecto-5'-nucleotidase), CD83, CD119 (IFN-g R a chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R), and / or the one or more markers of Group (ii) are selected from CD96 (TACTILE). In some embodiments, the one or more markers are the markers listed in Table 1. Table 1 lists the markers, alternative names, and a description of the marker and its role. Table 1: Markers and their descriptions

[0155] In some embodiments, the one or more markers are non-canonical markers. In some embodiments, the one or more markers are markers that are not typically used to assess T cell activation (e.g., non-canonical markers). In some embodiments, the one or more markers are identified by comparing the expression or percentage of positive cells of the one or more markers to the expression or percentage of positive cells expressing canonical activation markers, indicating that the one or more markers are non-canonical activation markers. B. Binding Agents

[0156] In some embodiments, the T cell activation status is determined based on the amount or percentage of cells that bind to a binding agent. In some embodiments, one or more binding agents specifically bind to the one or more markers. In some embodiments, the one or more binding agents are antibodies. In some embodiments, one or more antibodies are uniquely labeled. In some embodiments, the one or more antibodies are each uniquely fluorescently labeled. In some embodiments, measuring the unique label of the antibodies is used to quantify surface expression of the one or more antibodies. In some embodiments, the unique fluorescent label is used to quantify surface expression of the one or more antibodies. In some embodiments, the one or more antibodies are commercially available antibodies. 1. Antibodies

[0157] In some embodiments, the marker is detected using any of the substances for detecting a marker selected from the group consisting of CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (OX40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Bone marrow stromal cell antigen 2), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, integrin b7, mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain l, IgM, CD119 (IFN-g R a chain), CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Fn14, TWEAKCD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcR1 (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin-1), CD49d, CD73 (ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, TSLPR (TSLP-R), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CD11b, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), CD49f, a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR.

[0158] In some embodiments, the one or more binding agents is one or more antibodies. As is well known in the art, an "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide through at least one epitope recognition site, situated in the variable region of the immunoglobulin (Ig) molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as dAbs, Fabs, Fab', F(ab')2, Fv), single chain (scFv), synthetic variants, naturally occurring variants, fusion proteins comprising an antibody portion with required specificity, chimeric antibodies, nanobodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site or fragment (epitope recognition site) with the desired specificity. Also included are minibodies comprising a scFv linked to a CH3 domain (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See, e.g., Ward, E. S. et al., Nature 341, 544-546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); PCT / US92 / 09965; WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996.

[0159] In some embodiments, each of the one or more binding agents (e.g., antibodies) comprises a means for binding (e.g., specifically or preferentially binding) to a respective marker. In some embodiments, binding of a binding agent (e.g., antibody) to a marker can be used to detect one or more markers on the surface of a T cell, e.g., by flow cytometry, such as to determine T cell activation. In some embodiments, each of the one or more binding agents (e.g., antibodies) comprises a means for detecting one or more markers to determine T cell activation.

[0160] A binding agent (such as an antibody) that "specifically binds" or "preferentially binds" to a marker (the terms are used interchangeably herein) is a term well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with greater duration, and / or with greater affinity with a particular marker target than it does with alternative markers. An antibody specifically binds or preferentially binds a target if it binds the target with greater affinity, avidity, more readily, and / or for a longer duration than it binds other substances. By reading this definition, it will also be understood that specific binding or preferential binding does not necessarily require (although it can include) exclusive binding. Methods of determining such specific or preferential binding are also well known in the art, such as immunoassays.

[0161] In particular embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a humanized antibody, a single chain antibody, an antibody fragment, and combinations thereof. In some embodiments, the antibody is a full-length IgG antibody. In some embodiments, the antibody is a Fab antibody fragment. In some embodiments, the one or more antibodies are monoclonal. In some embodiments, the one or more antibodies are polyclonal.

[0162] In some embodiments, the antibody is detectably labeled. In some embodiments, the detectable label is a fluorescent label, a radioactive label, or an enzymatic label. In some embodiments, the one or more antibodies are fluorescently labeled with one or more fluorescent moieties.

[0163] Any of a variety of antibody binding reagents are known and can be used to detect any one or more of the above markers. Various commercial sources of antibody reagents include, but are not limited to, Thermo Fisher, Biolegend, BD Biosciences, Abeam, Bio X Cell, Invitrogen, Sigma-Aldrich, Miltenyi Biotec, Beckman, LifeSpan BioSciences Inc, Santa Cruz Bioscience, and / or Novus Biologicals.

[0164] In some embodiments, the one or more binding agents can include one or more of the following antibodies: anti-human CD107b (LAMP-2) (e.g., clone H4B4, clone 8E2F2, clone 6A10H10, clone 02, or clone AF488), anti-human CD120b (e.g., clone 3G7A02, clone MR2-1, clone 22221, clone utrl, clone 80M2, clone 7G8B6, or 2H11CR), anti-human CD357 (GITR) (e.g., clone 108-17, clone 2H4, clone 621, clone OTI9G8, clone ANC7D6, clone ANC5E3, AIT 158D, clone 4H2D6, or clone aa26-162), anti-human CD83 (e.g., clone HB15e, clone 3G10-1F4, clone 8A4C11, or clone 6H4G10), anti-human CD200 (OX2) (e.g., clone A18042B, clone OX-104, or clone 6E8B11), anti-human CD134 (OX40) (e.g., clone Ber-ACT35, clone W4-3, clone UMAB276, clone 3G5G7, or clone OTI2F12A2), anti-human CD155 (PVR) (e.g., clone TX24, clone aa314-342, clone aa220-345, clone ANC2B2, or clone ANC6A3), anti-human CD74 (e.g., clone LN2, clone BU45, clone PIN.1, B318, clone CDLA74-1, clone 2D1B11, clone M-B741, or clone 2D1B3), anti-human CD170 (Siglec-5) (e.g., clone 1A5, clone 110, clone 3F5A3, or clone 11), anti-human Notch 1 (e.g., clone MHN1-519, clone OTI3E12, clone A6, clone mN1A, or clone 4G1), anti-human Notch 2 (e.g., clone MHN2-25, clone NOD-15, clone 8A1, clone OTI3E12, clone A6, clone OTI2E7, or clone 487CT6.9.2), anti-human CD166 (e.g., clone 3A6, clone 3F8B12, clone 10F1G12, clone 4h9A5, or clone 8E12C7), anti-human CD107a (LAMP-1) (e.g., clone H4A3, clone 6E2, clone 5H6, clone Ly1C6, or clone OTI8B1), anti-human CD71 (e.g., clone CY1G4, clone 1E6, clone H68.4. Clones of 3G291, DF1513, 10F11, 1A1B2, MEM-189, or SOM4D10; anti-human CD245 (p220 / 240) (e.g., clones of DY12 or OTI3F7); anti-human CD154 (e.g., clones of 24-31, 2E2, 8H10F5, 1H4, 301, or 5A3A9); anti-human CD165 (e.g., clones of SN2 or AD2); anti-human CD355 (CRTAM) (e.g., clones of Cr...). 24.1, clone 12, clone 08, clone REA1225 or clone 06), anti-human GPR56 (e.g. clone CG4, clone REA467 or clone CG4.rMAB), anti-human CD49f (e.g. clone GoH3, clone 129CD49.6.5, clone BQ16 or clone 6B4), anti-human CD124 (IL-4Rα) (e.g. clone G077F6, clone 25463, clone 1D3, clone R401, clone R001 or clone Hil4r-M57), anti-human CCRL2 (e.g. Anti-human CD217 (e.g., clones of K097F7, 12K19, or 1B2), anti-human CD192 (CCR2) (e.g., clones of K036C2, 7A7, 3B6B1, 4D12, 2A9-a, or 48607), and anti-human CD96 (TACTILE) (e.g., clones of NK92.39, 6f9, 1C8, 5E6C12, or 8A11F8). Anti-human CD127 (IL-7Rα) (e.g., clones A019D5, AbD11590, HIL-7R-M21, IL7R / 2751, ANC8F2, or 3F5D9), anti-human CD11b (e.g., clones ICRF44, REA713, M1 / 70.15, CBRM1 / 5, M1 / 70, X-5, or 3A10H5), and anti-human CX3CR1 (e.g., clones 2A9-1, 2B11, or REA385).

[0165] In provided embodiments, the binding agent (e.g., antibody) is conjugated to a fluorescent label, such as a fluorophore. For example, cells can be incubated with one or more fluorescently labeled antibodies. In some embodiments, any fluorescent label or fluorophore suitable for use in flow cytometry analysis can be used. Some non-limiting examples of fluorescent labels include fluorescent proteins (e.g., GFP, YFP, RFP), fluorescent moieties (e.g., fluorescein isothiocyanate) (FITC), phycoerythrin (PE), allophycocyanin (APC), Alexa Fluor (AF)), nucleic acid stains (e.g., 4',6-diamidino-2-phenylindole (DAPI), SYT016, propidium iodide (PI), cell membrane stains (e.g., FMl-43), cell function dyes (e.g., Fluo-4, Indo-1), and synthetic dyes (e.g., Brilliant Violet (BV)).Exemplary fluorophores include, but are not limited to, hydroxycoumarin, Cascade Blue, Dylight 405 Pacific Orange, Alexa Fluor 430, fluorescein, Oregon Green, Alexa Fluor 488, BODIPY 493, 2,7-dichlorofluorescein, ATTO 488, Chromeo 488, Dylight 488, HiLyte 488, Alexa Fluor 532, Alexa Fluor 555, ATTO 550, BODIPY TMR-X, CF 555, Chromeo 546, Cy3, TMR, TRITC, Dy547, Dy548, Dy549, HiLyte 555, Dylight 550, BODIPY 564, Alexa Fluor 568, Alexa Fluor 594, Rhodamine, Texas Red, Alexa Fluor 610, Alexa Fluor 633, Dylight 633, Alexa Fluor 647, APC, ATTO 655, CF633, CF640R, Chromeo 642, Cy5, Dylight 650, Alexa Fluor 680, IRDye 680, Alexa Fluor 700 (AF700), Cy5.5, ICG, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7, PE-Cy7, Cy7.5, Alexa Fluor 790, Dylight 800, IRDye 800, BV421, BV510, BV570, BV605, BV650, BV711, BV750, BV785, Qdot® 525, Qdot® 565, Qdot® 605, Qdot® 655, Qdot® 705, or Qdot® 800.

[0166] In some embodiments, the one or more antibodies are labeled with one or more of the following fluorescent moieties: DyLight 405, Alexa Fluor 405, Pacific Blue, Alexa Fluor 488, fluorescein (FITC), DyLight 550, phycoerythrin (PE), allophycocyanin (APC), Alexa Fluor 647, DyLight 650, Peridinin-chlorophyll-protein (PerCP), Alexa Fluor 700, StarBright Violet 440, StarBright Violet 515, StarBright Violet 610, StarBright Violet 670, StarBright Violet 700, PE-Alexa Fluor® 647, PE-Cy5, PerCP-Cy5.5, PE-Cy5.5, PE-Alexa Fluor® 750, PE-Cy7, APC-Cy7, cyan fluorescent protein (CFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), mCHERRY, Cy5, and Cy7. In some embodiments, the one or more antibodies are labeled with phycoerythrin (PE).

[0167] In some embodiments, multiple color staining or labeling is performed using multiple fluorophores, where multiple antibodies against different markers are incubated with the cells. In some embodiments, the fluorescent labels conjugated to such antibodies are chosen to minimize energy transfer between them, such as avoiding or minimizing overlapping emission and absorption spectra. In some embodiments, each fluorescent label has a different emission spectrum. In some embodiments, multiple fluorescent labels can be excited with a single wavelength or multiple wavelengths, but detection occurs in regions where the peak emission spectra do not overlap. In some embodiments, one or more of the fluorescent labels can be excited by a single wavelength or the same wavelength of light, but emit different wavelengths of light therefrom.

[0168] In some embodiments, the one or more antibodies are each labeled with a unique fluorescent moiety. In some embodiments, the one or more fluorescent labels each individually comprise a fluorophore selected from PE-Cy7, APC, AF700, BV421, Aqua, and BV605. C. Measuring Marker Surface Expression

[0169] In some embodiments, the cells are incubated with one or more binding agents for staining or detecting the level or percentage of cells expressing the one or more markers. In some embodiments, the cell staining involves incubation with an antibody or binding agent that specifically binds to such markers, in some embodiments, followed by a washing step, and cells that have bound the antibody or binding partner are separated from those that have not bound the antibody or binding partner. In some aspects of such processes, a volume of cells is mixed with an amount of a desired staining reagent and incubated under conditions to stain the cells. In some embodiments, the staining or labeling is performed at a temperature between 0 °C and 25 °C, such as at or about 4 °C. In some embodiments, the staining or labeling is performed for greater than 5 minutes, typically greater than 15 minutes. In some embodiments, the staining or labeling is performed for a time between 15 minutes and 6 hours, such as between 30 minutes and 2 hours. In some embodiments, the staining or labeling is performed for a time that is, for example, at or about 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, or any value between any of the foregoing values. In some embodiments, the labeling with the one or more staining reagents is performed simultaneously. In some embodiments, one or more washing steps are performed prior to measuring or determining the level of the one or more markers or the percentage of cells positive for the one or more markers.

[0170] In some embodiments, the one or more markers are measured by quantifying a binding agent that binds the one or more markers. In some embodiments, the binding agent is quantified by measuring a fluorescent tag on the binding agent. In some embodiments, the binding agent is a fluorescently labeled antibody that selectively binds the one or more markers. In some embodiments, the T cells within the cell composition are incubated with the antibody. In some embodiments, the T cells are fixed after incubation with the antibody. In some embodiments, the T cells are viable after incubation with the antibody. In some embodiments, the T cells are incubated with an antibody that recognizes a marker other than the one or more markers. In some embodiments, the additional marker is a canonical marker that helps to identify a population of cells within the cell composition.

[0171] In some embodiments, the antibody bound to the one or more markers is measured using flow cytometry. In some embodiments, the antibody bound to the one or more markers is measured using infinite flow cytometry. In some embodiments, the antibody is measured by immunohistochemistry (IHC). In some embodiments, the one or more markers are measured in a tissue. In some embodiments, the cells of the cell composition are fixed and stained with an antibody prior to measurement with IHC.

[0172] In some embodiments, the measured values of surface markers are analyzed in FloJo. In some embodiments, after quantifying expression levels with flow cytometry, the population is labeled as positive or negative based on the cell's gating. In some embodiments, a cell of a cell composition is determined to be positive if it is in the group with higher expression levels in a bimodal distribution of cells. In some embodiments, a cell of a cell composition is determined to be positive if it is in the group with higher expression levels compared to other cells in the composition. D. Assessing the activation status of T cells

[0173] In some embodiments, the activation of T cells within a cell composition is assessed by comparing the surface expression level of the one or more activation markers on the T cells to the surface expression level of the one or more activation markers on an unstimulated control. In some embodiments, the unstimulated control consists of T cells. In some embodiments, the unstimulated control is a cell composition treated in the same manner as the cell composition whose T cells are being assessed, except that the unstimulated control is not exposed to any of the stimulation reagents described in Section III. In some embodiments, the unstimulated control can consist of any cell composition as described in Section II, but the cell composition is not exposed to any stimulation reagents (such as those described in Section III). In some embodiments, the unstimulated control is from a patient or donor. In some embodiments, the unstimulated control is from the same patient or donor as the cell composition being assessed.

[0174] In some embodiments, activation of T cells in the composition of cells is assessed by comparing the level of surface expression of the one or more markers on the cells of the composition to the surface expression of the one or more markers on an unstimulated control, wherein the surface level of the one or more markers is measured on the cell composition and the unstimulated control at the same time. In some embodiments, activation of T cells is assessed by comparing the level of surface expression of the one or more markers on the T cells to the surface expression of the one or more markers on an unstimulated control, wherein the surface level of the one or more markers is measured on the cell composition and the unstimulated control at different times. In some embodiments, activation of T cells in the composition of cells is assessed by determining whether the level of each of the one or more markers or the percentage that is positive for each of the one or more markers of the T cells within the cell composition is higher than the level of each of the one or more markers or the percentage that is positive for each of the one or more markers of the cells of an unstimulated control. In some embodiments, activation of T cells in the composition of cells is assessed by determining whether the level of each of the one or more markers or the percentage that is positive for each of the one or more markers of the T cells within the cell composition is lower than the level of each of the one or more markers or the percentage that is positive for each of the one or more markers of the cells of an unstimulated control.

[0175] In some embodiments, activation of T cells within the cell composition is assessed by comparing the level or percentage of surface expression of the one or more activation markers on the T cells within the cell composition to the level or percentage of surface expression of the one or more activation markers in a reference. In some embodiments, activation of T cells within the cell composition is assessed by comparing the level or percentage of surface expression of the one or more activation markers on the T cells within the cell composition to the level or percentage of surface expression of the one or more activation markers in a reference, wherein a higher level or higher percentage of the one or more activation markers on the T cells compared to the reference indicates activation. In some embodiments, activation of T cells within the cell composition is assessed by comparing the level or percentage of surface expression of the one or more activation markers on the T cells within the cell composition to the level or percentage of surface expression of the one or more activation markers in a reference, wherein a lower level or lower percentage of the one or more activation markers on the T cells compared to the reference indicates activation.

[0176] In some embodiments, the reference is a combination of unstimulated cell compositions from several donors or patients. In some embodiments, the reference is an average expression level or average percentage of positive cells across a plurality of cell compositions from a patient or donor, wherein the cell compositions are unstimulated. In some embodiments, the reference is a median expression level or median percentage of positive cells across a plurality of cell compositions from a patient or donor, wherein the cell compositions are unstimulated. In some embodiments, the reference is a plurality of cell compositions, each obtained from a different donor or patient. In some embodiments, the reference is a gating scheme, wherein the gating scheme is determined based on the expression level or amount of positive cells in a plurality of cell compositions, wherein each cell composition is obtained from a different patient or donor.

[0177] In some embodiments, T cell surface expression of the one or more markers is determined to be "low," "lower," or "decreased" if the percentage of cells positive for the one or more markers is lower than the percentage of cells positive for the one or more markers in a reference (e.g., an unstimulated control cell composition, an average percentage of positive cells in a plurality of cell compositions, a median percentage of positive cells in a plurality of cell compositions, a maximum percentage of positive cells in a plurality of cell compositions, a minimum percentage of positive cells in a plurality of cell compositions, etc.). In some embodiments, T cell surface expression of the one or more markers is determined to be "low," "lower," or "decreased" if the surface level expression of the one or more markers is lower than the surface level expression of the one or more markers in a reference (e.g., a mean or median fluorescence intensity of an unstimulated control cell composition, an average mean or median fluorescence intensity across a plurality of cell compositions, a median mean or median fluorescence intensity across a plurality of cell compositions, a maximum mean or median fluorescence intensity across a plurality of cell compositions, a minimum mean or median fluorescence intensity across a plurality of cell compositions, etc.).

[0178] In some embodiments, T cell surface expression of the one or more markers is determined to be "high," "higher," or "increased" if the percentage of cells positive for the one or more markers is higher than the percentage of cells positive for the one or more markers in a reference (e.g., the mean percentage of positive cells in an unstimulated control cell composition, the median percentage of positive cells in a plurality of cell compositions, the maximum percentage of positive cells in a plurality of cell compositions, the minimum percentage of positive cells in a plurality of cell compositions, etc.). In some embodiments, T cell surface expression of the one or more markers is determined to be "high," "higher," or "increased" if the surface level expression of the one or more markers is higher than the surface level expression of the one or more markers in a reference (e.g., the mean or median fluorescence intensity of an unstimulated control cell composition, the average mean or median fluorescence intensity across a plurality of cell compositions, the median mean or median fluorescence intensity across a plurality of cell compositions, the maximum mean or median fluorescence intensity across a plurality of cell compositions, the minimum mean or median fluorescence intensity across a plurality of cell compositions, etc.).

[0179] In some embodiments, T cell activation is assessed by T cells that are positive for the one or more markers (marker+or marker 阳性 ) or express a high level of the one or more markers (marker 高 ), or that are negative for the one or more markers or express a relatively low level of the one or more markers (marker 阴性 ). Thus, it should be understood that the terms positive (positive, pos) or + are used interchangeably herein with respect to one of the one or more markers. Likewise, it should be understood that the terms negative (negative, neg) or - are used interchangeably herein with respect to one of the one or more markers. Further, it should be understood that reference to a cell that is marker 阴性 may refer herein to a cell that is negative for the marker as well as a cell that expresses a relatively low level of the marker (e.g., a level that is not readily detectable as compared to a control or background level). In some cases, such markers are those that are not present or are expressed at a relatively low level on certain T cell populations, but are present or are expressed at a relatively higher level on certain other lymphocyte populations (e.g., NK cells). In some cases, such markers are those that are present or are expressed at a relatively higher level on certain T cell populations, but are not present or are expressed at a relatively low level on certain other lymphocyte populations (e.g., NK cells or subsets thereof). E. Utilization of Activation Status

[0180] In some embodiments, the method comprises incubating the cell composition with a stimulating reagent, such as those described in Section III. In some embodiments, the method comprises incubating the cell composition with a stimulating reagent prior to detecting T cell activation using one or more markers. In some embodiments, the method comprises incubating the cell composition with a stimulating reagent after detecting T cell activation using one or more markers. In some embodiments, the method comprises determining the time for which to incubate the cell composition with a stimulating reagent based on determining T cell activation with one or more markers prior to stimulation. In some embodiments, the method comprises determining the dose of stimulating reagent to incubate with the cell composition based on determining T cell activation with one or more markers.

[0181] In some embodiments, the method comprises engineering the T cell after determining T cell activation using one or more markers, as described in Section III. In some embodiments, the method comprises administering a vector or virus based on the determination of the activation state of the T cell. In some embodiments, the method consists of incubating the T cell with a vector for a duration determined based on detection of one or more markers on the surface of the T cell. In some embodiments, the method consists of incubating the T cell with a vector for a duration determined based on an assessment of the activation state of the T cell.

[0182] In some embodiments, the method comprises culturing and or expanding the cell composition after assessing T cell activation. In some embodiments, the method comprises determining the length of time to culture the cell composition based on the T cell activation assessed using one or more markers.

[0183] In some embodiments, based on the T cell activation assessed by the methods disclosed herein, a population of T cells is enriched from the sample by the provided methods, such as by isolation or selection. In some embodiments, T cells that are positive for the one or more markers (Marker+or Marker 阳性 ) or express high levels of the one or more markers (Marker 高 ), or that are negative for the one or more markers or express relatively low levels of the one or more markers (Marker- or Marker 阴性 ).

[0184] In some embodiments, any known method for separation based on the one or more markers can be used. In some embodiments, the separation is affinity-based or immunoaffinity-based separation. For example, in some aspects, the separation comprises separating cells and cell populations based on the expression or expression level of the one or more markers, typically cell surface markers, for example by incubation with an antibody or binding partner that specifically binds to such markers, followed by typically a washing step and separating cells that have bound the antibody or binding partner from those that have not bound to the antibody or binding partner. In some embodiments, the incubation is static (without mixing). In some embodiments, the incubation is dynamic (with mixing). II. Cell Compositions A. Pharmaceutical Products

[0185] In some embodiments, the cells of the cell composition are engineered to express a recombinant receptor, such as those listed in Section IV. In some embodiments, the recombinant receptor is a CAR. In some embodiments, the method for assessing T cell activation is used to determine the potency of the cells of the cell composition. In some embodiments, the method for assessing T cell activation is used to assess whether the cells of the cell composition were successfully engineered by any of the methods described in Section III. In some embodiments, the cell composition is a pharmaceutical product. In some embodiments, the pharmaceutical product consists of cells engineered using any of the methods listed in Section III. In some embodiments, the pharmaceutical product is a therapeutic composition, including any of the compositions presented in Section III E. In some embodiments, the pharmaceutical product is delivered to a patient after assessing T cell activation. In some embodiments, the cells of the composition are administered to a patient after assessing T cell activation. B. Engineered T Cells

[0186] In some embodiments, the cells of the cell composition are engineered for use as a pharmaceutical product. In some embodiments, the cells of the cell composition are cells stimulated using any of the methods described in Section III B. In some embodiments, the method for assessing T cell activation is performed after stimulating the cells of the cell composition to assess whether the cells are ready to be engineered. In some embodiments, the engineering is performed if the T cells are determined to be activated by detecting the level of a surface marker using any of the methods described in Section III C.

[0187] In some embodiments, the cell composition is evaluated for T cell activation prior to transduction or engineering of the T cells. In some embodiments, the cell composition is isolated from a patient for treatment of a disease. In some embodiments, the cell composition is a sample obtained using any of the methods in Section II. In some embodiments, the cell composition is isolated from a patient and evaluated for T cell activation prior to engineering the cells to assess the ability of the cells to be engineered. In some embodiments, the cell composition is a sample obtained using any of the methods in Section II, which is stimulated prior to evaluating T cell activation to after engineering the cell composition using any of the stimulating reagents and methods in Section II. C. Peripheral Blood Mononuclear Cells

[0188] In some embodiments, the cell composition in the method of evaluating T cell activation consists of peripheral blood mononuclear cells (PBMCs). In some embodiments, the method for evaluating T cell activation can be used in conjunction with the isolation of PBMCs. In some embodiments, the method for evaluating T cell activation is used to evaluate the activation of T cells within PBMCs. In some embodiments, the PBMCs are incubated with a stimulating reagent, such as those listed in Section III B. In some embodiments, after incubating the PBMCs with the stimulating reagent, the surface expression level of one or more markers is detected to quantify T cell activation within the PBMCs.

[0189] PBMCs used in the methods described herein can be isolated by any standard means. In some embodiments, the PBMCs can be isolated from a patient. In some embodiments, the PBMCs can be isolated from a healthy donor. In some embodiments, the PBMCs can be isolated from a patient prior to when they need treatment. In some embodiments, the PBMCs can be isolated from a patient after they need treatment. In some embodiments, the PBMCs can be isolated from a donor and the T cell activity evaluated to determine how to use them. III. Methods for Generating Engineered T Cells

[0190] In some embodiments, the methods of assessing activation of T cells within a cell composition provided herein can be used in conjunction with the generation of a therapeutic composition (e.g., an output composition) of engineered cells (e.g., engineered CD4+ T cells and / or engineered CD8+ T cells) expressing a recombinant protein, e.g., a recombinant receptor such as a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the methods provided herein are used in conjunction with manufacturing, generating, or producing a cell therapy, and can be used in conjunction with additional processing steps, such as steps of isolating, separating, selecting, activating or stimulating, transducing, washing, suspending, diluting, concentrating, and / or formulating the cells. In some embodiments, the method of generating or producing engineered cells, e.g., engineered CD4+ T cells and / or engineered CD8+ T cells, comprises one or more of the following: isolating cells from a subject, preparing cells, processing cells, incubating cells under stimulatory conditions, and / or engineering (e.g., transducing) cells. In some embodiments, the method comprises processing steps performed in the following order: first isolating, such as selecting or separating, input cells, e.g., primary cells, from a biological sample; incubating the input cells under stimulatory conditions, engineering the input cells with a vector particle, e.g., a viral vector particle, to introduce a recombinant polynucleotide into the cells, e.g., by transduction or transfection; culturing the engineered cells (e.g., transduced cells), such as to expand the cells; and collecting, harvesting, all or a portion of the cells, and / or filling a container with the cells to formulate the cells as an output composition. In some embodiments, CD4+ and CD8+ T cells are manufactured independently of one another, e.g., in separate input compositions, but the manufacturing processes include the same processing steps. In some embodiments, CD4+ and CD8+ T cells are manufactured together, e.g., in the same input composition. In some embodiments, the cells of the generated output composition (e.g., a therapeutic cell composition) are re-introduced into the same subject prior to or after cryopreservation. In some embodiments, the output composition (e.g., a therapeutic cell composition) of engineered cells is suitable for use in a therapy (e.g., an autologous cell therapy, an allogeneic cell therapy). Exemplary manufacturing methods are described in published International Patent Application Publication No. WO 2019 / 089855, the contents of which are incorporated herein by reference in their entirety. A. Sample and Cell Preparation

[0191] In particular embodiments, the provided methods are used in combination with isolating, selecting, and / or enriching cells from a biological sample to generate one or more input compositions enriched for cells, e.g., T cells. In some embodiments, the provided methods include isolating cells or compositions thereof from a biological sample, e.g., those biological samples obtained or derived from a subject, e.g., a subject having a particular disease or condition or in need of or to be administered a cell therapy. In some aspects, the subject is a human, such as a subject who is a patient in need of a particular therapeutic intervention, e.g., an adoptive cell therapy in which cells are isolated, manipulated, and / or engineered for use in the adoptive cell therapy. Thus, in some embodiments, the cells are primary cells, e.g., primary human cells. The samples include tissues, fluids, and other samples taken directly from a subject. The biological sample can be a sample obtained directly from a biological source or a processed sample. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue and organ samples, including processed samples derived therefrom.

[0192] In some aspects, the sample is a blood or blood-derived sample, or is or is derived from apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testicle, ovary, tonsil, or other organ and / or cells derived therefrom. In the context of cell therapy, e.g., adoptive cell therapy, samples include those from autologous and allogeneic sources.

[0193] In some instances, cells from circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. In some aspects, the sample contains lymphocytes (including T cells, monocytes, granulocytes, B cells), other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects contains cells other than red blood cells and platelets.

[0194] In some embodiments, the blood cells collected from a subject are washed, for example, to remove the plasma fraction, and the cells are placed in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, the washing step is accomplished by a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, after washing, the cells are resuspended in a variety of biocompatible buffers (e.g., PBS without Ca ++ / Mg ++ In certain embodiments, components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

[0195] In some embodiments, the method of preparation includes a step of freezing (e.g., cryopreservation) the cells prior to, or after, isolation, selection and / or enrichment, and / or incubation for transduction and engineering, and / or after incubation and / or harvesting of the engineered cells. In some embodiments, the freezing and subsequent thawing steps remove granulocytes, and to some extent monocytes, from the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., after a washing step, to remove plasma and platelets. In some aspects, any of a variety of known freezing solutions and parameters can be used. In some embodiments, the cells are frozen, e.g., cryopreserved, in a medium and / or solution having a final concentration of, or of about, 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or 5.0% DMSO, or between 1% and 15%, between 6% and 12%, between 5% and 10%, or between 6% and 8% DMSO. In particular embodiments, the cells are frozen, e.g., cryopreserved, in a medium and / or solution having a final concentration of, or of about, 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5%, or 0.25% HSA, or between 0.1% and -5%, between 0.25% and 4%, between 0.5% and 2%, or between 1% and 2% HSA. One example involves the use of PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1 : 1 with media, such that the final concentrations of DMSO and HSA are 10% and 4%, respectively. Cells are then typically frozen at a rate equal to or about 1 ° / minute to equal to or about -80ºC and stored in the gas phase of a liquid nitrogen storage tank.

[0196] In some embodiments, the isolation of the cells or population includes one or more preparative and / or non-affinity based cell separation steps. In some examples, the cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, e.g., to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, the cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity and / or resistance to particular components. In some embodiments, the method includes a density-based cell separation method, such as preparing white blood cells from peripheral blood by lysing red blood cells and centrifuging through a Percoll or Ficoll gradient.

[0197] In some embodiments, at least a portion of the selecting step comprises incubating the cells with a selection reagent. Incubation with one or more selection reagents, for example as part of a selection method, can be performed using one or more selection reagents for selecting one or more different cell types based on the expression or presence of one or more particular molecules, such as a surface marker (e.g., a surface protein), an intracellular marker, or a nucleic acid, in or on the cell. In some embodiments, any known method of separation that utilizes one or more selection reagents based on such markers can be used. In some embodiments, the one or more selection reagents results in a separation that is based on affinity or immunoaffinity. For example, in some aspects, the selecting comprises incubation with one or more reagents for separating cells and cell populations based on the expression or level of expression of one or more markers, typically a cell surface marker, for example by incubation with an antibody or binding partner that specifically binds to such a marker, followed by typically a washing step and separation of cells that have bound the antibody or binding partner from those that have not bound to the antibody or binding partner.

[0198] In some aspects of such processes, a volume of cells is mixed with an amount of a desired affinity-based selection reagent. Immunoaffinity-based selection can be performed using any system or method that results in a favorable energetic interaction between the cells being separated and a molecule that specifically binds to a marker on the cells, for example an antibody or other binding partner on a solid surface (e.g., a particle). In some embodiments, the method is performed using particles such as beads (e.g., magnetic beads) that are coated with a selection agent (e.g., an antibody) that is specific for a marker of the cells. The particles (e.g., beads) can be incubated or mixed with the cells in a container (such as a tube or bag) while being shaken or mixed, with a constant ratio of cell density to particles (e.g., beads) to help facilitate the energetically favorable interaction. In other cases, the method comprises selecting cells, wherein all or a portion of the selection is performed in the interior cavity of a centrifugal chamber, for example under centrifugal rotation. In some embodiments, the incubation of the cells with a selection reagent, such as an immunoaffinity-based selection reagent, is performed in a centrifugal chamber. In certain embodiments, the separation or partitioning is performed using a system, device, or apparatus described in International Patent Application Publication No. WO 2009 / 072003 or US 20110003380 Al. In one example, the system is a system as described in International Publication No. WO 2016 / 073602.

[0199] In some embodiments, by performing such selection steps or portions thereof (e.g., incubation with antibody-coated particles (e.g., magnetic beads)) in the cavities of the centrifugal chambers, the user is able to control certain parameters, such as the volumes of various solutions, the addition of solutions during processing and their timing, which can provide a variety of advantages compared to other available methods. For example, the ability to reduce the volume of liquid in the chamber during incubation can increase the concentration of particles (e.g., bead reagents) used in the selection, thereby increasing the chemical potential of the solution without affecting the total number of cells in the chamber. This, in turn, can enhance the pairwise interactions between the cells being processed and the particles used for selection. In some embodiments, for example, when associated with systems, circuitry, and controls as described herein, performing the incubation steps in the chambers allows the user to achieve agitation of the solutions at one or more desired times during incubation, which can also improve the interactions.

[0200] In some embodiments, at least a portion of the selection steps are performed in the centrifugal chambers, which include incubating the cells with selection reagents. In some aspects of such processes, a volume of cells is mixed with an amount of affinity-based selection reagents desired for the selection, which is substantially less than the amount typically employed in a tube or vessel-based incubation for performing a similar selection for selecting the same number of cells and / or the same volume of cells according to the manufacturer’s instructions. In some embodiments, the amount of selection reagent(s) employed is no more than 5%, no more than 10%, no more than 15%, no more than 20%, no more than 25%, no more than 50%, no more than 60%, no more than 70%, or no more than 80% of the amount of the same selection reagent(s) used for selecting cells in a tube or vessel-based incubation according to the manufacturer’s instructions for the same number of cells and / or the same volume of cells.

[0201] In some embodiments, for selection of cells, e.g., selection based on immunoaffinity, the cells are incubated in the chamber cavity in a composition that also contains a selection buffer with a selection reagent, e.g., a molecule, e.g., an antibody, that specifically binds to a surface marker on the cells that one wishes to enrich and / or deplete (but not on other cells in the composition), optionally coupled to a scaffold (e.g., a polymer or surface, e.g., a bead, e.g., a magnetic bead, e.g., a magnetic bead coupled to a monoclonal antibody specific for CD4 and CD8). In some embodiments, the selection reagent is added to the cells in the chamber cavity in an amount that is significantly less (e.g., no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the amount typically used or would be needed to achieve approximately the same or similar selection efficiency for the same number of cells or the same volume of cells as when the selection is performed in a tube that is shaken or spun). In some embodiments, the incubation is performed with the addition of selection buffer to the cells and selection reagent to achieve a target volume of incubation of, e.g., 10 mL to 200 mL, such as at least or about at least or about 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 reagent. In some embodiments, the selection buffer and selection reagent are pre-mixed prior to addition to the cells. In some embodiments, the selection buffer and selection reagent are added to the cells separately. In some embodiments, the selection incubation is performed under conditions of periodic gentle mixing, which can help to promote energetically favorable interactions, allowing for the use of less total selection reagent while achieving high selection efficiency.

[0202] In some embodiments, the total duration of incubation with the selection reagent is from 5 minutes to 6 hours or from about 5 minutes to about 6 hours, such as 30 minutes to 3 hours, e.g., at least or about at least 30 minutes, 60 minutes, 120 minutes, or 180 minutes.

[0203] In some embodiments, the incubation is typically performed under mixing conditions, such as in the presence of rotation, typically at a relatively low force or speed, such as a speed lower than that used to pellet the cells, such as from 600 rpm to 1700 rpm or from about 600 rpm to about 1700 rpm (e.g., at or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm), such as at a certain RCF at the sample or wall of the chamber or other container, the RCF being from 80 g to 100 g or from about 80 g to about 100 g (e.g., at or about or at least 80 g, 85 g, 90 g, 95 g, or 100 g). In some embodiments, the rotation is performed using a repeating interval of rotation at such low speed and a rest period thereafter, such as rotation and / or rest for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, such as rotation for about 1 or 2 seconds followed by rest for about 5, 6, 7, or 8 seconds.

[0204] In some embodiments, such a process is performed within a fully closed system that is integral with the chamber. In some embodiments, this process (and in some aspects one or more additional steps, such as a pre-wash step to wash a sample containing cells, such as an apheresis sample) is performed in an automated manner, such that cells, reagents, and other components are drawn into and pushed out of the chamber at appropriate times and centrifuged so as to complete the washing and binding steps in a single closed system using an automated program.

[0205] In some embodiments, after incubating and / or mixing the cells with one and / or more selection reagents, the incubated cells are subjected to separation to select cells based on the presence or absence of one or more particular reagents. In some embodiments, the separation is performed in the same closed system in which the cells were incubated with the selection reagents. In some embodiments, after incubation with the selection reagents, the incubated cells, including those in which the selection reagents have bound, are transferred to a system for separating cells based on immunoaffinity. In some embodiments, the system for separation based on immunoaffinity is or contains a magnetic separation column.

[0206] Such separation steps can be based on positive selection (in which cells that have bound a reagent, such as an antibody or binding partner, are retained for further use) and / or negative selection (in which cells that have not bound a reagent, such as an antibody or binding partner, are retained). In some instances, both fractions are retained for further use. In some aspects, negative selection can be particularly useful in the absence of antibodies that can be used to specifically identify a cell type in a heterogeneous population, such that separation is preferably based on markers expressed by cells other than the desired population.

[0207] In some embodiments, the process steps further comprise performing negative and / or positive selection on the incubated cells, e.g., using a system or apparatus that can perform affinity-based selection. In some embodiments, the isolation is performed by enriching for a particular cell population via positive selection, or depleting a particular cell population via negative selection. In some embodiments, the positive or negative selection is accomplished by incubating the cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers that are expressed (marker+) or expressed at a relatively higher level (marker- ) on the cells being positively or negatively selected, respectively. The antibodies or other binding agents can be conjugated to a label, e.g., a fluorescent label, that can be detected by a system or apparatus that can perform affinity-based selection. In some embodiments, the system or apparatus that can perform affinity-based selection is a fluorescence-activated cell sorter (FACS) or a magnetic-activated cell sorter (MACS). In some embodiments, the system or apparatus that can perform affinity-based selection is a FACS. 高 Multiple rounds of the same selection step (e.g., positive or negative selection step) can be performed. In certain embodiments, the positively or negatively selected fraction is subjected to the selection process, e.g., by repeating the positive or negative selection step. In some embodiments, the selection is repeated two, three, four, five, six, seven, eight, nine, or more than nine times. In certain embodiments, the same selection is performed up to five times. In certain embodiments, the same selection step is performed three times.

[0208] The isolation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment for a particular type of cell, e.g., those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in the complete absence of cells that do not express the marker. Likewise, negative selection, removal, or depletion of a particular type of cell, e.g., those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in the complete removal of all such cells.

[0209] In some examples, multiple rounds of separation steps are performed, in which a positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating the cells with multiple antibodies or binding partners, each antibody or binding partner specific for a marker targeted for negative selection. Likewise, multiple cell types can be positively selected simultaneously by incubating the cells with multiple antibodies or binding partners expressed on various cell types. In certain embodiments, one or more separation steps are repeated and / or performed more than once. In some embodiments, a positively or negatively selected fraction resulting from a separation step is subjected to the same separation step, such as by repeating a positive or negative selection step. In some embodiments, a single separation step is repeated and / or performed more than once, for example to increase the yield of positively selected cells, to increase the purity of negatively selected cells, and / or to further remove positively selected cells from a negatively selected fraction. In certain embodiments, one or more separation steps are performed and / or repeated two, three, four, five, six, seven, eight, nine, ten, or more than ten times. In certain embodiments, the one or more selection steps are performed and / or repeated between one and ten times, between one and five times, or between three and five times. In certain embodiments, one or more selection steps are repeated three times.

[0210] For example, in some aspects, a particular subpopulation of T cells is isolated by positive or negative selection techniques, such as cells that are positive for or express high levels of one or more surface markers, such as CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells. In some embodiments, such cells are selected by incubation with one or more antibodies or binding partners that specifically bind such markers. In some embodiments, the antibodies or binding partners can be conjugated (such as directly or indirectly) to a solid support or matrix, such as magnetic or paramagnetic beads, to effect selection. For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander and / or ExpACT® beads).

[0211] In some embodiments, T cells are isolated from a PBMC sample by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes, such as CD14. In some aspects, CD4+ or CD8+ selection steps are used to isolate CD4+ helper T cells and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively high degree on one or more naive T cell, memory T cell, and / or effector T cell subpopulations.

[0212] In some embodiments, CD8+ T cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulations. In some embodiments, enrichment is performed for central memory T (TCM) cells to increase efficacy, such as to improve long-term survival, expansion, and / or engraftment following administration, which in some aspects is particularly robust in such subpopulations. See Terakura et al. (2012) Blood. 1 :72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TCM-enriched CD8+ T cells with CD4+ T cells further enhances efficacy.

[0213] In embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted for CD62L-CD8+ and / or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.

[0214] In some embodiments, enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of a TCM cell-enriched CD8+ population is performed by depletion of cells expressing CD4, CD14, CD45RA and positive selection or enrichment of cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is performed starting from a negatively selected cell fraction based on CD4 expression, which is subjected to negative selection based on expression of CD14 and CD45RA and positive selection based on CD62L.

[0215] In some aspects this selection is performed simultaneously, while in other aspects sequentially in any order. In some aspects the same CD4 expression based selection step for preparing a CD8+ T cell population or subpopulation is also used to generate a CD4+ T cell population or subpopulation, such that both the positive and negative fractions from the CD4 based isolation are retained and used in subsequent steps of the method, optionally after one or more other positive or negative selection steps. In some embodiments, selection of a CD4+ T cell population and selection of a CD8+ T cell population are performed simultaneously. In some embodiments, selection of a CD4+ T cell population and selection of a CD8+ T cell population are performed sequentially in either order. In some embodiments, the methods for selecting cells can include those described in published US application number US 20170037369. In some embodiments, the selected CD4+ T cell population and the selected CD8+ T cell population can be combined after selection. In some aspects, the selected CD4+ T cell population and the selected CD8+ T cell population can be combined in a bioreactor bag as described herein. In some embodiments, the selected CD4+ T cell population and the selected CD8+ T cell population are separately processed as per the provided methods, whereby the selected CD4+ T cell population is enriched for CD4+ T cells and incubated with a stimulating reagent (e.g. anti-CD3 / anti-CD28 magnetic beads), transduced with a viral vector encoding a recombinant protein (e.g. a CAR), and incubated under conditions to expand T cells; and the selected CD8+ T cell population is enriched for CD8+ T cells and incubated with a stimulating reagent (e.g. anti-CD3 / anti-CD28 magnetic beads), transduced with a viral vector encoding a recombinant protein (e.g. a CAR) (such as the same recombinant protein as used to engineer the CD4+ T cells from the same donor), and incubated under conditions to expand T cells.

[0216] In particular embodiments, a biological sample (e.g. a sample of PBMCs or other white blood cells) is subjected to selection for CD4+ T cells, with both negative and positive fractions being retained simultaneously. In certain embodiments, CD8+ T cells are selected from the negative fraction. In some embodiments, a biological sample is subjected to selection for CD8+ T cells, with both negative and positive fractions being retained simultaneously. In certain embodiments, CD4+ T cells are selected from the negative fraction.

[0217] In particular examples, a sample of PBMCs or other white blood cells is subjected to selection for CD4+ T cells, with both negative and positive fractions being retained. The negative fraction is then negatively selected based on expression of CD14 and CD45RA or CD19, and positively selected based on a marker profile of central memory T cells (such as CD62L or CCR7), with the positive and negative selection being performed in any order.

[0218] By identifying cell populations having cell surface antigens, CD4+ T helper cells can be sorted into naive, central memory, and effector cells. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, or CD4+ T cells. In some embodiments, central memory CD4+ T cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ T cells are CD62L- and CD45RO-.

[0219] In one example, to enrich for CD4+ T cells by negative selection, a cocktail of monoclonal antibodies typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding partners are bound to a solid support or matrix, such as magnetic or paramagnetic beads, to allow for cell separation for positive and / or negative selection. For example, in some embodiments, immunomagnetic (or affinity magnetic) separation techniques are used to separate or isolate cells and cell populations (reviewed in Methods in Molecular Medicine, Volume 58: Metastasis Research Protocols, Volume 2: Cell Behavior In Vitro and In Vivo, pp. 17-25 S.A. Brooks and U. Schumacher eds. © Humana Press Inc., Totowa, NJ).

[0220] In some aspects, the incubated cell sample or composition to be isolated is incubated with a selection reagent containing small magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., Dynabeads or MACS® beads). The magnetically responsive material (e.g., particles) is typically directly or indirectly attached to a binding partner (e.g., an antibody) that specifically binds to a molecule (e.g., a surface marker) present on a cell, cells, or population of cells that one wishes to isolate (e.g., one wishes to negatively or positively select).

[0221] In some embodiments, the magnetic particles or beads comprise a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. A number of well-known magnetically responsive materials for use in magnetic separation methods are known, such as those described in Molday, U.S. Patent No. 4,452,773 and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal-sized particles can also be used, such as those described in Owen U.S. Patent No. 4,795,698 and Liberti et al., U.S. Patent No. 5,200,084.

[0222] Incubation is typically carried out under conditions whereby the antibody or binding partner, or a molecule that specifically binds to such an antibody or binding partner attached to a magnetic particle or bead (such as a secondary antibody or other reagent), specifically binds to a cell surface molecule, if present on the cells within the sample.

[0223] In certain embodiments, the magnetically responsive particles are coated in a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, the magnetic particles are attached to the cells by coating with a primary antibody specific for one or more markers. In certain embodiments, the cells are labeled with a primary antibody or binding partner rather than the beads, and then a cell type-specific secondary antibody or other binding partner (e.g., streptavidin)-coated magnetic particle is added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

[0224] In some aspects, separation is achieved in a procedure in which the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, the cells that are attracted to the magnet are retained; for negative selection, the cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, in which both the positive and negative fractions are retained and further processed or subjected to additional separation steps.

[0225] In some embodiments, affinity-based selection is performed via magnetic activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetic activated cell sorting (MACS) (e.g., CliniMACS system) enables high purity selection of cells with magnetized particles attached to them. In certain embodiments, MACS is operated in a mode in which non-target and target species are sequentially eluted after application of an external magnetic field. That is, cells attached to magnetized particles remain in place while unattached species are eluted. Then, after the first elution step is complete, the species that were captured in the magnetic field and prevented from eluting are released in such a way that they can be eluted and recovered. In certain embodiments, the non-target cells are labeled and depleted from the heterogeneous cell population.

[0226] In some embodiments, the magnetically responsive particles remain attached to the cells, which are then incubated, cultured, and / or engineered; in some aspects, the particles remain attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.

[0227] In some embodiments, the isolating and / or selecting results in one or more input compositions enriched for T cells, e.g., CD3+ T cells, CD4+ T cells, and / or CD8+ T cells. In some embodiments, two or more separate input compositions are isolated, selected, enriched, or obtained from a single biological sample. In some embodiments, separate input compositions are isolated, selected, enriched, and / or obtained from separate biological samples collected, obtained, and / or derived from the same subject.

[0228] In certain embodiments, the one or more input compositions are or include a composition of enriched T cells comprising 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%, at least 99.5%, at least 99.9%, or at or about 100% CD3+ T cells. In particular embodiments, the input composition of enriched T cells consists essentially of CD3+ T cells.

[0229] In certain embodiments, the one or more input compositions are or include a composition enriched for CD4+ T cells, the composition comprising 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%, at least 99.5%, at least 99.9%, or is or is about 100% CD4+ T cells. In certain embodiments, the input composition of CD4+ T cells contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and / or is free of CD8+ T cells, and / or is free or substantially free of CD8+ T cells. In some embodiments, the composition enriched for T cells consists essentially of CD4+ T cells.

[0230] In certain embodiments, the one or more compositions are or include a composition of CD8+ T cells, the composition being or comprising 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%, at least 99.5%, at least 99.9%, or is or is about 100% CD8+ T cells. In certain embodiments, the composition of CD8+ T cells contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and / or is free of CD4+ T cells, and / or is free or substantially free of CD4+ T cells. In some embodiments, the composition enriched for T cells consists essentially of CD8+ T cells.

[0231] In some embodiments, the one or more input compositions of enriched T cells are frozen, e.g., cryopreserved and / or cryofrozen, after isolation, selection, and / or enrichment. In some embodiments, the one or more input compositions are frozen, e.g., cryopreserved and / or cryofrozen, prior to any step of incubating, activating, stimulating, engineering, transducing, transfecting, culturing, expanding, harvesting, and / or formulating a composition of the cells. In particular embodiments, the one or more cryofrozen input compositions are stored at or about -80°C for 12 hours to 7 days, 24 hours to 120 hours, or 2 days to 5 days. In particular embodiments, the one or more cryofrozen input compositions are stored at or about -80°C for an amount of time that is less than 10 days, 9 days, 8 days, 7 days, 6 days, or 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the one or more cryofrozen input compositions are stored at or about -80°C for about 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days. B. Activation and stimulation of cells

[0232] In some embodiments, the provided methods are used in conjunction with incubating the cells under stimulating conditions. In some embodiments, stimulating conditions include conditions that activate or stimulate and / or are capable of activating or stimulating a signal in the cells (e.g., CD4+ T cells or CD8+ T cells), such as a signal generated from a TCR and / or a co-receptor. In some embodiments, stimulating conditions include one or more steps of culturing, culturing, incubating, activating, propagating the cells with and / or in the presence of a stimulating reagent (e.g., a reagent that activates or stimulates and / or is capable of activating or stimulating a signal in the cells). In some embodiments, the stimulating reagent stimulates and / or activates a TCR and / or a co-receptor. In particular embodiments, the stimulating reagent is a reagent described in Section II-B-l.

[0233] In certain embodiments, one or more compositions of enriched T cells are incubated under stimulating conditions prior to genetically engineering the cells, e.g., transfecting and / or transducing the cells by the techniques provided in Section II-C. In particular embodiments, one or more compositions of enriched T cells are incubated under stimulating conditions after the one or more compositions have been isolated, selected, enriched, or obtained from a biological sample. In particular embodiments, the one or more compositions are input compositions. In particular embodiments, the one or more input compositions have been previously cryofrozen and stored, and thawed prior to incubation.

[0234] In certain embodiments, the one or more compositions of enriched T cells are or include two separate compositions of enriched T cells, e.g., separate input compositions. In particular embodiments, the two separate compositions of enriched T cells, e.g., two separate compositions of enriched T cells selected, isolated, and / or enriched from the same biological sample, are separately incubated under stimulatory conditions. In certain embodiments, the two separate compositions include a composition of enriched CD4+ T cells. In particular embodiments, the two separate compositions include a composition of enriched CD8+ T cells. In some embodiments, the two separate compositions of enriched CD4+ and CD8+ T cells are separately incubated under stimulatory conditions.

[0235] In some embodiments, a single composition of enriched T cells is incubated under stimulatory conditions. In certain embodiments, the single composition is a composition of enriched CD4+ T cells. In some embodiments, the single composition is a composition of enriched CD4+ and CD8+ T cells that has been combined from separate compositions prior to incubation.

[0236] In some embodiments, a composition of enriched CD4+ T cells incubated under stimulatory conditions comprises 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%, at least 99.5%, at least 99.9%, or is or is about 100% CD4+ T cells. In certain embodiments, a composition of enriched CD4+ T cells incubated under stimulatory conditions comprises less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and / or is free of CD8+ T cells, and / or is free or substantially free of CD8+ T cells.

[0237] In some embodiments, a composition of enriched CD8+ T cells incubated under stimulatory conditions comprises 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%, at least 99.5%, at least 99.9%, or is or is about 100% CD8+ T cells. In certain embodiments, a composition of enriched CD8+ T cells incubated under stimulatory conditions comprises less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and / or is free of CD4+ T cells, and / or is free or substantially free of CD4+ T cells.

[0238] In some embodiments, separate compositions of enriched CD4+ and CD8+ T cells are combined into a single composition and incubated under stimulating conditions. In certain embodiments, separate stimulated compositions of enriched CD4+ and enriched CD8+ T cells are combined into a single composition after the incubation has been performed and / or completed. In some embodiments, separate stimulated compositions of stimulated CD4+ T and stimulated CD8+ T cells are treated separately after the incubation has been performed and / or completed, whereby a stimulated CD4+ T cell population (e.g., incubated with stimulating anti-CD3 / anti-CD28 magnetic bead stimulating reagents) is transduced with a viral vector encoding a recombinant protein (e.g., a CAR) and incubated under conditions to expand T cells, and a stimulated CD8+ T cell population (e.g., incubated with stimulating anti-CD3 / anti-CD28 magnetic bead stimulating reagents) is transduced with a viral vector encoding a recombinant protein (e.g., a CAR) (such as the same recombinant protein used to engineer CD4+ T cells from the same donor) and incubated under conditions to expand T cells, as per the methods provided.

[0239] In some embodiments, incubation under stimulating conditions can include culturing, incubating, stimulating, activating, propagating, including by incubation in the presence of stimulating conditions, e.g., conditions designed to induce proliferation, expansion, activation, and / or survival of cells in the population, mimic antigen exposure, and / or prime cells for genetic engineering (e.g., for introduction of a recombinant antigen receptor). In particular embodiments, stimulating conditions can include one or more of: a particular medium, temperature, oxygen content, carbon dioxide content, time, agents (e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors (such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells)).

[0240] In some aspects, stimulation and / or incubation under stimulating conditions is performed according to a variety of techniques, such as those described in U.S. Patent No. 6,040,177 to Riddell et al.; Klebanoff et al. (2012) J Immunother. 35(9): 651-660; Terakura et al. (2012) Blood. 1:72-82; and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0241] In some embodiments, cells (e.g., T cells), cell compositions, and / or cell populations, such as CD4 + and CD8 +T cells or compositions, populations, or subpopulations thereof: adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs) (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded) to a culture starting composition; and incubating the culture (e.g., for a time sufficient to expand the number of the T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to the culture medium prior to the addition of the population of T cells.

[0242] In some embodiments, the stimulating conditions include a temperature suitable for the growth of human T lymphocytes, such as at least about 25 degrees Celsius, typically at least about 30 degrees Celsius, and typically at or about 37 degrees Celsius. In some embodiments, a temperature shift is achieved during the culturing, such as from 37 degrees Celsius to 35 degrees Celsius. Optionally, the incubation can further comprise the addition of non-dividing EBV-transformed lymphoblastoid cells (LCLs) as feeder cells. The LCLs can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. In some aspects, the LCL feeder cells are provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10: 1.

[0243] In embodiments, populations of antigen-specific CD4 + and CD8 + T cells can be obtained by stimulating naive or antigen-specific T lymphocytes with an antigen. For example, antigen-specific T cell lines or clones can be generated against cytomegalovirus antigens by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen. Naive T cells can also be used.

[0244] In particular embodiments, the stimulating conditions include incubating, culturing, and / or growing the cells with a stimulating reagent. In particular embodiments, the stimulating reagent is a reagent described in Section II-B-l. In certain embodiments, the stimulating reagent contains or comprises a bead. An exemplary stimulating reagent is or comprises anti-CD3 / anti-CD28 magnetic beads. In certain embodiments, the initiation and / or start of incubating, culturing, and / or growing the cells under stimulating conditions occurs when the cells are contacted with and / or incubated with the stimulating reagent. In particular embodiments, the cells are incubated before, during, and / or after genetically engineering the cells (e.g., introducing a recombinant polynucleotide into the cells, such as by transfection or transduction).

[0245] In some embodiments, the ratio of stimulatory reagent and / or beads (e.g., anti-CD3 / anti-CD28 magnetic beads) to cells for the enriched T cell composition is at or about 3: 1, 2.5: 1, 2: 1, 1.5: 1, 1.25: 1, 1.2: 1, 1.1: 1, 1: 1, 0.9: 1, 0.8: 1, 0.75: 1, 0.67: 1, 0.5: 1, 0.3: 1, or 0.2: 1. In particular embodiments, the ratio of stimulatory reagent and / or beads to cells is between 2.5: 1 and 0.2: 1, between 2: 1 and 0.5: 1, between 1.5: 1 and 0.75: 1, between 1.25: 1 and 0.8: 1, between 1.1: 1 and 0.9: 1. In particular embodiments, the ratio of stimulatory reagent to cells is about 1: 1 or is 1: 1. In some embodiments, the ratio is determined after evaluating T cell activation using the methods provided herein.

[0246] In particular embodiments, incubating cells at a ratio of less than 3: 1 or less than 3 stimulatory reagents (e.g., anti-CD3 / anti-CD28 magnetic beads) per cell, such as a ratio of 1: 1, reduces the amount of cell death (e.g., due to activation-induced cell death) that occurs during incubation. In some embodiments, the cells are incubated with the stimulatory reagent (e.g., anti-CD3 / anti-CD28 magnetic beads) at a ratio of less than 3 (or 3: 1 or less than 3 beads per cell) of beads to cells. In particular embodiments, incubating the cells at a ratio of less than 3: 1 or less than 3 beads per cell, such as a ratio of 1: 1, reduces the amount of cell death (e.g., such as due to activation-induced cell death) that occurs during incubation.

[0247] In particular embodiments, the enriched T cell composition is incubated with the stimulating reagent (e.g., anti-CD3 / anti-CD28 magnetic beads) at a ratio of stimulating reagent and / or beads to cells of less than 3: 1, such as a ratio of 1: 1, and at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the T cells survive, e.g., are viable and / or do not undergo necrosis, programmed cell death, or apoptosis, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days or at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days after completion of the incubation. In particular embodiments, the enriched T cell composition is incubated with the stimulating reagent at a ratio of stimulating reagent and / or beads to cells of less than 3: 1, such as a ratio of 1: 1, and less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% of the cells undergo activation-induced cell death during the incubation.

[0248] In certain embodiments, an enriched T cell composition is incubated with a stimulating reagent (e.g., anti-CD3 / anti-CD28 magnetic beads) at a ratio of beads to cells of less than 3: 1, such as a ratio of 1: 1, and the cells of the composition have a survival rate that 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%, at least 100%, at least 150%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, or at least 100-fold greater than cells that undergo an exemplary and / or alternative process in which an enriched T cell composition is incubated with a stimulating reagent at a ratio of 3: 1 or greater.

[0249] In some embodiments, the enriched T cell composition incubated with the stimulating reagent comprises from 1.0 x 10 5 cells / mL to 1.0 x 10 8 cells / mL or from about 1.0 x 10 5 cells / mL to about 1.0 x 10 8 cells / mL, such as at least or about at least or about 1.0 x 10 5 cells / mL, 5 x 10 5 cells / mL, 1 x 10 6 cells / mL, 5 x 10 6 cells / mL, 1 x 107 about 0.5 x 10 7 about 1 x 10 8 about 1.5 x 10 6 about 2 x 10 6 about 2.5 x 10 6 about 3 x 10 6 about 3.5 x 10 6 about 4 x 10 6 about 4.5 x 10 6 about 5 x 10 6 about 5.5 x 10 6 about 6 x 10 6 about 6.5 x 10 6 about 7 x 10 6 about 7.5 x 10 6 about 8 x 10 6 about 8.5 x 10 6 about 9 x 10 6 about 9.5 x 10 6 about 10 x 10 6 about 10.5 x 10 6 about 11 x 10 6 about 11.5 x 10 6 about 12 x 10

[0250] In some embodiments, the composition of enriched T cells is incubated with the stimulating reagent at a temperature from about 25ºC to about 38ºC, such as from about 30ºC to about 37ºC, for example at or at about 37ºC ± 2ºC. In some embodiments, the composition of enriched T cells is incubated with the stimulating reagent at a CO2 level from about 2.5% to about 7.5%, such as from about 4% to about 6%, for example at or at about 5% ± 0.5%. In some embodiments, the composition of enriched T cells is incubated with the stimulating reagent at a temperature at or at about 37ºC and / or at a CO2 level at or at about 5%.

[0251] In particular embodiments, the stimulating conditions comprise incubating, culturing, and / or culturing the T cell-enriched composition with one or more cytokines and / or in the presence of one or more cytokines. In particular embodiments, the one or more cytokines is a recombinant cytokine. In some embodiments, the one or more cytokines is a human recombinant cytokine. In certain embodiments, the one or more cytokines binds and / or is capable of binding a receptor expressed by T cells and / or endogenous to T cells. In particular embodiments, the one or more cytokines is or comprises a member of the 4-alpha-helix bundle family of cytokines. In some embodiments, the member of the 4-alpha-helix bundle family of cytokines comprises, but is not limited to, interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 7 (IL-7), interleukin 9 (IL-9), interleukin 12 (IL-12), interleukin 15 (IL-15), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF). In some embodiments, the one or more cytokines is or comprises IL-15. In particular embodiments, the one or more cytokines is or comprises IL-7. In particular embodiments, the one or more cytokines is or comprises IL-2. In some embodiments, the stimulating conditions comprise incubating a T cell-enriched composition, such as an enriched CD4+ T cell or an enriched CD8+ T cell, in the presence of a stimulating reagent as described (anti-CD3 / anti-CD28 magnetic beads) and in the presence of one or more recombinant cytokines.

[0252] In particular embodiments, the composition of enriched CD4+ T cells is incubated with IL-2, e.g., recombinant IL-2. Without wishing to be bound by theory, particular embodiments contemplate that CD4+ T cells obtained from some subjects do not produce or do not produce sufficient amounts of IL-2 that allow for growth, division, and expansion throughout the process for producing a composition of output cells, e.g., engineered cells suitable for use in cell therapy. In some embodiments, incubating a composition of enriched CD4+ T cells in the presence of recombinant IL-2 under stimulating conditions increases the probability or likelihood that the CD4+ T cells of the composition will continue to survive, grow, expand, and / or activate during the incubation step and throughout the process. In some embodiments, incubating the composition of enriched CD4+ T cells in the presence of recombinant IL-2 increases the probability and / or likelihood of producing an output composition of enriched CD4+ T cells, e.g., engineered CD4+ T cells suitable for cell therapy, from the composition of enriched CD4+ T cells by at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, 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 150%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, or at least 100-fold compared to an alternative and / or exemplary method of incubating the composition of enriched CD4+ T cells without incubation in the presence of recombinant IL-2.

[0253] In certain embodiments, the amount or concentration of the one or more cytokines is measured and / or quantified in international units (IU). International units can be used to quantify vitamins, hormones, cytokines, vaccines, blood products, and similar biologically active substances. In some embodiments, IU are or include a unit of measure by potency of a biological agent compared to an international reference standard having a specific weight and strength (e.g., WHO 1st International Standard for Human IL-2, 86 / 504 for IL-2). International units are the only recognized and standardized method of reporting units of biological activity that are published and derived from international cooperative research efforts. In particular embodiments, IU of a composition, sample, or source of a cytokine can be obtained by product comparison testing with similar WHO standard products. For example, in some embodiments, IU / mg of a composition, sample, or source of human recombinant IL-2, IL-7, or IL-15 are compared to WHO standard IL-2 product (NIBSC Code: 86 / 500), WHO standard IL-17 product (NIBSC Code: 90 / 530), and WHO standard IL-15 product (NIBSC Code: 95 / 554), respectively.

[0254] In some embodiments, biological activity in IU / mg is equivalent to an ED 50 ) -1 x 10 6 In particular embodiments, an ED 50 of recombinant human IL-2 or IL-15 is equivalent to the concentration required for half-maximal stimulation of cell proliferation using CTLL-2 cells (XTT cleavage). In certain embodiments, an ED 50 of recombinant human IL-7 is equivalent to the concentration required for half-maximal stimulation of PHA-activated human peripheral blood lymphocyte proliferation. Details related to the determination and calculation of IU for IL-2 are discussed in Wadhwa et al., Journal of Immunological Methods (2013), 379 (1-2): 1-7; and Gearing and Thorpe, Journal of Immunological Methods (1988), 114 (1-2): 3-9; and details related to the determination and calculation of IU for IL-15 are discussed in Soman et al. Journal of Immunological Methods (2009) 348 (1-2): 83-94; which are hereby incorporated by reference in their entirety.

[0255] In particular embodiments, the composition enriched for CD8+ T cells is incubated in the presence of IL-2 and / or IL-15 under stimulatory conditions. In certain embodiments, the composition enriched for CD4+ T cells is incubated in the presence of IL-2, IL-7, and / or IL-15 under stimulatory conditions. In some embodiments, the IL-2, IL-7, and / or IL-15 is recombinant. In certain embodiments, the IL-2, IL-7, and / or IL-15 is human. In particular embodiments, the one or more cytokines is or includes human recombinant IL-2, IL-7, and / or IL-15. In some aspects, the incubation of the enriched T cell composition further includes the presence of a stimulatory reagent, such as anti-CD3 / anti-CD28 magnetic beads.

[0256] In some embodiments, the cells are incubated with a cytokine, such as a recombinant human cytokine, at a concentration between 1 IU / ml and 1,000 IU / ml, between 10 IU / ml and 50 IU / ml, between 50 IU / ml and 100 IU / ml, between 100 IU / ml and 200 IU / ml, between 100 IU / ml and 500 IU / ml, between 250 IU / ml and 500 IU / ml, or between 500 IU / ml and 1,000 IU / ml. In some embodiments, the IU / mL of cytokine incubated with the cells is based on the assessment of T cell activation with any of the methods provided herein.

[0257] In some embodiments, the enriched T cell composition is incubated with IL-2, e.g., human recombinant IL-2, at a concentration of between 1 IU / ml and 200 IU / ml, between 10 IU / ml and 200 IU / ml, between 10 IU / ml and 100 IU / ml, between 50 IU / ml and 150 IU / ml, between 80 IU / ml and 120 IU / ml, between 60 IU / ml and 90 IU / ml, or between 70 IU / ml and 90 IU / ml. In particular embodiments, the enriched T cell composition is incubated with recombinant IL-2 at a concentration of, or of about, 50 IU / ml, 55 IU / ml, 60 IU / ml, 65 IU / ml, 70 IU / ml, 75 IU / ml, 80 IU / ml, 85 IU / ml, 90 IU / ml, 95 IU / ml, 100 IU / ml, 110 IU / ml, 120 IU / ml, 130 IU / ml, 140 IU / ml, or 150 IU / ml. In some embodiments, the enriched T cell composition is incubated in the presence of recombinant IL-2 at, or at about, 85 IU / ml. In some embodiments, the composition incubated with recombinant IL-2 is enriched for a population of T cells, e.g., CD4+ T cells and / or CD8+ T cells. In some embodiments, the T cell population is a CD4+ T cell population. In some embodiments, the enriched T cell composition is an enriched CD8+ T cell composition. In particular embodiments, the enriched T cell composition is enriched for CD8+ T cells, wherein CD4+ T cells are not enriched and / or wherein CD4+ T cells are negatively selected or depleted from the composition. In some embodiments, the enriched T cell composition is an enriched CD4+ T cell composition. In particular embodiments, the enriched T cell composition is enriched for CD4+ T cells, wherein CD8+ T cells are not enriched and / or wherein CD8+ T cells are negatively selected or depleted from the composition. In some embodiments, an enriched CD4+ T cell composition incubated with recombinant IL-2 can also be incubated with recombinant IL-7 and / or recombinant IL-15, e.g., in the amounts described. In some embodiments, an enriched CD8+ T cell composition incubated with recombinant IL-2 can also be incubated with recombinant IL-15, e.g., in the amounts described.

[0258] In some embodiments, the enriched T cell composition is incubated with recombinant IL-7, e.g., human recombinant IL-7, at a concentration between 100 IU / ml and 2,000 IU / ml, between 500 IU / ml and 1,000 IU / ml, between 100 IU / ml and 500 IU / ml, between 500 IU / ml and 750 IU / ml, between 750 IU / ml and 1,000 IU / ml, or between 550 IU / ml and 650 IU / ml. In particular embodiments, the enriched T cell composition is incubated with recombinant IL-7 at a concentration of, or about, 50 IU / ml, 100 IU / ml, 150 IU / ml, 200 IU / ml, 250 IU / ml, 300 IU / ml, 350 IU / ml, 400 IU / ml, 450 IU / ml, 500 IU / ml, 550 IU / ml, 600 IU / ml, 650 IU / ml, 700 IU / ml, 750 IU / ml, 800 IU / ml, 750 IU / ml, 750 IU / ml, 750 IU / ml, or 1,000 IU / ml. In particular embodiments, the enriched T cell composition is incubated in the presence of recombinant IL-7 at, or about, 600 IU / ml. In some embodiments, the composition incubated with recombinant IL-7 is enriched for a population of T cells, e.g., CD4+ T cells. In some embodiments, the enriched CD4+ T cell composition incubated with recombinant IL-7 can also be incubated with recombinant IL-2 and / or recombinant IL-15, e.g., in the amounts described. In particular embodiments, the enriched T cell composition is enriched for CD4+ T cells, wherein CD8+ T cells are not enriched and / or wherein CD8+ T cells are negatively selected or depleted from the composition. In some embodiments, the enriched CD8+ T cell composition is not incubated with recombinant IL-7.

[0259] In some embodiments, the enriched T cell composition is incubated with recombinant IL-15, e.g., human recombinant IL-15, at a concentration between 0.1 IU / ml and 100 IU / ml, between 1 IU / ml and 100 IU / ml, between 1 IU / ml and 50 IU / ml, between 5 IU / ml and 25 IU / ml, between 25 IU / ml and 50 IU / ml, between 5 IU / ml and 15 IU / ml, or between 10 IU / ml and 100 IU / ml. In particular embodiments, the enriched T cell composition is incubated with recombinant IL-15 at a concentration of, or about, 1 IU / ml, 2 IU / ml, 3 IU / ml, 4 IU / ml, 5 IU / ml, 6 IU / ml, 7 IU / ml, 8 IU / ml, 9 IU / ml, 10 IU / ml, 11 IU / ml, 12 IU / ml, 13 IU / ml, 14 IU / ml, 15 IU / ml, 20 IU / ml, 25 IU / ml, 30 IU / ml, 40 IU / ml, or 50 IU / ml. In some embodiments, the enriched T cell composition is incubated in, or about, 10 IU / ml recombinant IL-15. In some embodiments, the composition incubated with recombinant IL-15 is enriched for a population of T cells, e.g., CD4+ T cells and / or CD8+ T cells. In some embodiments, the T cell population is a CD4+ T cell population. In some embodiments, the enriched T cell composition is an enriched CD8+ T cell composition. In particular embodiments, the enriched T cell composition is enriched for CD8+ T cells, wherein CD4+ T cells are not enriched and / or wherein CD4+ T cells are negatively selected or depleted from the composition. In some embodiments, the enriched T cell composition is an enriched CD4+ T cell composition. In particular embodiments, the enriched T cell composition is enriched for CD4+ T cells, wherein CD8+ T cells are not enriched and / or wherein CD8+ T cells are negatively selected or depleted from the composition. In some embodiments, an enriched CD4+ T cell composition incubated with recombinant IL-15 can also be incubated with recombinant IL-7 and / or recombinant IL-2, e.g., in the amounts described. In some embodiments, an enriched CD8+ T cell composition incubated with recombinant IL-15 can also be incubated with recombinant IL-2, e.g., in the amounts described.

[0260] In particular embodiments, the cells (e.g., enriched CD4+ T cells and / or enriched CD8+ T cells) are incubated with the stimulating reagent in the presence of one or more antioxidants. In some embodiments, the antioxidant includes, but is not limited to, one or more antioxidants including tocopherol, tocotrienol, a-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, a-tocotrienol, β-tocotrienol, a-tocopherolquinone, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), flavonoids, isoflavones, lycopene, β-carotene, selenium, ubiquinone, luetin, S-adenosylmethionine, glutathione, taurine, N-acetylcysteine (NAC), citric acid, L-carnitine, BHT, thioglycerol, ascorbic acid, propyl gallate, methionine, cysteine, homocysteine, gluthatione, cystamine, and cyststathionine, and / or glycine-glycine-histidine. In some aspects, the incubation of the enriched T cell composition (e.g., enriched CD4+ T cells and / or enriched CD8+ T cells) with the antioxidant further includes the presence of a stimulating reagent (e.g., anti-CD3 / anti-CD28 magnetic beads) and one or more recombinant cytokines (e.g., as described).

[0261] In some embodiments, the one or more antioxidants are or include sulfur-containing antioxidants. In certain embodiments, the sulfur-containing antioxidant can include a thiol-containing antioxidant and / or an antioxidant that exhibits one or more sulfur moieties, e.g., within a ring structure. In some embodiments, the sulfur-containing antioxidant can include, e.g., N-acetylcysteine (NAC) and 2,3-dimercapto propanol (DMP), L-2-oxo-4-thiazolidine carboxylate (OTC), and thioctic acid. In particular embodiments, the sulfur-containing antioxidant is a glutathione precursor. In some embodiments, the glutathione precursor is a molecule that can be modified in one or more steps within a cell to a derivative of glutathione. In particular embodiments, the glutathione precursor can include, but is not limited to, N-acetylcysteine (NAC), L-2-oxothiazolidine-4-carboxylic acid (Procysteine), thioctic acid, S-allyl cysteine, or methylmethionine sulfonium chloride.

[0262] In some embodiments, incubating the cells (e.g., enriched CD4+ T cells and / or enriched CD8+ T cells) under stimulatory conditions comprises incubating the cells in the presence of one or more antioxidants. In particular embodiments, the cells are stimulated with the stimulatory reagents in the presence of one or more antioxidants. In some embodiments, the cells are incubated in the presence of between 1 ng / ml and 100 ng / ml, between 10 ng / ml and 1 µg / ml, between 100 ng / ml and 10 µg / ml, between 1 µg / ml and 100 µg / ml, between 10 µg / ml and 1 mg / ml, between 100 µg / ml and 1 mg / ml, between 1 500 µg / ml and 2 mg / ml, between 500 µg / ml and 5 mg / ml, between 1 mg / ml and 10 mg / ml, or between 1 mg / ml and 100 mg / ml of one or more antioxidants. In some embodiments, the cells are incubated in the presence of at or about 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 µg / ml, 10 µg / ml, 100 µg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, 500 mg / ml of one or more antioxidants. In some embodiments, the one or more antioxidants is or comprises a sulfur-containing antioxidant. In particular embodiments, the one or more antioxidants is or comprises a glutathione precursor.

[0263] In some embodiments, the one or more antioxidants is or includes N-acetyl cysteine (NAC). In some embodiments, incubating the cells (such as the enriched CD4+ T cells and / or the enriched CD8+ T cells) under stimulating conditions includes incubating the cells in the presence of NAC. In particular embodiments, the cells are stimulated with the stimulating reagents in the presence of NAC. In some embodiments, the cells are incubated in the presence of between 1 ng / ml and 100 ng / ml, between 10 ng / ml and 1 pg / ml, between 100 ng / ml and 10 pg / ml, between 1 pg / ml and 100 pg / ml, between 10 pg / ml and 1 mg / ml, between 100 pg / ml and 1 mg / ml, between 1 -500 pg / ml and 2 mg / ml, between 500 pg / ml and 5 mg / ml, between 1 mg / ml and 10 mg / ml, or between 1 mg / ml and 100 mg / ml of NAC. In some embodiments, the cells are incubated in the presence of 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 pg / ml, 10 pg / ml, 100 pg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, 500 mg / ml, or about 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 pg / ml, 10 pg / ml, 100 pg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, 500 mg / ml of NAC. In some embodiments, the cells are incubated with or with about 0.8 mg / ml. In particular embodiments, incubating a composition of enriched T cells (such as enriched CD4+ T cells and / or enriched CD8+ T cells) in the presence of one or more antioxidants, such as NAC, reduces activation in the cells as compared to cells incubated in alternative and / or exemplary processes in the absence of an antioxidant.

[0264] In some embodiments, the composition or cells (e.g., enriched CD4+ T cells and / or enriched CD8+ T cells) are incubated in the presence of stimulatory conditions or stimulatory reagents as described. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, mimic antigen exposure, and / or prime cells for genetic engineering (e.g., for introduction of recombinant antigen receptors). Exemplary stimulatory reagents (e.g., anti-CD3 / anti-CD28 magnetic beads) are described below. Incubation with stimulatory reagents can also be performed in the presence of one or more stimulatory cytokines, such as in the presence of one or more of recombinant IL-2, recombinant IL-7, and / or recombinant IL-15, and / or in the presence of at least one antioxidant, such as NAC, as described above. In some embodiments, compositions of enriched CD4+ T cells are incubated in stimulatory conditions with stimulatory agents (i.e., recombinant IL-2, recombinant IL-7, recombinant IL-15) and NAC, such as in the amounts as described. In some embodiments, compositions of enriched CD8+ T cells are incubated in stimulatory conditions with stimulatory agents (i.e., recombinant IL-2, recombinant IL-15) and NAC, such as in the amounts as described.

[0265] In some embodiments, conditions for stimulation and / or activation can include one or more of the following: a particular medium, temperature, oxygen content, carbon dioxide content, time, agents (e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors (e.g., cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells)).

[0266] In some aspects, incubation is performed according to a variety of techniques, such as those described in U.S. Patent No. 6,040,177 to Riddell et al.; Klebanoff et al. (2012) J Immunother. 35(9):651-660; Terakura et al. (2012) Blood. 1:72-82; and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0267] In some embodiments, at least a portion of the incubation performed in the presence of one or more stimulating conditions or agents is performed in the interior cavity of a centrifugal chamber, e.g., under centrifugal rotation, as described in International Publication No. WO 2016 / 073602. In some embodiments, at least a portion of the incubation performed in the centrifugal chamber includes mixing with one or more reagents to induce stimulation and / or activation. In some embodiments, cells, e.g., selected cells, are mixed with stimulating conditions or agents in the centrifugal chamber. In some aspects of such processes, a volume of cells is mixed with an amount of one or more stimulating conditions or agents that is significantly less than the amount of stimulating conditions or agents typically used when performing similar stimulation in a cell culture plate or other system, e.g., to achieve approximately the same or similar selection efficiency for the same number of cells or the same volume of cells as would typically be used or would be required if the selection were performed without mixing in a centrifugal chamber.

[0268] In some embodiments, the stimulating agent is added to the cells in the cavity of the chamber in an amount that is significantly less than (e.g., no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of) the amount of stimulating agent that would typically be used or would be required to achieve approximately the same or similar selection efficiency for the same number of cells or the same volume of cells if the selection were performed without mixing in a centrifugal chamber, e.g., in a periodically oscillating or rotating tube or bag. In some embodiments, the incubation is performed with the addition of an incubation buffer to the cells and stimulating agent to achieve a target volume of reagents of, e.g., about 10 mL to about 200 mL or about 20 mL to about 125 mL (e.g., at least or at least about or about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 105 mL, 110 mL, 115 mL, 120 mL, 125 mL, 130 mL, 135 mL, 140 mL, 145 mL, 150 mL, 160 mL, 170 mL, 180 mL, 190 mL, or 200 mL). In some embodiments, the incubation buffer and stimulating agent are premixed prior to the addition of the cells. In some embodiments, the incubation buffer and stimulating agent are added separately to the cells. In some embodiments, the stimulating incubation is performed under periodic gentle mixing conditions, which can help to promote energetically favorable interactions and thereby allow for the use of less total stimulating agent while achieving stimulation and activation of the cells.

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

[0270] In some embodiments, the total duration of the incubation with the stimulatory agent, e.g., is between or between about 1 hour and 96 hours, 1 hour and 72 hours, 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours, 18 hours and 30 hours, or 12 hours and 24 hours, e.g., is at least or is at least about or is about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours. In some embodiments, the further incubation is for a time that is between or about between 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours, or 12 hours and 24 hours, inclusive.

[0271] In some embodiments, the cells are cultured, incubated, and / or incubated under stimulatory conditions prior to and / or during the step of introducing a polynucleotide, e.g., a polynucleotide encoding a recombinant receptor, into the cells, e.g., by transduction and / or transfection, as described in Section II-C. In certain embodiments, the cells are cultured, incubated, and / or incubated under stimulatory conditions for an amount of time that is between 30 minutes and 2 hours, between 1 hour and 8 hours, between 1 hour and 6 hours, between 6 hours and 12 hours, between 12 hours and 18 hours, between 16 hours and 24 hours, between 12 hours and 36 hours, between 24 hours and 48 hours, between 24 hours and 72 hours, between 42 hours and 54 hours, between 60 hours and 120 hours, between 96 hours and 120 hours, between 90 hours and between 1 day and 7 days, between 3 days and 8 days, between 1 day and 3 days, between 4 days and 6 days, or between 4 days and 5 days, prior to engineering. In some embodiments, the cells are incubated for 2 days or about 2 days prior to engineering.

[0272] In certain embodiments, the cells are incubated with and / or in the presence of a stimulating reagent prior to and / or during genetic engineering of the cells. In certain embodiments, the cells are incubated with and / or in the presence of a stimulating reagent for an amount of time that is between 12 hours and 36 hours, between 24 hours and 48 hours, between 24 hours and 72 hours, between 42 hours and 54 hours, between 60 hours and 120 hours, between 96 hours and 120 hours, between 90 hours and between 2 days and 7 days, between 3 days and 8 days, between 1 day and 8 days, between 4 days and 6 days, or between 4 days and 5 days. In particular embodiments, the cells are cultured, incubated, and / or incubated under stimulating conditions for an amount of time that is less than 10 days, 9 days, 8 days, 7 days, 6 days, or 5 days, 4 days, or for an amount of time that is less than 168 hours, 162 hours, 156 hours, 144 hours, 138 hours, 132 hours, 120 hours, 114 hours, 108 hours, 102 hours, or 96 hours prior to and / or during genetic engineering of the cells. In particular embodiments, the cells are incubated with and / or in the presence of a stimulating reagent for 4 days, 5 days, 6 days, or 7 days or about 4 days, 5 days, 6 days, or 7 days. In some embodiments, the cells are incubated with and / or in the presence of a stimulating reagent for 4 days or about 4 days. In particular embodiments, the cells are incubated with and / or in the presence of a stimulating reagent for 5 days or about 5 days. In certain embodiments, the cells are incubated with and / or in the presence of a stimulating reagent for less than 7 days.

[0273] In some embodiments, incubating the cells under stimulating conditions comprises incubating the cells with a stimulating reagent described in Section II-B-1. In some embodiments, the stimulating reagent contains or comprises beads, such as paramagnetic beads, and the cells are incubated with the stimulating reagent at a ratio of less than 3: 1 (bead:cell), such as a ratio of 1: 1. In particular embodiments, the cells are incubated with the stimulating reagent in the presence of one or more cytokines and / or one or more antioxidants. In some embodiments, a composition of enriched CD4+ T cells is incubated with the stimulating reagent at a ratio of 1: 1 (bead:cell) in the presence of recombinant IL-2, IL-7, IL-15, and NAC. In certain embodiments, a composition of enriched CD8+ T cells is incubated with the stimulating reagent at a ratio of 1: 1 (bead:cell) in the presence of recombinant IL-2, IL-15, and NAC. In some embodiments, the stimulating reagent is removed and / or separated from the cells at, within, or about 6 days, 5 days, or 4 days from the start or initiation of incubation, e.g., from the time the stimulating reagent is added to or contacted with the cells. 1. a stimulatory reagent

[0274] In some embodiments, incubating the composition of enriched cells under stimulatory conditions is or comprises incubating and / or contacting the composition of enriched cells with a stimulatory reagent capable of activating and / or expanding T cells. In some embodiments, the stimulatory reagent is capable of stimulating and / or activating one or more signals in the cells. In some embodiments, the one or more signals are mediated by a receptor. In particular embodiments, the one or more signals are changes in the level or amount of signal transduction and / or a second messenger (e.g., cAMP and / or intracellular calcium), changes in the amount, cellular localization, conformation, phosphorylation, ubiquitination, and / or truncation of one or more cellular proteins, and / or changes in cellular activity (e.g., transcription, translation, protein degradation, cell morphology, activation state, and / or cell division), or are associated with such changes. In particular embodiments, the stimulatory reagent activates and / or is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules.

[0275] In certain embodiments, the stimulatory reagent contains a particle (e.g., a bead) conjugated or linked to one or more agents (e.g., biomolecules) capable of activating and / or expanding cells (e.g., T cells). In some embodiments, the one or more agents are bound to the bead. In some embodiments, the bead is biocompatible, i.e., composed of materials suitable for biological use. In some embodiments, the bead is non-toxic to cultured cells (e.g., cultured T cells). In some embodiments, the bead can be any particle capable of attaching agents in a manner that permits interaction between the agents and the cells.

[0276] In some embodiments, the stimulatory reagent contains one or more agents capable of activating and / or expanding T cells, e.g., a pan-T cell activating reagent. In some embodiments, the pan-T cell activating reagent comprises anti-CD3 / anti-CD28 beads. In some embodiments, the pan-T cell activating reagent comprises anti-CD3 / anti-CD28 streptavidin oligomerization reagents.

[0277] In some embodiments, the stimulatory reagent contains one or more agents capable of activating and / or expanding cells (e.g., T cells), which are bound to or otherwise attached to a bead, e.g., bound to or attached to the surface of a bead. In certain embodiments, the bead is a non-cellular particle. In particular embodiments, the bead can include a colloidal particle, a microsphere, a nanoparticle, a magnetic bead, etc. In some embodiments, the bead is a Sepharose bead. In certain embodiments, the bead is a Sepharose gel bead.

[0278] In certain embodiments, the stimulatory reagent contains monodisperse beads. In certain embodiments, the monodisperse beads comprise a size dispersion of less than 5% standard deviation in diameter from one another.

[0279] In some embodiments, the beads contain one or more agents, such as agents coupled, conjugated or linked (directly or indirectly) to the surface of the beads. In some embodiments, the agents, as contemplated herein, can include, but are not limited to, RNA, DNA, proteins (e.g., enzymes), antigens, polyclonal antibodies, monoclonal antibodies, antibody fragments, carbohydrates, lectins or any other biological molecule that has affinity for a desired target. In some embodiments, the desired target is a T cell receptor and / or a component of a T cell receptor. In certain embodiments, the desired target is CD3. In certain embodiments, the desired target is a T cell costimulatory molecule, such as CD28, CD137 (4-1-BB), OX40 or ICOS. The one or more agents can be attached to the beads directly or indirectly by various methods known and available in the art. The attachment can be covalent, non-covalent, electrostatic or hydrophobic, and can be achieved by various attachment means, including, for example, chemical means, mechanical means or enzymatic means. In some embodiments, a biological molecule (e.g., a biotinylated anti-CD3 antibody) can be attached to the beads indirectly via another biological molecule (e.g., an anti-biotin antibody) that is directly attached to the beads.

[0280] In some embodiments, the stimulatory reagent contains beads and one or more agents that directly interact with a macromolecule on the surface of a cell. In certain embodiments, the beads (e.g., paramagnetic beads) interact with a cell via one or more agents (e.g., antibodies) that are specific for one or more macromolecules (e.g., one or more cell surface proteins) on the cell. In certain embodiments, the beads (e.g., paramagnetic beads) are labeled with a first agent (e.g., a primary antibody (e.g., an anti-biotin antibody) or other biological molecule) as described herein, and then a second agent (e.g., a secondary antibody (e.g., a biotinylated anti-CD3 antibody) or other second biological molecule (e.g., streptavidin)) is added, whereby the secondary antibody or other second biological molecule specifically binds to such primary antibody or other biological molecule on the particle.

[0281] In some embodiments, the stimulatory reagent contains one or more agents (e.g., antibodies) attached to a bead (e.g., a paramagnetic bead) and specifically bind to one or more of the following macromolecules on a cell (e.g., a T cell): CD2, CD3, CD4, CD5, CD8, CD25, CD27, CD28, CD29, CD31, CD44, CD45RA, CD45RO, CD54 (ICAM-1), CD127, MHC I, MHC II, CTLA-4, ICOS, PD-1, OX40, CD27L (CD70), 4-1BB (CD137), 4-1BBL, CD30L, LIGHT, IL-2R, IL-12R, IL-1R, IL-15R; IFN-gamma R, TNF-alpha R, IL-4R, IL-10R, CD18 / CD1 la (LFA-1), CD62L (L-selectin), CD29 / CD49d (VLA-4), Notch ligands (e.g., Delta-like 1 / 4, Jagged 1 / 2, etc.), CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, and CXCR3, or fragments thereof, including the corresponding ligands for these macromolecules or fragments thereof. In some embodiments, the agents (e.g., antibodies) attached to the bead specifically bind to one or more of the following macromolecules on a cell (e.g., a T cell): CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA, and / or CD45RO. In some embodiments, the one or more agents attached to the bead are antibodies. The antibodies can include polyclonal antibodies, monoclonal antibodies (including full-length antibodies with an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab')2, and Fv). In some embodiments, the stimulatory reagent is an antibody fragment (including an antigen-binding fragment), such as a Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. It will be appreciated that constant regions of any isotype can be used in the antibodies contemplated herein, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be obtained from any human or animal species (e.g., a murine species).

[0282] In some embodiments, the agent is an antibody that binds and / or recognizes one or more components of the T cell receptor. In particular embodiments, the agent is an anti-CD3 antibody. In certain embodiments, the agent is an antibody that binds and / or recognizes a co-receptor. In some embodiments, the stimulatory agent comprises an anti-CD28 antibody. In particular embodiments, the stimulatory agent contains an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the antibody is a Fab. In some embodiments, the stimulatory agent contains an anti-CD3 Fab and an anti-CD28 Fab.

[0283] In some embodiments, the stimulatory agent is an anti-CD3 / anti-CD28 streptavidin oligomerization agent, such as described in PCT Publication No. WO / 2015 / 158868 or WO 2019 / 197949. In some embodiments, the streptavidin is recombinant Streptactin, which is a mutant streptavidin. In some embodiments, the Streptactin is a mutant streptavidin that contains the mutations Val-Thr-Ala-Arg or Ile-Gly-Ala-Arg at positions 44-47 of wild-type streptavidin, replacing the wild-type amino acids Glu-Ser-Ala-Val. In some embodiments, the stimulatory agent is soluble. In some embodiments, the agent is a soluble agent of anti-CD3 / CD28 Fab fragments linked to a recombinant Streptactin backbone. In some embodiments, the stimulatory agent is Expamer™.

[0284] In some embodiments, the stimulatory agent is an anti-CD3 / anti-CD28 bead (e.g., DYNABEADS® M-450 CD3 / CD28 T cell expander and / or ExpACT® beads).

[0285] In some embodiments, the bead has a diameter greater than about 0.001 pm, greater than about 0.01 pm, greater than about 0.1 pm, greater than about 1.0 pm, greater than about 10 pm, greater than about 50 pm, greater than about 100 pm, or greater than about 1000 pm and no more than about 1500 pm. In some embodiments, the bead has a diameter of about 1.0 pm to about 500 pm, about 1.0 pm to about 150 pm, about 1.0 pm to about 30 pm, about 1.0 pm to about 10 pm, about 1.0 pm to about 5.0 pm, about 2.0 pm to about 5.0 pm, or about 3.0 pm to about 5.0 pm. In some embodiments, the bead has a diameter of about 3 pm to about 5 pm. In some embodiments, the bead has a diameter of at least or at least about or about 0.001 pm, 0.01 pm, 0.1 pm, 0.5 pm, 1.0 pm, 1.5 pm, 2.0 pm, 2.5 pm, 3.0 pm, 3.5 pm, 4.0 pm, 4.5 pm, 5.0 pm, 5.5 pm, 6.0 pm, 6.5 pm, 7.0 pm, 7.5 pm, 8.0 pm, 8.5 pm, 9.0 pm, 9.5 pm, 10 pm, 12 pm, 14 pm, 16 pm, 18 pm, or 20 pm. In certain embodiments, the bead has a diameter of at or about 4.5 pm. In certain embodiments, the bead has a diameter of at or about 2.8 pm.

[0286] In some embodiments, the density of the bead is greater than 0.001 g / cm 3 , greater than 0.01 g / cm 3 , greater than 0.05 g / cm 3 , greater than 0.1 g / cm 3 , greater than 0.5 g / cm 3 , greater than 0.6 g / cm 3 , greater than 0.7 g / cm 3 , greater than 0.8 g / cm 3 , greater than 0.9 g / cm 3 , greater than 1 g / cm 3 , greater than 1.1 g / cm 3 , greater than 1.2 g / cm 3 , greater than 1.3 g / cm 3 , greater than 1.4 g / cm 3 , greater than 1.5 g / cm 3 , greater than 2 g / cm 3 , greater than 3 g / cm 3 , greater than 4 g / cm 3or greater than 5 g / cm 3 . In some embodiments, the density of the bead is between about 0.001 g / cm 3 and about 50 g / cm 3 . In some embodiments, the density of the bead is between about 0.01 g / cm 3 and about 50 g / cm 3 . In some embodiments, the density of the bead is between about 0.1 g / cm 3 and about 10 g / cm 3 . In some embodiments, the density of the bead is between about 0.1 g / cm 3 and about.5 g / cm 3 . In some embodiments, the density of the bead is between about 0.5 g / cm 3 and about 1 g / cm 3 . In some embodiments, the density of the bead is between about 0.5 g / cm 3 and about 1.5 g / cm 3 . In some embodiments, the density of the bead is between about 1 g / cm 3 and about 1.5 g / cm 3 . In some embodiments, the density of the bead is between about 1 g / cm 3 and about 2 g / cm 3 . In some embodiments, the density of the bead is about 1 g / cm 3 . In some embodiments, the density of the bead is between about 1 g / cm 3 and about 5 g / cm 3 . In some embodiments, the density of the bead is about 0.5 g / cm 3 , about 0.6 g / cm 3 , about 0.7 g / cm 3 , about 0.8 g / cm 3 , about 0.9 g / cm 3 , about 1.0 g / cm 3 , about 1.1 g / cm 3 , about 1.2 g / cm 3 , about 1.3 g / cm 3 , about 1.4 g / cm 3 , about 1.5 g / cm 3 , about 1.6 g / cm 3 , about 1.7 g / cm 3 , about 1.8 g / cm 3 , about 1.9 g / cm 3 , or about 2.0 g / cm 3 . In certain embodiments, the density of the bead is about 1.6 g / cm 3 . In particular embodiments, the density of the bead or particle is about 1.5 g / cm 3 . In certain embodiments, the density of the particle is about 1.3 g / cm 3 .

[0287] In certain embodiments, the plurality of beads have a uniform density. In certain embodiments, the uniform density comprises a standard deviation of density that is less than 10%, less than 5%, or less than 1% of the average bead density.

[0288] In some embodiments, the surface area of the beads is between about 0.001 m 2 / g of the particles (m 2 / g) to about 1,000 m 2 / g, about.010 m 2 / g to about 100 m 2 / g, about 0.1 m 2 / g to about 10 m 2 / g, about 0.1 m 2 / g to about 1 m 2 / g, about 1 m 2 / g to about 10 m 2 / g, about 10 m 2 / g to about 100 m 2 / g, about 0.5 m 2 / g to about 20 m 2 / g, about 0.5 m 2 / g to about 5 m 2 / g, or about 1 m 2 / g to about 4 m 2 / g. In some embodiments, the surface area of the particles or beads is about 1 m 2 / g to about 4 m 2 / g.

[0289] In some embodiments, the beads react in a magnetic field. In some embodiments, the beads are magnetic beads. In some embodiments, the magnetic beads are paramagnetic. In particular embodiments, the magnetic beads are superparamagnetic. In certain embodiments, the beads do not exhibit any magnetic properties unless they are exposed to a magnetic field.

[0290] In particular embodiments, the bead comprises a magnetic, paramagnetic, or superparamagnetic core. In some embodiments, the magnetic core contains a metal. In some embodiments, the metal can be, but is not limited to, iron, nickel, copper, cobalt, gadolinium, manganese, tantalum, zinc, zirconium, or any combination thereof. In certain embodiments, the magnetic core comprises a metal oxide (e.g., iron oxide), a ferrite (e.g., a manganese ferrite, a cobalt ferrite, a nickel ferrite, etc.), hematite, and a metal alloy (e.g., CoTaZn). In some embodiments, the magnetic core comprises one or more of a ferrite, a metal, a metal alloy, iron oxide, or chromium dioxide. In some embodiments, the magnetic core comprises elemental iron or a compound thereof. In some embodiments, the magnetic core comprises one or more of magnetite (Fe304), maghemite (yFe203), or greigite (Fe3S4). In some embodiments, the inner core comprises iron oxide (e.g., Fe304).

[0291] In certain embodiments, the bead contains a magnetic, paramagnetic, and / or superparamagnetic core that is covered by a surface-functionalized coat or coating. In some embodiments, the coat can contain a material that can include, but is not limited to, a polymer, a polysaccharide, silica, a fatty acid, a protein, carbon, agarose, Sepharose, or a combination thereof. In some embodiments, the polymer can be polyethylene glycol, poly(lactic-co-glycolic acid), polyglutaraldehyde, polyurethane, polystyrene, or polyvinyl alcohol. In certain embodiments, the outer coat or coating comprises polystyrene. In particular embodiments, the outer coat or coating is surface-functionalized.

[0292] In some embodiments, the stimulatory reagent comprises a bead that contains a metal oxide core (e.g., an iron oxide core) and a coat, wherein the metal oxide core comprises at least one polysaccharide (e.g., dextran), and wherein the coat comprises at least one polysaccharide (e.g., aminodextran), at least one polymer (e.g., polyurethane), and silica. In some embodiments, the metal oxide core is a colloidal iron oxide core. In certain embodiments, the one or more agents comprise an antibody or antigen-binding fragment thereof. In particular embodiments, the one or more agents comprise an anti-CD3 antibody and an anti-CD28 antibody or antigen-binding fragment thereof. In some embodiments, the stimulatory reagent comprises an anti-CD3 antibody, an anti-CD28 antibody, and an anti-biotin antibody. In some embodiments, the stimulatory reagent comprises an anti-biotin antibody. In some embodiments, the bead has a diameter of about 3 μιη to about 10 μιη. In some embodiments, the bead has a diameter of about 3 μιη to about 5 μιη. In certain embodiments, the bead has a diameter of about 3.5 μιη.

[0293] In some embodiments, the stimulatory reagent comprises one or more agents attached to a bead comprising a metal oxide core (e.g., an iron oxide core) and a coating (e.g., a protective coating), wherein the coating comprises polystyrene. In certain embodiments, the bead is a monodisperse paramagnetic (e.g., superparamagnetic) bead comprising a paramagnetic (e.g., superparamagnetic) iron core (e.g., a core comprising magnetite (Fe3C>4) and / or maghemite (yFe2C>3)) and a polystyrene coat or coating. In some embodiments, the bead is non-porous. In some embodiments, the bead contains a functionalized surface to which the one or more agents are attached. In certain embodiments, the one or more agents are covalently bound to the bead on the surface. In some embodiments, the one or more agents comprise an antibody or antigen-binding fragment thereof. In some embodiments, the one or more agents comprise an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the stimulatory reagent is or comprises an anti-CD3 / anti-CD28 magnetic bead. In some embodiments, the one or more agents comprise an anti-CD3 antibody and / or an anti-CD28 antibody, and an antibody or antigen fragment thereof capable of binding to a labeled antibody (e.g., a biotinylated antibody) such as a labeled anti-CD3 or anti-CD28 antibody. In certain embodiments, the bead has a density of about 1.5 g / cm3and a surface area of about 1 m2 / g to about 4 m2 / g. In particular embodiments; the bead is a monodisperse superparamagnetic bead having a diameter of about 4.5 pm and a density of about 1.5 g / cm3. In some embodiments, the bead is a monodisperse superparamagnetic bead having an average diameter of about 2.8 pm and a density of about 1.3 g / cm3. 3 2 2 In particular embodiments; the bead is a monodisperse superparamagnetic bead having a diameter of about 4.5 pm and a density of about 1.5 g / cm3. In some embodiments, the bead is a monodisperse superparamagnetic bead having an average diameter of about 2.8 pm and a density of about 1.3 g / cm3. 3 3

[0294] In some embodiments, the ratio of bead to cell is about 3 : 1, 2.5 : 1, 2 : 1, 1.5 : 1, 1.25 : 1, 1.2 : 1, 1.1 : 1, 1 : 1, 0.9 : 1, 0.8 : 1, 0.75 : 1, 0.67 : 1, 0.5 : 1, 0.3 : 1, or 0.2 : 1. In particular embodiments, the ratio of bead to cell is between 2.5 : 1 and 0.2 : 1, between 2 : 1 and 0.5 : 1, between 1.5 : 1 and 0.75 : 1, between 1.25 : 1 and 0.8 : 1, between 1.1 : 1 and 0.9 : 1. In particular embodiments, the ratio of stimulatory reagent to cell is about 1 : 1 or 1 : 1. C. Engineered cells ​​​​

[0295] In some embodiments, the provided methods involve engineering cells with a recombinant antigen receptor. Various methods for introducing genetically engineered components, such as recombinant receptors (e.g., CARs or TCRs), are well known and can be used with the provided methods and compositions. Exemplary methods include those for transferring nucleic acids encoding receptors, including by virus (such as retrovirus or lentivirus), transduction, transposon, and electroporation.

[0296] Cells expressing receptors and administered by the provided methods include engineered cells. Genetic engineering generally involves introducing a nucleic acid encoding a recombinant or engineered component into a composition containing cells, such as by retroviral transduction, transfection, or transformation.

[0297] In some embodiments, the methods provided herein are used in conjunction with one or more compositions of engineered enriched T cells. In certain embodiments, the engineering is or includes the introduction of a polynucleotide, such as a recombinant polynucleotide encoding a recombinant protein. In particular embodiments, the recombinant protein is a recombinant receptor, such as any of the receptors described in Section II. Introduction of nucleic acid molecules encoding recombinant proteins, such as recombinant receptors, into cells can be performed using any of a number of known vectors. Such vectors include viral and non-viral systems, including lentivirus and gamma retroviral systems, as well as transposon-based systems, such as PiggyBac or Sleeping Beauty-based gene transfer systems. Exemplary methods include those for transferring nucleic acids encoding receptors, including by virus (such as retrovirus or lentivirus), transduction, transposon, and electroporation. In some embodiments, the engineering results in one or more engineered compositions of enriched T cells.

[0298] In certain embodiments, one or more compositions of enriched T cells are engineered, such as transduced or transfected, prior to incubating the cells under conditions that promote proliferation and / or expansion, for example as provided by the methods of Section III-D. In particular embodiments, one or more compositions of enriched T cells are engineered after the one or more compositions have been stimulated, activated, and / or incubated under stimulatory conditions, as described in the methods provided by Section III-B. In particular embodiments, the one or more compositions are stimulated compositions. In particular embodiments, the one or more stimulated compositions have been previously cryopreserved and stored, and thawed prior to engineering.

[0299] In certain embodiments, the one or more compositions of stimulated T cells are or comprise two separate stimulated compositions of enriched T cells. In particular embodiments, the two separate compositions of enriched T cells, e.g., two separate compositions of enriched T cells that have been selected, isolated, and / or enriched from the same biological sample, are separately engineered. In certain embodiments, the two separate compositions comprise a composition of enriched CD4+ T cells. In particular embodiments, the two separate compositions comprise a composition of enriched CD8+ T cells. In some embodiments, the two separate compositions of enriched CD4+ T cells and enriched CD8+ T cells are separately genetically engineered following incubation under stimulation conditions as described above. In some embodiments, a single composition of enriched T cells is genetically engineered. In certain embodiments, the single composition is a composition of enriched CD4+ T cells. In some embodiments, the single composition is a composition of enriched CD4+ and CD8+ T cells that has been combined from separate compositions prior to engineering.

[0300] In some embodiments, a composition of engineered, e.g., transduced or transfected, enriched CD4+ T cells, such as stimulated CD4+ T cells, comprises 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%, at least 99.5%, at least 99.9%, or is or is about 100% CD4+ T cells. In certain embodiments, a composition of engineered enriched CD4+ T cells, such as stimulated CD4+ T cells, comprises less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and / or is free of CD8+ T cells, and / or is free or substantially free of CD8+ T cells.

[0301] In some embodiments, the composition of enriched CD8+ T cells (such as stimulated CD8+ T cells) that are engineered, e.g., transduced or transfected, comprises 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%, at least 99.5%, at least 99.9%, or is or is about 100% CD8+ T cells. In certain embodiments, the composition of engineered enriched CD8+ T cells (such as stimulated CD8+ T cells) comprises less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and / or is free of CD4+ T cells, and / or is free or substantially free of CD4+ T cells.

[0302] In some embodiments, separate compositions of enriched CD4+ and CD8+ T cells are combined into a single composition and genetically engineered, e.g., transduced or transfected. In certain embodiments, separate engineered compositions of enriched CD4+ and enriched CD8+ T cells are combined into a single composition after the genetic engineering has been performed and / or completed. In particular embodiments, separate compositions of enriched CD4+ and enriched CD8+ T cells (such as separate compositions of stimulated CD4+ and CD8+ T cells) are engineered separately, and treated separately after the genetic engineering has been performed and / or completed for the incubation and / or expansion of the T cells.

[0303] In some embodiments, introduction of a polynucleotide, e.g., a recombinant polynucleotide encoding a recombinant protein, is performed by contacting an enriched CD4+ or CD8+ T cell, e.g., a stimulated CD4+ or CD8+ T cell, with a viral particle containing the polynucleotide. In some embodiments, the contacting can be achieved by centrifugation, such as spinoculation (e.g., spin inoculation). In some embodiments, a composition containing the cells, viral particles, and reagents can be spun, typically at a relatively low force or speed, such as a speed that is lower than that used to pellet the cells, such as from 600 rpm to 1700 rpm or from about 600 rpm to about 1700 rpm (e.g., at or at about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm). In some embodiments, the spinning is performed at a force (e.g., relative centrifugal force) of from 100 g to 3200 g or from about 100 g to about 3200 g (e.g., at or at about or at least or at least about 100 g, 200 g, 300 g, 400 g, 500 g, 1000 g, 1500 g, 2000 g, 2500 g, 3000 g, or 3200 g), such as at or at about 693 g, as measured, for example, at the inner or outer walls of a chamber or cavity. The term “relative centrifugal force” or RCF is generally understood to be the effective force exerted on an object or mass (e.g., a cell, sample, or pellet and / or a point in the chamber or other container being spun) at a particular point in space, as compared to the axis of rotation, relative to the force of gravity of the Earth. The value can be determined using well-known formulas that take into account the force of gravity, the speed of rotation, and the radius of rotation (distance from the axis of rotation and the object, mass, or particle for which the RCF is being measured). In some embodiments, at least a portion of the contacting, incubation, and / or engineering of the cells (e.g., cells from a stimulated composition of enriched CD4+ T cells or enriched CD8+ T cells) with the virus is performed at a spin of between about 100 g and 3200 g, 1000 g and 2000 g, 1000 g and 3200 g, 500 g and 1000 g, 400 g and 1200 g, 600 g and 800 g, 600 g and 700 g, or 500 g and 700 g. In some embodiments, the spin is performed at between 600 g and 700 g, e.g., at or at about 693 g.

[0304] In certain embodiments, at least a portion of the engineering, transduction, and / or transfection is performed under rotation, e.g., spinoculation and / or centrifugation. In some embodiments, the rotation is performed for, or for about, or for at least or at least about 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or for at least 7 days. In some embodiments, the rotation is performed for, or for about, 60 minutes. In certain embodiments, the rotation is performed for about 30 minutes. In some embodiments, the rotation is performed at between 600 g to 700 g, e.g., at or at about 693 g, for about 30 minutes.

[0305] In certain embodiments, the number of viable cells to be engineered, transduced, and / or transfected ranges from about 5 x 10 6 cells to about 100 x 10 7 cells, such as from about 10 x 10 6 cells to about 100 x 10 6 cells, from about 100 x 10 6 cells to about 200 x 10 6 cells, from about 200 x 10 6 cells to about 300 x 10 6 cells, from about 300 x 10 6 cells to about 400 x 10 6 cells, from about 400 x 10 6 cells to about 500 x 10 6 cells, or from about 500 x 10 6 cells to about 100 x 10 7 cells. In particular examples, the number of viable cells to be engineered, transduced, and / or transfected is about or less than about 300 x 10 6 cells.

[0306] In certain embodiments, at least a portion of the engineering, transduction, and / or transfection is performed under a volume (e.g., spinoculation volume) ranging from about 5 mL to about 100 mL, such as from about 10 mL to about 50 mL, from about 15 mL to about 45 mL, from about 20 mL to about 40 mL, from about 25 mL to about 35 mL, or at or at about 30 mL. In certain embodiments, the volume of the cell pellet after spinoculation ranges from about 1 mL to about 25 mL, such as from about 5 mL to about 20 mL, from about 5 mL to about 15 mL, from about 5 mL to about 10 mL, or at or at about 10 mL.

[0307] In some embodiments, gene transfer is accomplished by first stimulating the cells, such as by combining them with a stimulus that induces a response (such as proliferation, survival, and / or activation), e.g., as measured by expression of cytokines or activation markers, then transducing the activated cells, and expanding in culture to a number sufficient for clinical use. In certain embodiments, gene transfer is accomplished by first incubating the cells under stimulating conditions, such as by any of the methods described in Section I-B.

[0308] In some embodiments, methods for genetic engineering are performed by contacting one or more cells of a composition with a nucleic acid molecule encoding a recombinant protein (e.g., a recombinant receptor). In some embodiments, the contacting can be achieved by centrifugation, such as spinoculation (e.g., centrifugal inoculation). Such methods include any of those described in International Publication No. WO 2016 / 073602. Exemplary centrifugal chambers include those produced and sold by Biosafe SA, including those for the Sepax® and Sepax® 2 systems, including A-200 / F and A-200 centrifugal chambers and various kits for such systems. Exemplary chambers, systems, and processing instruments and cabinets are described, e.g., in U.S. Patent No. 6,123,655, U.S. Patent No. 6,733,433, and published U.S. Patent Application Publication No. US 2008 / 0171951, and published International Patent Application Publication No. WO 00 / 38762, the contents of each of which are incorporated herein by reference in their entirety. Exemplary kits for such systems include, but are not limited to, disposable kits sold by BioSafe SA under the product names CS-430.1, CS-490.1, CS-600.1, or CS-900.2.

[0309] In some embodiments, the system is included with and / or associated with other instruments, including instruments for operating, automating, controlling, and / or monitoring aspects of the transduction step as well as one or more various other processing steps performed in the system (e.g., one or more processing steps that can be performed using or in conjunction with a centrifugal chamber system as described herein or in International Publication No. WO 2016 / 073602). In some embodiments, such instruments are housed in a cabinet. In some embodiments, the instruments include a cabinet comprising a housing containing control circuitry, a centrifuge, a hood, a motor, a pump, a sensor, a display, and a user interface. Exemplary apparatuses are described in U.S. Patent No. 6,123,655, U.S. Patent No. 6,733,433, and US 2008 / 0171951.

[0310] In some embodiments, the system comprises a series of containers, e.g., bags, tubing, stopcocks, clamps, connectors, and centrifugation chambers. In some embodiments, the containers, e.g., bags, include one or more containers, e.g., bags, that contain both cells to be transduced and viral vector particles in the same container or separate containers, e.g., the same bag or separate bags. In some embodiments, the system further includes one or more containers, e.g., bags, that contain media, e.g., diluent and / or wash solution, that is drawn into chambers and / or other components during the method to dilute, resuspend, and / or wash components and / or compositions. The containers can be connected at one or more locations in the system, e.g., at locations corresponding to input lines, diluent lines, wash lines, waste lines, and / or output lines.

[0311] In some embodiments, the chamber is associated with a centrifuge that is capable of effecting rotation of the chamber, e.g., rotation about its axis of rotation. Rotation can occur prior to, during, and / or after incubation in connection with transduction of cells and / or in one or more other processing steps. Thus, in some embodiments, one or more of the various processing steps are performed under rotation, e.g., under a particular force. The chamber is typically capable of being rotated upright or substantially upright, such that the chamber is placed upright during centrifugation, and the sidewall and axis are upright or substantially upright, and one or more end walls are horizontal or substantially horizontal.

[0312] In some embodiments, the composition containing cells and the composition containing viral vector particles, and optionally air, can be combined or mixed prior to providing the compositions to the cavity. In some embodiments, the composition containing cells and the composition containing viral vector particles, and optionally air, are provided separately in the cavity and combined and mixed therein. In some embodiments, the composition containing cells, the composition containing viral vector particles, and optionally air, can be provided to the interior cavity in any order. In any of such some embodiments, the composition containing cells and viral vector particles is an input composition that has been combined or mixed together, whether the input composition is combined and / or mixed inside or outside the centrifugation chamber, and / or whether the cells and viral vector particles are provided to the centrifugation chamber together or separately, e.g., simultaneously or sequentially.

[0313] In some embodiments, in the transduction method, intake of a volume of gas, e.g., air, occurs prior to incubation of the cells and viral vector particles, e.g., rotation. In some embodiments, in the transduction method, intake of a volume of gas, e.g., air, occurs during incubation, e.g., rotation, of the cells and viral vector particles.

[0314] In some embodiments, the volume of liquid, and optionally air, that constitutes the cell or viral vector particle of the transduction composition can be a predetermined volume. The volume can be a volume that is programmed into the system and / or controlled by circuitry associated with the system.

[0315] In some embodiments, the intake of the transduction composition and optionally gas, such as air, is controlled manually, semi-automatically, and / or automatically until a desired or predetermined volume has been taken into the internal cavity of the chamber. In some embodiments, sensors associated with the system can detect the liquid and / or gas flowing into and out of the chamber, for example, via its color, flow rate, and / or density, and can communicate with associated circuitry to stop or continue intake as needed until such a desired or predetermined volume of intake has been achieved. In some aspects, a sensor that is programmed or simply capable of detecting liquid but not gas, such as air, in the system can be enabled to allow gas, such as air, to pass into the system without stopping intake. In some such embodiments, a piece of opaque tubing can be placed in the line near the sensor when gas, such as air, intake is needed. In some embodiments, the intake of gas, such as air, can be controlled manually.

[0316] In aspects of the provided methods, the internal cavity of the chamber is subjected to high speed rotation. In some embodiments, the rotation is achieved prior to, concurrently with, after, or intermittently with the intake of the liquid input composition and optionally air. In some embodiments, the rotation is achieved after the intake of the liquid input composition and optionally air. In some embodiments, the rotation is by centrifugation of the chamber at a relative centrifugal force at or about or at least or at least about 800 g, 1000 g, 1100 g, 1500, 1600 g, 1800 g, 2000 g, 2200 g, 2500 g, 3000 g, 3500 g, or 4000 g at the inner surface of the sidewall of the internal cavity and / or at the surface layer of the cells. In some embodiments, the rotation is by centrifugation at a force greater than or about 1100 g, for example, by a force greater than or about 1200 g, greater than or about 1400 g, greater than or about 1600 g, greater than or about 1800 g, greater than or about 2000 g, greater than or about 2400 g, greater than or about 2800 g, greater than or about 3000 g, or greater than or about 3200 g. In some embodiments, the rotation is by centrifugation at a force at or about 1600 g.

[0317] In some embodiments, the transduction method includes rotating or centrifuging the transduction composition and optionally air in the centrifugation chamber for greater than or about 5 minutes, such as greater than or about 10 minutes, greater than or about 15 minutes, greater than or about 20 minutes, greater than or about 30 minutes, greater than or about 45 minutes, greater than or about 60 minutes, greater than or about 90 minutes, or greater than or about 120 minutes. In some embodiments, the transduction composition and optionally air are rotated or centrifuged in the centrifugation chamber for greater than 5 minutes but not more than 60 minutes, not more than 45 minutes, not more than 30 minutes, or not more than 15 minutes. In particular embodiments, the transduction includes rotating or centrifuging for or for about 60 minutes.

[0318] In some embodiments, the transduction method includes rotating or centrifuging the transduction composition and optional air in the centrifugation chamber for a time that is at or between about 10 minutes and 60 minutes, 15 minutes and 60 minutes, 15 minutes and 45 minutes, 30 minutes and 60 minutes, or 45 minutes and 60 minutes, each inclusive, and the rotating or centrifuging is performed at a force at the inner cavity side wall inner surface and / or at the cell layer of at least or greater than or about 1000 g, 1100 g, 1200 g, 1400 g, 1500 g, 1600 g, 1800 g, 2000 g, 2200 g, 2400 g, 2800 g, 3200 g, or 3600 g. In particular embodiments, the transduction method includes rotating or centrifuging the transduction composition (e.g., cells and viral vector particles) at or about 1600 g for or about 60 minutes.

[0319] In some embodiments, the gas (such as air) in the cavity of the chamber is evacuated from the chamber. In some embodiments, the gas (such as air) is evacuated to a container that is operatively connected to the centrifugation chamber as part of a closed system. In some embodiments, the container is a free or empty container. In some embodiments, the air (such as gas) in the cavity of the chamber is evacuated through a filter that is operatively connected to the inner cavity of the chamber via a sterile tubing set. In some embodiments, the air is evacuated using a manual, semi-automated, or automated process. In some embodiments, the air is evacuated from the cavity prior to, concurrently with, intermittently, or subsequent to expressing an output composition containing the incubated cells and viral vector particles (e.g., cells that have begun transduction or cells that have been transduced with a viral vector) from the cavity of the chamber.

[0320] In some embodiments, transduction and / or other incubation is performed as a continuous or semi-continuous process or as part of such a continuous or semi-continuous process. In some embodiments, a continuous process involves continuous intake of cells and viral vector particles, e.g., a transduction composition (as a single pre-existing composition, or by continuous pipetting into the same vessel (e.g., cavity), thereby mixing portions thereof), and / or continuous delivery or removal of liquid from the vessel, and optionally removal of gas (e.g., air), during at least a portion of the incubation (e.g., while centrifuging). In some embodiments, continuous intake and continuous delivery are performed at least partially simultaneously. In some embodiments, continuous intake occurs during a portion of the incubation, e.g., during a portion of centrifugation, and continuous delivery occurs during a separate portion of the incubation. Both can be performed alternately. Thus, continuous intake and delivery can allow for processing (e.g., transduction) of a larger total volume of sample while performing the incubation.

[0321] In some embodiments, the incubation is part of a continuous process, the method comprising, during at least a portion of the incubation, effecting continuous intake of the transduction composition into the cavity during rotation of the chamber and continuous delivery of liquid from the cavity through the at least one opening and optionally removal of gas (e.g., air) from the cavity during rotation of the chamber.

[0322] In some embodiments, semi-continuous incubation is performed by alternating between effecting intake of a composition into a cavity, incubation, delivery of liquid from the cavity and optionally removal of gas (e.g., air) from the cavity, e.g., into an output container, then intake of a subsequent (e.g., second, third, etc.) composition containing more cells and other reagents (e.g., viral vector particles) for processing, and repeating the process. For example, in some embodiments, the incubation is part of a semi-continuous process, the method comprising, prior to the incubation, effecting intake of a transduction composition into the cavity through the at least one opening, and, after the incubation, effecting delivery of fluid from the cavity; effecting intake of another transduction composition comprising cells and viral vector particles into the interior cavity; and incubating the other transduction composition in the interior cavity under conditions whereby cells in the other transduction composition are transduced by the vector. The process can continue in an iterative manner for many additional rounds. In this regard, semi-continuous or continuous methods can allow for production of even larger volumes and / or numbers of cells.

[0323] In some embodiments, a portion of the transduction incubation is performed in a centrifugation chamber, which is performed under conditions including rotation or centrifugation.

[0324] In some embodiments, the method comprises an incubation, wherein another portion of the incubation of the cells and viral vector particles is performed without rotation or centrifugation, which is generally performed after the at least one portion of the incubation that includes rotation or centrifugation of the chamber. In certain embodiments, the incubation of the cells and viral vector particles is performed without rotation or centrifugation for at least 1 hour, 6 hours, 12 hours, 24 hours, 32 hours, 48 hours, 60 hours, 72 hours, 90 hours, 96 hours, 3 days, 4 days, 5 days, or more than 5 days. In certain embodiments, the incubation is for 72 hours or about 72 hours.

[0325] In some such embodiments, the further incubation is effected under conditions to allow integration of the viral vector into the host genome of the one or more cells. It is within the level of skill of the artisan to empirically determine whether the incubation has resulted in integration of the viral vector particles into the host genome, and thus the conditions for further incubation. In some embodiments, integration of the viral vector into the host genome can be assessed or determined by measuring the level of expression of a recombinant protein, such as a heterologous protein, encoded by the nucleic acid contained in the viral vector particle genome following incubation. A variety of well-known methods can be used to assess the level of expression of a recombinant molecule, for example in the case of a cell surface protein, for example by detection by an affinity-based method, such as an immunoaffinity-based method, for example by flow cytometry. In some examples, expression is measured by detection of a transduction marker and / or reporter construct. In some embodiments, a nucleic acid encoding a truncated surface protein is included within the vector and used as a marker for its expression and / or enhancement.

[0326] In some embodiments, the composition containing cells, vectors (e.g., viral particles), and reagents can be spun, typically at a relatively low force or speed, such as a speed lower than that used to pellet cells, such as from 600 rpm to 1700 rpm or from about 600 rpm to about 1700 rpm (e.g., at or at about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm). In some embodiments, the spinning is at a force (e.g., relative centrifugal force), such as measured at the inner or outer walls of the chamber or cavity, of from 100 g to 3200 g or from about 100 g to about 3200 g (e.g., at or at about or at least or at least about 100 g, 200 g, 300 g, 400 g, 500 g, 1000 g, 1500 g, 2000 g, 2500 g, 3000 g, or 3200 g). The term “relative centrifugal force” or RCF is generally understood to be the effective force exerted on an object or substance (e.g., cells, sample or pellets and / or a point in the chamber or other container being spun) at a particular point in space, such as compared to the axis of rotation, relative to the force of gravity of the Earth. The value can be determined using well-known formulas that take into account the force of gravity, the speed of rotation, and the radius of rotation (distance from the axis of rotation and the object, substance, or particle for which the RCF is being measured).

[0327] In some embodiments, during at least a portion of the genetic engineering (e.g., transduction), and / or after the genetic engineering, the cells are transferred to a bioreactor bag assembly for culturing the genetically engineered cells, e.g., for incubating or expanding the cells, as described above.

[0328] In certain embodiments, the composition of enriched T cells is engineered in the presence of a transduction adjuvant, e.g., transduction or transfection. In some embodiments, the composition of enriched T cells is engineered in the presence of one or more polycations. In some embodiments, the composition of enriched T cells is transduced in the presence of one or more transduction adjuvants, e.g., incubation of the composition of enriched T cells with viral vector particles. In particular embodiments, the composition of enriched T cells is transfected in the presence of one or more transduction adjuvants, e.g., incubation of the composition of enriched T cells with non-viral vectors. In certain embodiments, the presence of one or more transduction adjuvants increases the efficiency of gene delivery as evidenced by an increase in the amount, fraction, and / or percentage of cells in the composition that are engineered (e.g., transduced or transfected). In certain embodiments, the presence of one or more transduction adjuvants increases the efficiency of transfection. In certain embodiments, the presence of one or more transduction adjuvants increases the efficiency of transduction. In particular embodiments, at least 25%, 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 at least 99% of the cells engineered in the presence of a polycation contain or express a recombinant polynucleotide. In some embodiments, 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 150%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, or at least 100-fold more cells in the composition are engineered to contain or express a recombinant transduction adjuvant in the presence of a polycation as compared to an alternative and / or exemplary method of engineering cells in the absence of a transduction adjuvant.

[0329] In some embodiments, the composition of enriched cells is engineered in the presence of less than 100 pg / ml, less than 90 pg / ml, less than 80 pg / ml, less than 75 pg / ml, less than 70 pg / ml, less than 60 pg / ml, less than 50 pg / ml, less than 40 pg / ml, less than 30 pg / ml, less than 25 pg / ml, less than 20 pg / ml, or less than pg / ml, less than 10 pg / ml of a transduction adjuvant. In certain embodiments, transduction adjuvants suitable for use in the provided methods include, but are not limited to, polycations, fibronectin or fragments or variants derived from fibronectin, RetroNectin, and combinations thereof.

[0330] In some embodiments, the cells are engineered in the presence of a cytokine, e.g., a recombinant human cytokine, at a concentration of between 1 IU / ml and 1,000 IU / ml, between 10 IU / ml and 50 IU / ml, between 50 IU / ml and 100 IU / ml, between 100 IU / ml and 200 IU / ml, between 100 IU / ml and 500 IU / ml, between 250 IU / ml and 500 IU / ml, or between 500 IU / ml and 1,000 IU / ml.

[0331] In some embodiments, the composition of enriched T cells is engineered in the presence of IL-2, e.g., human recombinant IL-2, at a concentration of between 1 IU / ml and 200 IU / ml, between 10 IU / ml and 100 IU / ml, between 50 IU / ml and 150 IU / ml, between 80 IU / ml and 120 IU / ml, between 60 IU / ml and 90 IU / ml, or between 70 IU / ml and 90 IU / ml. In particular embodiments, the composition of enriched T cells is engineered in the presence of recombinant IL-2 at a concentration of, or of about, 50 IU / ml, 55 IU / ml, 60 IU / ml, 65 IU / ml, 70 IU / ml, 75 IU / ml, 80 IU / ml, 85 IU / ml, 90 IU / ml, 95 IU / ml, 100 IU / ml, 110 IU / ml, 120 IU / ml, 130 IU / ml, 140 IU / ml, or 150 IU / ml. In some embodiments, the composition of enriched T cells is engineered in the presence of, or of about, 85 IU / ml. In some embodiments, the population of T cells is a population of CD4+ T cells. In particular embodiments, the composition of enriched T cells is enriched for CD4+ T cells, wherein CD8+ T cells are not enriched and / or wherein CD8+ T cells are negatively selected or depleted from the composition. In particular embodiments, the composition of enriched T cells is a composition of enriched CD8+ T cells. In particular embodiments, the composition of enriched T cells is enriched for CD8+ T cells, wherein CD4+ T cells are not enriched and / or wherein CD4+ T cells are negatively selected or depleted from the composition.

[0332] In some embodiments, the composition of enriched T cells is engineered in the presence of recombinant IL-7, e.g., human recombinant IL-7, at a concentration of between 100 IU / ml and 2,000 IU / ml, between 500 IU / ml and 1,000 IU / ml, between 100 IU / ml and 500 IU / ml, between 500 IU / ml and 750 IU / ml, between 750 IU / ml and 1,000 IU / ml, or between 550 IU / ml and 650 IU / ml. In particular embodiments, the composition of enriched T cells is engineered in the presence of IL-7 at a concentration of, or of about, 50 IU / ml, 100 IU / ml, 150 IU / ml, 200 IU / ml, 250 IU / ml, 300 IU / ml, 350 IU / ml, 400 IU / ml, 450 IU / ml, 500 IU / ml, 550 IU / ml, 600 IU / ml, 650 IU / ml, 700 IU / ml, 750 IU / ml, 800 IU / ml, 750 IU / ml, 750 IU / ml, 750 IU / ml, or 1,000 IU / ml. In particular embodiments, the composition of enriched T cells is engineered in the presence of IL-7 at, or at about, 600 IU / ml. In some embodiments, the composition engineered in the presence of recombinant IL-7 is enriched for a population of T cells, e.g., CD4+ T cells. In particular embodiments, the composition of enriched T cells is enriched for CD4+ T cells, wherein CD8+ T cells are not enriched and / or wherein CD8+ T cells are negatively selected or depleted from the composition.

[0333] In some embodiments, the composition of enriched T cells is engineered in the presence of recombinant IL-15, e.g., human recombinant IL-15, at a concentration of between 0.1 IU / ml and 100 IU / ml, between 1 IU / ml and 50 IU / ml, between 5 IU / ml and 25 IU / ml, between 25 IU / ml and 50 IU / ml, between 5 IU / ml and 15 IU / ml, or between 10 IU / ml and 100 IU / ml. In particular embodiments, the composition of enriched T cells is engineered in the presence of IL-15 at a concentration of, or of about, 1 IU / ml, 2 IU / ml, 3 IU / ml, 4 IU / ml, 5 IU / ml, 6 IU / ml, 7 IU / ml, 8 IU / ml, 9 IU / ml, 10 IU / ml, 11 IU / ml, 12 IU / ml, 13 IU / ml, 14 IU / ml, 15 IU / ml, 20 IU / ml, 25 IU / ml, 30 IU / ml, 40 IU / ml, or 50 IU / ml. In some embodiments, the composition of enriched T cells is engineered in, or in about, 10 IU / ml IL-15. In some embodiments, the composition of enriched T cells is incubated in, or in about, 10 IU / ml recombinant IL-15. In some embodiments, the composition engineered in the presence of recombinant IL-15 is enriched for a population of T cells, e.g., CD4+ T cells and / or CD8+ T cells. In some embodiments, the composition of enriched T cells is a composition of enriched CD8+ T cells. In particular embodiments, the composition of enriched T cells is enriched for CD8+ T cells, wherein CD4+ T cells are not enriched and / or wherein CD4+ T cells are negatively selected or depleted from the composition. In some embodiments, the composition of enriched T cells is a composition of enriched CD4+ T cells. In particular embodiments, the composition of enriched T cells is enriched for CD4+ T cells, wherein CD8+ T cells are not enriched and / or wherein CD8+ T cells are negatively selected or depleted from the composition.

[0334] In particular embodiments, the composition of enriched CD8+ T cells is engineered in the presence of IL-2 and / or IL-15. In certain embodiments, the composition of enriched CD4+ T cells is engineered in the presence of IL-2, IL-7, and / or IL-15. In some embodiments, the IL-2, IL-7, and / or IL-15 is recombinant. In certain embodiments, the IL-2, IL-7, and / or IL-15 is human. In particular embodiments, the one or more cytokines is or includes human recombinant IL-2, IL-7, and / or IL-15.

[0335] In particular embodiments, the cells are engineered in the presence of one or more antioxidants. In some embodiments, the antioxidant includes, but is not limited to, one or more antioxidants including tocopherol, tocotrienol, a-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, a-tocotrienol, β-tocotrienol, a-tocopherolquinone, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), flavonoids, isoflavones, lycopene, β-carotene, selenium, ubiquinone, methionine, S-adenosylmethionine, glutathione, taurine, N-acetylcysteine (NAC), citric acid, L-carnitine, BHT, thioglycerol, ascorbic acid, propyl gallate, methionine, cysteine, homocysteine, glutathione, cystamine, and cysteine and / or glycine-glycine-histidine.

[0336] In some embodiments, the one or more antioxidants is or includes a sulfur-containing antioxidant. In certain embodiments, the sulfur-containing antioxidant can include a sulfur-containing thiol-containing antioxidant and / or an antioxidant that exhibits one or more sulfur moieties, e.g., within a ring structure. In some embodiments, the sulfur-containing antioxidant can include, e.g., N-acetylcysteine (NAC) and 2,3-dimercapto propanol (DMP), L-2-oxo-4-thiazolidine carboxylate (OTC), and lipoic acid. In particular embodiments, the sulfur-containing antioxidant is a glutathione precursor. In some embodiments, the glutathione precursor is a molecule that can be modified in one or more steps within a cell to a derivative of glutathione. In particular embodiments, the glutathione precursor can include, but is not limited to, N-acetylcysteine (NAC), L-2-oxothiazolidine-4-carboxylic acid (Procysteine), lipoic acid, S-allyl cysteine, or methyl methionine sulfonium chloride.

[0337] In some embodiments, the cells are engineered in the presence of one or more antioxidants. In some embodiments, the cells are engineered in the presence of between 1 ng / ml and 100 ng / ml, between 10 ng / ml and 1 pg / ml, between 100 ng / ml and 10 pg / ml, between 1 pg / ml and 100 pg / ml, between 10 pg / ml and 1 mg / ml, between 100 pg / ml and 1 mg / ml, between 500 pg / ml and 2 mg / ml, between 500 pg / ml and 5 mg / ml, between 1 mg / ml and 10 mg / ml, or between 1 mg / ml and 100 mg / ml of one or more antioxidants. In some embodiments, the cells are engineered in the presence of 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 pg / ml, 10 pg / ml, 100 pg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, 500 mg / ml, or about 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 pg / ml, 10 pg / ml, 100 pg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, 500 mg / ml of the one or more antioxidants. In some embodiments, the one or more antioxidants is or includes a sulfur-containing antioxidant. In particular embodiments, the one or more antioxidants is or includes a glutathione precursor.

[0338] In some embodiments, the cells are engineered in the presence of NAC. In some embodiments, the cells are engineered in the presence of between 1 ng / ml and 100 ng / ml, between 10 ng / ml and 1 pg / ml, between 100 ng / ml and 10 pg / ml, between 1 pg / ml and 100 pg / ml, between 10 pg / ml and 1 mg / ml, between 100 pg / ml and 1 mg / ml, between 1,500 pg / ml and 2 mg / ml, between 500 pg / ml and 5 mg / ml, between 1 mg / ml and 10 mg / ml, or between 1 mg / ml and 100 mg / ml of NAC. In some embodiments, the cells are engineered in the presence of at or at about 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 pg / ml, 10 pg / ml, 100 pg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, 500 mg / ml of NAC. In some embodiments, the cells are engineered with or with about 0.8 mg / ml.

[0339] In some embodiments, a composition of enriched T cells (such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells) is engineered in the presence of one or more polycations. In some embodiments, a composition of enriched T cells (such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells) is transduced, e.g., incubated with viral vector particles, in the presence of one or more polycations. In particular embodiments, a composition of enriched T cells (such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells) is transfected (e.g., incubated with a non-viral vector) with a non-viral vector in the presence of one or more polycations. In certain embodiments, the presence of one or more polycations increases the efficiency of gene delivery, e.g., by increasing the amount, fraction, and / or percentage of cells in the composition that are engineered (e.g., transduced or transfected). In certain embodiments, the presence of one or more polycations increases the efficiency of transfection. In certain embodiments, the presence of one or more polycations increases the efficiency of transduction. In particular embodiments, at least 25%, 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 at least 99% of the cells engineered in the presence of polycations contain or express a recombinant polynucleotide. In some embodiments, 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 150%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, or at least 100-fold more cells in the composition are engineered to contain or express a recombinant polynucleotide in the presence of polycations as compared to alternative and / or exemplary methods of engineering cells in the absence of polycations.

[0340] In certain embodiments, for example, a composition of enriched cells, e.g., a composition of enriched CD4+ T cells or enriched CD8+ T cells (such as stimulated T cells thereof), is engineered in the presence of a low concentration or amount of a polycation, e.g., relative to exemplary and / or alternative methods of engineering cells in the presence of a polyanion. In certain embodiments, a composition of enriched cells, such as stimulated T cells (e.g., stimulated CD4+ T cells or stimulated CD8+ T cells), is engineered in the presence of an amount or concentration of a polycation that is less than 90%, less than 80%, less than 75%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% of the amount of polycation used in an exemplary and / or alternative process for engineering cells. In some embodiments, a composition of enriched cells, such as stimulated T cells (e.g., stimulated CD4+ T cells or stimulated CD8+ T cells), is engineered in the presence of less than 100 µg / ml, less than 90 µg / ml, less than 80 µg / ml, less than 75 µg / ml, less than 70 µg / ml, less than 60 µg / ml, less than 50 µg / ml, less than 40 µg / ml, less than 30 µg / ml, less than 25 µg / ml, less than 20 µg / ml, or less than µg / ml, less than 10 µg / ml of the polycation. In particular embodiments, a composition of enriched cells, such as stimulated T cells (e.g., stimulated CD4+ T cells or stimulated CD8+ T cells), is engineered in the presence of 1 µg / ml, or about 1 µg / ml, 5 µg / ml, 10 µg / ml, 15 µg / ml, 20 µg / ml, 25 µg / ml, 30 µg / ml, 35 µg / ml, 40 µg / ml, 45 µg / ml, or 50 µg / ml of the polycation.

[0341] In particular embodiments, engineering the enriched cells, such as a composition of stimulated T cells (e.g., stimulated CD4+ T cells or stimulated CD8+ T cells), in the presence of a polycation reduces the amount of cell death (e.g., due to necrosis, programmed cell death, or apoptosis). In some embodiments, the composition of enriched T cells, such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, is engineered in the presence of a low amount of polycation (e.g., less than 100 µg / ml, 50 µg / ml, or 10 µg / ml), and at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the cells survive, e.g., do not undergo necrosis, programmed cell death, or apoptosis, during or for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days after completion of the engineering step. In some embodiments, the composition is engineered in the presence of a low concentration or amount of polycation, as compared to alternative and / or exemplary methods of engineering cells in the presence of a higher amount or concentration of polycation (e.g., more than 50 µg / ml, 100 µg / ml, 500 µg / ml, or 1,000 µg / ml), and the cells of the composition have at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, at least a 95%, at least a 100%, at least a 150%, at least a 1-fold, at least a 2-fold, at least a 3-fold, at least a 4-fold, at least a 5-fold, at least a 10-fold, at least a 25-fold, at least a 50-fold, or at least a 100-fold greater survival rate as compared to cells that undergo the exemplary and / or alternative processes.

[0342] In some embodiments, the polycation is positively charged. In certain embodiments, the polycation reduces the repulsion between a cell and a vector (e.g., a viral or non-viral vector), and mediates contact and / or binding of the vector to the cell surface. In some embodiments, the polycation is polybrene, DEAE-dextran, protamine sulfate, poly-L-lysine, or a cationic liposome.

[0343] In particular embodiments, the polycation is protamine sulfate. In some embodiments, the composition of enriched T cells (such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells) is engineered in the presence of less than or about 500 pg / ml, less than or about 400 pg / ml, less than or about 300 pg / ml, less than or about 200 pg / ml, less than or about 150 pg / ml, less than or about 100 pg / ml, less than or about 90 pg / ml, less than or about 80 pg / ml, less than or about 75 pg / ml, less than or about 70 pg / ml, less than or about 60 pg / ml, less than or about 50 pg / ml, less than or about 40 pg / ml, less than or about 30 pg / ml, less than or about 25 pg / ml, less than or about 20 pg / ml, or less than or about 15 pg / ml, or less than or about 10 pg / ml of protamine sulfate. In particular embodiments, the composition of enriched cells, such as stimulated T cells (e.g., stimulated CD4+ T cells or stimulated CD8+ T cells) is engineered in the presence of at or about 1 pg / ml, 5 pg / ml, 10 pg / ml, 15 pg / ml, 20 pg / ml, 25 pg / ml, 30 pg / ml, 35 pg / ml, 40 pg / ml, 45 pg / ml, 50 pg / ml, 55 pg / ml, 60 pg / ml, 75 pg / ml, 80 pg / ml, 85 pg / ml, 90 pg / ml, 95 pg / ml, 100 pg / ml, 105 pg / ml, 110 pg / ml, 115 pg / ml, 120 pg / ml, 125 pg / ml, 130 pg / ml, 135 pg / ml, 140 pg / ml, 145 pg / ml, or 150 pg / ml of protamine sulfate.

[0344] In some embodiments, the engineered composition of enriched CD4+ T cells, such as stimulated T cells (e.g., stimulated CD4+ T cells) comprises at least 40%, 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%, at least 99.5%, at least 99.9%, or is or is about 100% CD4+ T cells. In certain embodiments, the engineered composition of enriched CD4+ T cells, such as stimulated T cells (e.g., stimulated CD4+ T cells) comprises less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and / or is free of CD8+ T cells, and / or is free or substantially free of CD8+ T cells.

[0345] In some embodiments, the engineered composition of enriched CD8+ T cells, such as stimulated T cells (e.g., stimulated CD8+ T cells) comprises 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%, at least 99.5%, at least 99.9%, or is or is about 100% CD8+ T cells. In certain embodiments, the engineered composition of enriched CD8+ T cells, such as stimulated T cells (e.g., stimulated CD8+ T cells) comprises less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and / or is free of CD4+ T cells, and / or is free or substantially free of CD4+ T cells.

[0346] In some embodiments, engineering the cells comprises culturing, contacting, or incubating with a vector (e.g., a viral vector or a non-viral vector). In certain embodiments, the engineering comprises culturing, contacting, and / or incubating the cells with the vector for, for about, or for at least 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 30 hours, 36 hours, 40 hours, 48 hours, 54 hours, 60 hours, 72 hours, 84 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more than 7 days. In particular embodiments, the engineering comprises culturing, contacting, and / or incubating the cells with the vector for or for about 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, or 84 hours, or for or for about 2 days, 3 days, 4 days, or 5 days. In some embodiments, the engineering step is performed or performed for about 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, or 84 hours. In certain embodiments, the engineering is performed for about 60 hours or about 84 hours, for or for about 72 hours, or for or for about 2 days.

[0347] In some embodiments, the engineering is performed at a temperature from about 25ºC to about 38ºC, such as from about 30ºC to about 37ºC, from about 36ºC to about 38ºC, or at or at about 37ºC ± 2ºC. In some embodiments, the composition of enriched T cells is engineered at a CO2 level from about 2.5% to about 7.5%, such as from about 4% to about 6%, for example at or at about 5% ± 0.5%. In some embodiments, the composition of enriched T cells is engineered at a temperature at or at about 37ºC and / or at a CO2 level at or at about 5%.

[0348] In some embodiments, the cells are incubated after performing one or more steps for genetically engineering, e.g., transducing or transfecting, the cells (e.g., CD4+ and / or CD8+ T cells) to contain a polynucleotide encoding a recombinant receptor. In some embodiments, incubating can comprise culturing, incubating, stimulating, activating, expanding, and / or propagating. In some such embodiments, the further incubation is effected under conditions to allow integration of the viral vector into the host genome of the cell or cells. Incubating and / or engineering can be performed in a culture vessel, such as a unit, chamber, well, column, tube, tube set, valve, vial, dish, bag, or other container for culturing or incubating cells. In some embodiments, the composition or cells are incubated in the presence of a stimulating condition or stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime cells for genetic engineering, such as for introduction of a recombinant antigen receptor.

[0349] In some embodiments, the further incubation is performed at a temperature above room temperature, e.g., above or above about 25ºC, e.g., generally above or above about 32ºC, 35ºC, or 37ºC. In some embodiments, the further incubation is at a temperature at or at about 37ºC ± 2ºC, e.g., at or at about 37ºC.

[0350] In some embodiments, the further incubation is performed under conditions for stimulating and / or activating the cells, which can include one or more of the following: a particular medium, temperature, oxygen content, carbon dioxide content, time, agents (e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells).

[0351] In some embodiments, the stimulating conditions or stimulating agents include one or more agents (e.g., stimulatory and / or accessory agents) capable of activating the intracellular signaling domains of the TCR complex, e.g., a ligand. In some aspects, the agents turn on or initiate the TCR / CD3 intracellular signaling cascade in the T cells, e.g., are agents suitable for delivering a primary signal to initiate activation of ITAM-induced signals (e.g., those specific to TCR components), and / or agents that facilitate a costimulatory signal (e.g., a costimulatory signal specific to a T cell costimulatory receptor), e.g., anti-CD3, anti-CD28, or anti-41-BB (e.g., optionally bound to a solid support such as a bead) and / or one or more cytokines. The stimulating agents include anti-CD3 / anti-CD28 beads (e.g., DYNABEADS® M-450 CD3 / CD28 T cell expander and / or ExpACT® beads). Optionally, the expansion method can further include a step of adding anti-CD3 and / or anti-CD28 antibodies to the medium. In some embodiments, the stimulating agents include IL-2 and / or IL-15, e.g., an IL-2 concentration of at least about 10 units / mL.

[0352] In some embodiments, the stimulatory conditions or stimulatory agents include one or more agents (e.g., ligands) capable of activating the intracellular signaling domains of the TCR complex. In some aspects, the agents turn on or initiate the TCR / CD3 intracellular signaling cascade in the T cells. Such agents can include, for example, antibodies bound to a solid support (such as a bead), such as those specific for TCR components and / or costimulatory receptors (e.g., anti-CD3, anti-CD28); and / or one or more cytokines. Optionally, the expansion method can further include the step of adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulatory agents include IL-2 and / or IL-15, e.g., at a concentration of at least about 10 units / mL, at least about 50 units / mL, at least about 100 units / mL, or at least about 200 units / mL.

[0353] The conditions can include one or more of the following: a particular medium, temperature, oxygen content, carbon dioxide content, time, agents (e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agent intended to activate the cells).

[0354] In some aspects, the incubation is performed according to a variety of techniques, such as those described in U.S. Patent No. 6,040,177 to Riddell et al.; Klebanoff et al. (2012) J Immunother. 35(9): 651-660; Terakura et al. (2012) Blood. 1:72-82; and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0355] In some embodiments, the further incubation is performed in the same vessel or device in which the contacting is performed. In some embodiments, the further incubation is performed without rotation or centrifugation, typically after the at least a portion of the incubation that is performed under rotation (e.g., in combination with centrifugation or spinoculation). In some embodiments, the further incubation is performed outside of a stationary phase, e.g., outside of a chromatographic matrix, e.g., in solution.

[0356] In some embodiments, the further incubation is performed in a different vessel or device from the vessel or device in which the contacting is performed, e.g., by transferring (e.g., automatically transferring) the cell composition into a different vessel or device after contacting with the viral particles and reagents.

[0357] In some embodiments, the further culturing or incubation is for greater than or greater than about 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days, e.g., to facilitate ex vivo expansion. In some embodiments, the further culturing or incubation is for no more than 6 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days, or no more than 24 hours.

[0358] In some embodiments, the total duration of incubation, e.g., with a stimulatory agent, is at or between about 1 hour and 96 hours, 1 hour and 72 hours, 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours, or 12 hours and 24 hours, such as at least or about at least or about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours. In some embodiments, the further incubation is for a time at or about between 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours, or 12 hours and 24 hours, inclusive.

[0359] In some embodiments, the methods provided herein do not include further culturing or incubation, e.g., do not include an ex vivo expansion step, or include a significantly shorter ex vivo expansion step.

[0360] In some embodiments, the stimulatory agent is removed and / or isolated from the cells prior to engineering. In particular embodiments, the stimulatory agent is removed and / or isolated from the cells after engineering. In certain embodiments, the stimulatory agent is removed and / or isolated from the cells after engineering and prior to incubating the engineered cells, e.g., under conditions that promote proliferation and / or expansion. In certain embodiments, the stimulatory agent is a stimulatory agent described in Section I-B-1. In particular embodiments, the stimulatory agent is removed and / or isolated from the cells as described in Section I-B-2. 1. Vectors and Methods

[0361] In some embodiments, cells (e.g., T cells) are genetically engineered to express a recombinant receptor. In some embodiments, engineering is performed by introducing one or more polynucleotides encoding a recombinant receptor or a portion or component thereof. Also provided are polynucleotides encoding a recombinant receptor, as well as vectors or constructs containing such nucleic acids and / or polynucleotides.

[0362] In particular embodiments, the vector is a viral vector, a non-viral vector. In some cases, the vector is a viral vector, such as a retroviral vector, e.g., a lentiviral vector or a gammaretroviral vector.

[0363] In some embodiments, the polynucleotide encoding the recombinant receptor contains at least one promoter operably linked to control expression of the recombinant receptor. In some instances, the polynucleotide contains two, three, or more promoters operably linked to control expression of the recombinant receptor. In some embodiments, the polynucleotide can contain regulatory / control elements specific to the type of host (e.g., bacterial, fungal, plant, or animal) into which the polynucleotide will be introduced, as appropriate and taking into account whether the polynucleotide is DNA-based or RNA-based. In some embodiments, the polynucleotide can contain regulatory / control elements such as promoters, enhancers, introns, polyadenylation signals, kozak consensus sequences, internal ribosome entry sites (IRES), 2A sequences, and splice acceptors or donors. In some embodiments, the polynucleotide can contain a non-native promoter operably linked to the nucleotide sequence encoding the recombinant receptor and / or one or more additional polypeptides. In some embodiments, the promoter is selected from an RNA pol I, pol II, or pol III promoter. In some embodiments, the promoter is recognized by RNA polymerase II (e.g., CMV, SV40 early region, or adenovirus major late promoter). In another embodiment, the promoter is recognized by RNA polymerase III (e.g., U6 or H1 promoter). In some embodiments, the promoter can be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long terminal repeat of murine stem cell virus. Other known promoters are also contemplated.

[0364] In some embodiments, the promoter is or comprises a constitutive promoter. Exemplary constitutive promoters include, for example, the simian virus 40 early promoter (SV40), the cytomegalovirus immediate early promoter (CMV), the human ubiquitin C promoter (UBC), the human elongation factor 1 alpha promoter (EF1a), the mouse phosphoglycerate kinase 1 promoter (PGK), and the chicken beta-actin promoter coupled to the CMV early enhancer (CAGG). In some embodiments, the constitutive promoter is a synthetic or modified promoter. In some embodiments, the promoter is or comprises the MND promoter, which is a synthetic promoter containing a modified U3 region of the MoMuLV LTR with the myeloblastosis-associated virus enhancer (see Challita et al. (1995) J. Virol. 69(2):748-755). In some embodiments, the promoter is a tissue-specific promoter. In another embodiment, the promoter is a viral promoter. In another embodiment, the promoter is a non-viral promoter. In some embodiments, exemplary promoters can include, but are not limited to, the human elongation factor 1 alpha (EF1a) promoter or modified versions thereof or the MND promoter.

[0365] In another embodiment, the promoter is a regulated promoter (e.g., an inducible promoter). In some embodiments, the promoter is an inducible promoter or a repressible promoter. In some embodiments, the promoter comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, or a doxycycline operator sequence, or is an analog thereof or is capable of being bound or recognized by a Lac repressor or a tetracycline repressor or an analog thereof. In some embodiments, the polynucleotide does not include a regulatory element, e.g., a promoter.

[0366] In some cases, the nucleic acid sequence encoding a recombinant receptor (e.g., a chimeric antigen receptor (CAR)) contains a signal sequence that encodes a signal peptide. Non-limiting exemplary examples of signal peptides include a GMCSFRa chain signal peptide set forth in SEQ ID NO: 10 and encoded by the nucleotide sequence set forth in SEQ ID NO: 9, a CD8a signal peptide set forth in SEQ ID NO: 11, or a CD33 signal peptide set forth in SEQ ID NO: 12.

[0367] In some embodiments, the polynucleotide contains nucleic acid sequences encoding one or more additional polypeptides (e.g., one or more markers and / or one or more effector molecules). In some embodiments, the one or more markers include a transduction marker, a surrogate marker, and / or a resistance marker or selection marker. The additional nucleic acid sequences introduced, e.g., encoding one or more additional polypeptides, include nucleic acid sequences that can improve the efficacy of a therapy, e.g., by promoting the viability and / or function of the transferred cells; nucleic acid sequences that provide a genetic marker for selection and / or evaluation of cells, such as assessing in vivo survival or localization; nucleic acid sequences that increase safety, e.g., by making the cells susceptible to negative selection in vivo, as described in Lupton S. D. et al., Mol. and Cell Biol., 11 :6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also WO 1992008796 and WO 1994028143 (describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker), and U.S. Patent No. 6,040,177.

[0368] In some embodiments, the marker is a transduction marker or surrogate marker. A transduction marker or surrogate marker can be used to detect cells that have been introduced with a polynucleotide, e.g., a polynucleotide encoding a recombinant receptor. In some embodiments, a transduction marker can indicate or confirm modification of a cell. In some embodiments, a surrogate marker is a protein made to be co-expressed on the surface of a cell with a recombinant receptor, e.g., a CAR. In particular embodiments, such a surrogate marker is a surface protein that has been modified to have little or no activity. In certain embodiments, a surrogate marker is encoded by the same polynucleotide that encodes a recombinant receptor. In some embodiments, the nucleic acid sequence encoding a recombinant receptor is operably linked to the nucleic acid sequence encoding a marker, optionally separated by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosomal skipping, such as a 2A sequence. In some cases, an extrinsic marker gene can be associated with an engineered cell for allowing detection or selection of the cell, and in some cases, can also be used to facilitate cell elimination and / or cell suicide.

[0369] Exemplary surrogate markers can include truncated forms of a cell surface polypeptide, such as a truncated form that is non-functional and does not transduce or cannot transduce a signal or a signal that is normally transduced by a full-length form of the cell surface polypeptide, and / or does not internalize or cannot internalize. Exemplary truncated cell surface polypeptides include truncated forms of a growth factor or other receptor, such as a truncated human epidermal growth factor receptor 2 (tHER2), a truncated epidermal growth factor receptor (tEGFR, exemplary tEGFR sequences are set forth in SEQ ID NO: 2 or 3), or prostate specific membrane antigen (PSMA) or a modified form thereof (such as a truncated PSMA (tPSMA)). In some aspects, a tEGFR can contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibody or binding molecule, which can be used to identify or select cells that have been engineered with a tEGFR construct and encoded exogenous protein, and / or to eliminate or isolate cells expressing the encoded exogenous protein. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 Apr; 34(4): 430-434). In some aspects, a marker (e.g., surrogate marker) includes all or a portion (e.g., a truncated form) of CD34, NGFR, CD19, or a truncated CD19 (e.g., a truncated non-human CD19). Exemplary polypeptides of a truncated EGFR (e.g., tEGFR) include an amino acid sequence set forth in SEQ ID NO: 2 or 3 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 2 or 3.

[0370] In some embodiments, the marker is or comprises a detectable protein, such as a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP) (such as superfolder GFP (sfGFP)), red fluorescent protein (RFP) (such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed, or DsRed2), cyan fluorescent protein (CFP), blue-green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), and variants thereof, including species variants, monomeric variants, codon-optimized, stabilized, and / or enhanced variants of fluorescent proteins. In some embodiments, the marker is or comprises an enzyme (such as luciferase), the lacZ gene from E. coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT). Exemplary luminescent reporter genes include luciferase (luc), beta-galactosidase, chloramphenicol acetyltransferase (CAT), beta-glucuronidase (GUS), or variants thereof. In some aspects, expression of the enzyme can be detected by addition of a substrate that can be detected in accordance with expression and functional activity of the enzyme.

[0371] In some embodiments, the marker is a resistance marker or a selection marker. In some embodiments, the resistance marker or selection marker is or comprises a polypeptide that confers resistance to an exogenous agent or drug. In some embodiments, the resistance marker or ...

Claims

1. A method for evaluating T cell activation within a cellular composition, the method comprising: (a) Detecting the surface expression level of one or more markers or the percentage of cells positive for one or more markers in a composition containing T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein: Group (i) contains one or more markers selected from CD36L (SCARB1, SR-BI), CD120b, CD107b (LAMP-2), CD200 (OX2), CD357 (GITR), CD134 (OX40), CD83, CD155 (PVR), CD74, GPR56, Notch 1, CD119 (IFN-gRa chain), CD105 (endothelin), CD262 (DR5, TRAIL-R2), CD170 (Siglec-5), CD73 (extracellular 5'-nucleotidase), CD360 (IL-21R), CD20, CD107a (LAMP-1), CD109, CD132 (common g chain), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, binding agent), CD323 (JAM3), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD97, integrin b7, mouse IgG3k, Notch 2, CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, Ig light chain k, Ig light chain l, IgM, CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD266 (Fn14, TWEAK CD276 (B7-H3), CD326 (Ep-CAM), CD49e, CD87, CD8a, CD9, DcR1 (TRAIL-R3, CD263), ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (prions), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (neurofelin-1), CD49d, DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R); Furthermore, one or more markers in group (ii) are selected from CD192 (CCR2), CCRL2, CD96 (TACTILE), CD195 (CCR5), CD124 (IL-4Ra), CD49f, CD314 (NKG2D), KLRG1 (MAFA), CD96 (TACTILE), HLA-E, CD195 (CCR5), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CD11b, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR; (b) Compare the surface expression level or percentage of positive cells in the cell composition with the surface expression level or percentage of positive cells of each of the one or more markers in the reference, wherein a higher level or higher percentage of positive cells of the marker in (i) indicates that the T cells are activated compared with the reference, and a lower level or lower percentage of positive cells of the marker in (ii) indicates that the T cells are activated compared with the reference.

2. The method of claim 1, wherein the reference is composed of an unstimulated control cell composition.

3. The method of claim 1, wherein the reference is the expression level or percentage of positive cells across multiple cell compositions, wherein each cell composition is derived from a different patient, subject, or donor.

4. The method of claim 1, wherein the reference is the average expression level or average percentage of positive cells across multiple cell compositions, wherein each cell composition is derived from a different patient, subject, or donor.

5. The method of claim 1, wherein the reference is a median expression level or median percentage of positive cells across multiple cell compositions, wherein each cell composition is derived from a different patient, subject, or donor.

6. A method for evaluating the surface expression of T cell activation markers on T cells, the method comprising detecting the surface expression level of one or more markers or the percentage of cells positive for one or more markers in a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein: Group (i) contains one or more markers selected from CD36L (SCARB1, SR-BI), CD120b, CD107b (LAMP-2), CD200 (OX2), CD357 (GITR), CD134 (OX40), CD83, CD155 (PVR), CD74, GPR56, Notch 1, CD119 (IFN-gRa chain), CD105 (endothelin), CD262 (DR5, TRAIL-R2), CD170 (Siglec-5), CD73 (extracellular 5'-nucleotidase), CD360 (IL-21R), CD20, CD107a (LAMP-1), CD109, CD132 (common g chain), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, binding agent), CD323 (JAM3), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD97, integrin b7, mouse IgG3k, Notch 2, CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, Ig light chain k, Ig light chain l, IgM, CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD266 (Fn14, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD49e, CD87, CD8a, CD9, DcR1 (TRAIL-R3, CD263), ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (neurofelin-1), CD49d, DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R); and Group (ii) contains one or more markers selected from CD192 (CCR2), CCRL2, CD96 (TACTILE), CD195 (CCR5), CD124 (IL-4Ra), CD49f, CD314 (NKG2D), KLRG1 (MAFA), CD96 (TACTILE), HLA-E, CD195 (CCR5), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CD11b, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR.

7. The method of claim 6, wherein the surface expression level of one or more markers or the percentage of positive cells in (i) is positively correlated with T cell activation.

8. The method according to claim 6 or 7, wherein the surface expression level of one or more markers or the percentage of positive cells in (ii) is negatively correlated with T cell activation.

9. A method for comparing the activation of T cells in vivo, the method comprising: (a) Detecting the surface expression level of one or more markers or the percentage of cells positive for one or more markers in a composition containing T cells, wherein the one or more markers are selected from group (i) and / or group (ii).Group (i) consists of the following: CD36L (SCARB1, SR-BI), CD120b, CD107b (LAMP-2), CD200 (OX2), CD357 (GITR), CD134 (OX40), CD83, CD155 (PVR), CD74, GPR56, Notch 1, CD119 (IFN-gR a chain), CD105 (endothelin), CD262 (DR5, TRAIL-R2), CD170 (Siglec-5), CD73 (extracellular 5'-nucleotidase), CD360 (IL-21R), CD20, CD107a (LAMP-1), CD109, CD132 (common g chain), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, binding agent), CD323 (JAM3), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD97, integrin b7, mouse IgG3k, Notch 2, CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, Ig light chain k, Ig light chain l, IgM, CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD266 (Fn14, TWEAK CD276 (B7-H3), CD326 (Ep-CAM), CD49e, CD87, CD8a, CD9, DcR1 (TRAIL-R3, CD263), ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (prions), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (neurofelin-1), CD49d, DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R); Group (ii) consists of the following: CD192 (CCR2), CCRL2, CD96 (TACTILE), CD195 (CCR5), CD124 (IL-4Ra), CD49f, CD314 (NKG2D), KLRG1 (MAFA), CD96 (TACTILE), HLA-E, CD195 (CCR5), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CD11b, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR; (b) Compare the surface expression level or the percentage of positive cells with the level or percentage of each of the one or more markers in the unstimulated cell composition, wherein a higher level or higher percentage of positive cells of the marker in (i) indicates that the T cells are activated compared with the unstimulated cell composition, and a lower level or lower percentage of positive cells of the marker in (ii) indicates that the T cells are activated compared with the unstimulated cell composition.

10. The method according to any one of claims 1-9, wherein prior to the detection, the composition containing T cells is incubated with a T cell stimulator under conditions that induce T cell activation.

11. The method according to any one of claims 1-9, wherein the method comprises incubating the composition with a T-cell stimulant prior to the detection.

12. The method according to any one of claims 1-9, wherein the method comprises incubating the composition with a T-cell stimulant after the detection.

13. The method according to any one of claims 10-12, wherein the incubation with the T-cell stimulant is performed in the body of a subject.

14. The method according to any one of claims 10-12, wherein the incubation with the T cell stimulant is performed in vitro or ex vivo.

15. The method according to any one of claims 10-14, wherein the incubation with the T cell stimulant is performed for 12-72 hours.

16. The method according to any one of claims 10-15, wherein the incubation with the T cell stimulant is performed for about 24 hours.

17. The method according to any one of claims 1-16, wherein one or more markers of group (i) are selected from CD20, CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD105 (endothelin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (OX40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, binding agent), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, integrin b7, mouse IgG3k, Notch 1, Notch 2. CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain l, IgM, CD116, CD334 (FGFR4), CD66a / c / e and TSLPR (TSLP-R); Furthermore, one or more markers in group (ii) are selected from CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD172g (SIRPg), CD127 (IL-7Ra), CD49f, a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR.

18. The method according to any one of claims 10-15, wherein the incubation with the T cell stimulant is performed for about 48 hours.

19. The method according to any one of claims 1-15 and 18, wherein one or more markers of group (i) are selected from CD20, CD105 (endothelin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (OX40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, binding agent), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, integrin b7, mouse IgG3k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain l, IgM, CD119 (IFN-γ). a chain), CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Fn14, TWEAK CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcR1 (TRAIL-R3, CD263), ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD230 (prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (neurofelin-1), CD49d, CD73 (extracellular 5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), and GARP (LRRC32). Furthermore, one or more markers of group (ii) are selected from CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD11b, CX3CR1, NKp80, CD127 (IL-7Ra), and CD49f.

20. The method according to any one of claims 1-19, wherein one or more markers of group (i) are selected from CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM), and GPR56. Furthermore, one or more markers of group (ii) are selected from CD49f, CD124 (IL-4Rα), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Rα), CD11b, and CX3CR1.

21. The method according to any one of claims 1-20, wherein the one or more markers are (i) and selected from CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM), and GPR56.

22. A method for identifying activated T cells, the method comprising detecting cell surface expression of one or more markers in cells comprising a composition of T cells, wherein the one or more markers are selected from CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM), and GPR56, and wherein cells expressing high levels of the one or more markers are activated T cells.

23. The method according to any one of claims 1-22, wherein the one or more markers are selected from group (i) and consist of: CD200 (OX2), CD357 (GITR), CD120b, CD155 (PVR), CD107b (LAMP-2).

24. The method according to any one of claims 1-20, wherein the one or more markers are (ii) and selected from CD49f, CD124 (IL-4Rα), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Rα), CD11b and CX3CR1.

25. A method for identifying activated T cells, the method comprising detecting cell surface expression of one or more markers in cells comprising a composition containing T cells, wherein the one or more markers are selected from CD49f, CD124 (IL-4Rα), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Rα), CD11b, and CX3CR1, and wherein cells expressing low levels of the one or more markers are activated T cells.

26. The method according to any one of claims 1-25, wherein the detection is the detection of CD4+ or CD8+ T cells in the composition comprising T cells.

27. The method according to any one of claims 1-26, wherein the one or more markers from group (i) are selected from CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), CD355 (CRTAM), and GPR56. Furthermore, one or more markers from group (ii) are selected from CD49f, CD124 (IL-4Rα), CCRL2, CD217, CD192 (CCR2), CD96 (TACTILE), and CD195 (CCR5).

28. The method according to any one of claims 1-25, wherein the detection is the detection of CD4+ T cells in the composition comprising T cells.

29. The method of claim 28, wherein the one or more markers from group (i) are selected from CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, and CD165. And the one or more markers from group (ii) are selected from CD49f, CD124 (IL-4Rα), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE) and CD127 (IL-7Rα).

30. A method for evaluating the surface expression of T cell activation markers in CD4+ T cells, the method comprising detecting the surface expression level of one or more markers or the percentage of cells positive for one or more markers in CD4+ T cells comprising a composition of T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein group (i) comprises: CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2. CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154 and CD165, Group (ii) consists of the following: CD49f, CD124 (IL-4Rα), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), and CD127 (IL-7Rα).

31. The method of claim 30, wherein the surface expression level of one or more markers or the percentage of positive cells in said (i) is positively correlated with CD4+ T cell activation.

32. The method according to claim 30 or 31, wherein the surface expression level of one or more markers or the percentage of positive cells in said (ii) is negatively correlated with CD4+ T cell activation.

33. A method for assessing the activation of CD4+ T cells, the method comprising: (a) Detecting the surface expression level of one or more markers or the percentage of cells positive for one or more markers in CD4+ T cells comprising a composition containing T cells, wherein said one or more markers are selected from group (i) and / or group (ii), wherein group (i) consists of: CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, and CD165. And group (ii) consists of the following: CD49f, CD124 (IL-4Rα), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), and CD127 (IL-7Rα); and (b) Compare the surface expression level or the percentage of positive cells to the level or percentage of each of the one or more markers in the cells of an unstimulated control cell composition, wherein a higher level of the marker or a higher percentage of positive cells in (i) indicates that the CD4+ T cells are activated compared to the unstimulated control cell composition, and a lower level of the marker or a lower percentage of positive cells in (ii) indicates that the CD4+ T cells are activated compared to the unstimulated control cell composition.

34. The method according to any one of claims 1-25, wherein the detection is the detection of CD8+ T cells in the composition containing T cells.

35. The method of claim 34, wherein the one or more markers selected from group (i) are selected from CD120b, CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch 2. And the one or more markers from group (ii) are selected from CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CD11b, CX3CR1 and CD127 (IL-7Rα).

36. A method for evaluating the surface expression of T cell activation markers in CD8+ T cells, the method comprising detecting the surface expression level of one or more markers or the percentage of cells positive for one or more markers in CD8+ T cells comprising a composition of T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein group (i) comprises: CD120b, CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83 and Notch 2, And group (ii) consists of the following: CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CD11b, CX3CR1 and CD127 (IL-7Rα).

37. The method of claim 36, wherein the surface expression level of one or more markers or the percentage of positive cells in (i) is positively correlated with CD8+ T cell activation.

38. The method of claim 37, wherein the surface expression level of one or more markers or the percentage of positive cells in (ii) is negatively correlated with CD8+ T cell activation.

39. A method for assessing the activation of CD8+ T cells, the method comprising: (a) Detecting the surface expression level of one or more markers or the percentage of cells positive for one or more markers in CD8+ T cells comprising a composition of T cells, wherein said one or more markers are selected from group (i) and / or group (ii), wherein group (i) consists of: CD120b, CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch 2. And group (ii) consists of the following: CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CD11b, CX3CR1, and CD127 (IL-7Rα); and (b) Compare the surface expression level or the percentage of positive cells to the level or percentage of each of the one or more markers in the cells of an unstimulated control cell composition, wherein a higher level of the marker or a higher percentage of positive cells in (i) indicates that the CD8+ T cells are activated compared to the unstimulated control cell composition, and a lower level of the marker or a lower percentage of positive cells in (ii) indicates that the CD8+ T cells are activated compared to the unstimulated control cell composition.

40. The method according to any one of claims 1-39, wherein the composition comprising T cells comprises T cells genetically engineered to express a recombinant receptor.

41. The method of claim 27, wherein the one or more markers selected from group (i) consist of: CD120b, CD83, CD357 (GITR), CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD355 (CTRAM), CD155 (PVR), CD74, CD170 (Siglec-5) and GPR56, And the one or more markers selected from group (ii) consist of the following: CD49f, CCRL2, CD124 (IL-4Rα), CD217, CD192 (CCR2), CD195 (CCR5) and CD96 (TACTILE).

42. A method for assessing T cell activation, the method comprising: (a) Detecting the surface expression level of one or more markers or the percentage of cells positive for one or more markers in CD8+ T cells comprising a composition of T cells, wherein said one or more markers are selected from group (i) and / or group (ii), wherein group (i) consists of: CD120b, CD83, CD357 (GITR), CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD355 (CTRAM), CD155 (PVR), CD74, CD170 (Siglec-5), and GPR56. Group (ii) consists of the following: CD120b, CD83, CD357 (GITR), CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD355 (CTRAM), CD155 (PVR), CD74, CD170 (Siglec-5), and GPR56.

43. The method according to claim 41 or claim 42, wherein one or more markers from group (i) are selected from CD134 (OX40), CD107b (LAMP-2), CD155 (PVR), CD74 and CD170 (Siglec-5). Furthermore, the one or more markers selected from group (ii) are selected from CD49f, CCRL2, CD124 (IL-4Rα), CD217, CD355 (CRTAM), GPR56 and CD96 (TACTILE).

44. The method according to any one of claims 41-43, wherein the detection is the detection of recombinant receptor-expressing CD4+ T cells in the composition comprising T cells.

45. The method of claim 44, wherein the one or more markers from group (i) are selected from CD134 (OX40), CD107b (LAMP-2), CD155 (PVR), and CD74. And / or one or more markers selected from group (ii) are selected from CD49f, CCRL2 and CD124 (IL-4Rα).

46. ​​The method according to any one of claims 41-43, wherein the detection is the detection of recombinant receptor-expressing CD8+ T cells in the composition comprising T cells.

47. The method of claim 46, wherein the one or more markers from group (i) are selected from CD200 (OX2), CD107b (LAMP-2), CD155 (PVR), CD355 (CRTAM), and GPR56. And / or one or more markers selected from group (ii) are selected from CCRL2, CD217, CD96 (TACTILE).

48. The method according to any one of claims 1-16, wherein one or more markers of group (i) are selected from CD36L (SCARB1, SR-BI), CD262 (DR5, Trail-R2), CD105 (endothelial linkerin), CD73 (extracellular 5'-nucleotidase), CD83, CD119 (IFN-gRa chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R). Furthermore, one or more markers of group (i) are selected from CD96 (TACTILE).

49. A method for assessing T cell activation, the method comprising: (a) Detecting the surface expression level of one or more markers in a T cell composition or the percentage of cells positive for one or more markers, said T cell composition comprising T cells expressing a recombinant receptor, wherein said one or more markers are selected from group (i) and / or group (ii), wherein group (i) consists of: CD36L (SCARB1, SR-BI), CD262 (DR5, Trail-R2), CD105 (endothelin), CD73 (extracellular 5'-nucleotidase), CD83, CD119 (IFN-γ Ra chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R). Furthermore, group (ii) consists of CD96 (TACTILE).

50. The method of claim 48, wherein the composition comprising T cells comprises cells expressing a recombinant receptor.

51. The method of claim 49 or claim 50, wherein the detection is the detection of recombinant receptor-expressing cells of the T-cell composition.

52. The method according to any one of claims 49-51, wherein, on cells expressing the recombinant receptor, the surface expression of one or more markers of group (i) is increased compared with cells not expressing the recombinant receptor.

53. The method according to any one of claims 49-52, wherein, on cells expressing the recombinant receptor, the surface expression of one or more markers of group (ii) is reduced compared with cells not expressing the recombinant receptor.

54. The method according to any one of claims 3-47, wherein the composition comprising T cells comprises T cells genetically engineered to express a recombinant receptor, and wherein the T cell stimulant is a recombinant receptor stimulant that induces recombinant receptor-dependent T cell activation.

55. The method according to any one of claims 27-54, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

56. The method of claim 54 or claim 55, wherein the recombinant receptor stimulator comprises a recombinant target antigen recognized by the recombinant receptor.

57. The method of claim 54 or claim 55, wherein the recombinant receptor stimulator is an antibody specific to the extracellular antigen-binding domain of the recombinant receptor.

58. The method of claim 54 or claim 55, wherein the recombinant receptor stimulator is an anti-idiotype antibody specific for the extracellular antigen-binding domain of the recombinant receptor.

59. The method according to any one of claims 54-58, wherein the recombinant receptor stimulant is fixed or attached to a solid support.

60. The method of claim 59, wherein the solid support is the surface of a vessel, optionally a hole in a microplate or a flask.

61. The method of claim 59, wherein the solid support is a bead.

62. The method of claim 54 or claim 55, wherein the recombinant receptor stimulant is an antigen-expressing cell, optionally wherein the cell is a clone, derived from a cell line, or a primary cell taken from the subject.

63. The method of claim 62, wherein the antigen-expressing cell is a cell line.

64. The method of claim 63, wherein the cell line is a tumor cell line.

65. The method of claim 63, wherein the antigen-expressing cell is a cell that has been engineered to express the antigen of the recombinant receptor.

66. The method according to any one of claims 27-65, wherein the detection is the detection of recombinant receptor-expressing T cells in the composition comprising T cells.

67. The method according to any one of claims 1-26, wherein the one or more markers from group (i) are selected from CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, CD165, CD120b, CD83, CD357 (GITR), CD200 (OX2), and CD134 (OX40). And / or one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CD192 (CCR2), CD11b and CX3CR1.

68. The method according to any one of claims 1-26, wherein the one or more markers from group (i) are selected from CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, CD165, and CD83. And / or one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CD11b and CX3CR1.

69. The method according to claim 67 or 68, wherein the detection is the detection of CD4+ T cells in the composition containing T cells.

70. The method of claim 69, wherein the one or more markers from group (i) are selected from CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, and CD165. And / or one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE) and CD127 (IL-7Ra).

71. The method of claim 67 or 68, wherein the detection is the detection of CD8+ T cells in the composition containing T cells.

72. The method of claim 71, wherein the one or more markers from group (i) are selected from CD71, Notch 1, CD107a (LAMP-1), CD166, Notch 2, CD165, and CD83. And / or one or more markers from group (ii) are selected from CD11b, CX3CR1 and CD127 (IL-7Ra).

73. The method according to any one of claims 67-72, wherein the T cell stimulant is a pan-T cell activator.

74. The method of claim 73, wherein the pan-T cell activating agent comprises an anti-CD3 antibody and an anti-CD28 antibody, optionally wherein the pan-T cell activating agent comprises anti-CD3 Fab and anti-CD28 Fab.

75. The method of claim 73 or claim 74, wherein the pan-T cell activating agent comprises anti-CD3 / anti-CD28 beads.

76. The method of claim 73 or claim 74, wherein the pan-T cell activating agent comprises a soluble anti-CD3 / anti-CD28 streptavidin oligomer.

77. The method according to any one of claims 1-76, wherein prior to the detection in step (a), the method comprises contacting the cells comprising the composition containing T cells with one or more binding agents that bind the one or more markers.

78. The method according to any one of claims 1-76, wherein prior to the detection in step (a), the method comprises contacting the cells comprising the composition containing T cells with one or more binding agents, the one or more binding agents comprising substances for binding the one or more markers.

79. The method of claim 77 or claim 78, wherein the one or more binding agents are one or more antibody or antigen-binding fragments.

80. The method according to any one of claims 77-79, wherein the one or more binders are detectably labeled.

81. The method of claim 80, wherein one or more binders are fluorescently labeled.

82. The method according to any one of claims 1-81, wherein the detection is performed by flow cytometry.

83. The method according to any one of claims 1-81, wherein the detection in step (a) is performed in conjunction with CITE-Seq or REAP-seq.

84. The method according to any one of claims 1-81, wherein the detection in step (a) is performed by immunohistochemistry, optionally by immunohistochemical fluorescence.

85. The method according to any one of claims 1-84, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 different markers are used for detection in step (a).

86. A kit for determining T cell activation, the kit comprising a binding agent comprising a substance for binding one or more markers in the method according to any one of claims 1-85.

87. The kit according to claim 86, wherein the substance used to detect each of the one or more markers is an antibody.

88. A kit for determining T cell activation, the kit comprising a binding agent containing a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) comprises: CD36L (SCARB1, SR-BI), CD120b, CD107b (LAMP-2), CD200 (OX2), CD357 (GITR), CD134 (OX40), CD83, CD155 (PVR), CD74, GPR56, Notch 1, CD119 (IFN-gRa chain), CD105 (endothelin), CD262 (DR5, TRAIL-R2), CD170 (Siglec-5), CD73 (extracellular 5'-nucleotidase), CD360 (IL-21R), CD20, CD107a (LAMP-1), CD109, CD132 (common g chain), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, binding agent), CD323 (JAM3), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD97, integrin b7, mouse IgG3k, Notch 2, CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, Ig light chain k, Ig light chain l, IgM, CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD266 (Fn14, TWEAK CD276 (B7-H3), CD326 (Ep-CAM), CD49e, CD87, CD8a, CD9, DcR1 (TRAIL-R3, CD263), ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (neurofelin-1), CD49d, DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R). Group (ii) consists of the following: CD192 (CCR2), CCRL2, CD96 (TACTILE), CD195 (CCR5), CD124 (IL-4Ra), CD49f, CD314 (NKG2D), KLRG1 (MAFA), CD96 (TACTILE), HLA-E, CD195 (CCR5), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CD11b, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR.

89. A kit for determining T cell activation, the kit comprising a binding agent containing a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) comprises: CD20, CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD105 (endothelin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (OX40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, binding agent), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, integrin b7, mouse IgG3k, Notch 1, Notch 2. CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain l, IgM, CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R). Group (ii) consists of the following: CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD172g (SIRPg), CD127 (IL-7Ra), CD49f, a / b T cell receptor, CD229 (Ly-9), CD84, and EGFR.

90. A kit for determining T cell activation, the kit comprising a binding agent containing a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) comprises: CD20, CD105 (endothelin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (OX40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, bindingins), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, integrin b7, mouse IgG3 k, Notch 1, Notch 2. CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain l, IgM, CD119 (IFN-gRa chain), CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Fn14, TWEAK) R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcR1 (TRAIL-R3, CD263), ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD230 (prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (neurofelin-1), CD49d, CD73 (extracellular 5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32). Group (ii) consists of the following: CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD11b, CX3CR1, NKp80, CD127 (IL-7Ra), and CD49f.

91. A kit for determining T cell activation, the kit comprising a binding agent containing a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) comprises: CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2. CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM) and GPR56, Group (ii) consists of the following: CD49f, CD124 (IL-4Rα), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Rα), CD11b, and CX3CR1.

92. A kit for determining T cell activation, the kit comprising a binding agent containing a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) comprises: CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), CD355 (CRTAM) and GPR56, And group (ii) consists of the following: CD49f, CD124 (IL-4Rα), CCRL2, CD217, CD192 (CCR2), CD96 (TACTILE) and CD195 (CCR5).

93. A kit for determining T cell activation, the kit comprising a binding agent containing a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) comprises: CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (OX40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2. CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154 and CD165, Group (ii) consists of the following: CD49f, CD124 (IL-4Rα), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), and CD127 (IL-7Rα).

94. A kit for determining T cell activation, the kit comprising a binding agent containing a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) comprises: CD120b, CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83 and Notch 2, And group (ii) consists of the following: CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CD11b, CX3CR1 and CD127 (IL-7Rα).

95. A kit for determining T cell activation, the kit comprising a binding agent containing a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) comprises: CD120b, CD83, CD357 (GITR), CD200 (OX2), CD134 (OX40), CD107b (LAMP-2), CD155 (PVR), CD74, And the one or more markers selected from group (ii) consist of the following: CD49f, CCRL2, CD124 (IL-4Rα), CD217, CD192 (CCR2), CD355 (CRTAM), GPR56, CD195 (CCR5) and CD96 (TACTILE).

96. A kit for determining T cell activation, the kit comprising a binding agent containing a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) comprises: CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, CD165, CD120b, CD83, CD357 (GITR), CD200 (OX2) and CD134 (OX40), And / or one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CD192 (CCR2), CD11b and CX3CR1.

97. A kit for determining T cell activation, the kit comprising a binding agent containing a substance for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) comprises: CD36L (SCARB1, SR-BI), CD262 (DR5, Trail-R2), CD105 (endothelin), CD73 (extracellular 5'-nucleotidase), CD83, CD119 (IFN-γ Ra chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R). And / or one or more markers from group (ii) are selected from CD96 (TACTILE).

98. The kit according to any one of claims 86-97, wherein the binder is one or more antibody or antigen-binding fragments.

99. The kit according to any one of claims 86-98, wherein the binder is detectably labeled.

100. The kit of claim 99, wherein the binder is fluorescently labeled.

101. A method for isolating activated T cells, the method comprising identifying a population of activated T cells and isolating the population according to any one of claims 1-85.

102. A method for enriching activated T cells, the method comprising identifying a population of activated T cells and selecting the population according to any one of claims 1-85, thereby obtaining a cell population rich in activated T cells.

103. A method for depleting a population of activated T cells, the method comprising identifying a population of activated T cells and depleting the population of activated T cells according to any one of claims 1-85.

104. A population of T cells generated by the method according to any one of claims 101-103.

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